WO2024145451A2 - Methods for restoring regenerative potential of aged lung alveoli and aged adult stem cell compartments - Google Patents

Abstract

The present disclosure provides methods for restoring the regenerative potential of aged lung alveoli or aged adult stem cell compartments in a subject in need thereof comprising administering to the subject an effective amount of an inhibitor of NUPR1/LCN2-2 axis or iron/transferrin. In some embodiments, the subject is diagnosed with or suffers from a lung insufficiency such as COPD, COVID, influenza, and pneumonia sequelae. Also disclosed herein are methods for preventing or treating lung cancer in young (non-aged) subjects comprising administering an inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent.

Description

METHODS FOR RESTORING REGENERATIVE POTENTIAL OF AGED LUNG ALVEOLI AND AGED ADULT STEM CELL COMPARTMENTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/435,914, filed December 29, 2022, the contents of which are incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure generally relates to methods for restoring the regenerative potential of aged lung alveoli or aged adult stem cell compartments in a subject in need thereof comprising administering to the subject an effective amount of an inhibitor of NUPR1/LCN2-2 axis or iron/transferrin. In some embodiments, the subject is diagnosed with or suffers from a lung insufficiency such as COPD, COVID, influenza, and pneumonia sequelae. Also disclosed herein are methods for preventing or treating lung cancer in young (non-aged) subjects comprising administering an inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent.

GOVERNMENT SUPPORT

[0003] This invention was made with government support under CA008748 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] Aging is the most important risk factor for cancer development¹. However, overall cancer incidence begins to decline after 75-85 years of age, with the median age of decline depending on cancer type². The dramatic increase in cancer incidence with aging has been attributed to accumulation of somatic mutations¹, yet the reasons underpinning the decline in cancer incidence in the very aged population are poorly understood. Studies employing transplantation of syngeneic cancer cell lines into aged vs. young mice have greatly advanced our understanding of changes in the aged host tumor microenvironment, which have pronounced effects on tumor growth and progression¹¹. For example, the aged lung microenvironment facilitates a permissive niche for efficient outgrowth of dormant disseminated melanoma cells. This is in contrast to the skin primary site where age-related changes suppress melanoma growth but drive dissemination¹². Although powerful, the transplantation-based approaches are biased towards investigation of advanced tumors and microenvironmental effects. As such, our understanding of how aging impacts the early stages of tumorigenesis remains limited. In particular, how the age of the cell of origin impacts tumor initiation and progression has been little studied.

[0006] Cancer predominantly arises from tissue stem cells or progenitors¹³. Aging is associated with reduced number and output of tissue stem cells³'⁶. Telomere attrition, genomic instability, cellular senescence, dysregulated cell-cell communication, and epigenetic alterations are “hallmarks of aging” that, among multiple effects in tissues, degrade sternness^7,8 - the potential of stem cells to proliferate and differentiate. Among the molecular hallmarks of aging, stereotypic changes in DNA methylation patterns involving global hypomethylation of CpGs and focal hypermethylation of CpG islands at gene promoters represent the most ubiquitous biomarker of aging across species and cell types⁸. A recent in vitro study demonstrated that aged colon epithelial cells undergo methylation of tumor suppressor gene promoters more rapidly than young cells, rendering the aged cells more susceptible for transformation¹⁴. However, whether the aging-associated loss of sternness and changes in DNA methylation impact the tumorigenic potential of epithelial stem cells, the origin of the majority of human cancers, has not been investigated in vivo.

SUMMARY OF THE PRESENT TECHNOLOGY

[0007] In one aspect, the present disclosure provides a method for restoring regenerative potential (sternness) of alveolar stem cells in a subject in need thereof comprising administering to the subject an effective amount of a Nuclear Protein 1 (NUPR1) inhibitor or a lipocalin-2 (LCN2) inhibitor. In some embodiments, the subject is diagnosed with or suffers from a lung insufficiency. Examples of lung insufficiency include, but are not limited to, COPD (chronic obstructive pulmonary disease), cystic fibrosis, pneumonia, pulmonary embolism, COVID-19; conditions that affect the nerves and muscles that control breathing, such as amyotrophic lateral sclerosis (ALS), muscular dystrophy, spinal cord injuries, and stroke; problems with the spine, such as scoliosis (a curve in the spine); damage to the tissues and ribs around the lungs; drug or alcohol overdose; or inhalation injuries, such as from inhaling smoke (from fires) or harmful fumes. The subject may be an immunocompromised subject or a geriatric subject.

[0008] In one aspect, the present disclosure provides a method for preventing or treating lung cancer in a subject in need thereof comprising administering to the subject an effective amount of a Nuclear Protein 1 (NUPR1) inhibitor or a lipocalin-2 (LCN2) inhibitor, wherein the subject is a pediatric subject, an adolescent subject, or a young adult subject (e.g., < 55 years old). Also disclosed herein is a method for preventing or treating lung cancer in a subject in need thereof comprising administering to the subject an effective amount of a ferroptosis-inducing agent, wherein the subject is a pediatric subject, an adolescent subject, or a young adult subject (e.g., < 55 years old). In any and all embodiments of the methods disclosed herein, the young adult subject is about 54, about 53, about 52, about 51, about 50, about 49, about 48, about 47, about 46, about 45, about 44, about 43, about 42, about 41, about 40, about 39, about 38, about 37, about 36, about 35, about 34, about 33, about 32, about 31, about 30, about 29, about 28, about 27, about 26, about 25, about 24, about 23, about 22, about 21, or about 20 years old. The ferroptosis-inducing agent may be a class 1 ferroptosis inducer (system X_c“ inhibitor) or a class 2 ferroptosis inducer (glutathione peroxidase 4 (GPx4) inhibitor). Examples of ferroptosis-inducing agents include, but are not limited to, an inhibitory nucleic acid that targets GPX4, erastin, erastin derivatives (e.g., MEII, PE, AE, imidazole ketone erastin (IKE)), DPI2, BSO, SAS, lanperisone, SRS13-45, SRS13-60, RSL3, DPI7, DPI10, DPI12, DPI13, DPI17, DPI18, DPI19, ML160, sorafenib, artemisinin derivatives, artesunate, BAY87-2243, cisplatin, ironomycin, lanperisone, salinomycin, sulfasalazine, temozolomide, and lapatinib in combination with siramesine. In any of the preceding embodiments of the methods disclosed herein, the lung cancer is lung adenocarcinoma (LU AD). In certain embodiments, the lung cancer comprises a mutation in KRAS or TP 53.

[0009] Additionally or alternatively, in some embodiments of the methods disclosed herein, the NUPR1 inhibitor is a small molecule, an NUPR1 -specific inhibitory nucleic acid, or an anti-NUPRl neutralizing antibody. In certain embodiments, the NUPR1 inhibitor is trifluoperazine (TFP), ZZW-115, ZZW-129, ZZW-130, ZZW-131, ZZW-132, ZZW-142, ZZW-143, ZZW-144, ZZW-145, or ZZW-148.

[0010] Additionally or alternatively, in certain embodiments of the methods disclosed herein, the LCN2 inhibitor is a small molecule, an LCN2-specific inhibitory nucleic acid, or an anti- LCN2 neutralizing antibody. In some embodiments, the LCN2 inhibitor is ZINC00784494, ZINC00640089, ZINC00230567, or ZINC00829534.

[0011] In any and all embodiments of the methods disclosed herein, the NUPR1 -specific inhibitory nucleic acid, the LCN2-specific inhibitory nucleic acid or the inhibitory nucleic acid that targets GPX4 is a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA.

[0012] In another aspect, the present disclosure provides a method for restoring regenerative potential (sternness) of alveolar stem cells in a subject in need thereof comprising administering to the subject an effective amount of iron or transferrin. In some embodiments, the iron is unbound iron. In other embodiments, the iron is bound to transferrin or is complexed with vitamin C. In some embodiments, the subject is diagnosed with or suffers from a lung insufficiency. Examples of lung insufficiency include, but are not limited to, COPD (chronic obstructive pulmonary disease), cystic fibrosis, pneumonia, pulmonary embolism, COVID-19; conditions that affect the nerves and muscles that control breathing, such as amyotrophic lateral sclerosis (ALS), muscular dystrophy, spinal cord injuries, and stroke; problems with the spine, such as scoliosis (a curve in the spine); damage to the tissues and ribs around the lungs; drug or alcohol overdose; or inhalation injuries, such as from inhaling smoke (from fires) or harmful fumes. The subject may be an immunocompromised subject or a geriatric subject.

[0013] In one aspect, the present disclosure provides a method for restoring regenerative potential (sternness) of aged adult stem cell compartments in a subject comprising administering to the subject an effective amount of a Nuclear Protein 1 (NUPR1) inhibitor or a lipocalin-2 (LCN2) inhibitor. In another aspect, the present disclosure provides a method for restoring regenerative potential (sternness) of aged adult stem cell compartments in a subject comprising administering to the subject an effective amount of iron or transferrin. Additionally or alternatively, in some embodiments, the aged adult stem cell compartments comprise one or more of neural adult subventricular zone (SVZ) stem cells, hair follicle stem cells, mesenchymal stem cells, keratinocyte stem cells, or intestinal stem cells.

[0014] Additionally or alternatively, in some embodiments of the methods disclosed herein, the NUPR1 inhibitor is a small molecule, an NUPR1 -specific inhibitory nucleic acid, or an anti-NUPRl neutralizing antibody. In certain embodiments, the NUPR1 inhibitor is trifluoperazine (TFP), ZZW-115, ZZW-129, ZZW-130, ZZW-131, ZZW-132, ZZW-142, ZZW-143, ZZW-144, ZZW-145, or ZZW-148.

[0015] Additionally or alternatively, in certain embodiments of the methods disclosed herein, the LCN2 inhibitor is a small molecule, an LCN2-specific inhibitory nucleic acid, or an anti- LCN2 neutralizing antibody. In some embodiments, the LCN2 inhibitor is ZINC00784494, ZINC00640089, ZINC00230567, or ZINC00829534.

[0016] Additionally or alternatively, in certain embodiments of the methods disclosed herein, the iron is unbound iron. In other embodiments, the iron is bound to transferrin or is complexed with vitamin C.

[0017] In any and all embodiments of the methods described herein, the NUPR1 inhibitor, LCN2 inhibitor, ferroptosis-inducing agent, iron and/or transferrin is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, iontophoretically, transmucosally, or intramuscularly. In any of the preceding embodiments of the methods described herein, the subject is human.

[0018] In another aspect, the present disclosure provides kits for the prevention and/or treatment of lung cancer in young (non-aged) subjects, comprising one or more inhibitors of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agents disclosed herein. Also provided herein are kits for restoring regenerative potential (sternness) of aged alveolar stem cells/aged adult stem cell compartments in a subject comprising one or more of iron, transferrin, or an inhibitor of NUPR1/LCN2-2 axis. In some embodiments, the subject is diagnosed with or suffers from a lung insufficiency. Examples of lung insufficiency include, but are not limited to, COPD (chronic obstructive pulmonary disease), cystic fibrosis, pneumonia, pulmonary embolism, COVID-19; conditions that affect the nerves and muscles that control breathing, such as amyotrophic lateral sclerosis (ALS), muscular dystrophy, spinal cord injuries, and stroke; problems with the spine, such as scoliosis (a curve in the spine); damage to the tissues and ribs around the lungs; drug or alcohol overdose; or inhalation injuries, such as from inhaling smoke (from fires) or harmful fumes. The subject may be an immunocompromised subject or a geriatric subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGs. 1A-1H: Aged AT2 cells exhibit reduced cell-intrinsic potential for proliferation and tumorigenesis. FIG. 1A: Experimental scheme. KP: Kraslox-stop- lox(LSL)-G12D/+;Trp53fl/fl„ KPT: KrasLSL-^G12D/+ ;Trp53ff-;Rosa26^LSL~^tdTomato/+, KP-RIK: Kra^^jSL~^G12D/+;Trp53 f;Rosa26^LSL~^rtTA3~^IRES~^mKate/+. SPC: surfactant protein-C gene promoter. FIG. IB: Survival of aged vs. young KP LU AD mice post-tumor initiation (p = 0.0007, logrank test), n = 7 aged mice and n = 13 young mice. FIG. 1C: Representative images of hematoxylin-eosin staining of tumor-bearing lungs from aged and young KP mice 17 weeks post-tumor initiation. Scale bars: 1 mm (left panel) and 50 pm (right panel). FIG. ID: Fold change of tumor burden in aged vs. young KP mice at 4, 8, 12, and 17 weeks post-tumor initiation. N= 4, 3, 3, and 8 young mice and 3, 3, 3, and 10 aged mice at 4, 8, 12, and 17 weeks, respectively. FIG. IE: Proliferation of AT2 cells during homeostasis (baseline) and alveolar regeneration in response to hyperoxia damage at days 0, 3, and 10 following 48-hour exposure to hyperoxia. Representative images of proliferating (Ki67+, green) AT2 cells (SPC+, red) are shown on the right. N= 3, 3, 3, and 4 young mice and 2, 3, 3 and 3 aged mice at baseline, day 0, 3 and 10 post-hyperoxia. Scale bar: 20 pm. FIG. IF: Schematic representation of alveolar organoid formation assay (left). Primary and secondary alveolar organoid formation derived from aged vs. young primary AT2 cells (right). N= 15 replicates from 3 young and aged mice for primary organoid passage and n = 5 and 4 replicates from 3 young and aged mice for secondary organoid passage. Scale bar: 100 pm. FIG. 1G: Schematic summary of ex vivo transformation assay using primary AT2 cells isolated from KP-Cas9 (Kra^^SL~^G12D/+ TrpSSff; Rosa26LSL-Cas9-2a-GFP/+) mice. Gray background in plot indicates alveolar organoid formation efficiency between aged vs. young KP-Cas9 AT2 cells without Cre recombinase; green background indicates number of GFP+ tumor organoids transformed by lentiviral delivery of Cre recombinase. Representative images of organoids are shown on the right; arrowheads indicate transformed organoids. PGK: ubiquitously active human phosphoglycerate kinase- 1 promoter. Scale bar: 200 pm. (n = 8 technical replicates from 3 young and 3 aged mice). FIG. 1H: Transformation efficiency of aged vs. young KP- Cas9 AT2 cells calculated as the ratio of GFP+ transformed organoids/non-transformed alveolar organoids (n = 8 technical replicates from 3 young and 3 aged mice). Mean with SD is shown in FIG. IE. Y: young, A: aged. *p < 0.05; **p < 0.01; and ***p < 0.001. Student’s /-test was used in FIGs. 1D-1H).

[0020] FIGs. 2A-2L: Delayed histological and molecular progression of lung tumors in aged mice. FIG. 2A: Quantification of KP LU AD tumor size in aged vs. young mice at 4, 8, 12, and 17 weeks post-tumor initiation. Number of cancer cells per tumor nodule was quantified in KPT mice using tdTomato to mark cancer cells at the 4 and 8 week time points; tumor diameter was quantified in KP mice from HE stained sections for at the 12 and 17 week time points. N= 109, 60, 371, and 358 tumors from young mice and n = 95, 66, 121, and 144 tumors from aged mice at 4, 8, 12, and 17 weeks. FIG. 2B: Quantification of proliferating tumor cells at different stages of tumor development. The proportion of Ki67 positive cells per total tumor cells was calculated and normalized to the average of young tumors at the corresponding time point. N= 55, 62, 127, and 306 tumors from young mice and n = 32, 62, 25, and 237 tumors from aged mice at 4, 8, 12 and 17 weeks. FIG. 2C: Histopathological grade (G) of lung tumors at 12 and 17 weeks post-tumor initiation, as quantified by Aiforia digital pathology algorithm (FIG. 9D). The proportion of G1-G4 tumor area per total tumor area is shown. FIG. 2D: Uniform manifold approximation projection (UMAP) embedding of LU AD single-cell transcriptomes isolated from young and aged KP tumors labeled based on previously defined transcriptionally distinct subsets²³, at 4, 12, and 17 weeks post-tumor initiation. The numbers indicate order of cell state progression⁷⁷. WT AT2: wild-type AT2 cells, the cell of origin (“0”). FIG. 2E: MetaCell neighborhoods, groups of similar cells representing discrete cell states⁶³, of 4-week-old KP LU AD tumors projected onto the UMAP introduced in FIG. 2D. Circle size corresponds to the number of cells, while color signifies the fraction of young and aged cells forming a MetaCell neighborhood (blue: enriched in young; red: enriched in aged). Note enrichment of young cancer cells at the high-plasticity cell state (HPCS, dashed blue oval). Validation of higher proportion of young cancer cells in HPCS by immunofluorescence for the HPCS marker integrin a2 (green) at 4 weeks post-tumor initiation. tdTomato (red) marks cancer cells. N = 54 and 125 tumors from 3 young and 3 aged mice, respectively. Scale bar: 50 pm. FIG. 2F: MetaCell analysis of KP LU AD tumors at 17 weeks post-tumor initiation. Note enrichment of young cancer cells at the endoderm-like state (dashed blue circle), which is validated by immunohistochemical staining for the endoderm-like state marker HNF4a (brown). The proportion of tumors containing >5% HNF4a+ cells per the total numbers of tumors is shown. N= 10 young and 11 aged tumor-bearing mice. Scale bar: 100 pm. FIG. 2G: Schematic summary of experiment to evaluate molecular progression of KP LU AD cells in vitro. Briefly, AT2 cells were isolated from aged vs. young KP-Cas9 mice and transformed in vitro by lentiviral Cre recombinase (P0, FIG. 1G). Bulk mRNA sequencing (RNA-seq) was performed over eight organoid passages (P1-P8). FIG. 2H: Projection of bulk RNA-seq data from ex vivo transformed young and aged AT2 cells in diffusion pseudotime space. Upper panel: samples colored according to diffusion pseudotime using untransformed AT2 cells as the starting point (0). Lower panels: samples colored based on age (blue: young; aged: red). Note enrichment of young tumor spheres at later pseudotime points (blue dashed oval), whereas aged spheres are enriched at the midway point (dashed red circle). FIG. 21: Boxplots showing diffusion pseudotime distribution of young (blue) and aged (red) ex vivo transformed cells stratified according to passage (P). Individual samples are represented by black dots (p = 0.0014, Wilcoxon ranked sum test). N= 5 young and 5 aged ex vivo transformed tumor sphere lines. FIG. 2J: Heatmap showing the mean expression of AT2 marker genes in young and aged ex vivo transformed cells stratified according to passage. The colormap shows the log2-fold change compared to sample mean for each gene harmonized for young and aged samples. FIG. 2K: Pearson correlation between the model-based analysis of single-cell transcriptomics (MAST) differentially expressed genes (DEGs) coefficient⁶³ aged vs. young in normal AT2 cells (x-axis) and all LU AD cells (y-axis). The inset shows the percentage of genes in each quadrant, which is significantly different from random (binomial test p < 2.2’ ¹⁶). The regression line (blue) and its confidence interval (light grey) were plotted using a linear model. FIG. 2L: Heatmap displaying the MAST DEG coefficient of the top 25 most consistently upregulated and downregulated genes across normal and tumor cell types. The top 25 upregulated and top 25 downregulated genes with aging were selected based on (i) significance in normal AT2 with FDR < 0.1; (ii) similar gene expression change trend across AT2 cells and all LU AD cell states; (iii) the sum of absolute DEG coefficient across AT2 cells and all LU AD cell states. The color bar indicates the MAST coefficient value. The genes are ordered from most downregulated (dark blue) to the most upregulated (dark red) in aged across the x-axis. Mean with SEM is shown in FIGs. 2A, 2B, 2E and 2F. Y: young; A: aged. *p < 0.05; **p < 0.01; and ***p < 0.001. Student’s / test was used in FIGs. 2A-2C and

2E-2F

[0021] FIGs. 3A-3I: Elevated expression of Nuprl results in loss of tumorigenic potential in the aged lung. FIG. 3A: Dot plot showing enrichment of iron metabolism- related gene sets in the aged signature defined in FIG. 2L. Combined score = log(p) z, where p is the p-value computed using the Fisher exact test, and z is the z-score computed by assessing the deviation from the expected rank. FIGs. 3B-3C: Quantification of Nuprl mRNA level (red dots) by in situ hybridization in AT2 cells (FIG. 3B) (n = 1126 and 425 AT2 cells from 6 young and 6 aged wild-type mouse lungs, respectively) and LU AD cancer cells at 12 weeks post-tumor initiation (FIG. 3C) (n = 32 and 12 tumors from 3 aged and 3 young tumor-bearing mice at 12 weeks post-tumor initiation, respectively). Surfactant protein-C (Sftpc) mRNA (green) was used to mark AT2 and cancer cells. FIG. 3D: Schematic summary of lentiviral Nuprl gene-targeting strategy in the context of LU AD tumorigenesis in vivo. FIG. 3E: Quantification of tumor burden in lungs of aged and young KP-Cas9 mice with non-targeting control or Nuprl -targeting sgRNAs (from left to right, n = 3, 3, 5, and 4 mice in each of the groups in the plot). Representative images of tumor-bearing lungs are shown on the right (HE staining). Scale bar: 100 pm. FIGs. 3F-3G: Quantification of tumor area (FIG. 3F) (n = 87, 40, 146, and 121 tumors for Young-sgControl, Young- sgNuprl, Aged-sgControl, and Aged-sgNuprl) and proportion of proliferating (Ki67+) cancer cells (FIG. 3G) (n = 71, 134, 40, and 93 tumors for Young-sgControl, Young- sgNuprl, Aged-sgControl, and Aged-sgNuprl) in the experiment described in FIG. 3E. FIG. 3H: Bulk RNA-seq analysis of sorted KP-Cas9 cells from LU AD tumors harboring the indicated sgRNAs at 10 weeks post-tumor initiation. Enrichment of KP-LUAD cell state signatures (FIG. 2D) that define progression is shown. FIG. 31: Ex vivo transformation of AT2 cells isolated from aged and young KP-Cas9 mice with the indicated lentiviral vectors (Fig. 3d) delivering Cre + control sgRNA or two independent sgRNAs targeting Nuprl (n = 4 technical repeats in each condition). Representative images of transformed tumor spheres are shown on the right. Scale bar: 100 pm. Mean with SEM is shown in FIGs. 3B-3C, 3F and 3G. Mean with SD is shown in FIG. 31. Y: young; A: aged. *p < 0.05; **p < 0.01; and ***p < 0.001. Student’s t test was used in FIGs. 3B-3E and one-way ANOVA was used in FIGs.

3F-3G and FIG. 31

[0022] FIGs. 4A-4H: Aging-induced expression of NUPR1 disrupts iron homeostasis in AT2 cells. FIG. 4A: Ex vivo transformation of aged vs. young primary AT2 cells isolated from KP-Cas9 mice. Cells were transduced by lentiviral delivery of Cre + a control sgRNA or an sgRNA targeting Nuprl, followed by culture in the presence or absence of the iron chelator deferoxamine (DFO, 2 pM; n = 3-4 technical replicates/condition). FIG. 4B: Ex vivo transformation of AT2 cells isolated from aged vs. young KP-RIK mice with lentiviral PGK-Cre, with or without supplementation with 50 pg/ml ironbound recombinant mouse transferrin (n = 4 technical replicates/condition). Representative images of the tumor spheres are shown on the right. Scale bar: 100 pm. FIG. 4C: Fold change in iron concentration in tumor spheres established from ex vivo transformed KP-Cas9 AT2 cells with sgRNAs targeting Nuprl or non-targeting control sgRNA with or without 2 pM DFO. Fold change is calculated from the average of the control spheres. Each bar represents one replicate. FIG. 4D: Percentage of lipocalin-2+ cells in SPC+ KP-Cas9 LU AD cells in tumors at 12 weeks post-tumor initiation. Representative images of lipocalin-2 immunofluorescence are shown on the right (n = 31, 30, 10, and 31 tumors for each condition, left to right). Note that SPC staining is not shown for clarity. Scale bar: 20 pm. FIG. 4E: Quantification of lipocalin-2 immunofluorescence intensity (n = 23 and 25 high power fields of view from 3 young and aged mice). Representative images are shown on the right. A. U.: arbitrary units. FIG. 4F: Lcn2 mRNA levels in aged vs. young AT2 organoids with or without the NUPR1 inhibitor ZZW-115 (ZZW, 2 pM; n = 3 technical replicates). FIG. 4G: Alveolar organoid formation by aged vs. young primary AT2 cells in the presence of vehicle control, ZZW (2 pM), or ZZW+ 2 pM DFO (n = 3-4 technical replicates/condition). Scale bar: 100 pm. FIG. 4H: Organoid formation by primary mouse AT2 cells stimulated with 50 pg/ml iron-bound recombinant mouse transferrin or vehicle control (n = 3-4 technical replicates/condition). Mean with SEM is shown in FIGs. 4D-4E. Mean with SD is shown in FIGs. 4A-B and 4F- 4H.Y: young; A: aged. *p < 0.05; **p < 0.01; and ***p < 0.001. One-way ANOVA was used in FIGs. 4A-4B, 4D and 4G-4H); Student’s t test was used in FIGs. 4E-4F.

[0023] FIGs. 5A-5L: DNA demethylation underpins aging-associated overactivation of the NUPR1 -lipocalin-2 axis. FIG. 5A: Heatmap illustrating the correlation between differentially methylated cytosines (DMCs) and differentially expressed genes (DEGs) in AT2 cells (FDR < 0.05) in each AT2 histone mark category. The color bar indicates the Pearson correlation coefficient. *Pearson p value< 0.05 ; **Pearson p value < 0.01; ***Pearson p value < 0.001. FIG. 5B: The mean differential methylation change of AT2 DMCs at histone marks at Lcn2 and Nuprl. Grey means no DMC was detected. The color bar indicates the methylation change. Purple-blue color indicates demethylation in aged compared to young mice. The asterisks indicate the CpGs at the indicated histone marks are significantly differentially methylated (p < 0.05). FIG. 5C: The distribution of the mean promoter methylation level at Lcn2 and Nuprl in aged and young AT2 cells. The promoter is defined as 1 kb upstream and 200 bp downstream from the transcriptional start site (TSS). Each dot is one biological replicate (n = 4). Statistical significance was assessed by the Wilcoxon test. FIG. 5D: Scatter plot showing mean differential methylation level of promoter regions (1 kb upstream and 200 bp downstream from the TSS) of the top 25 upregulated and downregulated genes among the aging-associated signature shared by both AT2 and LU AD cells (FIG. 2L). Pearson correlation was calculated. The regression line (blue) and its confidence interval (light grey) were plotted using a linear model. Genes transcriptionally upregulated in the aged AT2 and LU AD cells are shown in red; genes upregulated in the young are marked in blue. FIG. 5E: Experimental design of DNMT1 inhibition (DNMTl-i, orange) with GSK-3484862 over 8 days in vivo. FIG. 5F: Contour plot showing the correlation between the fold change (FC) of DEGs in AT2 cells isolated from young mice administered DNMTl-i or vehicle (x-axis) vs. aged or young vehicle- treated mice (y-axis). The grayscale represents the probability distribution. The Pearson correlation was calculated. The table showed the percentage of genes in each quadrant, which is significantly different from random (binomial test p < 2.2'¹⁶). The regression line (blue) and its confidence interval (light grey) were plotted using a linear model. FIG. 5G: Scatter plot showing the correlation between the fold change of DEGs from the signature shared by AT2 cells and LU AD cells (FIG. 2L) in AT2 cells isolated from young mice administered DNMTl-i or vehicle (x-axis) vs. aged or young vehicle-treated mice (y-axis). Pearson correlation was calculated. The regression line (blue) and its confidence interval (light grey) were plotted using a linear model. FIG. 5H: Quantification of Nuprl mRNA molecules in Sftpc+ AT2 cells in young mice administered vehicle (blue) or DNMTl-i (orange) for 8 days, compared to aged mice that received vehicle (red) (n = 353, 438, and 429 AT2 cells per condition, respectively). FIG. 51: Gene expression and promoter methylation change of genes among the top 25 upregulated aging signature genes (FIG. 2L) in AT2 and LU AD cells. The top row displays the shared DEG change in AT2 and LU AD (red indicates upregulation in the aged compared to the young). The second row indicates the DEG change in young mice administered DNMTl-i or vehicle (red indicates upregulation - an aged-like change - in mice that received DNMTl-i compared to young vehicle controls). The last two rows show the direction of differential methylation in aged vs young AT2 and LU AD cells (light blue: demethylated, pink: more methylated in aged compared to the young). FIG. 5J: Experimental design of DNMT1 inhibition in the KP-Cas9 ex vivo transformation assay. FIG. 5K: Contour plot showing the correlation between the fold change of DEGs in young transformed tumor spheres subjected to DNMTl-i or vehicle (x-axis) vs. aged or young vehicle controls (y-axis). The grayscale represents the probability distribution. Pearson correlation was calculated. The inset shows the percentage of genes in each quadrant, which is significantly different from random based on a binomial test (p < 2.2'¹⁶). The regression line (blue) and its confidence interval (light grey) were plotted using a linear model. FIG. 5L: Nuprl gene expression in tumor sphere cultures established from primary A7U LUAD tumors (n = 4 replicates from two established cell lines established from young LU AD tumors). Mean with SEM is shown in FIG. 5H. Mean with SD is shown in FIG. 5L.*/? < 0.05; **/? < 0.01; ***/? < 0.001. One-way ANOVA was used in FIG. 5H; Student’s t test was used in FIG. 5L.

[0024] FIGs. 6A-6F: Aging-induced changes in AT2 cells and LU AD are conserved in humans. FIG. 6A: Quantification of AT2 cell density in healthy lungs of aged (>70 years old) and young (<50 years old) humans (n = 23 young and 25 aged patients, respectively). The proportion of HTII-280 positive AT2 cells (orange) over total lung cell number. FIGs. 6B-6C: Quantification of NUPR1 (FIG. 6B) (n = 10 young cases and 24 aged cases) and lipocalin-2 (FIG. 6C) (n = 14 young cases and 15 aged cases,) protein level in normal lung tissues of aged and young patients. The intensity of NUPR1 (red) or lipocalin-2 (red) immunofluorescence was quantified only in cells expressing either the HTII-280 or SPC AT2 cell marker (green). A. U.: arbitrary units. FIG. 6D: Fold change in organoids formed by primary human AT2 cells in response to stimulation with recombinant human transferrin normalized to the number of control organoids. Scale bar: 50 gm. FIG. 6E: Correlation of young mouse LU AD and aged mouse LU AD gene expression signatures with patient age in The Cancer Genome Atlas data. All tumors had a KRAS mutation. FIG. 6F: NUPR1 immunofluorescence in LU AD tissue obtained from aged (>80 years old, n = 35) and young (<55 years old, n = 36) patients. NUPR1 signal intensity was measured only in Pan- cytokeratin (CK) positive cancer cells. Y: young; A: aged. *p < 0.05; **p < 0.01; and ***p < 0.001. Student’s / test was used in FIGs. 6A-6D and 6F.

[0025] FIGs. 7A-7I. FIG. 7A: Representative images of LU AD tumors in aged and young KPT (4 and 8 weeks) or KP (12 weeks) mice. At 4 and 8 weeks post-tumor initiation, tumor cells were visualized by tdTomato immunohistochemistry. Scale bar: 200 gm. FIG. 7B: Quantification of AT2 cells in aged and young mouse lungs. AT2 cells were identified by surfactant protein-C (SPC) (n = 18 high-power field of views from 6 aged and 6 young mice). FIG. 7C: FACS strategy for isolating mouse AT2 cells. AT2 cells are identified as DAPI- /Lineage (CD45, CD1 lb, CD11c, F4/80 and Teri 19)-/EpCAM+/MHCII+ (P7). FIG. 7D: Validation of sort purity by qPCR for AT2 marker gene Sftpc. Note lack of detectable Sftpc expression in non-AT2 lineage-negative cells (P8 and P9). FIG. 7E: Quantification of the percentage of AT2 cells over total EpCAM+ epithelial cells in young (12-16 weeks old), middle-aged (52 weeks old), and aged (104-130 weeks old) wild-type mice (n = 5 mice from young, middle-aged and aged group). FIG. 7F: AdSPC-Cre transduction efficiency of AT2 cells in lungs of aged vs. young Rosa26mTmG/+ mice, where Cre induces a switch from tdTomato to GFP fluorescence. The percentage of transduced AT2 cells (GFP) in the total AT2 cell population, defined by the same surface marker panel as in FIGs. 7C-7D (n = 3 per condition). FIGs. 7G-7H: Representative images (FIG. 7G) and quantification (FIG. 7H) of senescent cells identified by C12RG fluorescence-based detection of senescence-associated P-galactosidase activity. Note that C12RG positive senescent cells do not express the AT2 cell marker SPC (n = 17 and 29 high-power fields from 3 young and 3 aged mice were examined, respectively). FIG. 71: Quantification of lentiviral transduction efficiency of aged and young AT2 cells in the ex vivo transformation assay. AT2 cells were isolated from both aged and young KP mice and transduced with lenti-PGK-GFP. Transduction efficiency was defined as the percentage of GFP+ cells over total number of AT2 cells (n = 3 technical replicates/condition). Mean with SD is shown in FIGs. 7D, 7F and 71. Y: young; A: aged. *p < 0.05; **p < 0.01; and ***p < 0.001. Student’s t test was used in FIGs. 7B, 7D, 7F, 7H and 71; one-way ANOVA was used in (e).

[0026] FIGs. 8A-8D. FIG. 8A: Schematic summary of Eml4-Alk LU AD model (image representing chromosomal rearrangement reproduced from Maddalo et al.³⁴). Lungs of aged (104-130 weeks old) and young (12-16 weeks old) wild-type C57BL6/J mice were intratracheally transduced with adeno-sgEml4-sgAlk-Cas9 virus, which induces an Eml4-Alk gene fusion via an intra-chromosomal inversion on chromosome 17. FIGs. 8B-8D: Representative HE images (FIG. 8B) and LU AD tumor burden, quantified as tumor number/cross section (FIG. 8C) and percentage of tumor area over total tissue area (FIG. 8D) (n = 18 and 8 mice for young and aged conditions, respectively). Scale bar: 1 mm. *p < 0.05 and **p < 0.01. Student’s t test was used in FIGs. 8C-8D.

[0027] FIGs. 9A-9D. FIGs. 9A-9C: Quantification of senescent cancer cells in young vs. aged KP LU AD tumors at 12 weeks post-tumor initiation. The senescent cells were identified by pl6 (FIGs. 9A-9B) (n = 30 and 23 high power fields in young and aged KP tumors, respectively) and C12RG staining (FIG. 9C) (n = 11 and 9 tumors from 3 young and aged mice). Representative images of pl6 staining are shown in FIG. 9A. FIG. 9D: Histopathological grading of KP LU AD tumors derived from aged vs. young mice at 12 and 17 weeks post-tumor initiation. Representative images of the grading by an automated deep neural network (Aiforia Technologies) are shown. Scale bar: 1 mm. Mean with SD is shown in FIG. 9B. Student’s t test was used in FIGs. 9B-9C.

[0028] FIGs. 10A-10D. FIG. 10A: Plots of gene set enrichment analysis (GSEA) showing the enrichment of gene sets linked to iron metabolism in age-related signatures introduced in FIG. 2L ordered according to fold change. Red indicates enrichment in aged, blue enrichment in young. FIG. 10B: Violin plots of showing the expression of Nuprl in young and aged AT2 cells and LU AD cell states. Rank-sum tests on single-cell gene expression vector were performed for significance testing. FIG. 10C: Lentiviral transduction efficiency of AT2 cells in aged and young mice in vivo. Lungs of Kras^+/+ ; Trp53A^ littermates of KP mice were intratracheally transduced with 50,000 units of PGK-mScarlet lentivirus. The percentage of transduced AT2 cells was measured by flow cytometry (mScarlet+/MCHII+/EpCAM+/lineage-) and total transduced AT2 cell numbers = (mScarlet+ AT2 cell) % x total cell number n = 3 young and 3 aged mice). FIG. 10D: Ex vivo transformation of AT2 cells isolated from aged and young KP-RIK mice, with or without treatment with the NUPR1 inhibitor ZZW-115 (ZZW, 2 uM) (n = 3 technical replicates). Mean with SD is shown in FIG. 10D. *p < 0.05 and ***p < 0.001. Student’s t test was used in FIG. 10C and one-way ANOVA was used in FIG. 10D.

[0029] FIGs. 11A-11F. FIG. 11 A: Ex vivo transformation of AT2 cells isolated from aged and young KP-RIK mice by lentiviral PGK-Cre with or without treatment with ZZW- 115 (ZZW, 2 pM) and deferoxamine (DFO, 2 pM) (n = 4 technical replicates). FIG. 11B: Ex vivo transformation of AT2 cells isolated from aged and young KP-Cas9 mice using lentiviral vectors delivering Cre recombinase and an sgRNA targeting Nuprl or a control sgRNA with or without transferrin supplementation (50 pg/ml; n = 4 technical replicates/condition). FIG. 11C: Violin plots showing expression of Lcn2 in young and aged AT2 cells and LU AD cell states. Rank-sum tests on single-cell gene expression vector were performed for significance testing. FIGs. 11D-11E: Lcn2 mRNA level in transformed AT2 cells from experiment in FIG. 31 (n = 3 technical replicates) (FIG. 11D) and FIG. 11A n = 3 technical replicates) (FIG. HE). FIG. HF: Ex vivo transformation of AT2 cells isolated from aged vs. young KP-RIK mice with lentiviral PGK-Cre, with or without stimulation with 100 ng/ml recombinant mouse lipocalin-2 (n = 4 technical repeats). Mean with SD is shown in FIGs. 11A-11B and 11D-11F. *p < 0.05 and ***p < 0.001. One-way ANOVA was used in FIGs. 11A-11B and 11D-11F

[0030] FIGs. 12A-12H. FIG. 12A: Density plot showing Enzymatic Methyl-Seq sequencing coverage in primary AT2 cell samples. FIG. 12B: Density plot of the methylation proportion in AT2 cell samples. FIG. 12C: Heatmap showing unsupervised clustering of aged vs. young AT2 cells based on DNA methylation profiles. FIG. 12D: Number of differentially methylated cytosines (DMCs) in aged vs. young AT2 cells based on location of CpG residues. FIG. 12E: Density plot showing Enzymatic Methyl-Seq sequencing coverage in KP LU AD cell samples. FIG. 12F: Density plot of the methylation proportion of KP LU AD samples. FIG. 12G: Heatmap showing unsupervised clustering of aged vs. young KP LU AD cells based on DNA methylation profiles. FIG. 12H: Number of differentially methylated cytosines (DMCs) in aged vs. young KP LU AD cells based on location of CpG residues. [0031] FIGs. 13A-13B. FIG. 13A: The number of DMCs at sites marked by distinct histone modifications in AT2 cells. The height of the bars indicates higher methylation level in young (blue) or higher methylation in aged (red). The number of methylated CpCs detected is shown. FIG. 13B: Pearson correlation between DMCs and DEGs in aged vs. young AT2 cells in each category of histone modification (FDR < 0.05). The regression line (blue) and its confidence interval (light grey) were plotted using a linear model.

[0032] FIGs. 14A-14B: Aging induces Nuprl in AT2 cells and lung cancer cells arising from them. FIG. 14A: Scatter plot showing the correlation between the fold change of DEGs from the signature shared by AT2 cells and LU AD cells. Pearson correlation was calculated. The regression line (blue) and its confidence interval (light grey) were plotted using a linear model. FIG. 14B: Quantification of single Nuprl mRNA molecules in Sftpc+ positive cells in lungs of aged vs. young mice. Note induction of Nuprl expression in aged and young AT2 cells.

[0033] FIGs. 15A-15B: Nuprl cooperates with transformation in the young but is a tumor suppressor in the aged. FIG. 15A: Schematic summary of approach to test the aging-associated role of Nuprl in vivo. FIG. 15B: Quantification of tumor burden in lungs of aged and young KP-Cas9 mice with non-targeting control or Nuprl -targeting sgRNAs.

[0034] FIGs. 16A-16B: Young AT2 cells are sensitive to ferroptosis but aged cells are resistant. FIG. 16A: Cartoon summarizing mechanism by which aging-associated induction of the NUPRl-lipocalin-2 axis causes a functional iron insufficiency in AT2 and lung cancer cells, while protecting the aged cells from ferroptosis. FIG. 16B: Ex vivo AT2 transformation assay showing young cells benefit from protection from ferroptosis (liproxstatin-1) and are sensitive to ferroptosis inductin (by RSL-3 or erastin). Conversely, no effect is seen in aged cells.

[0035] FIGs. 17A-17B: Ferroptosis suppresses tumorigenesis in young but aged cells are resistant. FIG. 17A: Cartoon summarizing mechanism by which aging-associated induction of the NUPRl-lipocalin-2 axis causes a functional iron insufficiency in AT2 and lung cancer cells, while protecting the aged cells from ferroptosis. FIG. 17B: Quantification of tumor burden in lungs of aged and young mice administered liproxstatin-1 or control. Note increased tumor burden in young mice following liproxstatin-1 administration compared to vehicle and no difference in aged mice.

[0036] FIGs. 18A-18B: Aging-associated Nuprl induction suppresses AT2 sternness and tumorigenic potential by reprogramming iron homeostasis. Aging-associated induction of NUPR1 renders AT2 cells iron insufficient (FIG. 18A), whereas targeting NUPR1 or iron supplementation rejuvenates aged AT2 cells (FIG. 18B).

[0037] FIG. 19: Nuprl is the only iron homeostasis gene showing age context-specific function in AT2 regenerative potential. Ex vivo AT2 transformation assay showing both aged and young cells develop a growth detriment following knockout of iron storage proteins (Fthl, Ftll), iron uptake proteins (Trf Tfrc. Cd44 . and iron export (Slc40al). This is in contrast to Nuprl knockout, which is detrimental in young cells and beneficial to growth of aged AT2 cells.

[0038] FIG. 20: Free iron complexed with vitamin C (FAC) rescues sternness of aged AT2 cells, but is detrimental to young AT2 cells. Ex vivo AT2 transformation assay showing aged AT2 cells benefit from supplementation with iron associated with vitamin C (FAC). No additional benefit is observed by combining FAC with iron-loaded transferrin.

[0039] FIG. 21. NUPR1 drives iron insufficiency in aged AT2 cells via lipocalin-2. Ex vivo AT2 transformation assay showing aged AT2 cells benefit from Nuprl knockout, whereas overexpression of Lcn2 in the context of Nuprl knockout completely reverses the benefit. We also find that Lcn2 knockout alone promotes growth of aged AT2 cells.

[0040] FIG. 22. NUPR1 drives iron insufficiency in aged AT2 cells via lipocalin-2. Ex vivo AT2 transformation assay showing aged AT2 cells benefit from Nuprl knockout, whereas overexpression of Lcn2 in the context of Nuprl knockout completely reverses the benefit. This is a repeat of the experiment in FIG. 20.

[0041] FIG. 23. Graphical summary of findings of this study.

[0042] FIG. 24. Aging induces the NUPR1 -lipocalin-2 axis in other adult stem cell compartments. Gene expression data showing change in gene expression in different adult stem cell compartments. Note induction of Nuprl and Lcn2 in most adult stem cell compartments. DETAILED DESCRIPTION

[0043] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0044] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir ’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0045] Aging is one of the most important risk factors for cancer development; yet the incidence of most cancers begins to decline in very advanced age^1,2. Among multiple molecular and cellular alterations aging is associated with dysregulation of DNA methylation and loss of stem cell function³'⁸. However, the impact of these age-associated changes on tumorigenesis in vivo is poorly understood. Here, using autochthonous genetically engineered mouse models^9,10, we show that aging suppresses lung cancer development, which we attribute to a decline in sternness of the alveolar cell of origin. Aging induced expression of the iron homeostasis rheostat Nuprl, which was a prominent component of an age- associated gene expression signature that was shared between the aged cell of origin and lung tumors in aged mice and human patients. Genetic or pharmacologic inactivation of NUPR1 or supplementation with transferrin-bound iron rescued sternness and tumor-initiation potential in aged alveolar cells, which was blunted by iron chelation. The age-associated gene signature and Nuprl upregulation correlated with DNA demethylation in the aged alveolar cells and was induced in young alveolar cells by pharmacologic inhibition of DNA methylation.

[0046] Our findings demonstrate that aging-associated DNA demethylation promotes iron insufficiency by inducing NUPR1 expression, which leads to loss of sternness and tumorigenic potential in lung alveoli. Remarkably, a significant fraction of the conserved aging-associated alterations in gene expression in the alveolar cell of origin are inherited by tumors; such alterations may constitute age-dependent cancer vulnerabilities. These results provide an explanation for the decline in lung cancer incidence in very aged individuals and cast iron supplementation and inhibition of NUPR1 as attractive therapeutic strategies for rejuvenating regenerative potential of aged lung alveoli.

Definitions

[0047] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0048] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). [0049] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and the administration by another.

[0050] The terms “complementary” or “complementarity” as used herein with reference to polynucleotides (z.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) refer to the base-pairing rules. The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in “antiparallel association.” For example, the sequence “5'-A-G-T-3'” is complementary to the sequence “3'-T-C-A-5 ” Certain bases not commonly found in naturally-occurring nucleic acids may be included in the nucleic acids described herein. These include, for example, inosine, 7- deazaguanine, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA). Complementarity need not be perfect; stable duplexes may contain mismatched base pairs, degenerative, or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs. A complementary sequence can also be an RNA sequence complementary to the DNA sequence or its complementary sequence, and can also be a cDNA.

[0051] As used herein, the term “biological sample” means sample material derived from living cells. Biological samples may include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present within a subject. Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, renal tissue, the uterine cervix, the endometrium, the head or neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostate fluid, seminal fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or from cancers. Biological samples can be obtained from subjects for diagnosis or research or can be obtained from non-diseased individuals, as controls or for basic research. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.

[0052] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0053] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0054] As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.

[0055] As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0056] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by =HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological activity. Two sequences are deemed “unrelated” or “non-homologous” if they share less than 40% identity, or less than 25% identity, with each other.

[0057] The term “hybridize” as used herein refers to a process where two substantially complementary nucleic acid strands (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex through formation of hydrogen bonds between complementary base pairs. Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, and the thermal melting point (T_m) of the formed hybrid. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al., 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, N.J. In some embodiments, specific hybridization occurs under stringent hybridization conditions. An oligonucleotide or polynucleotide (e.g., a probe or a primer) that is specific for a target nucleic acid will “hybridize” to the target nucleic acid under suitable conditions.

[0058] As used herein, the terms “identical” or percent “identity”, when used in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding an antibody described herein or amino acid sequence of an antibody described herein)), when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (e.g., NCBI web site). Such sequences are then said to be “substantially identical.” This term also refers to, or can be applied to, the complement of a test sequence. The term also includes sequences that have deletions and/or additions, as well as those that have substitutions. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or 50-100 amino acids or nucleotides in length. [0059] As used herein, the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient or subject is a human.

[0060] As used herein, the terms “lung insufficiency” or “respiratory insufficiency” or “pulmonary insufficiency” refers to a condition in which the lungs cannot take in sufficient oxygen or expel sufficient carbon dioxide to meet the needs of the cells in the body of a subject. These conditions may affect the muscles, nerves, bones, or tissues that support breathing or may affect the lungs directly. Examples of these conditions include: Diseases that affect the lungs, such as COPD (chronic obstructive pulmonary disease), cystic fibrosis, pneumonia, pulmonary embolism, and COVID- 19; Conditions that affect the nerves and muscles that control breathing, such as amyotrophic lateral sclerosis (ALS), muscular dystrophy, spinal cord injuries, and stroke; Problems with the spine, such as scoliosis (a curve in the spine); Damage to the tissues and ribs around the lungs; Drug or alcohol overdose; or Inhalation injuries, such as from inhaling smoke (from fires) or harmful fumes.

[0061] The term “NUPR1 inhibitor” as used herein refers to an agent that inhibits the expression and/or activity of Nuclear Protein 1, Transcriptional Regulator (NUPR1). The NUPR1 inhibitors of the present disclosure inhibit at least one biological activity of NUPR1.

[0062] As used herein, “oligonucleotide” refers to a molecule that has a sequence of nucleic acid bases on a backbone comprised mainly of identical monomer units at defined intervals. The bases are arranged on the backbone in such a way that they can bind with a nucleic acid having a sequence of bases that are complementary to the bases of the oligonucleotide. The most common oligonucleotides have a backbone of sugar phosphate units. A distinction may be made between oligodeoxyribonucleotides that do not have a hydroxyl group at the 2' position and oligoribonucleotides that have a hydroxyl group at the 2' position. Oligonucleotides may also include derivatives, in which the hydrogen of the hydroxyl group is replaced with organic groups, e.g., an allyl group. One or more bases of the oligonucleotide may also be modified to include a phosphorothioate bond (e.g., one of the two oxygen atoms in the phosphate backbone which is not involved in the intemucleotide bridge, is replaced by a sulfur atom) to increase resistance to nuclease degradation. The exact size of the oligonucleotide will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including, for example, chemical synthesis, DNA replication, restriction endonuclease digestion of plasmids or phage DNA, reverse transcription, PCR, or a combination thereof. The oligonucleotide may be modified e.g., by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides.

[0063] As used herein, the term “pharmaceutically-acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration. Pharmaceutically-acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20^th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

[0064] As used herein, the term “polynucleotide” or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and doublestranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

[0065] As used herein, the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. [0066] As used herein, “prevention” or “preventing” of a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0067] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0068] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0069] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0070] The term “specific” as used herein in reference to an oligonucleotide means that the nucleotide sequence of the oligonucleotide has at least 12 bases of sequence identity with a portion of a target nucleic acid when the oligonucleotide and the target nucleic acid are aligned. An oligonucleotide that is specific for a target nucleic acid is one that, under the stringent hybridization or washing conditions, is capable of hybridizing to the target nucleic acid of interest and not substantially hybridizing to nucleic acids which are not of interest. Higher levels of sequence identity are desirable and include at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity.

[0071] The term “stringent hybridization conditions” as used herein refers to hybridization conditions at least as stringent as the following: hybridization in 50% formamide, 5xSSC, 50 mM NaH2PO4, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5x Denhart's solution at 42° C. overnight; washing with 2x SSC, 0.1% SDS at 45° C; and washing with 0.2x SSC, 0.1% SDS at 45° C. In another example, stringent hybridization conditions should not allow for hybridization of two nucleic acids which differ over a stretch of 20 contiguous nucleotides by more than two bases.

[0072] As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0073] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0074] It is also to be appreciated that the various modes of treatment or prevention of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

Inhibitors of NUPR1/ Lipocalin-2 Axis

[0075] NUPR1 (UniProtKB 060356), or nuclear protein 1, is an 82-resi due-long (8 kDa), highly basic intrinsically disordered protein that localizes throughout the whole cell, and is involved in the development and progression of several tumors. The NUPR1 inhibitor may be a small molecule, an NUPR1 -specific inhibitory nucleic acid (e.g., an antisense oligonucleotide, a shRNA, a sgRNA or a ribozyme), or an anti-NUPRl neutralizing antibody. Examples of small molecule inhibitors of NUPR1 include, but are not limited to, trifluoperazine (TFP), ZZW-115, ZZW-129, ZZW-130, ZZW-131, ZZW-132, ZZW-142, ZZW-143, ZZW-144, ZZW-145, and ZZW-148. See Rizzuti et al., Biomolecules. 2021, 11(10): 1453.

[0076] Lipocalin-2 (LCN2) (UniprotKB X6R8F3) is a secreted glycoprotein involved in transporting hydrophobic ligands across the cell membrane, modulating the immune response during bacterial infection, and promoting epithelial cell differentiation and iron homeostasis. LCN2 is aberrantly upregulated in cancerous tissues derived from the pancreas, colon, ovaries, and breast. Overexpression of LCN2 is also associated with the progression of aggressive forms of endometrial carcinoma, pancreas, and breast cancers. The LCN2 inhibitor may be a small molecule, an LCN2-specific inhibitory nucleic acid (e.g., an antisense oligonucleotide, a shRNA, a sgRNA or a ribozyme), or an anti- LCN2 neutralizing antibody. Examples of small molecule inhibitors of LCN2 include, but are not limited to, ZINC00784494, ZINC00640089, ZINC00230567, and ZINC00829534. See Santiago- Sanchez et al., IntJMol Sci. 2021 : 22(16): 8581.

[0077] The present disclosure also provides an antisense nucleic acid comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of an NUPR1 mRNA or LCN2 mRNA, thereby reducing or inhibiting expression of NUPR1 or LCN2. The antisense nucleic acid may be antisense RNA, or antisense DNA. Antisense nucleic acids based on the known gene sequences of NUPR1 or LCN2 can be readily designed and engineered using methods known in the art. In some embodiments, the antisense nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or a complement thereof.

[0078] Antisense nucleic acids are molecules which are complementary to a sense nucleic acid strand, e.g., complementary to the coding strand of a double-stranded DNA molecule (or cDNA) or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can form hydrogen bonds with a sense nucleic acid. The antisense nucleic acid can be complementary to an entire NUPR1 or LCN2 coding strand, or to a portion thereof, e.g., all or part of the protein coding region (or open reading frame). In some embodiments, the antisense nucleic acid is an oligonucleotide which is complementary to only a portion of the coding region of NUPR1 or LCN2 mRNA. In certain embodiments, an antisense nucleic acid molecule can be complementary to a noncoding region of the NUPR1 or LCN2 coding strand. In some embodiments, the noncoding region refers to the 5' and 3' untranslated regions that flank the coding region and are not translated into amino acids. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NUPR1 or LCN2. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. [0079] An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fhiorouracil, 5-bromouracil, 5-chlorouracil, 5-hodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2- thouridine, 5-carboxymethylaminometh-yluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3 -methylcytosine, 5-metnylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5 '-methoxy carboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopenten-yladenine, uracil-5-oxyacetic acid (v), wybutosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thlouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-cxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3- N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (z.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

[0080] The antisense nucleic acid molecules may be administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding the protein of interest to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can occur via Watson-Crick base pairing to form a stable duplex, or in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.

[0081] In some embodiments, the antisense nucleic acid molecules are modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. In some embodiments, the antisense nucleic acid molecule is an alpha-anomeric nucleic acid molecule. An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual P- units, the strands run parallel to each other (Gaultier el al.. Nucleic Acids. Res. 15:6625- 6641(1987)). The antisense nucleic acid molecule can also comprise a 2'-0 - methylribonucleotide (Inoue et al., Nucleic Acids Res. 15:6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al., FEBS Let. 215:327-330 (1987)).

[0082] The present disclosure also provides a short hairpin RNA (shRNA) or small interfering RNA (siRNA) comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of an NUPR1 or LCN2 mRNA, thereby reducing or inhibiting NUPR1 or LCN2 expression. In some embodiments, the shRNA or siRNA is about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 base pairs in length. Double-stranded RNA (dsRNA) can induce sequence-specific post-transcriptional gene silencing (e.g., RNA interference (RNAi)) in many organisms such as C. elegans, Drosophila, plants, mammals, oocytes and early embryos. RNAi is a process that interferes with or significantly reduces the number of protein copies made by an mRNA. For example, a double-stranded siRNA or shRNA molecule is engineered to complement and hydridize to a mRNA of a target gene. Following intracellular delivery, the siRNA or shRNA molecule associates with an RNA- induced silencing complex (RISC), which then binds and degrades a complementary target mRNA (such as NUPR1 or LCN2 mRNA). In some embodiments, the shRNA or siRNA comprises the nucleic acid sequence of SEQ ID NO: 1, or SEQ ID NO: 2.

[0083] The present disclosure also provides a ribozyme comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of an NUPR1 or LCN2 mRNA, thereby reducing or inhibiting NUPR1 or LCN2 expression. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a complementary single-stranded nucleic acid, such as an mRNA. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach, Nature 334:585-591 (1988))) can be used to catalytically cleave NUPR1 or LCN2 transcripts, thereby inhibiting translation of NUPR1 or LCN2.

[0084] A ribozyme having specificity for a NUPR1 or ZCA2-encoding nucleic acid can be designed based upon NUPR1 or LCN2 gene nucleic acid sequence. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a NUPR1 or LCN2 -encoding mRNA. See, e.g., U.S. Pat. No. 4,987,071 and U.S. Pat. No. 5, 116,742. Alternatively, NUPR1 or LCN2 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261 : 1411-1418, incorporated herein by reference.

[0085] The present disclosure also provides a synthetic guide RNA (sgRNA) comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of an NUPR1 or LCN2 mRNA. Guide RNAs for use in CRISPR-Cas systems are typically generated as a single guide RNA comprising a crRNA segment and a tracrRNA segment. The crRNA segment and a tracrRNA segment can also be generated as separate RNA molecules. The crRNA segment comprises the targeting sequence that binds to a portion of an NUPR1 or LCN2 mRNA, and a stem portion that hybridizes to a tracrRNA. The tracrRNA segment comprises a nucleotide sequence that is partially or completely complementary to the stem sequence of the crRNA and a nucleotide sequence that binds to the CRISPR enzyme. In some embodiments, the crRNA segment and the tracrRNA segment are provided as a single guide RNA. In some embodiments, the crRNA segment and the tracrRNA segment are provided as separate RNAs. The combination of the CRISPR enzyme with the crRNA and tracrRNA make up a functional CRISPR-Cas system. Exemplary CRISPR-Cas systems for targeting nucleic acids, are described, for example, in WO20 15/089465.

[0086] In some embodiments, a synthetic guide RNA is a single RNA represented as comprising the following elements: 5'-Xl-X2-Y-Z-3' where XI and X2 represent the crRNA segment, where XI is the targeting sequence that binds to a portion of an NUPR1 or LCN2 mRNA, X2 is a stem sequence the hybridizes to a tracrRNA, Z represents a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to X2, and Y represents a linker sequence. In some embodiments, the linker sequence comprises two or more nucleotides and links the crRNA and tracrRNA segments. In some embodiments, the linker sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. In some embodiments, the linker is the loop of the hairpin structure formed when the stem sequence hybridized with the tracrRNA. [0087] In some embodiments, a synthetic guide RNA is provided as two separate RNAs where one RNA represents a crRNA segment: 5'-Xl-X2-3' where XI is the targeting sequence that binds to a portion of a NUPR1 or LCN2 mRNA, X2 is a stem sequence the hybridizes to a tracrRNA, and one RNA represents a tracrRNA segment, Z, that is a separate RNA from the crRNA segment and comprises a nucleotide sequence that is partially or completely complementary to X2 of the crRNA.

[0088] Exemplary crRNA stem sequences and tracrRNA sequences are provided, for example, in WO/2015/089465, which is incorporated by reference herein. In general, a stem sequence includes any sequence that has sufficient complementarity with a complementary sequence in the tracrRNA to promote formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the stem sequence hybridized to the tracrRNA. In general, degree of complementarity is with reference to the optimal alignment of the stem and complementary sequence in the tracrRNA, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the stem sequence or the complementary sequence in the tracrRNA. In some embodiments, the degree of complementarity between the stem sequence and the complementary sequence in the tracrRNA along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the stem sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the stem sequence and complementary sequence in the tracrRNA are contained within a single RNA, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. In some embodiments, the tracrRNA has additional complementary sequences that form hairpins. In some embodiments, the tracrRNA has at least two or more hairpins. In some embodiments, the tracrRNA has two, three, four or five hairpins. In some embodiments, the tracrRNA has at most five hairpins.

[0089] In a hairpin structure, the portion of the sequence 5' of the final “N” and upstream of the loop corresponds to the crRNA stem sequence, and the portion of the sequence 3' of the loop corresponds to the tracrRNA sequence. Further non-limiting examples of single polynucleotides comprising a guide sequence, a stem sequence, and a tracr sequence are as follows (listed 5' to 3'), where “N” represents a base of a guide sequence (e.g. a modified oligonucleotide provided herein), the first block of lower case letters represent stem sequence, and the second block of lower case letters represent the tracrRNA sequence, and the final poly-T sequence represents the transcription terminator: (a) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataa ggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 4); (b) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccg aaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 5); (c) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccg aaatcaacaccctgtcattttatggcagggtgtTTTTTT (SEQ ID NO: 6); (d) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaactt gaaaaagtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 7); (e) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaac ttgaaaaagtgTTTTTTT (SEQ ID NO: 8); and (f) NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTT

[0090] Selection of suitable oligonucleotides for use in as a targeting sequence in a CRISPR Cas system depends on several factors including the particular CRISPR enzyme to be used and the presence of corresponding proto-spacer adjacent motifs (PAMs) downstream of the target sequence in the target nucleic acid. The PAM sequences direct the cleavage of the target nucleic acid by the CRISPR enzyme. In some embodiments, a suitable PAM is 5'- NRG or 5'-NNGRR (where N is any Nucleotide) for SpCas9 or SaCas9 enzymes (or derived enzymes), respectively. Generally the PAM sequences should be present between about 1 to about 10 nucleotides of the target sequence to generate efficient cleavage of the target nucleic acid. Thus, when the guide RNA forms a complex with the CRISPR enzyme, the complex locates the target and PAM sequence, unwinds the DNA duplex, and the guide RNA anneals to the complementary sequence on the opposite strand. This enables the Cas9 nuclease to create a double-strand break. In some embodiments, the sgRNA comprises the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0091] A variety of CRISPR enzymes are available for use in conjunction with the disclosed guide RNAs of the present disclosure. In some embodiments, the CRISPR enzyme is a Type II CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage. In some embodiments, the CRISPR enzyme catalyzes RNA cleavage. In some embodiments, the CRISPR enzyme is any Cas9 protein, for instance any naturally-occurring bacterial Cas9 as well as any chimeras, mutants, homologs or orthologs. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified variants thereof. In some embodiments, the CRISPR enzyme cleaves both strands of the target nucleic acid at the Protospacer Adjacent Motif (PAM) site. In some embodiments, the CRISPR enzyme is a nickase, which cleaves only one strand of the target nucleic acid.

[0092] Accumulation of phospholipid peroxides can lead to ferroptotic death. In mammalian cells, phospholipid peroxides are effectively neutralized by glutathione peroxidase-4 (GPX4), and blockage of GPX4 enzyme often triggers ferroptosis. As GPX4 requires the reducing agent glutathione to function, deprivation of cysteine, the essential building block of glutathione, via approaches such as cystine starvation or pharmacological inhibition of system xc- cystine/glutamate antiporter, can also trigger ferroptosis.

[0093] A ferroptosis-inducing agent may be a class 1 ferroptosis inducer (system X_c inhibitor) or a class 2 ferroptosis inducer (glutathione peroxidase 4 (GPx4) inhibitor). Examples of ferroptosis-inducing agents include, but are not limited to, erastin, erastin derivatives (e.g., MEII, PE, AE, imidazole ketone erastin (IKE)), DPI2, BSO, SAS, lanperisone, SRS13-45, SRS13-60, RSL3, DPI7, DPI10, DPI12, DPI13, DPI17, DPI18, DPI19, ML160, sorafenib, artemisinin derivatives, artesunate, BAY87-2243, cisplatin, ironomycin, lanperisone, salinomycin, sulfasalazine, temozolomide, lapatinib in combination with siramesine, and the like. In some embodiments, the ferroptosis inducing agent is an inhibitory nucleic acid e.g., an antisense oligonucleotide, a shRNA, a sgRNA or a ribozyme) that targets GPX4. Iron/Transferrin

[0094] Transferrin is a plasma glycoprotein capable of tightly but reversibly binding two atoms of iron and transporting them to proliferating cells for the synthesis of hemoglobin. Upon binding to the transferrin receptors on the cell surface, the transferrin is internalized within an endocytic vesicle. After the irons are dissociated from transferrin, irons pass through the endosomal membrane and enter into cytosol.

Formulations Including Iron/Transferrin, Inhibitors of NUPR1/ Lipocalin-2 Axis, and/or Ferroptosis Inducing Agents of the Present Technology

[0095] The pharmaceutical compositions of the present technology can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, or emulsifying processes, among others. Compositions may be produced in various forms, including granules, precipitates, or particulates, powders, including freeze dried, rotary dried or spray dried powders, amorphous powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. Formulations may optionally contain solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, pH modifiers, isotonic agents, thickening or emulsifying agents, stabilizers and preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. In certain embodiments, the compositions disclosed herein are formulated for administration to a mammal, such as a human.

[0096] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, cyclodextrins, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. [0097] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Compositions formulated for parenteral administration may be injected by bolus injection or by timed push, or may be administered by continuous infusion.

[0098] In order to prolong the effect of a compound of the present disclosure, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0099] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents such as phosphates or carbonates.

[00100] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[00101] The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Methods of Treatment of the Present Technology

[00102] In one aspect, the present disclosure provides a method for restoring regenerative potential (sternness) of alveolar stem cells in a subject in need thereof comprising administering to the subject an effective amount of a Nuclear Protein 1 (NUPR1) inhibitor or a lipocalin-2 (LCN2) inhibitor. In some embodiments, the subject is diagnosed with or suffers from a lung insufficiency. Examples of lung insufficiency include, but are not limited to, COPD (chronic obstructive pulmonary disease), cystic fibrosis, pneumonia, pulmonary embolism, COVID-19; conditions that affect the nerves and muscles that control breathing, such as amyotrophic lateral sclerosis (ALS), muscular dystrophy, spinal cord injuries, and stroke; problems with the spine, such as scoliosis (a curve in the spine); damage to the tissues and ribs around the lungs; drug or alcohol overdose; or inhalation injuries, such as from inhaling smoke (from fires) or harmful fumes. The subject may be an immunocompromised subject or a geriatric subject.

[00103] In one aspect, the present disclosure provides a method for preventing or treating lung cancer in a subject in need thereof comprising administering to the subject an effective amount of a Nuclear Protein 1 (NUPR1) inhibitor or a lipocalin-2 (LCN2) inhibitor, wherein the subject is a pediatric subject, an adolescent subject, or a young adult subject (e.g., < 55 years old). Also disclosed herein is a method for preventing or treating lung cancer in a subject in need thereof comprising administering to the subject an effective amount of a ferroptosis-inducing agent, wherein the subject is a pediatric subject, an adolescent subject, or a young adult subject (e.g., < 55 years old). In any and all embodiments of the methods disclosed herein, the young adult subject is about 54, about 53, about 52, about 51, about 50, about 49, about 48, about 47, about 46, about 45, about 44, about 43, about 42, about 41, about 40, about 39, about 38, about 37, about 36, about 35, about 34, about 33, about 32, about 31, about 30, about 29, about 28, about 27, about 26, about 25, about 24, about 23, about 22, about 21, or about 20 years old. The ferroptosis-inducing agent may be a class 1 ferroptosis inducer (system X_c“ inhibitor) or a class 2 ferroptosis inducer (glutathione peroxidase 4 (GPx4) inhibitor). Examples of ferroptosis-inducing agents include, but are not limited to, an inhibitory nucleic acid that targets GPX4, erastin, erastin derivatives (e.g., MEII, PE, AE, imidazole ketone erastin (IKE)), DPI2, BSO, SAS, lanperisone, SRS13-45, SRS13-60, RSL3, DPI7, DPI10, DPI12, DPI13, DPI17, DPI18, DPI19, ML160, sorafenib, artemisinin derivatives, artesunate, BAY87-2243, cisplatin, ironomycin, lanperisone, salinomycin, sulfasalazine, temozolomide, and lapatinib in combination with siramesine. In any of the preceding embodiments of the methods disclosed herein, the lung cancer is lung adenocarcinoma (LU AD). In certain embodiments, the lung cancer comprises a mutation in KRAS or TP 53.

[00104] Additionally or alternatively, in some embodiments of the methods disclosed herein, the NUPR1 inhibitor is a small molecule, an NUPR1 -specific inhibitory nucleic acid, or an anti-NUPRl neutralizing antibody. In certain embodiments, the NUPR1 inhibitor is trifluoperazine (TFP), ZZW-115, ZZW-129, ZZW-130, ZZW-131, ZZW-132, ZZW-142, ZZW-143, ZZW-144, ZZW-145, or ZZW-148.

[00105] Additionally or alternatively, in certain embodiments of the methods disclosed herein, the LCN2 inhibitor is a small molecule, an LCN2-specific inhibitory nucleic acid, or an anti- LCN2 neutralizing antibody. In some embodiments, the LCN2 inhibitor is ZINC00784494, ZINC00640089, ZINC00230567, or ZINC00829534.

[00106] In any and all embodiments of the methods disclosed herein, the NUPR1 -specific inhibitory nucleic acid, the LCN2-specific inhibitory nucleic acid or the inhibitory nucleic acid that targets GPX4 is a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA.

[00107] In another aspect, the present disclosure provides a method for restoring regenerative potential (sternness) of alveolar stem cells in a subject in need thereof comprising administering to the subject an effective amount of iron or transferrin. In some embodiments, the iron is unbound iron. In other embodiments, the iron is bound to transferrin or is complexed with vitamin C. In some embodiments, the subject is diagnosed with or suffers from a lung insufficiency. Examples of lung insufficiency include, but are not limited to, COPD (chronic obstructive pulmonary disease), cystic fibrosis, pneumonia, pulmonary embolism, COVID-19; conditions that affect the nerves and muscles that control breathing, such as amyotrophic lateral sclerosis (ALS), muscular dystrophy, spinal cord injuries, and stroke; problems with the spine, such as scoliosis (a curve in the spine); damage to the tissues and ribs around the lungs; drug or alcohol overdose; or inhalation injuries, such as from inhaling smoke (from fires) or harmful fumes. The subject may be an immunocompromised subject or a geriatric subject. [00108] In one aspect, the present disclosure provides a method for restoring regenerative potential (sternness) of aged adult stem cell compartments in a subject comprising administering to the subject an effective amount of a Nuclear Protein 1 (NUPR1) inhibitor or a lipocalin-2 (LCN2) inhibitor. In another aspect, the present disclosure provides a method for restoring regenerative potential (sternness) of aged adult stem cell compartments in a subject comprising administering to the subject an effective amount of iron or transferrin. Additionally or alternatively, in some embodiments, the aged adult stem cell compartments comprise one or more of neural adult subventricular zone (SVZ) stem cells, hair follicle stem cells, mesenchymal stem cells, keratinocyte stem cells, or intestinal stem cells.

[00109] Additionally or alternatively, in some embodiments of the methods disclosed herein, the NUPR1 inhibitor is a small molecule, an NUPR1 -specific inhibitory nucleic acid, or an anti-NUPRl neutralizing antibody. In certain embodiments, the NUPR1 inhibitor is trifluoperazine (TFP), ZZW-115, ZZW-129, ZZW-130, ZZW-131, ZZW-132, ZZW-142, ZZW-143, ZZW-144, ZZW-145, or ZZW-148.

[00110] Additionally or alternatively, in certain embodiments of the methods disclosed herein, the LCN2 inhibitor is a small molecule, an LCN2-specific inhibitory nucleic acid, or an anti- LCN2 neutralizing antibody. In some embodiments, the LCN2 inhibitor is ZINC00784494, ZINC00640089, ZINC00230567, or ZINC00829534.

[00111] Additionally or alternatively, in certain embodiments of the methods disclosed herein, the iron is unbound iron. In other embodiments, the iron is bound to transferrin or is complexed with vitamin C.

[00112] In any and all embodiments of the methods described herein, the NUPR1 inhibitor, LCN2 inhibitor, ferroptosis-inducing agent, iron and/or transferrin is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, iontophoretically, transmucosally, or intramuscularly. In any of the preceding embodiments of the methods described herein, the subject is human.

Modes of Administration and Effective Dosages

[00113] Any method known to those in the art for contacting a cell, organ or tissue with iron, transferrin, an inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent may be employed. Suitable methods include in vitro, ex vivo, or in vivo methods. In vivo methods typically include the administration of iron, transferrin, an inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent, such as those described herein, to a mammal, suitably a human. When used in vivo for therapy, the iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect). The dose and dosage regimen will depend upon the degree of the disease symptoms in the subject, the characteristics of the iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent, e.g., its therapeutic index, the subject, and the subject’s history.

[00114] The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount of iron, transferrin, an inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent useful in the methods may be administered to a mammal in need thereof by any of a number of well- known methods for administering pharmaceutical compounds. The iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent may be administered systemically or locally.

[00115] The iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent can be incorporated into pharmaceutical compositions for administration, singly or in combination, to a subject for the treatment or prevention of a disorder described herein. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[00116] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For convenience of the patient or treating physician, the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g., 7 days of treatment).

[00117] In some embodiments, the iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, or ferroptosis inducing agent described herein is administered by a parenteral route or a topical route.

[00118] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[00119] The iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent described herein can include a carrier, which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants can be included to prevent oxidation. In many cases, isotonic agents are included, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[00120] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00121] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[00122] For administration by inhalation, compositions including the iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent of the present technology can be delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.

[00123] Systemic administration of iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent of the present technology as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one embodiment, transdermal administration may be performed by iontophoresis.

[00124] The iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent of the present technology can be formulated in a carrier system. The carrier can be a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one embodiment, the therapeutic iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent is encapsulated in a liposome while maintaining structural integrity. As one skilled in the art would appreciate, there are a variety of methods to prepare liposomes. (See Lichtenberg et al. , Methods Biochem. Anal., 33:337-462 (1988); Anselem el al.. Liposome Technology, CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann. Pharmacother ., 34(7-8):915- 923 (2000)). An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.

[00125] The carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix. In one embodiment, the iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent can be embedded in the polymer matrix, while maintaining protein integrity. The polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly a-hydroxy acids. Examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is poly-lactic acid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Ann. Pharmacother ., 34(7- 8):915-923 (2000)). A polymer formulation for human growth hormone (hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).

[00126] Examples of polymer microsphere sustained release formulations are described in PCT publication WO 99/15154 (Tracy et al.), U.S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale et al.), PCT publication WO 96/40073 (Zale et al.), and PCT publication WO 00/38651 (Shah et al.). U. S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.

[00127] In some embodiments, the iron, transferrin, an inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent are prepared with carriers that will protect the iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using known techniques. The materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[00128] The iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent can also be formulated to enhance intracellular delivery. For example, liposomal delivery systems are known in the art, see, e.g., Chonn and Cullis, “Recent Advances in Liposome Drug Delivery Systems,” Current Opinion in Biotechnology 6:698- 708 (1995); Weiner, “Liposomes for Protein Delivery: Selecting Manufacture and Development Processes,” Immunomethods, 4(3):201-9 (1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery: Progress and Problems,” Trends Biotechnol., 13(12):527-37 (1995). Mizguchi et al., Cancer Lett., 100:63-69 (1996), describes the use of fusogenic liposomes to deliver a protein to cells both in vivo and in vitro. [00129] Dosage, toxicity and therapeutic efficacy of the iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. In some embodiments, the iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent exhibit high therapeutic indices. While the iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[00130] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any of the iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[00131] Typically, an effective amount of the iron, transferrin, inhibitor of NUPR1/LCN2- 2 axis, and/or ferroptosis inducing agent, sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Suitably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example, dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of iron, transferrin, an inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent ranges from 0.001-10,000 micrograms per kg body weight. In one embodiment, the iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent concentrations is in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[00132] In some embodiments, a therapeutically effective amount of iron, transferrin, an inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent may be defined as a concentration of iron, transferrin, an inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent at the target tissue of 10'¹² to 10'⁶ molar, e.g., approximately 10'⁷ molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg/kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue. In some embodiments, the doses are administered by single daily or weekly administration, but may also include continuous administration (e.g., parenteral infusion or transdermal application). In some embodiments, the dosage of the iron, transferrin, inhibitor of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agent of the present technology is provided at a “low,” “mid,” or “high” dose level. In one embodiment, the low dose is provided from about 0.0001 to about 0.5 mg/kg/h, suitably from about 0.001 to about 0.1 mg/kg/h. In one embodiment, the mid-dose is provided from about 0.01 to about 1.0 mg/kg/h, suitably from about 0.01 to about 0.5 mg/kg/h. In one embodiment, the high dose is provided from about 0.5 to about 10 mg/kg/h, suitably from about 0.5 to about 2 mg/kg/h.

[00133] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments. [00134] The mammal treated in accordance present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In some embodiments, the mammal is a human.

Kits

[00135] The present disclosure also provides kits for the prevention and/or treatment of lung cancer in young (non-aged) subjects, comprising one or more inhibitors of NUPR1/LCN2-2 axis, and/or ferroptosis inducing agents disclosed herein. Optionally, the above described components of the kits of the present technology are packed in suitable containers and labeled for the prevention and/or treatment of lung cancer in young (non-aged, e.g., <55 years old) subjects. In some embodiments, the lung cancer is lung adenocarcinoma (LU AD). Additionally or alternatively, in some embodiments, the lung cancer comprises a mutation in KRAS or TP53.

[00136] Also disclosed herein are kits for restoring regenerative potential (sternness) of aged alveolar stem cells/aged adult stem cell compartments in a subject comprising one or more of iron, transferrin, or an inhibitor of NUPR1/LCN2-2 axis. In some embodiments, the subject is diagnosed with or suffers from a lung insufficiency, including but not limited to, chronic obstructive pulmonary disease (COPD), COVID, influenza, and pneumonia sequelae. In some embodiments, the aged adult stem cell compartments comprise one or more of neural adult subventricular zone (SVZ) stem cells, hair follicle stem cells, mesenchymal stem cells, keratinocyte stem cells, or intestinal stem cells.

[00137] The above-mentioned components may be stored in unit or multi-dose containers, for example, sealed ampoules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution. The kit may further comprise a second container which holds a diluent suitable for diluting the pharmaceutical composition towards a higher volume. Suitable diluents include, but are not limited to, the pharmaceutically acceptable excipient of the pharmaceutical composition and a saline solution. Furthermore, the kit may comprise instructions for diluting the pharmaceutical composition and/or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper which may be pierced by a hypodermic injection needle). The kit may further comprise more containers comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts. The kits may optionally include instructions customarily included in commercial packages of therapeutic products, that contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and/or warnings concerning the use of such therapeutic products.

[00138] The kit can also comprise, e.g., a buffering agent, a preservative or a stabilizing agent. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present technology may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit. In certain embodiments, the use of the reagents can be according to the methods of the present technology.

EXAMPLES

[00139] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.

Example 1: Materials and Methods

[00140] Mice

[00141] All animal studies were approved by the MSKCC Institutional Animal Care and Use Committee (protocol # 17-11-008). MSKCC guidelines for the proper and humane use of animals in biomedical research were followed. All genetically engineered mice were maintained on a mixed C57BL/6J x Svl29 background. Similar numbers of female and male aged and young mice of the following genetically engineered strains were used: Kra^^SL~^G12D Trp53^{2lo:: 2o:}'^: (K )¹¹’¹², with or without reporter alleles including Rosa26^LSL~^Tomato (7)⁵⁰, Rosa2 (> SL-rTTA-IIUS-mKate2 RIK)5 , Rosa26^LSL~^Luc,ferase (E)⁵², and R26LSL-eGFP-Ca_S9 _{Cas9 wild}_ type C57BL/6J mice were purchased from Jackson Laboratories (strain #000664) at 10 and 77-78 weeks old and housed at the Sloan Kettering Institute animal facility until they reached 14 or at least 104 weeks to start experiments (young and aged, respectively). All mice were housed in non-pathogenic conditions, controlled temperature (20-25 °C), and a 12-hour light-dark cycle with 30-70% relative humidity. Food and water were provided ad libitum. Mice were genotyped at ~2 weeks of age.

[00142] Tumor initiation

[00143] Lung tumors were initiated in KP, KPT, KPL, and KP-RIK mice by intratracheal administration of 1.5-5 x 10⁸ plaque-forming units (pfu) of AdSPC-Cre (University of Iowa, #Berns-l 168) as previously described²⁶. In the survival experiment (FIG. IB) mice were euthanized upon humane endpoint (respiratory distress, extreme weight loss, lethargy).

Alternatively Eml4-Alk gene fusion-driven lung tumors were induced in young and aged wild type C57BL6/J mice (strain #000664) by intratracheal administration of 5 x 10⁸ pfu of adenoviral vectors encoding U6-sgAlk-U6-sgEml4-CMV-Cas9, as previously described³⁰.

[00144] Hyperoxia lung injury

[00145] Alveolar injury was induced in aged and young C57BL/6J mice by 66 hours of exposure to >85% 02 in hyperoxia chamber (BioSpherix). The fractional inspired oxygen concentration in the chamber was monitored by an in-line oxygen analyzer and maintained with a constant flow of gas (~3 L/min). Mice were euthanized on days 0, 3, and 10 after the 66-hour exposure, followed by collection of lungs for histological analysis. Proliferating AT2 cells were identified by co-staining for AT2 marker surfactant protein-C (SPC, EMD Millipore, #AB3786) and proliferation maker Ki67 (Invitrogen, #14-5698-82).

[00146] Lung tissue harvest

[00147] Mice were euthanized by CO2 asphyxiation followed by systemic perfusion with PBS to clear lungs of blood. For histological analysis, lung tissues were fixed in 10% neutral- buffered formalin (Sigma Aldrich, #HT501128) overnight and further processed and embedded in paraffin using standard protocols. Five micrometer sections were prepared by the Molecular Cytology Core Facility (MCCF) at MSKCC for hematoxylin and eosin (HE) staining, immunofluorescence, and immunohistochemistry. [00148] Quantification of tumor burden

[00149] Tumor burden was quantified using HE or immunohistochemical detection in 5 pm formalin-fixed paraffin-embedded (FFPE) sections of lung tissues. For early-stage tumors (4- and 8-week post-tumor initiation), cancer cells were identified by the immunohistochemistry staining of tdTomato reporter allele (anti-RFP, Rockland, 600-401- 379). Late-stage tumors (12- and 17-week post-tumor initiation) were identified by HE staining. Tumor burden was defined by the number of nodules in the cross section of tumorbearing lungs, normalized by the size of lung tissue. Tumor area, tumor diameter and tissue area were determined using Caseviewer (3DHISTECH).

[00150] Histological analysis

[00151] Histological classification of mouse lung tumor grades was performed on the HE- stained sections by two methods: (i) an automated deep neural network (Aiforia Technologies, NSCLC_v25 algorithm), as and (ii) an independent classification by a board- certified veterinary pathologist, who was blinded to the sample group identifiers. Established histopathological criteria to evaluate mouse models of lung cancer were used^11,45. Tumor grades in pulmonary lobes ranged from 1 to 4 with grade 1 being composed by uniform histomorphology of the neoplastic cells without nuclear atypia and grade 4 being composed by pleomorphic neoplastic cells exhibiting increased nuclear atypia, mitoses and/or occasionally binucleated to multinucleated cells. The following tumor subtypes were identified in the lungs of young and aged mice: solid adenomas and adenocarcinomas, papillary adenomas and adenocarcinoma, and mixed tumor subtypes containing both papillary and solid structures.

[00152] Immunohistochemistry

[00153] Immunohistochemistry was performed on 5 pm FFPE sections using standard staining protocols. Briefly, sections were de-paraffmized and heat-induced antigen retrieval was performed by EDTA antigen retrieval buffer (Sigma Aldrich, #E1161). Sections were blocked by BLOXALL solution (Vector laboratories, #SP-6000-100) at room temperature for 30 minutes and incubated with primary antibody at 4 °C overnight. IgG controls (Thermo Fisher Scientific, #02-6102, #02-6202 and #10400C) from the corresponding species of primary antibody were used as negative controls. [00154] Signal development was performed by ImmPRESS Polymer Detection Kits (Vector Laboratories, #MP-7401-50) and ImmPACT DAB Substrate Kit, Peroxidase (HRP) (Vector Laboratories, #SK-4105) following the manufacturer’s protocol. The sections were counterstained with hematoxylin (Thermo Fischer Scientific, #72404) and mounted with coverslips. Mounted slides were digitally scanned by the Mirax Midi-Scanner (Carl Zeiss AG). Image analysis was performed by Fiji⁵³.

[00155] Immunofluorescence

[00156] Immunofluorescence was performed on 5 pm FFPE sections. Briefly, sections were de-paraffinized and heat-induced antigen retrieval was performed by EDTA antigen retrieval buffer (Sigma Aldrich, #E1161). Sections were blocked by donkey immunomix [0.2% BSA (Sigma, #810533), 5% donkey serum (Thermo Fisher Scientific, #31874) and 0.3% Triton-X (Fisher Scientific, #BP151-100) in PBS (Gibco, #10010-023)] at room temperature for 30 minutes. Incubation of primary antibodies diluted in donkey immunomix was performed at 4 °C overnight. IgG controls (Thermo Fisher Scientific, #02-6102, #02- 6202, and #10400C) from the corresponding species of primary antibody were used as negative controls. AlexaFluor secondary antibodies raised in donkey were used for signal detection (Thermo Fisher Scientific #A-31571, #A-21207, #A32795, #A-11058, #A32787). Finally, slides were counterstained with 1 pg/mL DAPI (Sigma Aldrich, #D9542) for 10 min, with coverslips using Mowiol mounting reagent (EMD Millipore, #475904). Mounted slides were digitally scanned by the Mirax Midi-Scanner (Carl Zeiss AG). Image analysis was performed by Fiji⁵³.

[00157] Detection of senescence-associated beta-galactosidase activity

[00158] Fluorescent senescence-associated (SA)-P-gal labeling was performed according to manufacturer’s instructions using the ImaGene Red C12RG lacZ Gene Expression Kit (Invitrogen, #12906). The tissues used for staining were flash frozen by liquid nitrogen and directly embedded in O.C.T medium (Fisher Healthcare, #4585). Cryoblocks were immediately sectioned at 5 pm thickness. Sections were placed on SuperFrost microscope slides (Fischer Scientific, #12-550-15) and immediately used for staining. For staining, sections were incubated with 1% chloroquine at 37 °C for 30 minutes. Chloroquine was removed by two washes using pre-cooled PBS. Sections were incubated with C12RG substrate (1 :50 dilution) at 37 °C for 2 hours, followed by an immediate rinse by PBS and incubation in PETG (1 :50) solution at room temperature for 20 minutes. Sections were fixed in 4% PFA for 20 minutes on ice. To identify AT2 cells and tumor cells derived from AT2 cells, we performed SPC immunofluorescence after C12RG staining, as described above. Slides were digitally scanned using the Mirax Midi-Scanner (Carl Zeiss AG) and C12RG positive senescent cells were quantified using Fiji⁵³.

[00159] Single-molecule mRNA in situ hybridization

[00160] In situ hybridization was performed on freshly sectioned 5 pm FFPE sections using the RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, #323100) following manufacturer’s protocol. Probes against mouse Sftpc (Advanced Cell Diagnostics, #314101-C2) an Nuprl (Advanced Cell Diagnostics, 434811) were used. Probe signals were developed using Opal-520 and Opal-570 dyes (Akoya Biosciences, #OP-001001 and #OP-001003). Slides were digitally scanned by Mirax Midi-Scanner (Carl Zeiss AG). AT2 and LU AD cells were identified by Sftpc positivity and Nuprl expression was quantified using Fiji⁵³ as number of individual dots overlapping with the nuclear DAPI staining.

[00161] Lung dissociation

[00162] For isolation of normal AT2 cells and tumor cells derived from 4-, 8- and 12-week time points, lungs were inflated with 3 ml digestion buffer [S-MEM (Thermo Fisher Scientific, #11380037) with 1.7 Ul/ml dispase (Coming, #354235), 0.5 UEpl collagenase IV (Thermo Fisher Scientific, #17104019) and 10 pg/ml DNase I (StemCell Technologies, #07900) through the trachea and then finely minced. For the 17-week time point, tumors were micro-dissected, finely minced, and suspended in ~2-3 volumes of digestion buffer. Tissues were dissociated in a 37 °C oven with gentle agitation for 45-60 minutes. The dissociated tissue cells were filtered using a 100 pm strainer (Coming, #431752) and spun at 1500 rpm for 5 minutes at 4 °C. The supernatant was removed by aspiration and red blood cell lysis was performed using BD PharmLyse (BD Biosciences, #555899) following manufacturer’s protocol. Cells were washed with S-MEM (Thermo Fisher Scientific, #11380037) with 2% heat-inactivated fetal bovine serum (HI-FBS, Hyclone, #SH30910.03), filtered through a 40 pm strainer (Corning, #431750), and pelleted by spinning at 1500 rpm for 5 minutes at 4 °C. Cell pellets were resuspended in 2% HI-FBS S-MEM for antibody staining. [00163] Human lung tissues were obtained from 4 patients undergoing pulmonary resections. Normal tissue was obtained from outside the tumor margin, as determined by intraoperative pathology examination. The human lung tissues were dissociated using a similar protocol as for the mice. Briefly, the lung tissue was minced into small cubic pieces and washed several times by pre-cooled PBS. Excessive PBS was drained and tissues were further minced and dissociated in 8-10 volumes of digestion buffer for 1-1.5 hours. Dissociated tissues were processed as described above and cell pellets were resuspended in 2% HI-FBS S-MEM for antibody staining.

[00164] Fluorescence activated cell sorting (FACS)

[00165] Single cell solutions of lung cells were incubated with mouse FcR block (BD Biosciences, #553142) on ice for 10 minutes, followed by 30 minutes of staining with desired antibody panels on ice. To sort cells for single-cell sequencing, cell-hashing method was used to maximize the number of biological replicates and control for batch effect in each singlecell sequencing lane. To do this, the cell suspension derived from each mouse was labelled by a different hashtag antibody (2 pl/test, Biolegend, TotalSeq Panel B, #B0301-B0310) together with the desired antibody panel, following the manufacturer’s protocol. After the 30- minute staining period, cells were centrifuged at 1500 rpm for 5 minutes at 4 °C and washed twice with 2% HI-FBS S-MEM. Cell pellets were resuspended in PBS with 2% HI-FBS (Hyclone, #SH30910.03) containing 1 pg/mL DAPI (Sigma Aldrich, #D9542) to detect dead cells. Samples were sorted using a BD FACS Aria Sorter using 4-way purity mode. AT2 cells were defined as MHCII+/EpCAM+/Scal-/Podoplanin-/lineage-(CD45, CD31, CDl lb, CD11c, F4/80 and Teri 19)/D API- cells. Seal and podoplanin were included to maximize the purity of AT2 cells. Early-stage cancer cells (4, 8, and 12 weeks post-tumor initiation) were isolated by selecting tdTomato+ (for KPT mice), mKate2+ (for KP-RIK mice), or GFP+ (for KP-Cas9 mice) and EpCAM+/lineage-/DAPI- cells. For later-stage tumors, EpCAM+/lineage-/DAPI- cancer cells were isolated from micro-dissected tumors. For the isolation of human AT2 cells, single-cell solution of lung cells was incubated with Human TruStain FcX (1 :20, Biolegend, #422302) for 10 minutes, followed by staining with antihuman CD45 (Biolegend, #368538), CD31 (Biolegend, #303116), EpCAM (Biolegend, #324222) and HTII-280 (marker for human AT2 cells⁵⁴, 1 :40, Terrace Biotech, #TB- 27AHT2-280) for 30 minutes on ice. Cells were washed once with 2% HI-FBS S-MEM and further stained with PE-conjugated anti-mouse IgM (Thermo Fisher Scientific, #12-5790-81) for 30 minutes to detect the anti-HTII-280 primary antibody. Cell samples were spun at 1500 rpm for 5 minutes at 4 °C and washed twice with 2% HI-FBS S-MEM. Cell pellets were resuspended in 2% HI-FBS PBS with 1 pg/mL DAPI (Sigma Aldrich, #D9542) to stain dead cells. Samples were sorted using a BD FACSAria Sorter on purity mode. AT2 cells were defined as HTII-280+/EpCAM+/CD45-/CD31-/DAPI-⁵⁴.

[00166] Alveolar organoid culture

[00167] FACS-purified AT2 cells were co-cultured with endothelial cells for alveolar organoid culture. Endothelial cells (CD31+/CD45-/DAPI-) were isolated from 4-week old Rosa26^mTmG mice⁵⁵ by FACS. Primary endothelial cells were expanded briefly using endothelial cell media [Advanced DMEM (Thermo Fisher Scientific, #12491015), 20% FBS (Hyclone, #SH30910.03), 1% GlutaMax (Thermo Fisher Scientific, #35050061), 1% Pen/Strep (Thermo Fisher Scientific, #15070063), 0.1 mg/ml Endothelial cell growth supplement (ECGS, Sigma Aldrich, #E2759, 0.1 mg/ml), heparin (Sigma Aldrich, #H3149, O. lmg/ml), and 25 mM HEPES (Thermo Fisher Scientific, #15630080)]; only cells from passages 2-3 were used. One to five thousand freshly sorted AT2 cells with 50,000 endothelial cells were resuspended in 50 pl alveolar organoid media [Ham’s F-12 (Thermo Fisher Scientific, #11765047), 10% FBS (Hyclone, #SH30910.03), 1% GlutaMax (Thermo Fisher Scientific, #35050061), 1% Pen/Strep (Thermo Fisher Scientific, #15070063), 1% ITS (Millipore Sigma, #13126) and 1% HEPES (Thermo Fisher Scientific, #15630080)] and mixed with 50 pl Matrigel (Fisher Scientific, #CB-40230C). Cell mix was placed in cell culture inserts (Thermo Fisher Scientific, #08-770). Alveolar organoid culture media (500 pl) was added in the 24-well companion plate (Thermo Fischer Scientific, #353504) and replaced every 3 days during culture. ZZW-115 (Cayman Chemical Company, #34974, 2 pM), Deferoxamine mesylate (DFO, Selleckchem, #S5742, 2 pM) and iron-loaded recombinant mouse transferrin (Rockland, #010-0134, 50 pg/ml) were added to the media on day 3 and refreshed every 3 days. Organoids were imaged with an EVOS M5000 Microscope and the number of organoids > 50 pm in diameter was counted at 2 weeks after plating. For secondary organoid cultures, primary organoids were digested with 5 lU/ml dispase (Coming, #354235) for 1 hour at 37 °C. Organoids released from Matrigel were collected by spining at 1000 rpm for 5 minutes. Organoids were further dissociated into single cell solution with TrypLE (Thermo Fisher Scientific, #12604013) for 10 minutes at 37 °C. Live AT2 cells were further purified from single-cell solution by sorting for MHCII+ZEpCAM+ZDAPI- and re-plated using the same protocol as for primary AT2 culture.

[00168] Ex vivo transformation assay

[00169] AT2 cells isolated from KP-RIK and KP-Cas9 mice were transduced by annotated lentivirus at multiplicity of infection (MOI) of 20 by spinfection (600 g, 37 °C, 30 minutes). One to five thousand AT2 cells were plated in the inserts with endothelial cells, as described above. Alveolar organoid culture media was used and replaced every 3 days. ZZW-115 (Cayman Chemical Company, #34974, 2 pM), Deferoxamine mesylate (DFO, Selleckchem, #S5742, 2 pM), GSK3685032 (Selleckchem, #E1046, 1 pM), iron-loaded recombinant mouse transferrin (Rockland, #010-0134, 50 pg/ml), and recombinant mouse lipocalin-2 (R&D Systems, #1857-LC-50, 100 ng/ml) were added to the media on day 3 and refreshed every 3 days. Transformed tumor spheres were identified by mKate2 or GFP fluorescence. Organoids were imaged with an EVOS M5000 microscope and tumor spheres > 50 pm in size were counted 2 weeks after plating.

[00170] Tumor sphere culture

[00171] Freshly isolated tumor cells from aged and young KP LUAD 17 weeks post-tumor initiation were seeded in 3D culture plugs, as before²⁵. Eight hundred pl of tumor sphere media [Advanced DMEM/F-12 (Thermo Fisher Scientific, #12634028) with 2% HI-FBS (Hyclone, #SH30910.03), 1% GlutaMax (Thermo Fisher Scientific, #35050061), 1% Pen/Strep (Thermo Fisher Scientific, #15070063), 1% HEPES (Thermo Fisher Scientific, #15630080), 10 ug/mL Gentamicin (Thermo Fisher Scientific, 15750060)] was added to each well and refreshed every 3 days until the end of the experiment.

[00172] For serial passage of ex vivo transformed tumor spheres, Matrigel was digested with 5 lU/ml dispase (Corning, #354235) for 1 hour at 37 °C. Organoids released from Matrigel were collected by spining at 1000 rpm for 5 minutes. Organoids were further dissociated into single-cell solution with TrypLE (Thermo Fisher Scientific, #12604013) for 10 minutes at 37 °C. Cells were re-plated in Matrigel plugs at 3000 cells/plug and were grown in tumor sphere media without endothelial cells. Cells were grown for eight passages. Following each passage, a portion of cells were preserved in RLT-Plus buffer (Qiagen, #1053393) with 1% 2-mercaptoethanol (Sigma Aldrich, #M6250) and stored at -80 °C for RNA extraction. RNA was extracted with the RNeasy Mini Kit (Qiagen, #74104) following manufacturer’s protocol.

[00173] Human alveolar organoid culture

[00174] Human alveolar organoids were established from primary AT2 cells usign the insert culture system. One to four thousand AT2 cells were resuspended in 50 pl complete human alveolar organoid media [Advanced DMEM (Thermo Fisher Scientific, #12491015), 1% GlutaMax (Thermo Fisher Scientific, #35050061), 1% Pen/Strep (Thermo Fisher Scientific, #15070063), 1% HEPES (Thermo Fisher Scientific, #15630080), B27 supplement (1 :50, Thermo Fisher Scientific, #17504044), recombinant human FGF7 (100 ng/ml, Peprotech, 100-19), recombinant human FGF10 (100 ng/ml, Peprotech, #100-26), recombinant human Noggin, (100 ng/ml, Peprotech, #120-10C), recombinant human EGF (50 ng/ml, Thermo Fisher Scientific, #PHG0311), N-acetylcysteine (1 mM, Sigma Aldrich, #A9165), Nicotinamide (10 mM, Sigma Aldrich, #N0636), A083-01 (1 pM, Selleckchem, #S7692), CHIR99021 (3 pM, Sigma Aldrich, #SML1046), and Rspo3-Fc Fusion Protein Conditioned Medium (Immunopreci se, #R001-100ml, 2%)] and mixed with 50 pl Matrigel (Fisher Scientific, #CB-40230C). Human alveolar organoid media refreshed every 3 days until the end of the experiment. Iron-loaded recombinant human transferrin (Optiferrin Recombinant Transferrin, InVitria, #NC9954311, 100 pg/ml) was added to the media on day 3 and refreshed every 3 days. Organoids were imaged using an EVOS M5000 microscope and organoids > 30 pm in size were counted 3 weeks after plating.

[00175] Intracellular iron measurement

[00176] Cellular iron was measured by induction-coupled plasma mass spectrometry (ICP- MS; Agilent 7900) equipped with integrated sample introduction system (ISIS3) in high energy helium mode (10 ml/min; 1 point/peak, 4 replicates, 25 sweeps/replicate). The method was linear between 51000 ng/mL. Cell pellets (16,000-10,000,000 cells) were digested overnight in 100 uL of tetramethylammonium hydroxide. The cell pellet digestates were diluted 1 :80 in diluent (4% 1-butanol, 1% TMAH, 0.01% Triton X-100, 0.01% ammonium pyrrolidinedithiocarbamate) and quantified using the iron isotope 56 relative to a 7-point calibration and germanium as an internal standard. Results are reported relative to cell number.

[00177] Lentivirus production

[00178] HEK293T cells were transfected with custom-made shuttle vector plasmid and packaging plasmids pMD2.G (Addgene, #12259) and psPAX (Addgene, #12260) using TransIT-LTl Transfection Reagent (Minis Bio, #MIR 2305). Virus-containing media was collected 48 and 72 hours post-transfection, concentrated by ultracentrifugation (31500 rpm,

4 °C, 2 hours), resuspended overnight at 4 °C in D-MEM,and stored at -80 °C. Lentiviral vectors encoding Cre recombinase were titered using GreenGo Cre recombination reporter cells, as before²⁴.

[00179] Measurement of transduction efficiency

[00180] Adenoviral transduction efficiency in vivo was measured by intratracheal delivery

5 x 10⁸ pfu AdSPC-Cre to airways of aged and young Rosa26^LSL~^tdTomato and Rosa26^mTmG mice⁵⁵. Cells transduced by adenoviral Cre (University of Iowa Viral Vector Core, Berns- 1168) were quantified by flow cytometry for tdTomato or mGFP fluorescence, respectively. Lentiviral transduction efficiency was measured using a similar flow cytometry approach, in this case detecting phosphoglycerate kinase-1 promoter-driven mScarlet (PGK-mScarlet) following transduction of lung epithelial cells with lentivirus that was prepared and titered inhouse, as before^25,56. Five thousand pfu of PGK-mScarlet lentivirus was intratracheally delivered to lungs of Kras^+/+,. Trp53f^ox/f^ox mice, which were littermates of the Kras' ^GG(’^GD ,. Tr[)53'^lox "^ox mice used in the lung tumor studies. The fraction of mScarlet positive portion of total AT2 cells was evaluated to measure transduction efficiency. To measure the transduction efficiency of AT2 cells in the ex vivo transformation assay, PGK-GFP lentivirus produced and titered in-house was introduced to isolated wild-type aged vs. young primary AT2 cells by spinfection. The transduction efficiency was measured as ratio of GFP positive organoids in the total pool of alveolar organoids.

[00181] Quantitative PCR (qPCR)

[00182] RNA was extracted from sorted tumor and AT2 cells using the RNeasy Micro Kit (Qiagen, #74004) or TRIzol reagent (Invitrogen, #15596026) and standard chloroformisopropanol precipitation. cDNA was synthesized using the PrimeScript RT Reagent Kit (Clontech, #RR037B). Quantitative PCR was performed in triplicate with 30 ng of cDNA using the Powerup SYBR Green Master Mix (Applied Biosystems, #A25778) on the QuantStudio 7 Flex Real-Time PCR System. The AACT method was used to compare markers of interest and expression was normalized to Gapdh. The oligonucleotides used in this study are listed below

[00183] Single-cell mRNA sequencing (scRNA-seq)

[00184] Wild-type AT2 cells and KP LUAD tumor cells (4, 12, and 17 weeks post-tumor initiation) were isolated by FACS as described above. LUAD cells from tumors at 19-20 weeks post-initiation, representing all mature cancer cell states, were added to the analysis to facilitate unsupervised clustering and cell state identification. Ten to forty thousand freshly sorted cells were suspended in PBS with 0.04% BSA at the concentration of 1000 cells/pl for droplet-based scRNA-seq. Encapsulation and library preparation were performed using the 10X Genomics Chromium Single Cell 3’ Library & Gel bead Kit V3 according to manufacturer’s protocol. Libraries were sequenced using Nova-Seq 6000 platform (Illumina).

[00185] Bulk mRNA sequencing of AT2 cells isolated from mice administered DNMT1 inhibitor

[00186] DNMT1 inhibitor GSK3685032 (Selleckchem, #E1046) or vehicle [10% captisol (sulfobutylether-P-cyclodextrin, MedChemExpress, #HY-17031) adjusted to pH 4.5-5, stored for up to 1 week at 4 °C] was administered by intrapretoneal injection, twice daily, at the dose of 10 mg/kg body weight. After an eight-day treatment, AT2 cells were isolated from all mice by FACS, as described above. Cells were pelleted and resuspended in 350 pl Buffer RLT plus (Qiagen, #1053393) and stored at -80 °C. RNA extraction was performed using TRIzol Reagent (Thermo Fisher Scientific, #15596018). Phase separation in cells lysed in 1 mL TRIzol Reagent was induced with 200 pL chloroform. RNA was extracted from 350 pL of the aqueous phase using the RNeasy Mini Kit (Qiagen, #217004) on the QIAcube Connect (Qiagen) according to the manufacturer’s protocol. Samples were eluted in 35 pL RNase-free water. After RiboGreen quantification and quality control by Agilent BioAnalyzer, 2 ng total RNA with RNA integrity numbers ranging from 9.0 to 9.7 underwent amplification using the SMART-Seq v4 Ultra Low Input RNA Kit (Clonetech, #63488), with 12 cycles of amplification. Subsequently, 10 ng of amplified cDNA was used to prepare libraries with the KAPA Hyper Prep Kit (Kapa Biosystems, #KK8504) using 8 cycles of PCR. Samples were barcoded and run on a NovaSeq 6000 in a PEI 00 run, using the NovaSeq 6000 S2 Reagent Kit (200 Cycles) (Illumina). An average of 34 million paired reads were generated per sample and the percent of mRNA bases per sample ranged from 89% to 93%.

[00187] Methylome sequencing of AT2 cells and tumors from aged and young mice

[00188] Fifty to one hundred thousand wild-type mouse AT2 cells or LU AD tumor cells (12 weeks post-tumor initiation) were isolated by FACS, as described above. Genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen, #69504). NEB Next Enzymatic Methyl-seq (EM-seq, New England Biolabs, #E7120S) was used to identify 5-methylcytosine and 5-hydroxymethylcytosine bases and sequencing libraries were constructed by NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, #E7645S), following the manufacturer’s protocol. After PicoGreen quantification and quality control by Agilent TapeStation, libraries were pooled equimolar and run on a NovaSeq 6000 at PEI 50, using the NovaSeq 6000 S4 Reagent Kit (300 cycles) (Illumina). The loading concentration was 0.6- 0.7 nM and a 1% spike-in of PhiX was added to the run to increase diversity and for quality control purposes. The runs yielded on average 795 million reads per library.

[00189] Perturbation q/Auprl in vivo using CRISPR/Cas9

[00190] Lungs of aged and young KP-Cas9 mice were intratracheally transduced with U6- sgRNA-PGK-Cre lentivirus containing a guide RNA targeting Nuprl or a non-targeting control sgRNA²⁵. Lungs with tumors were harvested at 12 weeks post-tumor initiation. The whole lungs were paraffin embedded and stained for GFP (Abeam, #ab5450) and Ki67 (Thermo Fisher Scientific, #14-569882) to quantify the tumor burden and percentage of proliferating tumor cells, respectively. Additionally, some lung tumors were micro-dissected and dissociated for isolation of GFP+/EpCAM+/Lineage-/DAPL tumor cells for bulk mRNA sequencing

[00191] Human tissue

[00192] Archived and fresh human normal lung and LU AD tissues obtained under MSKCC Institutional Review Board approval (IRB #06-107 and IRB #12-245). Archived normal human lung tissue sections were obtained from both young (< 50 years old) and aged (>70 years old) patients. Areas chosen for analysis were evaluated by a board-certified pathologist at MSKCC to contain normal healthy lung.

[00193] Human LUAD tissue microarray (TMA) includes 35 young (<55 years old) and 37 aged (> 80 years old) cases. Tissue blocks from each case were examined to select for regions with maximal presence of tumor tissue and three cores (2.0 mm in diameter) of each case were taken to construct the TMAs. In the analysis, one aged case was excluded because the tissue sections detached from the glass slide.

[00194] Fresh human lung tissue to generate alveolar organoids was obtained with informed consent from patients under protocols approved by the MSKCC Institutional Review Board (IRB #12-245). The normal lung tissue was obtained from the distal tissue of surgical rescetions of patients with lung adenocarcinomas.

[00195] Computational Methods

[00196] Processing and analysis of single-cell RNA-sequencing data

[00197] FASTQ files of single-cell RNA-sequencing data generated on the 10X Chromium platform were processed using the standard CellRanger pipeline (version 5.0.0). Reads were aligned to a custom GRCm38 / mm 10 reference containing additional transgenes used in the study. The generated cell-gene count matrices were analyzed using a combination of published packages and custom scripts in either the scanpy / AnnData⁵⁷ (Python) or Seurat⁵⁸ (R) ecosystems. The specific analytic workflows employed are summarized below.

[00198] Pre-processing, quality control, and filtering of single-cell RNA-sequencing data [00199] Single-cell RNA-sequencing data from young / aged KP LU AD tumors (4 weeks, 12 weeks, 1718 weeks, and 19-20 weeks following initiation) and healthy lungs were dehashed and compiled into a combined count matrix. Cells with less than 500 UMIs, more than 20% mitochondrial UMIs and low complexity based on the number of detected genes vs. number of UMIs were removed. Doublets were filtered using scrublet⁵⁹. UMI counts were normalized using the size factor approach, as previously described⁶⁰.

[00200] Single-cell transcriptomes that passed filtering were subjected to an initial round of unsupervised clustering and low-dimensional embedding. Specifically, highly variable features were selected using a variance stabilizing transformation and dimensionality reduction was performed on normalized, log2 -transformed count data using either principal component analysis or non-negative matrix factorization. The dimensionality reduced count matrices were then used as input for unsupervised clustering with the leiden algorithm and two-dimensional embedding using UMAP; bbknn was used to control for batch effects. Epithelial and stromal clusters were identified using a set of previously published marker genes.

[00201] Clustering of cancer cells from young and aged KP LUAD tumors

[00202] Young and aged cells belonging to epithelial clusters (ATI cells, AT2 cells, and cancer cells) were selected. To ensure that the downstream analysis was not biased by any particular tumor stage or age, cells were randomly subsampled to approximately equalize cell counts between tumor stages and ages, resulting in the following cell numbers:

[00203] Unsupervised clustering and UMAP embedding of cancer cells was performed as described above. Cluster identities were assigned using marker genes identified in a previous study²³.

[00204] Analysis of enrichment of young or aged cells in KP LU AD tumors

[00205] To test whether young or aged cells were enriched in certain cancer cell populations, a meta-cell-based approach was employed. Meta-cells are neighborhoods of similar cells representing discrete cell states that are identified in high dimensional space and can subsequently be projected onto low-dimensional representations of the data. Meta-cells were identified separately for each tumor stage using the SEACells algorithm⁶¹ based on equal numbers of young and aged cells. To control for batch effects, Aarmcwy-corrected⁶² principal components were used as input for meta-cell generation. The ratio of young and aged cells in each meta-cell was determined and projected onto the UMAP.

[00206] Identification of age-related gene signatures

[00207] The differentially expressed gene analysis in normal AT2 and LU AD was performed using the following pipeline. The raw gene expression count was normalized using log2TPM and filtered with expression threshold >10% of cells. The differential gene expression analysis was performed with MAST⁶³ (1.16.0) in a two-part generalized linear model. DEGs were identified in all tumor cells with batch, sex, cell type and cellular detection rate added as covariates. Subsequently, the aging-related DEGs in all five LU AD cell states (AT2-like, ATI -like, high-plasticity state, endoderm-like, ribosomal) that molecularly define LU AD progression were performed separately with batch, sex, and cellular detection rate included in the model as covariates. A similar DEG analysis was also carried out in the healthy AT2 cells.

[00208] The inherited aging signature across wild-type AT2 cells and the five LU AD states was defined based on the following criteria: (1) significance in wild-type AT2 cells with FDR < 0.1; (2) same gene expression change trend across all cell types; (3) top 25 upregulated and downregulated based on the sum of absolute DEG MAST regression coefficient across all cell types.

[00209] Gene set enrichment analysis

[00210] Age-related gene signatures ( - value < 0.05; fold change > 0.1; n(young) = 1193 genes; n(aged) = 739 genes) in all tumor cells were tested for gene set enrichment using the EnrichR and PreRank functionalities implemented in the gseapy package⁶⁴. The following gene set repositories were used for enrichment analysis: MSigDB Hallmark (2020), KEGG mouse (2019), Reactome (2022), GO Biological Process (GOBP; 2021). In addition to unsupervised gene set enrichment analysis, iron metabolism related gene sets were a priori identified from the aforementioned repositories and tested on the aged signature.

[00211] Processing and analysis of bulk-cell RNA-sequencing data

[00212] FASTQ files of bulk-cell RNA-sequencing data were aligned to a custom GRCm38 / mmlO reference containing additional transgenes using STAR (version 2.7.5a)⁶⁵. Read counts were generated using the Python package HTSeq-count⁶⁶. The generated sample-gene count matrices were analyzed using a combination of published packages and custom scripts. The specific analytical workflows employed are summarized below.

[00213] Analysis of gene expression during ex vivo transformation

[00214] Bulk RNA-sequencing data from ex vivo transformation experiments of young and aged cells was compiled object and normalized using the size factor normalization function of DESeq2⁶'' . Highly variable features were identified using a variance stabilizing transformation. Diffusion maps were generated in scanpy using the top principal components from normalized, log2 -transformed count data. The pseudotime position of each sample was then calculated in scanpy in diffusion space using untransformed AT2 cells as root. To identify genes differentially expressed between young and aged ex vivo transformed cells independent of differentiation stage, gene expression models controlling for either passage or pseudotime were fitted in DESeq2.

[00215] DNA methylation data processing

[00216] The Bismark pipeline⁶⁸ was adopted to map DNA methylation sequencing reads and determine cytosine methylation states. Using Trim Galore (version O.6.4)⁶⁹’⁷⁰, raw reads with low-quality (less than 20) and adapter sequences were removed. The trimmed sequence reads were C(G) to T(A) converted and mapped to similarly converted reference mouse genome (mm 10) using default Bowtie 2⁷¹ settings implemented by Bismark. Duplicated reads were discarded. The remaining alignments were then used for cytosine methylation calling by Bismark methylation extractor. For AT2 cells, around 22.5M CpG sites were detected with an average coverage of 33x in the eight samples. For lung tumor cells, 23M CpG sites were recovered with an average coverage of 40x in nine samples.

[00217] Differential methylation analysis

[00218] Differentially methylated CpGs (DMCs) were identified using the DSS R package^72,73 on the basis of dispersion shrinkage method followed by Wald statistical test for beta-binomial distributions. Any CpGs with FDR < 0.05 and methylation percentage difference greater than 10% were considered significant DMCs. Pairwise comparisons were conducted between the aged group and young group for normal AT2 cells and LU AD cells, respectively.

[00219] DMCs correlate with gene expression changes at regulatory regions

[00220] To connect DMCs with promoters and enhancers in AT2 cells, we mapped DMCs to modifications of histone (H)3K4mel, H3K27ac, and H3K4me3⁴⁰. The correlation between differential methylation level at AT2 DMCs and differential gene expression change at AT2 DEG (FDR < 0.05) in each AT2 cell histone mark category was calculated using Pearson correlation test via the cor. test function in R.

[00221] Mean promoter methylation level at aging signatures in AT2 and LUAD [00222] The mean promoter methylation level at aging signature genes were calculated by taking the mean of all measured CpG sites overlapping the promoter region. The promoter is defined as Ikb upstream and 200 bp downstream 200 to the transcriptional start site (TSS). Wilcoxon test was performed to test the statistical significance of the difference in R. The correlation between the promoter differential methylation in AT2 and LU AD were measured using Pearson correlation via the cor. test function in R.

[00223] Analysis of gene expression change following DNMT1 inhibition in AT2 and LUAD

[00224] The raw gene expression count data were filtered with filterByExpr() command and normalized with the TMM method within the edgeR pipeline (version 3.34.1)⁷⁴ in R. Subsequently, the differentially expressed genes (DEGs) were identified with an overdispersed Poisson model adjusting for batch and sex using edgeR (version 3.34.1).

[00225] Module score method comparing mouse and human LUAD gene expression

[00226] To study the relevance of the aged and young KP mouse model signatures in patients, a correlative strategy leveraging the TCGA-LUAD cohort was used. Patients with at least one non-synonymous KRAS mutation and at least 50% tumor purity were selected (n = 120). First, significantly differentially expressed genes (DEGs) from malignant cells from the aged versus young mouse model tumors were selected. Next, human orthologs of these DEGs were obtained using the R package biomaRt¹⁵.

[00227] Using R, we transformed the FPKM data to TPM and performed quantile normalization. To focus on the effect of aging rather than microenvironment infiltration, we regressed out the tumor purity score for each sample by using linear regression to model each gene’s expression according to tumor purity and setting the residuals as the new data values. Next, we computed composite scores for the orthologs of the age-related mouse DEGs using the previously described module score method⁷⁶. Two module scores were computed for each patient for the aged and young DEGs respectively. Next, we performed correlation analysis of each patient’s module scores with their “age at index” variable using Pearson correlation via the cor. test function in R. To subselect genes from the mouse models that were especially strongly correlated with age in patients, we further utilized a jackknifing approach, leaving one gene at a time out of the module and re-computing the module score’s correlation with age. If the module score’s correlation with age was strengthened when leaving out a gene, then that gene was discarded. Pearson correlation of the jackknife-filtered aged and young module scores was reported at the end.

[00228] In addition to correlation, a multivariable regression approach was utilized to study the independent association of the aforementioned module scores with patient age in TCGA. Using the R Im function, the module score was provided as the dependent variable and the following clinical and molecular variables were provided as predictors: age; comprehensive tumor purity estimate; pack-years smoked; mutation burden; AJCC T, N and M scores; tumor lung site; and sex. Regression diagnostics were performed including tests for heteroscedasticity and normality of residuals, revealing no notable deviations. This regression approach was performed for the module scores derived from all the mouse DEGs and the jackknifed genes.

Data availability statement

[00229] Mouse lung AT2 cell chromatin immunoprecipitation data was obtained from the Gene Expression Omnibus (GEO) under accession code GSE1582O 5⁴⁰.

Example 2: Aged AT2 cells exhibit reduced cell-intrinsic potential for proliferation and tumorigenesis

[00230] To investigate the impact of aging on lung cancer development, we induced LU AD tumors in young (12-16 week-old) and aged (104-130 week-old) KP mice using adenoviral vectors delivering Cre recombinase placed under the control of an AT2-specific surfactant protein-C (SPC) promoter²⁷ (AdSPC-Cre, FIG. 1A). Surprisingly, aged mice exhibited a 37% increase in median survival when compared to young mice (FIG. IB,/? = 0.0007). To investigate the reason underlying this increase in survival, we examined tumor burden in lungs of KP mice at distinct stages of LU AD progression: atypical adenomatous hyperplasia (AAH, 4 weeks), adenoma (8 weeks), adenoma-to-adenocarcinoma transition (12 weeks), and fully formed adenocarcinoma (17 weeks) (FIG. 1A). We observed a dramatic decrease in the number of tumors in aged mice at all time points when compared to young counterparts (FIGs. 1C-1D; FIG. 7A), suggesting that aging suppresses tumor initiation. [00231] Tumor initiation potential is tightly linked to the number and regenerative capacity (sternness) of the cell of origin within tissues³. We detected a 36% reduction in the number of AT2 cells in lungs of aged wild-type mice compared to young lungs (FIG. 7B). Furthermore, aging led to a progressive reduction in the proportion of AT2 cells within the total lung epithelial pool (FIGs. 7C-7E), suggesting that AT2 cells are more susceptible to decline with aging than other lung epithelial cells. However, the proportion of AT2 cells transduced by the AdSPC-Cre vector was not different between aged and young mice (FIG. 7F). This suggested that qualitative changes underlie reduced tumor-initiation potential of aged AT2 cells.

[00232] To test the regenerative capacity of AT2 cells in vivo, we stimulated AT2 proliferation by hyperoxia injury^29,31 in aged and young wild-type mice. Compared to young mice, the aged mice displayed a 69% reduction in the proportion of proliferating (Ki67+) AT2 cells at three days following injury - the peak proliferative phase of alveolar regeneration²⁶. The proliferative response had subsided in both young and aged mice at 10 days post-injury (FIG. IE). To study the cell-intrinsic proliferative capacity of AT2 cells in the context of a controlled and consistent in vitro environment, we subjected isolated primary AT2 cells to a 3D alveolar organoid assay³². We found a 54% reduction in the number of organoids formed by the aged AT2 cells compared to young AT2 cells. The reduced potential for organoid growth persisted in secondary passage, implicating cell-intrinsic changes in decline of sternness in aged AT2 cells (FIG. IF). Of note, we did not detect senescence-associated beta-galactosidase activity in the AT2 cells in either aged or young mice, although an increase in non-epithelial senescent cells was observed in aged lungs (FIGs. 7G-7H)

[00233] To directly interrogate transformation capacity, we isolated primary AT2 cells from aged and young KP ;Rosa26^Cas9~^GFP/+ (hereafter KP-Cas9) mice³³ and induced transformation by lentivirally delivered Cre recombinase in an ex vivo tumor sphere formation assay. Similar to the in vivo experiments, the efficiency of transformation was significantly impaired in aged AT2 cells (FIG. 1G), even when adjusting for the baseline difference in alveolar organoid formation between aged and young AT2 cells (FIG. 1H). Importantly, this difference was not explained by susceptibility to lentiviral transduction, which was similar in both aged and young AT2 cells (FIG. 71). Interestingly, the 50-70% reduction in AT2 cell proliferative capacity in the in vitro (FIG. IF) and in vivo (FIG. IE) assays closely mirrored the overall -60% reduction in tumorigenic potential of the aged AT2 cells, suggesting that loss of AT2 sternness may underlie reduced tumor initiation in the aged lung.

[00234] To test whether these findings are relevant beyond the KP genotype we initiated lung tumors in aged vs. young wild-type mice using CRISPR-mediated engineering of Eml4- Alk fusions³⁴ (FIG. 8A), which represents another clinically relevant genetic LU AD subtype. Aged mice displayed a robust reduction in the number of tumors and overall tumor burden when compared to young mice (FIGs. 8B-8D), indicating that the loss of tumorigenic potential in the lung epithelium is independent of driver oncogene or p53 status.

Example 3: Delayed histological and molecular progression of luns tumors in aged mice

[00235] Although considerably fewer, some tumors formed in aged KP mice. Tumors in aged mice were reduced in size by 40-60% at 4, 8, and 17 weeks of tumor development when compared to young counterparts; however, no difference was observed at 12 weeks (FIG. 2A). The proliferative index of KP tumors was significantly lower in aged mice at all time points (FIG. 2B). Conversely, we did not detect a statistically significant difference in the number of senescent cells in the aged vs. young tumors (FIGs. 9A-9C). A negligible number of cleaved caspase-3 positive apoptotic cells were detected in the KP tumors in both young and aged mice (not shown). These results indicate that decreased proliferation rather than increased senescence or apoptotic activity explains the smaller tumor size in aged mice.

[00236] Besides the reduced proliferation, the aged KP lungs showed a higher proportion of grade (G) 1 AAH lesions at 12 weeks and G2 adenomas at 17 weeks post-tumor initiation when compared to the young KP tumors (FIG. 1C; FIG. 2C; FIG. 9D). Conversely, the young mice showed a higher fraction of G4 advanced adenocarcinomas than the aged mice at 17 weeks (FIG. 2C). These findings indicate delayed histological progression of aged KP tumors. To investigate the molecular evolution of LUAD tumors induced in aged vs. young mice, we analyzed cancer cells at 4, 12, and 17 weeks following tumor initiation using singlecell mRNA sequencing (scRNA-seq). AT2 cells from wild-type aged vs. young mice were also collected for analysis. The single-cell expression profiles spanned six clusters with distinct expression patterns by unsupervised clustering, where five clusters corresponded to cancer cell states that we had previously identified during LU AD evolution²³ and the wildtype AT2 cells formed their own cluster (FIG. 2D). While both the aged and young tumors progressed along a similar trajectory, the aged tumors displayed a significant delay in progression (FIGs. 2D-2F). For example, at 4 weeks a higher fraction of young KP cells had progressed to the high-plasticity cell state (HPCS) - an indicator of early progression²³ - than in the aged tumors (FIG. 2E). At 17 weeks more young KP cells were found in the endoderm-like state (FIG. 2F) - a transition that occurs late in progression^23,35 - when compared to the aged KP cells. We validated our scRNA-seq findings by immunostaining for markers of the HPCS (integrin a2)²³ and endoderm-like (HNF4a)³⁵ states at the early and late progression time points, respectively (FIGs. 2E-2F).

[00237] To evaluate the impact of age-associated AT2 cell-intrinsic changes on tumor evolution we returned to our ex vivo AT2 cell transformation assay (FIG. 1G) and captured tumor spheres for bulk RNA sequencing analysis over eight passages (~2 months); freshly isolated primary AT2 cells and primary AT2 organoid cultures from aged and young wildtype mice were analyzed as baseline comparators (FIG. IF; FIG. 2G). Although the transcriptomic features of the ex vivo KP tumor spheres are distinct from the LU AD tumors in vivo, tumor spheres initiated from aged AT2 cells displayed a significant delay in their progression along an unsupervised diffusion pseudotime trajectory (FIG. 2H, FIG. 21; p = 0.0014). This delay was exemplified by a slower downregulation of alveolar markers in aged tumor spheres compared to the young (FIG. 2J). These findings suggest that AT2 cell- intrinsic factors delay the progression of aged LU AD tumors.

[00238] To uncover molecular mechanisms underpinning loss of AT2 transformation potential, we performed differential gene expression analysis on our scRNA-seq data that encompassed aged vs. young AT2 cells and LU AD cells (FIG. 2D). Notably, we found a strong age-dependent correlation between differentially expressed genes (DEGs) in the AT2 cells and the LU AD cells (FIG. 2K). Taking this one step further, we identified a set of genes that showed a consistent change in expression in the AT2 cells as well as in all of the five transcriptional cell states that molecularly define LU AD progression (FIG. 2D; FIG. 2L). Our results suggest that a significant fraction of the aging-associated changes in gene expression are inherited from the AT2 cell of origin to LU AD tumors that arise from them, and that these changes persist in distinct cancer cell states during tumor progression. Example 4: Induction of Nuprl results in loss of tuniorigenic potential in the used lung

[00239] We hypothesized that the components of the inherited gene programs changed by aging in both AT2 and LU AD cells may underlie the reduction in tumor initiation and progression in aged mice. Gene set enrichment analysis revealed a consistent induction of genes involved in iron homeostasis (FIG. 3A; FIG. 10A). The top gene shared in these pathways that was induced by aging was nuclear protein, transcriptional regulator— 1 (Nuprl), a stress- induced transcriptional co-activator that was recently identified as a rheostat controlling iron homeostasis (FIGs. 2K-2L; FIG. 10B)^36,37. In addition to iron metabolism, NUPR1 participates in the regulation of a range of other cellular processes, including DNA repair, ER stress, oxidative stress response, cell cycle, autophagy, apoptosis, and chromatin remodeling^37,38. However, changes in these pathways were less pronounced or consistent. Of note, NUPR1 has been shown to be induced in a variety of cancers and targeting NUPR1 has anti-cancer effects in some contexts^37,38.

[00240] We confirmed induction of Nuprl mRNA in situ in AT2 cells in aged wild-type mice and in the aged LU AD tumors (FIGs. 3B-3C). We next functionally interrogated Nuprl in lung tumor initiation in vivo by lentiviral co-delivery of single guide RNAs (sgRNAs) and Cre recombinase into lungs of aged vs. young KP-Cas9 mice. In this system KP tumor initiation is coupled to activation of Cas9 and expression of an sgRNA targeting Nuprl or a non-targeting control sgRNA (FIG. 3D)²⁵. No difference was observed in the ability of the lentiviral vectors to transduce AT2 cells in vivo (FIG. 10C). Consistent with published literature^37,38 elucidating NUPR1 as an anti-cancer target ^37,38, inactivation of Nuprl suppressed tumorigenesis in young mice at 12 weeks post-tumor initiation. However, remarkably, Nuprl gene targeting in aged mice promoted tumor initiation (FIG. 3E). Further, targeting Nuprl in aged mice led to an increase in the average size of the lung tumors (FIG. 3F). Loss of Nuprl in the aged tumors increased cancer cell proliferation to a similar level to that seen in the young control tumors, whereas the opposite - suppression of proliferation - was observed in the young KP cancer cells (FIG. 3G). To evaluate effects on molecular progression we isolated KP-Cas9 cells from in vivo tumors subjected to Nuprl or control sgRNA for RNA-seq analysis. Loss of Nuprl permitted progression of KP lung tumors to the later molecular stages observed in young mice at this time point when compared to the aged controls, suggesting that Nuprl acts as a barrier to tumor progression in aged mice (FIG. 3H) Similar to our results in vivo, targeting NUPR1 by CRISPR/Cas9 or via a recently reported small molecule ZZW-115 that blocks NUPR1 nuclear translocation³⁹ increased tumor sphere formation of aged KP-Cas9 AT2 cells in the ex vivo transformation assay, whereas sphere formation by young KP-Cas9 AT2 cells was suppressed by NUPR1 inactivation (FIG. 31; FIG. 10D) Taken together, these results indicate that aging-associated induction of NUPR1 expression suppresses AT2 cell transformation via a cell-autonomous mechanism.

Example 5: Elevated expression ofNUPRl disrupts iron homeostasis in aged AT2 and LU AD cells

[00241] Given the established role ofNUPRl in controlling levels of cellular iron, we chelated iron using deferoxamine (DFO) in the context ofNUPRl loss-of-function in the ex vivo transformation assay. DFO blunted the increase in tumor sphere formation by aged KP- Cas9 AT2 cells in response to genetic or pharmacologic targeting ofNUPRl (FIG. 4A; FIG. 11A). Conversely, DFO did not have an effect on young tumor spheres in the context of NUPR1 inactivation (FIG. 4A; FIG. 11 A). These findings suggested that the aging- associated induction of Nuprl expression leads to iron deficiency in the AT2 cells. Consistent with this notion, supplementation of aged AT2 cells with transferrin-bound iron promoted tumor sphere formation in the ex vivo transformation assay, whereas no change was observed in the young AT2 cells (FIG. 4B). Supplementation of transferrin-bound iron did not change the effect of the Nuprl knockout in either the young or aged AT2 cells, suggesting either targeting NUPR1 or supplementation of transferrin is sufficient for rescuing the age- associated decline in tumorigenic potential (FIG. 11B). To directly test whether NUPR1 controls iron levels in transformed AT2 cells, we measured levels of cellular iron following Nuprl knockout in the AT2 ex vivo transformation assay. We found that loss of Nuprl promoted iron uptake by aged and young transformed AT2 cells, whereas the effect was less pronounced in the young cells (FIG. 4C). Interestingly, the baseline levels of iron were higher in the aged cells compared to the young, suggesting a higher requirement of iron for cell growth in the aged AT2 cells.

[00242] NUPR1 has been shown to protect pancreas cancer cells from excess iron by inducing expression of the iron sequestering protein lipocalin-2 (encoded by Lcn2)³⁶. Lcn2 was highly induced in the aged AT2 cells and LU AD cells (FIG. 2L; FIGs. 4D-4E; FIG. 11C). We also detected induction of lipocalin-2 protein in the aged LU AD tumors, whereas knockout of Nuprl restored lipocalin-2 to similar levels as in the young tumors (FIG. 4D). Similar effects were observed in the AT2 in ex vivo transformation assay in response to Nuprl knockout or pharmacologic targeting of NUPR1 with ZZW-115 (FIGs. 11D-11E). Supplementation of recombinant lipocalin-2 to transformed aged AT2 cell cultures blunted the increase in sphere formation in response to ZZW-115, but had no effect on the young tumor spheres (FIG. 11F). As in the tumor spheres, ZZW-115 suppressed Lcn2 expression in the alveolar organoids (FIG. 4F). Our results implicate NUPR1 as an upstream driver of Lcn2 expression in AT2 and LU AD cells, which, in light of the previous work³⁶, suggests that the relative iron insufficiency in aged AT2 cells is driven by overactivation of the NUPR1- lipocalin-2 axis, leading to loss of sternness and tumorigenic potential.

[00243] We next tested whether targeting NUPR1 could rejuvenate sternness of AT2 cells. ZZW-115 promoted organoid formation by aged AT2 cells in an iron-dependent manner. However, similar to the ex vivo transformation assay, a detrimental effect was observed in young AT2 cells (FIG. 4G). In contrast to NUPR1 inhibition, supplementation with transferrin-bound iron increased organoid formation by aged AT2 cells without detrimental effects on young AT2 cells (FIG. 4H).

Example 6: DNA demethylation underpins aging-associated induction of the NUPR1- lipocalin-2 axis

[00244] Given its prominence as a primary hallmark of aging¹⁵, we investigated the role of DNA methylation in the inheritance of age-associated changes in gene expression in AT2 cells and in cancer cells. We assessed DNA methylation on a genome-wide scale in young and aged AT2 cells and in age-matched LU AD tumors (FIG. 12). Consistent with previous findings in other cell types¹⁵'¹⁹, we observed a global DNA demethylation with CpG islandspecific hypermethylation in the aged AT2 and LU AD cells and found that age was the most significant driver of sample clustering (FIGs. 12C-12D, 12G-12H). To connect differentially methylated cytosines (DMCs) with promoters and enhancers in AT2 cells, we mapped DMCs to histone-3 (H3)K4mel, H3K27ac, and H3K4me3 modifications⁴⁰ (FIG. 13A). The most distinct difference in the number of hypermethylated and hypomethylated DMCs between aged and young lay at active enhancers marked by H3K4mel and H3K27ac (FIG. 13A). We observed statistically significant correlations between DNA demethylation at promoters and enhancers at genes that comprise the age-associated signature inherited from AT2 cells to LU AD tumors (FIG. 2L; FIG. 5A). In aged relative to young AT2 cells Nuprl was demethylated within an intron marked by H3K4me3 and H3K27ac and at a site marking an active enhancer (H3K4mel and H3K27ac), whereas Lcn2 was demethylated at an active enhancer site (H3K4me, H3K4me3, and H3K27ac) (FIG. 5B; FIG. 13B). Loss of DNA methylation at these key gene-regulatory regions is consistent with a de-repression of gene expression with age¹⁷. Notably, Nuprl and Lcn2 also showed a reduction in mean methylation level at the promoter region (-Ikb to +200bp from transcription start site) in primary LU AD cells (FIG. 5C). Similar to the AT2 cells, aged- associated DNA methylation levels between AT2 and LU AD were positively correlated at the genes that make up the inherited age-associated signature (FIG. 2L; FIG. 5D; Pearson correlation test, r = 0.35,/? = 0.015).

[00245] To functionally interrogate whether DNA demethylation underpins the induction of the age-associated gene expression signature in AT2 cells, we suppressed DNA methylation in young wild- type C57BL/6J mice by systemic administration of GSK- 3484862, a small molecule inhibitor of DNA methyltransferase- 1 (DNMT1), for 8 days (FIG. 5E). Young AT2 cells subjected to DNMT1 inhibition showed a strong correlation with the transcriptome observed in aged AT2 cells implicating global progressive DNA hypomethylation as a key mechanistic driver of the aging transcriptome in AT2 cells (FIG. 5F). Within the age-associated, inherited signature shared in AT2 and LU AD cells (FIG. 2L), Nuprl and Lcn2 were among the top genes induced by DNMT1 inhibition in the young AT2 cells (FIG. 5G). We confirmed induction of Nuprl in AT2 cells in situ in young mice administered DNMTl-i (FIG. 5H). Out of the top 25 genes showing inherited upregulation from AT2 to LU AD with aging, a total of 7 showed (i) induction in young cells with DNMT1 inhibition and were demethylated in aged (ii) AT2 and (iii) LU AD cells (FIG. 51), strongly suggesting de-repression of these genes via DNA demethylation in aged AT2 cells. These 7 genes include Nuprl and Lcn2 (FIG. 51). To test the importance of DNA methylation in cancer cells, we transformed young KP-Cas9 AT2 cells ex vivo and found that DNMT1 inhibition produced an aged-like transcriptome also in this context (FIGs. 5J-5K). Finally, DNMT1 blockade induced Nuprl expression in KP tumor sphere cultures established from young primary LU AD tumors (FIG. 5L). In conclusion, our results suggest that aging- associated DNA demethylation reprograms transcriptomes in AT2 cells. These gene regulatory networks are inherited in cancer cells that emerge from the reprogrammed AT2 cells supporting that cell-intrinsic mechanisms explain overactivation of the NUPR1- lipocalin-2 axis.

Example 7: Aging-induced changes in AT2 cells and LU AD are conserved in humans

[00246] Similar to our findings in mice, we observed a decline in the density of AT2 cells in aged human lungs (FIG. 6A). The aged human AT2 cells displayed elevated expression of both NUPR1 and lipocalin-2 (FIGs. 6B-6C). Notably, stimulation with iron-bound transferrin robustly promoted formation of organoids by human primary AT2 cells isolated from individuals 65-78 years of age, indicating that availability of iron is growth-limiting also in aged human AT2 cells (FIG. 6D). To examine conservation of age-associated gene expression in LU AD across mouse and man, we examined bulk RNA sequencing data in The Cancer Genome Atlas collection⁴¹. Orthologs of genes expressed significantly higher in young mouse LU AD tumors showed an inverse correlation with human LU AD patient age, whereas orthologs induced in aged LU AD tumors exhibited a positive correlation with human LU AD patient age (FIG. 6E). Similar to mouse LU AD, NUPR1 expression was higher in aged (>80-year old) patients when compared to young (<55-year old) patients upon examination of resected human LU AD tissues (FIG. 6F). Taken together, these findings demonstrate notable conservation of age-associated changes in AT2 cells and lung tumors across mice and humans.

[00247] We demonstrated that aged alveolar type 2 (AT2) cells and the lung cancers that arise from them share -86% of the gene expression changes that emerge with aging (FIG. 14A). Among these genes, Nuprl nd Lcn2 stood out as highly upregulated with aging. We confirmed the induction of both genes by co-detection of Sftpc (an AT2 cell marker) by in situ hybridization and Nuprl in aged vs. young mouse lungs (FIG. 14B and not shown). A similar change was also observed in human lungs.

[00248] We next functionally interrogated Nuprl in lung tumor initiation in vivo by lentiviral co-delivery of single guide RNAs (sgRNAs) and Cre recombinase into lungs of aged vs. young KP-Cas9 mice. In this system KP tumor initiation is coupled to activation of Cas9 and expression of an sgRNA targeting Nuprl or a non-targeting control sgRNA²⁶. We have now increased the number of mice in this experiment as well as utilized another Nuprl sgRNA to corroborate these findings (FIG. 15A). No difference was observed in the ability of the lentiviral vectors to transduce AT2 cells in vivo. Remarkably, inactivation of Nuprl in aged mice promoted tumor initiation, whereas the opposite - suppression of tumorigenesis - was observed in the young mice (FIG. 15B). Further, targeting Nuprl in aged mice led to an increase in the average size of the lung tumors. Loss of Nuprl in the aged tumors increased cancer cell proliferation to a similar level as in the young control tumors. Conversely, Nuprl knockout suppressed proliferation of the young KP cancer cells.

[00249] We had demonstrated that aged AT2 cells and the cancer cells that arise from them suffer from a functional iron insufficiency. Our findings demonstrate that aging- associated induction of NUPR1 expression leads to loss of sternness and tumorigenic potential in lung alveoli. We find that inhibition of NUPR1 and iron supplementation are attractive therapeutic strategies for rejuvenating regenerative potential of aged lung alveoli, e.g., in patients with low lung cancer risk.

Example 8: Young AT2 cells are sensitive to ferroptosis but aged cells are resistant

[00250] The reason for why young cells require NUPR1 and lipocalin-2 is because they are significantly more sensitive to ferroptosis, a non-apoptotic form of cell death dependent on iron (FIG. 16A). We find that young AT2 cells transformed ex vivo in a 3D tumor sphere assay benefit from supplementation with liproxstatin-1, a radical -trapping agent that targets cell membranes and thus protects cells from ferroptosis. Conversely, the young cells are highly sensitive to induction of ferroptosis by the GPX-4 inhibitor RSL-3 or cysteine uptake inhibitor erastin. Interestingly, aged AT2 cells do not benefit from liproxstatin-1 and are resistant to RSL3 or erastin stimulation, indicating aged AT2 cells are resistant to ferroptosis (FIG. 16B). We confirmed the sensitivity of young AT2 cells to ferroptosis by treating mice with genetically engineered lung tumors with liproxstatin-1 daily for 8 weeks: We observed an increase in tumorigenesis in the young mice upon liproxstatin-1 administration, whereas aged mice did not show a difference (FIG. 17). Based on these results, we conclude that ferroptosis is a viable target for preventing, intercepting, or treating lung cancer in young individuals (FIG. 18).

[00251] To investigate whether other iron metabolism genes would show a similar age context-dependence, we performed ex vivo transformation assays of aged vs. young AT2 cells with concomitant knockout of iron storage proteins (Fthl, Ftll iron uptake proteins (Trf, Tfrc, Cd44), and iron export (Slc40al). We found that knockout of all of these genes is equally detrimental to both young and aged AT2 cells. This is in contrast to Nuprl knockout, which is detrimental in young cells and beneficial to growth of aged AT2 cells (FIG. 19). In new data, we demonstrate that addition of free iron complexed to vitamin C recapitulates the effects of Nuprl knockout in aged and young AT2 cells (FIG. 20).

[00252] Our results implicate NUPR1 as an upstream driver of Lcn2 expression in AT2 and LU AD cells, which, suggested that iron depletion in aged AT2 cells is driven by overactivation of the NUPRl-lipocalin-2 axis, which leads to loss of sternness and tumorigenic potential. To directly test this, we performed two epistasis experiments (FIGs. 21-22). We found that knocking out Nuprl while simultaneously over-expressing Lcn2 cDNA completely rescues the effect of Nuprl knockout. Knockout of Lcn2 alone reproduced the effect of Nuprl knockout in both aged and young AT2 cells (FIG. 21). These data unequivocally establish a genetic interaction with Nuprl and Lcn2, whereby NUPR1 induction with aging induces lipocalin-2 expression. In light of the known function of lipocalin-2 in iron sequestering and export from cells, our results cast lipocalin-2 as another target for rejuvenating regenerative capacity of aged AT2 cells and targeting lung cancer in young patients (FIG. 23).

Example 9: Aging induces the NUPRl-lipocalin-2 axis in other adult stem cell compartments

[00253] Finally, we have explored the potential significance of our findings outside of the lung. To do this, we analyzed expression of Nuprl, Lcn2, and other iron metabolism genes in a published aged mouse single-cell mRNA-sequencing (scRNA-seq) dataset (“Tabula muris senis”) as well as several gene expression datasets focusing on the neural adult subventricular zone (SVZ) stem cells, hair follicle stem cells, and small intestinal stem cells. In this analysis, we found that -85% of all adult stem cell compartments showed an age-associated induction

-n- of Nuprl, Lcn2, or both (FIG. 24). In an ongoing experiment, we have explored iron supplementation and ferroptosis inducers in intestinal stem cells. Based on present results, it appears that aged intestinal stem cells can be rejuvenated by iron supplementation (transferrin, FAC). Furthermore, young intestinal stem cells are sensitive to ferroptosis, whereas the aged intestinal stem cells are not.

[00254] This data demonstrates that inhibiting NUPRl/lipocalin-2 or iron supplementation are strategies to rejuvenate stem cells in other stem cell compartments.

EQUIVALENTS

[00255] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[00256] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[00257] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[00258] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

REFERENCES

1. Rozhok, A. I. & DeGregori, J. The evolution of lifespan and age-dependent cancer risk. Trends Cancer 2, 552-560 (2016).

2. National. Cancer Institute: Cancer Statistics (2021).

3. White, A. C. & Lowry, W. E. Refining the role for adult stem cells as cancer cells of origin. Trends Cell Biol 25, 11-20 (2015).

4. Tomasetti, C., Li, L. & Vogelstein, B. Stem cell divisions, somatic mutations, cancer etiology, and cancer prevention. Science 355, 1330-1334 (2017).

5. Tomasetti, C. et al. Role of stem-cell divisions in cancer risk. Nature 548, E13-E14 (2017).

6. Oh, J., Lee, Y. D. & Wagers, A. J. Stem cell aging: mechanisms, regulators and therapeutic opportunities. Nat Med 20, 870-880 (2014).

7. Boyle, M., Wong, C., Rocha, M. & Jones, D. L. Decline in self-renewal factors contributes to aging of the stem cell niche in the Drosophila testis. Cell Stem Cell 1, 470-478 (2007).

8. Schultz, M. B. & Sinclair, D. A. When stem cells grow old: phenotypes and mechanisms of stem cell aging. Development 143, 3-14 (2016).

9. Pentinmikko, N. et al. Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium. Nature 571, 398-402 (2019).

10. Rozhok, A. & DeGregori, J. A generalized theory of age-dependent carcinogenesis. Elife 8 (2019).

11. Jackson, E. L. et al. The differential effects of mutant p53 alleles on advanced murine lung cancer. Cancer Res 65, 10280-10288 (2005).

12. Jackson, E. L. et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev 15, 3243-3248 (2001).

13. Lopez-Otin, C., Blasco, M. A., Partridge, L., Serrano, M. & Kroemer, G. The hallmarks of aging. Cell 153, 1194-1217 (2013).

14. 14 Singh, P. P., Demmitt, B. A., Nath, R. D. & Brunet, A. The Genetics of Aging: A Vertebrate Perspective. Cell 177, 200-220 (2019).

15. Booth, L. N. & Brunet, A. The Aging Epigenome. Mol Cell 62, 728-744 (2016).

16. Michalak, E. M., Burr, M. L., Bannister, A. J. & 1094 Dawson, M. A. The roles of DNA, RNA and histone methylation in ageing and cancer. Nat Rev Mol Cell Biol 20, 573-589 (2019). Seale, K., Horvath, S., Teschendorff, A., Eynon, N. & Voisin, S. Making sense of the ageing methylome. Nat Rev Genet 23, 585-605 (2022). Bocklandt, S. et al. Epigenetic predictor of age. PLoS One 6, el4821 (2011). Field, A. E. et al. DNA Methylation Clocks in Aging: Categories, Causes, and Consequences. Mol Cell 71, 882-895 (2018). Fane, M. & Weeraratna, A. T. How the ageing microenvironment influences tumour progression. Nat Rev Cancer 20, 89-106 (2020). Balducci, L. & Ershler, W. B. Cancer and ageing: a nexus at several levels. Nat Rev Cancer 5, 655-662 (2005). Liu, B. et al. Lung cancer in young adults aged 35 years or younger: A full-scale analysis and review. J Cancer 10, 3553-3559 (2019). Marj anovic, N. D. et al. Emergence of a High-Plasticity Cell State during Lung Cancer Evolution. Cancer Cell 38, 229-246 e213 (2020). Sanchez-Rivera, F. J. et al. Rapid modelling of cooperating genetic events in cancer through somatic genome editing. Nature 516, 428 (2014). Tammela, T. et al. A Wnt-producing niche drives proliferative potential and progression in lung adenocarcinoma. Nature 545, 355-359 (2017). Xu, X. et al. Evidence for type II cells as cells of origin of K-Ras-induced distal lung adenocarcinoma. Proc Natl Acad Set USA 109, 4910-4915 (2012). Sutherland, K. D. et al. Multiple cell s-of-ori gin of mutant K-Ras-induced mouse lung adenocarcinoma. Proc Natl Acad Set USA 111, 4952-4957 (2014). Mainardi, S. et al. Identification of cancer initiating cells in K-Ras driven lung adenocarcinoma. Proc Natl Acad Set USA 111, 255-260 (2014). Desai, T. J., Brownfield, D. G. & Krasnow, M. A. Alveolar progenitor and stem cells in lung development, renewal and cancer. Nature 507, 190-194 (2014). Lee, J. H. & Rawlins, E. L. Developmental mechanisms and adult stem cells for therapeutic lung regeneration. Dev Biol 433, 166-176 (2018). Altemeier, W. A., Hung, C. F. & Matute-Bello, G. m Acute Lung Injury and Repair: Scientific Fundamentals and Methods (eds Lynn M. Schnapp & Carol Feghali- Bostwick) 5-23 (Springer International Publishing, 2017). Lee, J.-H. et al. Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATcl-thrombospondin-l axis. Cell 156, 440-455 (2014). Platt, R. J. et al. CRISPR-Cas9 knockin mice for genome 1140 editing and cancer modeling. Cell 159, 440-455 (2014). Maddalo, D. et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516, 423-427 (2014). Winslow, M. M. et al. Suppression of lung adenocarcinoma progression by Nkx2-1. Nature 473, 101-104 (2011). Liu, J. et al. NUPR1 is a critical repressor of ferroptosis. Nat Commun 12, 647 (2021). Liu, S. & Costa, M. The role of NUPR1 in response to stress and cancer development. Toxicol Appl Pharmacol 454, 116244 (2022). Huang, C., Santofimia-Castano, P. & lovanna, J. NUPR1 : A Critical Regulator of the Antioxidant System. Cancers (Basel) 13 (2021). Lan, W. et al. ZZW-115-dependent inhibition ofNUPRl nuclear translocation sensitizes cancer cells to genotoxic agents. JCI Insight 5 (2020).

-SO- Little, D. R. et al. Differential chromatin binding of the lung lineage transcription factor NKX2-1 resolves opposing murine alveolar cell fates in vivo. Nat Commun 12, 2509 (2021). The Cancer Genome Atlas Research, N. Comprehensive molecular profiling of lung adenocarcinoma. Nature 511, 543-550 (2014). Alvarez, S. W. et al. NFS1 undergoes positive selection in lung tumours and protects cells from ferroptosis. Nature 551, 639-643 (2017). Jiang, X., Stockwell, B. R. & Conrad, M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol 22, 266-282 (2021). Marine, J. C., Dawson, S. J. & Dawson, M. A. Non-genetic mechanisms of therapeutic resistance in cancer. Nat Rev Cancer 20, 743-756 (2020). Faverio, P. et al. One-year pulmonary impairment after severe COVID-19: a prospective, multicenter follow-up study. Respir Res 23, 65 (2022). Lowery, E. M., Brubaker, A. L., Kuhlmann, E. & Kovacs, E. J. The aging lung. Clin Interv Aging 8, 1489-1496 (2013). Kim, M. & Costello, J. DNA methylation: an epigenetic mark of cellular memory. Exp Mol Med 49, e322 (2017). Tao, Y. et al. Aging-like Spontaneous Epigenetic Silencing Facilitates Wnt Activation, Sternness, and Braf(V600E)-Induced Tumorigenesis. Cancer Cell 35, 315-328 e316 (2019). Gaiti, F. et al. Epigenetic evolution and lineage histories of chronic lymphocytic leukaemia. Nature 569, 576-580 (2019). Madisen, L. et al. A robust and high-throughput Cre reporting 1184 and characterization system for the whole mouse brain. Nat Neurosci 13, 133-140 (2010). Dow, L. E. et al. Conditional reverse tet-transactivator mouse strains for the efficient induction of TRE-regulated transgenes in mice. PLoS One 9, e95236 (2014). Safiran, M. et al. Mouse reporter strain for noninvasive bioluminescent imaging of cells that have undergone Cre-mediated recombination. Mol Imaging 2, 297-302 (2003). Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676-682 (2012). Gonzalez, R. F., Allen, L., Gonzales, L., Ballard, P. L. & Dobbs, L. G. HTII-280, a biomarker specific to the apical plasma membrane of human lung alveolar type II cells. J Histochem Cytochem 58, 891-901 (2010). Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double fluorescent Cre reporter mouse. Genesis 45, 593-605 (2007). DuPage, M., Dooley, A. L. & Jacks, T. Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase. Nat Protoc 4, 1064-1072 (2009). Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol 19, 15 (2018). Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573-3587 e3529 (2021). Wolock, S. L., Lopez, R. & Klein, A. M. Scrublet: Computational Identification of Cell Doublets in Single-Cell Transcriptomic Data. Cell Syst 8, 281-291 e289 (2019). Lun, A. T., Bach, K. & Marioni, J. C. Pooling across cells to normalize single-cell RNA sequencing data with many zero counts. Genome Biol 17, 75 (2016). Persad, S. et al. SEACells: Inference of transcriptional and epigenomic cellular states from single-cell genomics data. biorXiv (2022). Korsunsky, I. et al. Fast, sensitive and accurate integration of single-cell data with Harmony. Nat Methods 16, 1289-1296 (2019). Finak, G. et al. MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data. Genome Biol 16, 278 (2015). Fang, Z., Liu, X. & Peltz, G. GSEApy: a comprehensive package for performing gene set enrichment analysis in Python. Bioinformatics (2022). Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21 (2013). Anders, S., Pyl, P. T. & Huber, W. HTSeq— 1228 a Python framework to work with high throughput sequencing data. Bioinformatics 31, 166-169 (2015). Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550 (2014). Krueger, F. & Andrews, S. R. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27, 1571-1572 (2011). Krueger, F. Trim Galore. (2022). Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17 (2022). Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357-359 (2012). Feng, H., Conneely, K. N. & Wu, H. A Bayesian hierarchical model to detect differentially methylated loci from single nucleotide resolution sequencing data. Nucleic Acids Res 42, e69 (2014). Park, Y. & Wu, H. Differential methylation analysis for BS-seq data under general experimental design. Bioinformatics 32, 1446-1453 (2016). Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139-140 (2010). Durinck, S., Spellman, P. T., Birney, E. & Huber, W. Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nat Protoc 4, 1184-1191 (2009). Tirosh, I. et al. Dissecting the multicellular ecosystem of metastatic melanoma by single cell RNA-seq. Science 352, 189-196 (2016). Baran, Y. et al. MetaCell: analysis of single-cell RNA-seq data using K-nn graph partitions. Genome Biol 20, 206 (2019).

Claims

WHAT IS CLAIMED IS

1. A method for restoring regenerative potential (sternness) of alveolar stem cells in a subject in need thereof comprising administering to the subject an effective amount of a Nuclear Protein 1 (NUPR1) inhibitor or a lipocalin-2 (LCN2) inhibitor.

2. The method of claim 1, wherein the subject is diagnosed with or suffers from a lung insufficiency, optionally wherein the lung insufficiency is selected from the group consisting of Chronic obstructive pulmonary disease (COPD), COVID, influenza, and pneumonia sequelae.

3. The method of any one of claims 1-2, wherein the subject is an immunocompromised subject or a geriatric subject.

4. A method for preventing or treating lung cancer in a subject in need thereof comprising administering to the subject an effective amount of a Nuclear Protein 1 (NUPR1) inhibitor or a lipocalin-2 (LCN2) inhibitor, wherein the subject is a pediatric subject, an adolescent subject, or a young adult subject (e.g., < 55 years old).

5. A method for preventing or treating lung cancer in a subject in need thereof comprising administering to the subject an effective amount of a ferroptosis-inducing agent, wherein the subject is a pediatric subject, an adolescent subject, or a young adult subject (e.g., < 55 years old).

6. The method of claim 5, wherein the ferroptosis-inducing agent is a class 1 ferroptosis inducer (system X_c“ inhibitor) or a class 2 ferroptosis inducer (glutathione peroxidase 4 (GPx4) inhibitor).

7. The method of claim 5 or 6, wherein the ferroptosis-inducing agent is selected from the group consisting of an inhibitory nucleic acid that targets GPX4, erastin, erastin derivatives (e.g., MEII, PE, AE, imidazole ketone erastin (IKE)), DPI2, BSO, SAS, lanperisone, SRS13-45, SRS13-60, RSL3, DPI7, DPI10, DPI12, DPI13, DPI17, DPI18, DPI19, ML160, sorafenib, artemisinin derivatives, artesunate, BAY87-2243, cisplatin, ironomycin, lanperisone, salinomycin, sulfasalazine, temozolomide, and lapatinib in combination with siramesine.

8. The method of any one of claims 4-7, wherein the lung cancer is lung adenocarcinoma (LU AD).

9. The method of of any one of claims 4-8, wherein the lung cancer comprises a mutation in KRAS or TP53.

10. The method of any one of claims 1-4 or 8-9, wherein the NUPR1 inhibitor is a small molecule, an NUPR1 -specific inhibitory nucleic acid, or an anti-NUPRl neutralizing antibody.

11. The method of claim 10, wherein the NUPR1 inhibitor is trifluoperazine (TFP), ZZW- 115, ZZW-129, ZZW-130, ZZW-131, ZZW-132, ZZW-142, ZZW-143, ZZW-144, ZZW-145, or ZZW-148.

12. The method of any one of claims 1-4 or 8-9, wherein the LCN2 inhibitor is a small molecule, an LCN2-specific inhibitory nucleic acid, or an anti- LCN2 neutralizing antibody.

13. The method of claim 12, wherein the LCN2 inhibitor is ZINC00784494, ZINC00640089, ZINC00230567, or ZINC00829534.

14. The method of any one of claims 7, 10 or 12, wherein the NUPR1 -specific inhibitory nucleic acid, the LCN2-specific inhibitory nucleic acid or the inhibitory nucleic acid that targets GPX4 is a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA.

15. A method for restoring regenerative potential (sternness) of alveolar stem cells in a subject in need thereof comprising administering to the subject an effective amount of iron or transferrin.

16. The method of claim 10, wherein the iron is unbound iron.

17. The method of claim 10, wherein the iron is bound to transferrin or is complexed with vitamin C.

18. The method of any one of claims 15-17, wherein the subject is diagnosed with or suffers from a lung insufficiency, optionally wherein the lung insufficiency is selected from the group consisting of Chronic obstructive pulmonary disease (COPD), COVID, influenza, and pneumonia sequelae.

19. The method of any one of claims 15-18, wherein the subject is an immunocompromised subject or a geriatric subject.

20. A method for restoring regenerative potential (sternness) of aged adult stem cell compartments in a subject comprising administering to the subject an effective amount of a Nuclear Protein 1 (NUPR1) inhibitor or a lipocalin-2 (LCN2) inhibitor.

21. A method for restoring regenerative potential (sternness) of aged adult stem cell compartments in a subject comprising administering to the subject an effective amount of iron or transferrin.

22. The method of claim 20 or 21, wherein the aged adult stem cell compartments comprise one or more of neural adult subventricular zone (SVZ) stem cells, hair follicle stem cells, mesenchymal stem cells, keratinocyte stem cells, or intestinal stem cells.

23. The method of claim 20 or 22, wherein the NUPR1 inhibitor is a small molecule, an NUPR1 -specific inhibitory nucleic acid, or an anti-NUPRl neutralizing antibody.

24. The method of claim 23, wherein the small molecule is trifluoperazine (TFP), ZZW- 115, ZZW-129, ZZW-130, ZZW-131, ZZW-132, ZZW-142, ZZW-143, ZZW-144, ZZW-145, or ZZW-148.

25. The method of claim 20 or 22, wherein the LCN2 inhibitor is a small molecule, an LCN2-specific inhibitory nucleic acid, or an anti- LCN2 neutralizing antibody.

26. The method of claim 25, wherein the LCN2 inhibitor is ZINC00784494, ZINC00640089, ZINC00230567, or ZINC00829534.

27. The method of claim 21 or 22, wherein the iron is unbound iron.

28. The method of claim 21 or 22, wherein the iron is bound to transferrin or is complexed with vitamin C.

29. The method of any one of claims 1-28, wherein the subject is human.

30. The method of any one of claims 1-4, 8-14, 20, 22-26 or 29, wherein the NUPR1 inhibitor or LCN2 inhibitor is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, iontophoretically, transmucosally, or intramuscularly.

31. The method of any one of claims 15-19, 21-22 or 27-29, wherein the iron or transferrin is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, iontophoretically, transmucosally, or intramuscularly.

32. The method of any one of claims 5-9, 14 or 29, wherein the ferroptosis-inducing agent is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, iontophoretically, transmucosally, or intramuscularly.

33. A kit comprising at least one NUPR1 inhibitor and/or LCN2 inhibitor and instructions for using the at least one NUPR1 inhibitor and/or LCN2 inhibitor to treat or prevent lung cancer or a lung insufficiency in a subject in need thereof, optionally wherein the lung insufficiency is selected from the group consisting of Chronic obstructive pulmonary disease (COPD), COVID, influenza, and pneumonia sequelae.

34. A kit comprising iron and/or transferrin and instructions for using the iron and/or transferrin to treat or prevent a lung insufficiency in a subject in need thereof, optionally wherein the lung insufficiency is selected from the group consisting of Chronic obstructive pulmonary disease (COPD), COVID, influenza, and pneumonia sequelae.

35. A kit comprising at least one ferroptosis inducing agent and instructions for using the at least one ferroptosis inducing agent to prevent or treat lung cancer in a young (nonaged) subject in need thereof.