WO2021262874A1 - Use of a dhodh inhibitor compound in combination cancer therapy - Google Patents

Abstract

Provided herein a method of treating cancer using a first anti-cancer agen in combination with a Chk1 inhibitor, Chk2 inhibitor, or a DNA-PK inhibitor, wherein the first anti-cancer agent is represented by Formula (I):

Description

USE OF A DHODH INHIBITOR

COMPOUND IN COMBINATION CANCER THERAPY

RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63/043,339, filed on June 24, 2020, the entire contents of which are expressly incorporated herein by reference in its entirety.

BACKGROUND

[0002] Dihydroorotate dehydrogenase (DHODH) is an enzyme that catalyzes one of the steps in the de novo pyrimidine nucleotide biosynthetic pathway. It catalyzes the only oxidation/reduction reaction in that pathway, which is the step of converting DHO (dihydroorotate) to orotate with the aid of flavin cofactor and an electron acceptor. Inhibitors of dihydroorotate dehydrogenase have been found to possess wider applications as chemotherapeutic agents.

[0003] 1 -methyl-5-(2 ’ -methyl- [ 1,1’ -biphenyl] -yl)- 1 H-bcn_/o\d\ [1,2,3] triazole-7 -carboxylic acid, hereinafter also referred to as the “compound of Formula (I)” (as shown below), has been characterized as an inhibitor of DHODH. See e.g., International Patent Application Publication Nos. WO 2014/128669 and WO 2019/164794 and US Patent No. 9,630,932, the contents of which are incorporated herein by reference.

[0004] The compound of Formula (I) was developed to treat conditions and disorders that would benefit from inhibition of DHODH, such as but not limited to solid cancers and hematological cancers.

[0005] However, there is a need for developing additional and even more effective anti-cancer therapies. SUMMARY

[0006] It has now been found that selective Chkl inhibitors and DNA-PK inhibitors exert synergistic effects in reducing cell viability when used independently in combination with the compound of Formula (I). For example, the combination of the compound of Formula (I) with the Chkl inhibitors LY2606368 (prexasertib) or SCH900776 or the DNA-PK inhibitors M3814 or NU7441 synergistically inhibits the growth of a panel of AML and DLBCL cell lines. See Example 3 below.

[0007] In one aspect, this application provides a method for treating a cancer in a subject, comprising administering to the subject an effective amount of a first anti-cancer agent and an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is represented by Formula (I):

or a pharmaceutically acceptable salt thereof; and wherein the second anti-cancer agent is selected from the group consisting of a Chkl inhibitor, a Chk2 inhibitor, and a DNA-PK inhibitor. In one embodiment, the pharmaceutically acceptable salt thereof is a tris(hydroxymethyl)aminomethane salt or a sodium salt.

[0008] In another aspect, this application provides the use of a first anti-cancer agent in the manufacture of a medicament for treating a cancer, wherein the medicament is used in combination with an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is represented by Formula (I):

or a pharmaceutically acceptable salt thereof; and wherein the second anti-cancer agent is selected from the group consisting of a Chkl inhibitor, a Chk2 inhibitor, and a DNA-PK inhibitor. In one embodiment, the pharmaceutically acceptable salt thereof is a tris(hydroxymethyl)aminomethane salt or a sodium salt.

[0009] In still another aspect, the application provides a first anti-cancer agent for use in treating a cancer in combination with a second anti-cancer agent, wherein the first anti-cancer agent is represented by Formula (I):

or a pharmaceutically acceptable salt thereof; and wherein the second anti-cancer agent is selected from the group consisting of a Chkl inhibitor, a Chk2 inhibitor, and a DNA-PK inhibitor. In one embodiment, the pharmaceutically acceptable salt thereof is a tris(hydroxymethyl)aminomethane salt or a sodium salt.

[0010] In a further aspect, the application provides a pharmaceutical composition comprising a first anti-cancer agent and a second anti-cancer agent, wherein the pharmaceutical composition additionally comprises an excipient, wherein the first anti-cancer agent is represented by Formula (I):

or a pharmaceutically acceptable salt thereof; and wherein the second anti-cancer agent is selected from the group consisting of a Chkl inhibitor, a Chk2 inhibitor, and a DNA-PK inhibitor. In one embodiment, the pharmaceutically acceptable salt thereof is a tris(hydroxymethyl)aminomethane salt or a sodium salt.

[0011] Other features or advantages will be apparent from the following detailed description of the drawings and several embodiments, and also from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1A and IB. CRISPR screen implicates a role for DNA damage/repair pathways in response to the compound of Formula (I). Figure 1A shows gene ontology (GO) analysis of significant hits from a genome wide CRISPR screen in A549 cells treated with the compound of Formula (I) compared to DMSO treated cells. GO terms associated with DNA repair or the DNA damage response are highlighted with boxes. Figure B shows the log2 fold change depletion of sgRNAs targeting a given gene versus the p-value in A549 cells treated with the compound of Formula (I). Genes included in the highlighted GO terms in A) are indicated.

[0013] Figure 2. GO terms of proteins upregulated in A549 cells by treatment with the compound of Formula (I). GO analysis of proteins upregulated at least 1.5-fold in A549 cells treated with 10 mM of the compound of Formula (I) compared to DMSO. GO terms associated with DNA repair or the DNA damage response are highlighted with boxes.

[0014] Figure 3. GO terms of proteins upregulated in OCIFY 19 cells. GO analysis of proteins upregulated at least 1.5-fold in OCIFY 19 cells treated with 1 mM the compound of Formula (I) compared to DMSO. GO terms associated with DNA repair or the DNA damage response are highlighted with a box.

[0015] Figures 4A and 4B. Combination of the compound of Formula (I) with either a Chkl or a DNA-PK inhibitor results in synergistic growth inhibition in a panel of AMF and DFBCF cell lines. Dose response matrix screening was carried out with the compound of Formula (I) in combination independently with a Chkl inhibitor (either FY2606368 (prexasertib) or SCH900776) and a DNA-PK inhibitor (either M3814 or NU7441). Figure 6A shows synergy scores and Best Cl (combination index) values for each combination in each cell line. Figure B provides a graphical depiction of the data shown above. The dotted lines represent the cutoff for likely combinatorial synergy using the different parameters (Cl < 0.7; synergy score >2.4).

[0016] Figures 5 A-C. Differential activation of DNA damage response and combinatorial efficacy of Chkl inhibition. Figure 5A shows Western blot of gH2AC levels in cells treated for the indicated times with 10 pM of the compound of Formula (I) or DMSO control. Figure 5B shows a CTG growth assay on cells treated with varying concentrations of the compound of Formula (I) and prexasertib in matrix format for 48 hours in advanced RPMI containing 10% dialyzed FBS. Relative growth rates (compared to DMSO control) for each combination are depicted and color-coded as indicated by the bar to the right of each graph. Negative values indicate cell killing. Figure 5C shows Western blot of gH2AC levels in cells treated with 10 mM the compound of Formula (I) and/or 100 nM prexasertib for 18 hours.

DETAILED DESCRIPTION

[0017] The invention is directed to a method for treating a cancer in a subject by administering an effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a Chkl inhibitor, a Chk2 inhibitor, or a DNA-PK inhibitor. The compound of Formula (I) acts agonistically with Chkl inhibitors, Chk2 inhibitors, or DNA-PK inhibitors against certain cancers.

[0018] In one aspect, the cancer being treated by the disclosed methods is responsive to inhibition of dihydroorotate dehydrogenase.

[0019] In one aspect, the cancer being treated by the disclosed methods is a hematological cancer. In some embodiments, the hematological cancer is selected from myeloma, lymphoma, and leukemia. In some embodiments, the hematological cancer is selected from acute myeloid leukemia, multiple myeloma, B-prolymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, follicular lymphoma, diffuse large B cell lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, lymphocytic lymphoma cancer of the bladder, and T-cell lymphoma. In one embodiment, the cancer is myelodysplastic/myeloproliferative neoplasms. In some embodiments, the hematological cancer is selected from chemotherapy-resistant acute myeloid leukemia, cytarabine-resistant acute myeloid leukemia, acute monocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, diffuse mixed cell lymphoma, myelodysplastic syndrome, primary effusion lymphoma, erythroleukemia, chronic myeloid leukemia, chronic monocytic leukemia, double hit diffuse large B cell lymphoma, and triple hit diffuse large B cell lymphoma. In some embodiments, the hematological cancer is selected from angioimmunoblastic lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma, blastic NK-cell lymphoma, cutaneous T-cell lymphoma, lymphoblastic lymphoma, MALT lymphoma, mediastinal large B-cell lymphoma, nodal marginal zone B-cell lymphoma, small lymphocytic lymphoma, thyroid lymphoma, follicular lymphoma, Waldenstrom's macroglobulinemia, essential thrombocythemia, chronic idiopathic myelofibrosis, and polyeythemia rubra vera. In some embodiments, the hematological cancer is acute myeloid leukemia or diffuse large B-cell lymphoma. In another aspect, the cancer being treated by the disclosed methods is a solid tumor. In some embodiments, the solid cancer is selected from lung cancer, breast cancer (e.g., triple negative breast cancer), melanoma, glioblastoma, prostate cancer, colon cancer, pancreatic cancer, bone cancer, cancer of the head or neck, skin cancer, cutaneous or intraocular malignant endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, an environmentally induced cancer, and a PTEN mutant cancer. In some embodiments, the cancer is selected from biliary tract cancer or cancer of the ampulla of Vater, non-small cell lung cancer, broncho alveolar carcinoma, liver cancer, cancer of the ovary, cancer of the upper aerodigestive tract, and a cancer induced by asbestos. In some embodiments, the cancer is selected from biliary tract cancer, cancer of the ampulla of Vater, non- small cell lung cancer, bronchoalveolar carcinoma, liver cancer, cancer of the ovary, and cancer of the upper aerodigestive tract. In some embodiments, the cancer is selected from triple negative breast cancer, melanoma, prostate cancer, and cancer of the esophagus. In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is colon cancer.

[0020] In one embodiment, the cancer being treated by the disclosed methods is a non- Hodgkin lymphoma or Hodgkin lymphoma. In one particular embodiment, the treated subject belongs to a subpopulation of non-Hodgkin lymphoma or Hodgkin lymphoma patients, where the non-Hodgkin lymphoma or Hodgkin lymphoma has progressed in spite of prior treatment, and for whom additional effective (curative or life-prolonging) standard therapy is not available. In other words, the treated subject belongs to a subpopulation of resistant or refractory non- Hodgkin lymphoma or Hodgkin lymphoma. In one embodiment, the standard curative therapy is high-dose chemotherapy and autologous stem cell transplantation (HD-ASCT). In one embodiment, the subject's non-Hodgkin lymphoma or Hodgkin lymphoma has relapsed after HD-ASCT. In other words, the treated subject belongs to a subpopulation of relapsed non- Hodgkin lymphoma or Hodgkin lymphoma (e.g., HD-ASCT-relapsed). In another embodiment, the subject is not eligible for HD-ASCT. In another embodiment, the subject has refused HD- ASCT. [0021] In one embodiment, the lymphoma being treated by the disclosed methods is a mature B-cell neoplasm, a mature T- and NK-cell neoplasm, a hodgkin lymphoma, or an Immunodeficiency-associated lymphoproliferative disorder.

[0022] As used herein the terms “subject” and “patient” may be used interchangeably, and mean a mammal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like). Typically, the subject is a human in need of treatment. [0023] An “effective amount” of the compound of Formula (I), or a pharmaceutically acceptable salt, is an amount sufficient to provide a therapeutic benefit in the treatment of a cancer or to delay or minimize one or more symptoms associated with the condition when combined with a second anti-cancer agent, such as a Chkl inhibitor, a Chk2 inhibitor, or a DNA- PK inhibitor. An “effective amount” of the second anti-cancer agent, as described herein, is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition when combined with the compound of Formula (I), or a pharmaceutically acceptable salt thereof. The term “therapeutically effective amount” and “effective amount” are used interchangeably. The term “effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, an effective amount is an amount sufficient for eliciting therapeutic effects in the treatment of a cancer (including solid tumors and hematological cancers as further described herein).

[0024] The precise amount of the compound of Formula (I) (or pharmaceutically acceptable salt thereof), a Chkl inhibitor, a Chk2 inhibitor, and/or a DNA-PK inhibitor administered to a subject will depend on various factors, such as the given drug, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. For example, determination of an effective amount will also depend on the degree, severity and type of cell proliferation. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. When co-administered with other therapeutic agents, e.g., when co administered with an anti-cancer agent, an “effective amount” of any additional therapeutic agent(s) will depend on the type of drug used. Suitable dosages are known for approved therapeutic agents and can be adjusted by the skilled artisan according to the condition of the subject, the type of condition(s) being treated and the amount of the compound of Formula (I) or pharmaceutically acceptable salt thereof. In cases where no amount is expressly noted, an effective amount should be assumed. Non-limiting examples of an effective amount of the compound of Formula (I) or pharmaceutically acceptable salt thereof are provided herein below. [0025] An effective amount of the compound of Formula (I), a pharmaceutically acceptable salt thereof, or a second anti-cancer agent is generally in the range from 0.1 pg to 100 mg/kg of body weight of the recipient (mammal) per day and particularly typically in the range from 1 to 10 mg/kg of body weight per day. Thus, the actual amount per day for an adult mammal weighing 70 kg is usually between 70 and 700 mg, where this amount can be administered as an individual dose per day or usually in a series of part-doses (such as, for example, two, three, four, five or six) per day, so that the total daily dose is the same. The effective dose of the compound of Formula (I) or pharmaceutically acceptable salt thereof to a subject can be 10 pg - 500 mg. Effective amounts of a Chkl inhibitor, a Chk2 inhibitor, and a DNA-PK inhibitor are known to those skilled in the art.

[0026] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. For example, the terms “treatment,” “treat,” and “treating” refer to reducing the size of a cancerous tumor or the number of cancer cells. Treatment may also be continued after symptoms have resolved, for example to reduce the likelihood of or delay their recurrence.

[0027] In this description, a “pharmaceutically acceptable salt” is a pharmaceutically acceptable, organic or inorganic acid or base salt of a compound described herein.

Representative pharmaceutically acceptable, organic or inorganic base salts include, e.g., alkali metal salts (e.g., sodium), alkali earth salts (e.g., calcium), and ammonium salts (e.g., tris(hydroxymethyl)aminomethane, hydrabamine, and N-methylglucamine ammonium salt). Representative pharmaceutically acceptable, organic or inorganic acid salts include, e.g., the acetate, amsonate (4,4-diaminostilbene-2, 2 -disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (l,l-methene-bis-2-hydroxy-3-naphthoate, einbonate), pantothenate, phosphate/diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts. A pharmaceutically acceptable salt can have more than one charged atom in its structure. In this instance the pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterions.

Methods of Treatment, Uses for Manufacture of Medicament, Salts for Use in Disease Treatment

[0028] In one aspect, the invention is directed to a method for treating a cancer in a subject, comprising administering to the subject an effective amount of a first anti-cancer agent, with an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is represented by Formula (I):

or a pharmaceutically acceptable salt thereof; and wherein the second anti-cancer agent is selected from the group consisting of a Chkl inhibitor, Chk2 inhibitor, and a DNA-PK inhibitor. [0029] In one embodiment, the second anti-cancer agent is a Chkl or a Chk2 inhibitor. As used herein, a “Chkl inhibitor” refers to an inhibitor of checkpoint kinase 1 and a “Chk2 inhibitor” refers to an inhibitor of checkpoint kinase 2. In another embodiment, the second anti-cancer agent is a Chkl inhibitor or a dual Chkl/Chk2 inhibitor. In another embodiment, the second anti-cancer agent is a Chkl inhibitor. Examples of a Chkl inhibitor include SCH 900776 (MK-8776), LY2606368 (prexasertib), SRA737, LY2603618 (rabusertib), PF477736, CCT245737 ((R)-5-((4-((morpholin-2-ylmethyl)amino)-5- (trifluoromethyl)pyridin-2-yl)amino)pyrazine-2-carbonitrile), GDC-0425, GDC-0575, UCN- 01 (7 -hydroxystaurosporine), PD-321852, SAR-020106, CCT-244747, S-024, S-144, D- 501036, V-158411, CHIR-124, and the like. Examples of dual Chkl and Chk2 inhibitors include AZD7762, XL-844 (EXEL-9844), and the like. In another embodiment, the second anti-cancer agent is a Chk2 inhibitor or a dual Chkl/Chk2 inhibitor. In another embodiment, the second anti-cancer agent is a Chk2 inhibitor. Examples of a Chk2 inhibitor include PV1019 (NSC 744039) (7-nitro-lH-indole-2-carboxylic acid {4-[l-(guanidinohydrazone)- ethyl]-phenyl} -amide), CCT241533, VRX0466617, C3742, ABI (2-arylbenzimidazole), hymenialdisine, debromohymenialdisine (DBH), indoloazepine, and NCS 109555 (4,4'- diacetyldiphenylurea-bis(guanylhydrazone), and the like.

[0030] In one embodiment, the Chkl inhibitor is selected from SCH 900776, LY2606368 (prexasertib), 3RA737, LY2603618 (rabuseitib), PF477736, CCT245737, ( DC-0425, GDC- 0575. UCN-01, PD-321852, SAR-020106, CCT-244747, S-024. S-144 D-501Q36, V-158411, CHIR-124, AZD7762, and XL-844. In another embodiment, the Chkl inhibitor is SRA737 or LY2603618 (rabuseitib). In another embodiment, the Chkl inhibitor is LY2606368 (prexasertib) or SCH900776 (MK-8776) whose structures are shown below:

In another embodiment, the dual Chkl/2 inhibitor is AZD7762 or XL-844. In another embodiment, the Chk2 inhibitor is selected from PV1019, CCT241533, VRX0466617, C3742, ABI, hymenialdisine, debromohymenialdisine, indoloazepine, and NCS 109555. [0031] In one embodiment, the method is for treating a hematological cancer in a subject, comprising administering to the subject an effective amount of a first anti-cancer agent, with an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is the compound of Formula (I) or a pharmaceutically acceptable salt thereof; wherein the second anti cancer agent is LY2606368 (prexasertib) or SCH900776 (MK-8776); and wherein the hematological cancer is acute myeloid leukemia or diffuse large B-cell lymphoma. In another embodiment, the method is for treating a solid tumor in a subject, comprising administering to the subject an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is the compound of Formula (I) or a pharmaceutically acceptable salt thereof; wherein the second anti-cancer agent is LY2606368 (prexasertib); and wherein the solid tumor is colon cancer or lung cancer.

[0032] In another embodiment, the second anti-cancer agent is a DNA-PK inhibitor. As used herein, a “DNA-PK inhibitor” refers to an inhibitor of the DNA-dependent protein kinase. Examples of a DNA-PK inhibitor include nedisertib (M3814/MSC2490484A) ((5)-[2-chloro-4- fluoro-5-(7-morpholinoquinazolin-4-yl)phenyl]-(6-methoxypyridazin-3-yl)methanol), NU7441 (KU-57788) (2-N- morpholino-8-dibcnzothiophcnyl-chromcn-4-onc), AZD7648 (7-methyl-2-[(7- methyl [ 1 ,2,4] triazolo [1,5- aj yridin- 6 -yl)amino] - 9 -(tetrahydro- 2H-pyran- 4 - yl) - 7 ,9 - dihydro-8 H - purin-8-one), VX-984 (8-[(l S)-2-[[6-(4, 6-didcutcrio-2-mcthylpyri midi n-5-yl)pyri midi n-4- yl] amino] -l-methylethyl]quinoline-4-carboxamide), IC60211 (2-hydroxy-4-morpholin-4-yl- benzaldehyde), SU-11752, CC-115 (a selective dual inhibitor of the mammalian target of rapamycin (mTOR) kinase and DNA-PK), avadomide (CC-122), wortmannin, vanillins, LY2094002, KU-0060648 (a dual inhibitor of DNA-PK and PI-3K), NU7026, NU7059, NU7427 (2- V-morpholino-8-dibenzofuranyl-chromen-4-one), and the like.

[0033] In one embodiment, the DNA-PK inhibitor is selected from nedisertib, NU7441, AZD7648, VX-984, IC60211, SU-11752, CC-115, avadomide, wortmannin, vanillins, LY2094002, KU-0060648, NU7026, NU7059, and NU7427. In another embodiment, the DNA- PK inhibitor is CC-115 or avadomide. In another embodiment, the DNA-PK inhibitor is nedisertib or NU7441 (KU-57788) whose structures are shown below:

[0034] In one embodiment, the method is for treating a hematological cancer in a subject, comprising administering to the subject an effective amount of a first anti-cancer agent, with an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is the compound of Formula (I) or a pharmaceutically acceptable salt thereof; wherein the second anti cancer agent is nedisertib (M3814) or NU7441 (KU-57788); and wherein the hematological cancer is acute myeloid leukemia or diffuse large B-cell lymphoma.

[0035] In one embodiment, the first anti-cancer agent and the second anti-cancer agent can be administered simultaneously in the same or different formulations. In an alternative embodiment, the first anti-cancer agent and the second anti-cancer agent can be administered sequentially, i.e., at diferent points in time. [0036] In some embodiments, the first anti-cancer agent is a pharmaceutically acceptable salt of the compound of Formula (I). In one embodiment, the first anti-cancer agent is the tris(hydroxymethyl)aminomethane (“Tris”) salt of the compound of Formula (I). In another embodiment, the first anti-cancer agent is the sodium salt of the compound of Formula (I).

[0037] In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt thereof is a solid state or crystalline form of the compound of Formula (I), as described in PCT/US2019/067897, filed December 20, 2019. The entire teachings of PCT/US2019/067897 are incorporated herein by reference. In one embodiment, the compound of Formula (I) is Form A, B, C, or D of the crystalline form of the compound of Formula (I). In one embodiment, the compound of Formula (I) is Form A, B, or C of the crystalline form of the Tris salt of the compound of Formula (I). In one embodiment, the compound of Formula (I) or the Tris salt thereof is in amorphous form.

[0038] In one embodiment, the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) once a week in one or more 28- day or 4- week cycles.

[0039] In one embodiment, the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for a period of 2 to 5 days each week, with 1 cycle of therapy defined as 4 consecutive weeks of treatment [0040] In one embodiment, the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for a period of 2 to 5 consecutive days followed by a 2 to 5 day break each week, in one or more 28-day or 4-week cycle.

[0041] In one embodiment, the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for 2 consecutive days followed by a 5-day break each week, in one or more 28-day or 4-week cycles. [0042] In one embodiment, the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof ( e.g ., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for 3 consecutive days followed by a 4-day break each week, in one or more 28-day or 4-week cycles.

[0043] In one embodiment, the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for 4 consecutive days followed by a 3-day break each week, in one or more 28-day or 4-week cycles.

[0044] In one embodiment, the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for 5 consecutive days followed by a 2-day break each week, in one or more 28-day or 4-week cycles.

[0045] In one embodiment, the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for a period of 2 to 5 consecutive days followed by a 2 to 5 day break each week, in one or more 28-day or 4-week cycle, where the number of consecutive days of treatment is increased in at least one of the weeks of the 28-day or 4-week cycle, in one or more 28-day or 4-week cycles.

[0046] In another embodiment, the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for a period of 2 to 5 consecutive days followed by a 2 to 5 day break each week, in one or more 28-day or 4-week cycle, where the number of consecutive days of treatment is decreased in at least one of the weeks of the 28-day or 4-week cycle, in one or more 28-day or 4-week cycles.

[0047] In another embodiment, the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) is administered to the subject for 2 consecutive days followed by a 5-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is increased in at least one of the weeks of the 28-day or 4-week cycle.

[0048] In another embodiment, the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof ( e.g ., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) is administered to the subject for 2 consecutive days followed by a 5-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is decreased in at least one of the weeks of a 28-day or 4-week cycle.

[0049] In another embodiment, the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) is administered to the subject for 3 consecutive days followed by a 4-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is increased in at least one of the weeks of a 28-day or 4-week cycle.

[0050] In another embodiment, the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) is administered to the subject for 3 consecutive days followed by a 4-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is decreased in at least one of the weeks of a 28-day or 4-week cycle.

[0051] In another embodiment, the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) is administered to the subject for 4 consecutive days followed by a 3-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is increased in at least one of the weeks of the 28-day or 4-week cycle.

[0052] In another embodiment, the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) is administered to the subject for 4 consecutive days followed by a 3-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is decreased in at least one of the weeks of a 28-day or 4-week cycle.

[0053] In another embodiment, the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) is administered to the subject for 5 consecutive days followed by a 2-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is increased in at least one of the weeks of the 28-day or 4-week cycle.

[0054] In another embodiment, the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof ( e.g ., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) is administered to the subject for 5 consecutive days followed by a 2-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is decreased in at least one of the weeks of a 28-day or 4-week cycle.

[0055] In one embodiment, the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) orally. In one embodiment, the effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) is administered as an oral capsule. In another embodiment, the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) is administered as an oral tablet.

[0056] In one embodiment, the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) once daily (QD). In another embodiment, the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) twice daily (BID).

[0057] The same dosing regimens described above also apply to the second anti-cancer agent if it is administered concurrently with the first anti-cancer agent (i.e., the compound of Formula (I), or a pharmaceutically acceptable salt thereof) either in the same pharmaceutical formulation or in separate pharmaceutical formulations. If the second anti-cancer agent is not administered concurrently with the first anti-cancer agent, those skilled in the art will be able to determine suitable dosing regimens using routine experimentation. Compositions

[0058] In a further aspect, the invention is directed to a pharmaceutical composition comprising a first anti-cancer agent and a second anti-cancer agent, wherein the pharmaceutical composition additionally comprises an excipient, wherein the first anti cancer agent is represented by Formula (I):

or a pharmaceutically acceptable salt thereof; and wherein the second anti-cancer agent is selected from the group consisting of a Chkl inhibitor, a Chk2 inhibitor, and a DNA-PK inhibitor.

In another embodiment, the second anti-cancer agent is a Chkl inhibitor or a dual Chkl/Chk2 inhibitor. In another embodiment, the second anti-cancer agent is a Chkl inhibitor. In one embodiment, the Chkl inhibitor is selected from 8CI I 900776, LY2606368 (prexasertib), 3RA737, LY2603618 (rabusertib), PE-477736, CCT245737, GDC-0425, GDC- 0575. UCN-01, PD-321852, SAR-020106, CCT-244747, S-024. S-144. D-501036, V-158411, CHIR-124, AZD7762, and XL-844. In another embodiment, the Chkl inhibitor is SRA737 or LY2603618 (rabusertib). In another embodiment, the Chkl inhibitor is LY2606368 (prexasertib) or SCH900776 (MK-8776). In one embodiment, the second anti-cancer agent is a dual Chkl/2 inhibitor. In another embodiment, the dual Chkl/2 inhibitor is AZD7762 or XL- 844. In one embodiment, the second anti-cancer agent is a Chk2 inhibitor. In another embodiment, the Chk2 inhibitor is selected from PV1019, CCT241533, VRX0466617, C3742, ABI, hymenialdisine, debromohymenialdisine, indoloazepine, and NCS 109555. [0059] In another embodiments, the second anti-cancer agent is a DNA-PK inhibitor. In one embodiment, the DNA-PK inhibitor is selected from nedisertib, NU7441, AZD7648, VX-984, IC60211, SU-11752, CC-115, avadomide, wortmannin, vanillins, LY2094002, KU-0060648, NU7026, NU7059, and NU7427. In another embodiment, the DNA-PK inhibitor is CC-115 or avadomide. In another embodiment, the DNA-PK inhibitor is nedisertib or NU7441 (KU- 57788). [0060] In some embodiments, the first anti-cancer agent is a pharmaceutically acceptable salt of the compound of Formula (I). In one embodiment, the first anti-cancer agent is the tris(hydroxymethyl)aminomethane salt of the compound of Formula (I). In another embodiment, the first anti-cancer agent is the sodium salt of the compound of Formula (I).

[0061] The first anti-cancer agent and/or second anti-cancer agent may be formulated together with a pharmaceutically acceptable carrier, adjuvant, or vehicle into pharmaceutical compositions prior to being administered to a subject.

[0062] The term “pharmaceutically acceptable carrier, adjuvant, or vehicle” refers to a carrier, adjuvant, or vehicle that may be administered to a subject, together with the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a second anti-cancer agent and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound. Those skilled in the art would know how to prepare formulations suitable for administration. The second anti-cancer agent optionally is part of the same formulation for the first anti-cancer agent (i.e., the compound of Formula (I), or a pharmaceutically acceptable salt thereof), if it is administered concurrently with the first anti-cancer agent, or, alternatively, the second anti-cancer agent can be administered separately.

[0063] Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d- a- tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

Cyclodextrins such as a-, b-, and g-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-P-cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a second anti-cancer agent. [0064] The pharmaceutical compositions may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or administration by injection. The pharmaceutical compositions may contain any conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0065] The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic 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 mannitol, water, Ringer’s solution 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 may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens or Spans and/or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

[0066] The pharmaceutical compositions may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried com starch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added. [0067] The pharmaceutical compositions may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a second anti cancer agent with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols. [0068] The pharmaceutical compositions may be administered topically to the skin. The pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of one aspect of this application include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with suitable emulsifying agents. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of one aspect of this application may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topical transdermal patches are also included in one aspect of this application.

[0069] The pharmaceutical compositions may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

[0070] The amount of active ingredient that may be combined with one or more pharmaceutical excipients to produce a single dosage form will vary depending upon the patient treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations contain from about 20% to about 80% active compound. In certain embodiments, the pharmaceutical composition comprises between about 10 mg to about 1500 mg of the compound of Formula (I) or a pharmaceutically acceptable salt thereof (based on the weight of the free form of the compound of Formula (I), apart from the weight of any conformer, salt former, water of hydration, solvent of solvation and the like). In some embodiments, the pharmaceutical composition comprises between about 90 mg and about 240 mg; between about 100 mg and about 240 mg; between about 10 mg and about 500 mg; or between about 10 mg and about 1000 mg of the compound of Formula (I) or a pharmaceutically acceptable salt. In some embodiments, the pharmaceutical composition comprises about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 125 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 175 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 225 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 275 mg, about 280 mg, about 290 mg, or about 300 mg of the compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition comprises about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, or about 1500 mg. In certain embodiments, the pharmaceutical composition is in the form of an orally acceptable dosage form, such as for example a capsule, and comprises about 50 mg, about 75 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 125 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 175 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 225 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 275 mg, about 280 mg, about 290 mg, or about 300 mg of the Tris salt of the compound of Formula

(I)·

[0071] As used herein, the terms “about” and “approximately” when used in combination with a numeric value or range of values mean the value or range of values may deviate to an extent deemed reasonable to one of ordinary skill in the art.

[0072] The compound of Formula (I) and formulations thereof can be prepared according to methods described in WO 2014/128669, WO 2019/164794, PCT/US2019/067897, and US Patent No. 9,630,932, the contents of which are incorporated herein by reference.

EXAMPLES

[0073] A CRISPR depletion screen and proteomics analyses were carried out to evaluate in an unbiased manner factors contributing to response to DHODH inhibition and pointed to DNA damage response and DNA double-strand break (DSB) repair pathways as likely adaptive mechanisms. In support, co-treatment with Chkl or DNA-PK inhibitors resulted in synergistic effects with the compound of Formula (I). [0074] Cell lines were purchased from ATCC or DSMZ and cultured in vendor-recommended media or Gibco advanced RPMI 1640 supplemented with 10% dialyzed FBS, 2mM glutamine and 25mM HEPES where indicated.

Example 1. CRISPR depletion screen points to DNA repair pathways in mediating response to the compound of Formula (I)

[0075] A genome-wide CRISPR depletion screen was performed in A549 cells in the presence or absence of the compound of Formula (I) to identify specific genes and pathways limiting response to the compound of Formula (I). Uridine was provided in the medium at a concentration of 5 mM, resulting in alternate periods of uridine availability and depletion over the course of the culture period.

[0076] A VSV-G pseudotype lentiviral plasmid library containing sgRNAs was generated using the Cellecta Three-Module Human Genome -Wide CRIPSR construct library with the pRSG16-U6-sg-HTS6C-UbiC-TagRFP-2A-Puro plasmid. A lentiviral plasmid encoding stable Cas9 from Cellecta (pR-CMV-Cas9-2A-Blast) was introduced into A549 cells. The sgRNA library was introduced into the Cas9-expressing cells at a multiplicity of infection of 0.3 and selected under antibiotics for 48 hours. A sample of cells was collected and then ImM the compound of Formula (I) or 0.05% DMSO was added. Cell culture media was changed every 2 days until the cells completed 12 doublings. Genomic DNA was extracted using phenol: chloroform followed by ethanol precipitation. sgRNAs were amplified by PCR using primers specific for the regions of the plasmid flanking the sgRNA (FwdU6-l: 5’- CAAGGCTGTTAGAGAGATAATTGG-3’ R2: 5 ’ -CGACAAC AACGC AGAAATTTTGAAT - 3’). The PCR product was sequenced using the Hiseq4000 sequencing platform with 80-100 million single-end 50 base pair reads.

[0077] The constant fixed sequence immediately before and after the 20 nucleotide CRISPR single guide RNA (sgRNA) sequence was first removed before read mapping using AWK command in Unix. To determine the read count value for each sgRNA, the trimmed reads were mapped to a custom sgRNA reference library using Bowtie2 (version 2.1.0) with option -L 20 -N 0 -k 1. These criteria retained only the perfectly matched reads for downstream analysis. The raw read count from each of three separate module Cellecta Human Genome-Wide CRIPSR was used to calculate gene level statistics using Mageck (version 0.5.4) and DrugZ (version 1.1.0.2) with default options. The gene level statistics from each module were merged together post Mageck and DrugZ processing. [0078] Gene ontology (GO) analysis of CRISPR screen hits with a p-value <0.05 was performed to identify pathways or processes impacting the compound of Formula (I) sensitivity. DNA double-strand break (DSB) repair/DNA damage response pathways stood out as enriched GO terms (Figure 1A). Gene targets involved in multiple different repair pathways, including single-strand annealing (OGGI, BLM), non-homologous end joining (RIF1, MAD2L2, SMCHD1) and homologous recombination (GEN1, RECQL5, SAMHD1) were among the significantly depleted hits (Figure IB).

Example 2. Proteomics analysis

[0079] Involvement of DNA repair pathways in response to the compound of Formula (I) was also implicated from an unbiased proteomics analysis on A549 cells treated +/- the compound of Formula (I).

[0080] Cells were cultured at le6/mL (OCILY 19) or 5e5/mL (A549) in standard culture media containing ImM the compound of Formula (I) or DMSO for 24 (OCILY 19) or 28 (A549) hours. Cells were collected, washed in PBS and snap frozen. Cells were treated for 28 hours under culture conditions that resulted in depletion of uridine from the medium within 16 hours; thus, the cells experienced a period of uridine availability followed by a period of uridine depletion (comparable to the conditions in the CRISPR screen).

[0081] Samples were processed and analyzed as described (Mahoney, et al. A chemical biology screen identifies a vulnerability of neuroendocrine cancer cells to SQLE inhibition. Nat Commun 2019; 10(1):96, the contents of which are hereby incorporated herein by reference). In brief, cell pellets were lysed in 8M urea and cysteines were reduced and alkylated. Proteins were sequentially digested with LysC (1:50 enzyme: substrate, Wako Chemicals) and trypsin (1:100 enzyme: substrate, ThermoFisher) and desalted by solid phase extraction. Peptide amounts were normalized and labelled with TMTIOplex Isobaric Label Reagents (Thermo Fisher). Pooled peptides were fractionated by basic pH reverse phase into a 96-well plate and fractions were consolidated into 24 samples in a checkerboard manner. Peptides were separated on a 50 mM C18 EASY-Spray column (Thermo Scientific) using a 70 min, 8-28% acetonitrile gradient and spectra were acquired on an Oribtrap Fusion (Thermo Scientific) using a TMT-MS3 method. All .RAW files were processed using Proteome Discoverer 2.1.0.81. Quantitation data from razor peptides were excluded and only unique peptides were used for protein quantitation. Each 10- plex contained the same pooled sample labelled with TMT-131 and all relative abundance data were normalized to this channel to compare relative protein abundance across 10-plexes. [0082] GO analysis of proteins with at least a 1.5-fold increase in expression in the presence of the compound of Formula (I) compared to DMSO identified DNA damage response and repair pathways as upregulated in response to the compound of Formula (I) in A549 cells (Figure 2). Any proteins that decreased in the DMSO-treated cells by 25% or more compared to TO were excluded from the analysis.

[0083] Proteomic analysis of the OCILY 19 lymphoma cell line treated with the compound of Formula (I) showed little overlap in upregulated DNA damage response related pathways by GO analysis (Figure 3).

Example 3. Drug combinations

[0084] Dose response matrix screening (9x9 format) in 384-well plates was carried out with the compound of Formula (I) in combination independently with the Chkl inhibitors LY2606368 and SCH900776 and the DNA-PK inhibitors M3814 and NU7441. Growth assays were carried out in vendor-recommended media with CTG readings performed at TO and T96. To measure combination effects in excess of additivity, a scalar measure derived by Horizon Discovery to characterize the strength of synergistic interaction (Synergy Score) was utilized as described (Schaffer, et al). Synergy scores >2.4 are considered to be substantially above the background noise of the assay. Scores from 2.4-9.3 are considered mild to moderate synergy, while scores >9.3 are considered strong synergy. Potency shifting was also scored using combination index (Cl) which is calculated as

C_x C_Y Cl = — + —

EC_X ECy where for a particular data point is the ratio of the X compound’s measured concentration to its effective concentration at the chosen effect level. The Cl is a rough estimate of how much drug was needed in combination relative to the single agent doses required to achieve the chosen effect level. Cl values 0.5-0.7 are typical for in vitro measurements of current clinical combinations. The Cl error (oCI) is calculated using standard error propagation through the Cl calculation based on the isobologram errors. Best Cl is the Cl with the largest signal-to-noise ratio level (1-CI)/ oCI.

[0085] For western blots, cells were plated at le6/mL (lymphoma lines) or 3e5/mL (solid tumor lines) in advanced RMPI containing 10% dialyzed FBS, 2mM glutamine and 25mM HEPES. Compounds were added at the indicated doses and incubation times. Protein was extracted in RIPA extraction buffer containing phosphatase and protease inhibitors. 30 pg of total protein was electrophoresed on 4-12% bis-tris NuPAGE gels (ThermoFisher). Proteins were transferred to a nitrocellulose membrane using the iBlot 2 dry transfer system, blocked in Odyssey TBS blocking buffer (LI-COR), and incubated with the indicated antibodies overnight (Cell Signaling Technologies) followed by a 1 hour incubation with the appropriate secondary antibody (LI-COR) and imaged on the LI-COR Odessey CLx system.

[0086] Cell growth assays (non-screen format) were performed using CTG as a readout of viable cells. For CTG assays cells were plated in 96-well tissue culture plates (Coming) at either 10,000 cells/well for cell lines of hematologic origin or 2000 cells/well for solid tumor-derived cell lines. Cells were incubated with Formula (I) or DMSO control in combination with LY2606368 (prexasertib) for 96 hours. CTG readings were performed at TO and at T96 using a Molecular Devices SpectraMAX Paradigm plate reader. Relative growth rates (p/pmax) were calculated using the CTG time 0 (TO) and 96-hour (T96) ATP measurements according to the following formula:

where T is the signal measure for the drug-treated arm at T96 and V is the DMSO vehicle treated control measure at T96. VO is the vehicle-treated control measure at TO. A value of 1 indicates no growth inhibition, 0 indicates complete growth inhibition, and a value <0 indicates cell death. [0087] Combination treatment decreased cell viability compared to either treatment alone in the solid tumor models and resulted in clear synergy in A549 cells (0=0.18; Figure 5B). In HCT116 cells, the combination also resulted in increased gH2AC compared to treatment with either agent alone (Figure 5C). The A549 and HCT116 cell lines used were insensitive or poorly sensitive to the compound of Formula (I) as a single agent whereas OCILY 19 and Z138 cell lines were sensitive to the compound of Formula (I) as a single agent. While prexasertib had limited impact on response of the highly sensitive Z138 and OCILY 19 cells to the compound of Formula (I) (Figure 5B), in an expanded panel of AML (n=3) and DLBCL (n=8) cell lines, prexasertib (LY2606368) demonstrated evidence of synergy in 9 out of the 11 lines (Figures 4A and 4B). An additional CHK1 inhibitor (SCH90076) and two DNA-PK inhibitors (M3814 and NU7441) also resulted in synergistic effects in combination with the compound of Formula (I) in this panel (Figures 4 A and 4B). [0088] Taken together, these results point to induction of DNA damage as a key component of response to the compound of Formula (I) and to potential combination strategies aimed at co targeting DNA damage response pathways.

Claims

What is claimed is:

1. A method for treating a cancer in a subject, comprising administering to the subject an effective amount of a first anti-cancer agent and an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is represented by Formula (I):

or a pharmaceutically acceptable salt thereof; and wherein the second anti-cancer agent is selected from the group consisting of a Chkl inhibitor, a Chk2 inhibitor, and a DNA-PK inhibitor.

2. The method of claim 1, wherein the cancer is selected from the group consisting of a hematological malignancy and a solid cancer.

3. The method of claim 1 or 2, wherein the cancer is selected from the group consisting of acute myeloid leukemia, multiple myeloma, B-prolymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin’s disease, non-Hodgkin’s lymphoma, follicular lymphoma, diffuse large B cell lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, lymphocytic lymphoma cancer of the bladder, T-cell lymphoma, lung cancer, breast cancer, triple negative breast cancer, melanoma, glioblastoma, prostate cancer, colon cancer, pancreatic cancer, bone cancer, cancer of the head or neck, skin cancer, cutaneous or intraocular malignant endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumours of childhood, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), tumour angiogenesis, spinal axis tumour, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers, and PTEN mutant cancers.

4. The method of claim 1 or 2, wherein the cancer is selected from the group consisting of acute myeloid leukemia, multiple myeloma, B-prolymphocytic leukemia, non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, triple negative breast cancer, melanoma, prostate cancer, and cancer of the esophagus.

5. The method of claim 1 or 2, wherein the cancer is selected from the group consisting of chemotherapy-resistant acute myeloid leukemia, cytarabine-resistant acute myeloid leukemia, acute monocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, diffuse mixed cell lymphoma, myelodysplastic syndrome, primary effusion lymphoma, erythroleukemia, chronic myeloid leukemia, chronic monocytic leukemia, double hit diffuse large B cell lymphoma, triple hit diffuse large B cell lymphoma, biliary tract cancer, cancer of the ampulla of Vater, non-small cell lung cancer, broncho alveolar carcinoma, liver cancer, cancer of the ovary, and cancer of the upper aerodigestive tract.

6. The method of claim 1 or 2, wherein the cancer is acute myeloid leukemia or diffuse large B-cell lymphoma.

7. The method of claim 1 or 2, wherein the cancer is lung cancer.

8. The method of claim 1 or 2, wherein the cancer is colon cancer.

9. The method of any one of claims 1-8, wherein the second anti-cancer agent is a Chkl inhibitor.

10. The method of claim 9, wherein the Chkl inhibitor is selected from a group consisting of SCH 900776, prexasertib, SRA737, rabusertib, PF477736, CCT245737, GDC-0425, GDC-0575, UCN-OL PD-321852, SAR-020I06, CCT-244747 S-024, S-144, D-501036, V- 158411, CH1R- 124, AZD7762, and XL-844.

11. The method of claim 10, wherein the Chkl inhibitor is prexasertib or SCH900776.

12. The method of any one of claims 1-8, wherein the second anti-cancer agent is a Chk2 inhibitor.

13. The method of any one of claims 1-8, wherein the second anti-cancer agent is a DNA-PK inhibitor.

14. The method of claim 13, wherein the DNA-PK inhibitor is selected from a group consisting of nedisertib, NU7441, AZD7648, VX-984, IC60211, SU-11752, CC-115, avadomide, wortmannin, vanillins, LY2094002, KU-0060648, NU7026, NU7059, and NU7427.

15. The method of claim 13, wherein the DNA-PK inhibitor is nedisertib or NU7441.

16. The method of any one of claims 1-15, wherein the first anti-cancer agent and the second anti-cancer agent are administered simultaneously in the same or different formulations.

17. The method of any one of claims 1-15, wherein the first anti-cancer agent and the second anti-cancer agent are administered sequentially.

18. The method of any one of claims 1-17, wherein the first anti-cancer agent is the tris(hydroxymethyl)aminomethane salt of the compound of Formula (I).

19. The method of any one of claims 1-17, wherein the first anti-cancer agent is the sodium of the compound of Formula (I).

20. A pharmaceutical composition comprising a first anti-cancer agent and a second anti cancer agent, wherein the pharmaceutical composition additionally comprises an excipient, wherein the first anti-cancer agent is represented by Formula (I):

or a pharmaceutically acceptable salt thereof; and wherein the second anti-cancer agent is selected from the group consisting of a Chkl inhibitor, a Chk2 inhibitor, and a DNA-PK inhibitor.

21. The pharmaceutical composition of claim 20, wherein the second anti-cancer agent is a Chkl inhibitor.

22. The pharmaceutical composition of claim 20 or 21, wherein the Chkl inhibitor is selected from a group consisting of SCH 900776, prexasertib, SRA737, rabuseitib, PF477736, CCT245737, GDC-0425, GDC-0575, UCN-01, PD-321852, SAR-020106, CCT-244747, S-024, S- 144, D- 501036, V 158411, CHIR-124, AZD7762, and XL-844.

23. The pharmaceutical composition of claim 21, wherein the Chkl inhibitor is prexasertib or SCH900776.

24. The pharmaceutical composition of claim 20, wherein the second anti-cancer agent is a Chk2 inhibitor.

25. The pharmaceutical composition of claim 20, wherein the second anti-cancer agent is a DNA-PK inhibitor.

26. The pharmaceutical composition of claim 20 or 25, wherein the DNA-PK inhibitor is selected from a group consisting of nedisertib, NU7441, AZD7648, VX-984, IC60211, SU- 11752, CC-115, avadomide, wortmannin, vanillins, LY2094002, KU-0060648, NU7026, NU7059, and NU7427.

27. The pharmaceutical composition of claim 25, wherein the DNA-PK inhibitor is nedisertib or NU7441.

28. The pharmaceutical composition of any one of claims 20-27, wherein the first anti-cancer agent is the tris(hydroxymethyl)aminomethane salt of the compound of Formula (I).

29. The pharmaceutical composition of any one of claims 20-27, wherein the first anti-cancer agent is the sodium salt of the compound of Formula (I)