WO2014092572A1 - Improved methods for cancer treatment with a genotoxic agent - Google Patents

Abstract

The invention relates to a susceptibility agent for rise in a method for treatment of an individual suffering from, cancer, whereby said susceptibility agent is combined with a genotoxic agent. Said genotoxic agent is preferably selected from gamma radiation, a. platinum-based compound and/or an anthracyciin, A preferred susceptibility agent inhibits expression and/or activity of a. product of a gene selected from the group consisting of AKIHL DUSP15, UBE1X, RPL7L1, USp8; 4EHP and PRKDL

Description

Title: Improved methods for cancer treatment with a genotoxic agent

Field: The invention relates to the field of cancer therapy, more specifically to methods and means for treating an individual suffering from cancer with a genotoxic agent. The methods and means of the invention will improve a response to treatment with a ge no toxic agent.

Introduction

D A damage provides the risk of toxicity and the accumulation of mutations and chromosomal instability, potentially resulting in malignant transformation (Ciccia and Elledge, 2010. Mol Cell 40: 179-2042010;

Jackson and Bartek, 2009, Nature 461: 1071-1078). To counteract these deleterious effects of DMA damage, cells are equipped with a highly complex signaling response termed the DNA damage response (DDR). The DDR activates effector components involved in protective pathways including DNA damage repair, cell cycle arrest, transcription, chromatin remodeling and cell death. In tandem with phosphorylation-mediated signaling, which is largely executed by the ΡΪ3Κ1 like kinases ATM, ATR and DNA PK, the checkpoint kinases Chkl and Chk2, and members of the MAPK family (Matsuoka et al. 2007, Science 316, 1160» 1166; Bernhardt and Yaffe, 2009. Curr Opin Cell Biol 21: 245-255), protein modifications by ubiquitm and ubiqnitin-like modifiers are crucial at all le vels of the DDR (Bergink and Jentech, 2009. Nature 458: 461-467).

Regulators of genome stability have been identified, including infrared- induced double strand brakes repair foci, and genotoxic stress-induced apoptosis (Arora et al 2010, Gynecol Oncol 118:220-227; Kolas et al. 2007, Science 318: 1837-1640; M&cKeigan et al. 2005, Nat Cell Biol 7: 591 -600; Paulsen et al, 2009. Mol Cell 35: 228-239). The DDE is a highl conserved process but somatic cells, ste cells, and cancer cells show variations on the theme through, developmental-, tissue specific-, or oncogenic alterations in expression of components of the DDR. For instance, p5B plays a major role in the DDR in somatic cells while its role in embryonic stem cells (ESC) is debated and p53 signaling is inactivated in the majority of cancer cells (Harper et al. 2007. MoL Cell 28: 739-745; Aladjem et aL 1998. Current Biology 8: 145 - 155). The large variety of genotoxieily-induced defects reflects the pleiotropic nature of DNA damage normally occurring through ongoing cellular metabolism and exposure to environmental mutagens,

Many cancer therapeutic age ts are genotoxie and induce DNA damage. These effects are counteracted in cancer cells at least in part by activation of the DNA damage response (DDR). Therefore, there is a need for

improvement of the response of cancer cells towards genotoxic agents.

The present invention provides a susceptibility agent, for use in a method for treatment of an individual suffering from cancer, whereby said

susceptibility agent is combined with a genotoxic agent.

It was found by the present inventors that a susceptibility agent according to the invention improves treatment of the individual by the genotoxic agent, compared to treatment with the genotoxic agent in the absence of a susceptibility agent.

The invention also provides a method for treatment of an individual suffering from cancer, comprising (a) providing said individual with a genotoxic agent; and (b) providing said individual with a susceptibility agent, whereby treatment is improved compared to treatment in the absence of said susceptibility agent.

The invention also provides use of a susceptibility agent, in the preparation of a medicament for treatment of an individual suffering from cancer, whereby said medicament is combined with, a genotoxic agent, whereby treatment of the individual is improved compared to treatment in the absence of said susceptibility agent.

The term "genotoxic agent" refers to an agent that induces damage in the genomic DNA of a cell that arrests the cell cycle, activates repair

mechanisms, and/or causes cell death or senescence. Said genomic DNA damage includes base modifications, single strand breaks, and crosslinks, such as intrastrand and interstrand cross-links. A preferred genotoxic agent is selected from an alkylating agent such as nitrogen mustard, e.g.

cyclophosphamide, mechlorethamine or in us tine, uramustine and/or uracil mustard, melphalan, chlorambucil, ifosfamide; nitrosourea, including carnmstine, lomustine, stre tozocin; an alkyl sulfonate such as bnsulfan, an ethylenime such as thiotepa and analogues thereof, a hydrazine/triazine such as dacarba^ine, altretamine, niito¾olomide, temoKolomide, altretamine, procarbazine, dacarba¾ine and temozolomide; an intercalating agent such as a platinum-based compound like cisplatin, carb platin, nedapiatin, oxaHplatin and sa raplatin; antbraeyelines such as doxorubicin,

daunorubicin, epirubicin and idarubicin; mitomycin- C, dactinomyein, bleomycin, adriamycin, and mithramycin, A further preferred genotoxic agent is provided by radiation, including ultraviolet radiation and gamma radiation.

A more preferred genotoxic agent is selected from gamma radiation, a platinum-based compound and or an anthracyclin, A most preferred genotoxic agent is cisplatin and/or doxorubicin.

Methods for providing a genotoxic agent to a patient in need thereof suffering from cancer are known in the art. For example, cisplatin is administered at 2 to 8 mg kg every 3 to 4 weeks or at 20 mg m2/day for 5 days every 3 to 4 weeks; at 40 mg-120 mg/m2 every 8 to 4 weeks. Cisplatin is preferably administered for treatment of advanced bladder cancer, malignant melanoma and osteogenic sarcomas. Cisplatin is preferably administered by injection or infusion, preferably by intravenous, intraarterial or intraperitoneal injection or infusion.

For example, antbracyclms sneh as doxorubicin, daunorabicin, epirnbicin and idarubiein are routinely administered at 40-75 mg m2, every 3 weeks for treatment of, for example, breast cancer, uterine cancer, ovarian cancer, and lung cancer.

For example, gamma radiation is administered in a dose that depends on the tumour type, whether radiation is given alone or with chemotherapy, before or after snrge y, the success of surgery as is known to the skilled person. For example, in case of curative treatment of a solid epithelial tumor radiation dose raging from 50-70 Gy is administered, In case of lymphomas 20-40 Gy is given. This dose is given in daily fraction called the fraction schedule. Typically adnlts receive 1,8-2 Gy per fraction. The typical treatment schedule is 5 days per week. These small frequent doses allow healthy cells time to grow back, repairing damage inflicted by the radiation. Daily fractions of radiation are given using an external source or an internal source such as implants.

Said individnal may snffer from breast cancer, ovarian cancer, lung cancer, liver cancer, head and neck cancer, squamous cell carcinoma, bladder cancer, colorectal cancer, cervical cancer, renal cell carcinoma, stomach cancer, prostate cancer, melanoma, brain cancer, and/or esophageal cancer. A susceptibility agent, as herein defined, is an agent that improves treatment of au individual suffering from cancer by a genotosde agent. By mechanisms that are yet unknown, a susceptibility agent makes cancer cells more sensible to the DNA damaging effects of a genotoxie agent Without being bound by theory, a susceptibility agent might hamper or inhibit the DNA damage response (DDR) in cancer cells, or parts of the DDR, and thereby improves the response of cancer cells towards a genotoxie agent.

A preferred susce ibility agent inhibits expression and/or activity of a product of a gene selected from the group consisting of FRKDl, BITSP15, ARIH1, UBE1X, RPL7L1, USPS and EHP.

1/ Protein kinase Bl (PRKD1) encodes serine/tkreonine-protem kinase Dl (EC 2.7.11,13). Alternative names for the serine/threonine -protein kinase are PKC-mu, PKB1, PRKCM1, PKC-mu, PKCM1, and PKC-MU.

2/ Dual specificity phosphatase 15 (DUSP15) encodes dual specificity phosphatase 15 (EC 8.1.3.483). Alternative names for the phosphatase are vaccinia virus VII 1 -related dual-specific protein phosphatase (VR^'Yl), dual specificity protein phosphatase 15, dual specificity phosphatase-like 15, and

3/ ARIHl (ariadne-i honiolog) encodes E3 uhiquitm-protem Kgase

(EC=6.3.2). -Alternative names for the ligase are H7 AP2, HHARI, monocyte protein 6 (MOP-6), UbeH7 -binding protein, IJbcM4 nteracting protein, ubiquitin-eonjugating enzyme E2~binding protein 1, Alternative gene names are ARI, ΜΌΡ6, and UBCH7BP.

After DNA damage, ongoing transcription and translation have to be adjusted to allow execution of stress-specific programs_., save energy, accomplish DNA repair and avoid the transcription and subsequent translation of potentially mutated genetic material (Reinhardt et al. 2011. Cell Cycle 10: 23-27). Genotoxie stress has been shown to induce a block in protein synthesis (Braunstein et al. 2009. Mol Cell Biol 29: 5645-5656;

Connolly et al. .2006, Mol Cell Biol 26: 3955-3965; Silvera et al. 2010. Nat Rev Cancer 10: 254-266). Eukaryotic niRNAs are mostly recruited to the ribosome through their 5' 7-methylguanosine cap (Kong and Lasko 2012. Nat Rev Genet 13; 383-394,). The rate -limiting step of eukaryotic cap- dependent translation initiation is the binding of the translation initiation factor eIF4F to the mRNA S'eap structure. EIF4F is composed of the eap5 binding protein eIF4E, the R A helicase eiF4A and the scaffold protein eiF4G (G ngras et al. 1999, Armu Rev Bioehera 68, 913-963: Gross et al. 2003. Cell 115, 739-750).

Silencing ARIHl, which additionally harbors XSGy!ase activity, sensitizes various cancer cells to genoi.ox.ic compotinds and γ-irradiation, irrespective of p53- or caspase-3 status. It was found that DNA damage triggers ARIHl accumulation and association with 4EHP, a competitive inhibitor of the translation initiation factor eI.F4E. Consequently, 4EHP translocates to the 5' mENA-cap and arrests mRNA translation upon DNA damage, in an ARIH 1 · dependent manner. It was found that in ARXIll-depleted cells, DNA damage-induced translation arrest, as well as resistance to genotoxic stress, are restored by the eiF2»alpha depbosphorylation inhibitor, salubrinal. These findings indicate that ARIHl is a mediator of a protective DNA damage response in cancer ceils that prevents aberrant translation, after genotoxic stress. A preferred susceptibility agent inhibits the expression and/or activity of ARIHl, and thereby improves the response of cancer cells towards a genotoxic agent.

4/ Eukaryotic translation initiation factor 4E family member 2 (4EHP or EIF4E2) encodes eukaryotic translation initiation factor 4E family member 2, Alternative names for the protein are eukaryotic translation initiation factor 4E ike 3 (EIF4EL3), mRNA cap-binding protein 4E11P, mRNA cap- ? binding protein type 3. and eukaryotie translation initiation factor 4E homologous protein, 4SHP is a competitive inhibitor of the translation initiation factor eIF4E. 4EHP translocates to the 5^s rnRNA-cap and arrests nsRNA translation upon DNA damage, in an ARIHl-dependent manner. Similar to downmodulation of AEIHl, downmodulation of 4EHF hampers or prevents DNA damage-induced translation arrest. Downmodulation of 4EHP improves the response of cancer cells to genotoxie stress. A preferred susceptibility agent inhibits the expression and/or activity of 4EHP and thereby improves the response of cancer cells towards a genotoxic agent,

5/ Ubiqui tin-activating enzyme El (UBE1X) encodes a protein that catalyzes the first step in iihiquitm conjugation to mark cellular proteins for degradation. Alternative names for the phosphatase are ubiquitin-like modifier activating enzyme 1 (IJBAI A1S9: A1ST; GXPL UBEl. A1S9T, AMCX1, POC2G, SMAX2, and UBA1A.

6/ Ribosomal protein L?~like 1 (RPL7L1) encodes ribosomal protein L7~like 1. An alternative name for the protein is 80S ribosomal protein L7 fke.

7/ Uluqxii tin- specific-processing protease 8 (USPS) encodes ubiquitin specific protease 8. Alternative names are ubiquitin thioesterase 8, UBPY1_?

HuniORFS. and hUBPy2. 81 2,

Methods for do wnmodulatin g and/or inhibiting expression and or activity of a product of a gene selected from the group consisting of AEIHl, DUSP15, UBEIX, RPL7L1, USpS, 4EHP and PRKD1 are known in the art. and include RNA antisense expression, RNA interference and introduction, of small inhibitor molecules. A preferred susceptibility agent according to the invention is an antisense RNA compound, a targeting RNA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of a gene selected from the group consisting of ARIH1, DUSF15, UBEXX, RPL7L1, USp8_s 4E.HP and PRKD1, A further preferred susceptibility agent according to the invention is an antisense RNA compound, a targeting RNA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of a gene selected from the group consisting of ARIHI, DUSP15, RPL7L1, USp8_s 4EHP and PRKD^'L Yet a further preferred susce tibility agent according to the invention is an anti sense RNA compound,, a targeting RNA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of a gene selected from the group consisting of ARIHI, DUSF15, HPL7L1, USp8 and PSKB!. Yet a further preferred susceptibility agent according to the invention is an antisense RNA compound, a targeting RNA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of a gene selected from the group consisting of ARIHI, DUSP15, RPL7L1 and PRKD1. Yet a farther preferred susceptibility agent according to the invention is an antisense RNA compound, a targeting RNA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of a gene selected from the group consisting of ARIH I, DUSP15 and PKKD L Yet a further preferred susceptibility agent according to the invention is an antisense SNA compound, a targeting RNA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of a gene selected from the group consisting of ARIHI and DUSPX5, Yet a further preferred susceptibility agent according to the invention is an antisense RNA compound, a targeting RNA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of ARIHI. A susceptibility agent according to the invention,, preferably an antisense RNA compound, a targeting ENA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of a gene selected from the group consisting of ARIH^'l, DUSP15, UBS IX, EPL7L1, USp8, 4ΕΪΪΡ and PRKD1, is preferably used in a method for treatment of a individual suffering from cancer, whereby said

susceptibilit agent is combined with a genotoxic agent,

RNA antisense expression.

Antisense RNA compounds a e commonly used as researcli reagents and diagnostics. For example, antisense oligonucleotides,, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. The specificity and sensitivity of antisense compounds is harnessed by those of skill in the art for therapeutic uses, Antisense oligonucleotides and antisense

polynucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides and antisense polynucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides and polynucleotides can be useful therapeutic modalities that can be configured to he useful in treatment regimes for treatment of cells, tissues and animals, especially humane.

In the context of this invention, the term "polynucleotide" refers to an polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DMA) or mimetics thereof. This term includes oligonucleotides composed of naturally- occurring nucleobases, sugars and cova!ent iutemucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occnrring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. A preferred polymer is or corresponds to more than 30 nucleotides, more preferred more than 50 nucleotides, more preferred more than 100 nucleotides, more preferred more than 200 nucleotides, more preferred more than 500 nucleotides.

In the context of this invention, the term "oligonucleotide" refers to an oligomer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term inehides oligonucleotides composed of naturally- occurring nucleobases, sugars and covalent intemucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

While antisense oligonucleotides and antisense polynucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric or polymeric antisense compounds, including but not limited to oligonucleotide mimetics and polynucleotide mimetics such as are described below. Preferrd oHgonucleotic antisense compounds in accordance with this invention preferably comprise from about 8 to about 30

nucleobases. Particularly preferred are antisense oligonucleotides

comprising from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides). As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines , Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2 3' or 5 ' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups eovalently link adjacent nucleosides to one another to form a linear polymeric compound.

Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a

phosphorus atom in their internncleoside backbone can also he considered to be oligonucleosides. Preferred modified oligonucleotide backbones include, for example, phosphorothioat.es, chiral phosphorothioates,

phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other a Iky I phosphonates including 3^!-alkyIene phosphonat.es and chiral phosphonates, phosphinates, phosphoramidates including 3 '-ammo phosphoramidate and aminoalkylphosphoramidates_}

thionophosphoramidates, thionoalkylphosphonatea,

thionoalkylphosphotriesters, and boranophosphates having normal 3' -5' linkages, 2' -5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are Hnked 3' ~5^! to 5' -3^f or 2" - 5^! to 5 '·2'.

Preferred modified oligonucleotide backbones that do not include a

phosphorus atom therein have backbones that are formed by short chain alkyl or cyeloa!kyl internncleoside linkages, mixed heteroatom and alky! or cycioalkyl internncleoside linkages, or one or more short chain heteroatomic or heterocyclic internncleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; form acetyl and thiofonnaeetyl backbones; methylene form acetyl and thioforxnacetyl

backbones ; a!kene containing backbones; sulfamate backbones;

methylenei ixxo and m eth l e e r asri o backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, 0, S and CH2 component parts.

In other preferred oligonucleotide mimetics, both the sugar and the internncleoside linkage, i.e.. the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that lias been shown to have excellent hybridisation properties, is referred to as a peptide nucleic acid (PNA) , In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an

aminoethylglyeine backbone. The nucieobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

Most preferred embodiments of the invention are oligonucleotides with phosphorotbioate backbones and oligonucleosides with heteroatom

backbones, and in particular CH2 H-0-CH2-, -CH2-N (CH3)-G-CR2~

[known as a methylene (methylimino) or MM! backbone], -CH2-0~K(€H3)- CH2- CH2-N(CH3)-N(CHS)-CH2- and -0-N (CH3) -CH2-CH2- [wherein the native phosphodiester backbone is represented as -0-P-0-CH2-], Modified oligonucleotides may also contain one or more substituted sugar moieties, Preferred oligonucleotides comprise one of the following at the 2' position ; OH; F; 0-, S-, or N-alkyl: Q- , S-, or N-alkenyl; 0^» , S- OF N-alkynyl; or O- alkyl-O alkyl, wherein the alkyl, alkenyl and alkyny). may he substituted or unsuhstituted C to CIO alkyl or 02 to CIO alkenyl and alkynyl. Particularly preferred are 0[(CH2)nQ]mCH3, 0(CH2)nOCH3, 0(CH2)nNH2,

0(CH2)nCH3, 0(CH2)nONH2, and O (CH2)nON [ (CH2)nCH3) ] 2, where a and m are. from 1 to about 10, Other preferred oligonucleotides comprise one of the following at the 2* position: CI to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkvL G-alkaryl or 0-araikyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3_f NH2, heterocycloalkyl heterocyeloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, a intercalator, a group for

improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties, A preferred modification includes 2 ' -methoxyethoxy (2 ' -0-CH2CH2OCH3, also known as 2 ' -0- (2- methoxyethyl) or 2 ' -MOE) (Martin et al. 1995, Helv. CM . Acta 78: 488-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2 ' ·· dimethylaminooxyethoxy, i.e., a O (CH2) 20 (CH3) 2 group, also known as 2^f-DMAOE, as described in examples herein below.

Other preferred modifications include 2 ^f -methoxy (2^f-G-CH8) , 2 ^! - aminopropoxy (2 ' -OCH2CH2CH2NH2) and 2 '-fluoro. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3° position of the sugar on the 3 ' terminal nucleotide or in 2^s -5' linked oligonucleotides and the 5^s position of 5' terminal nucleotide.

Oligonucleotides may also have sugar mimetics such as eyclob tyl moieties in place of the pentofuranosyl sugar.

Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein,

"unmodified" or "natural" nue!eobases include the purine bases adenine (A) and guanine (G) , and the pyrimidine bases thymine (T) , eytosine (C) and uracil (U) . Modified nucleohases include other synthetic and natural

mieleobases such as 5 · met yl eytosine (5-me-C) , 5-l.tydroxymetbyl cytosine, xanthine, hypoxanthine , 2-aminoadenme, 6 methyl and other alkyl derivatives of adenine and guanine, 2-propy). and other alky! derivatives of adenine and guanine, 2 thiouracil, 2-thiothymine and 2 hiocytosine, 5~ halouracil and cytoame, 5-propynyl uracil and eytosine, 6~azo uracil, eytosine and thymine, 5·· uracil (pseudouracil) ₅ 4-thiouracil , 8-halo, 8~amino_s 8-thioI. 8-thioaIkyI, 8-hydroxy.i. and other 8-substituted adenines and guanines, 5 halo particularly 5-faromo, 5-trii3uoromethyl and other 5- substituted uracils and eytosin.es, 7-methylguanine and 7-niethyladenine, 8- aza guanine and 8-azaadenine, 7~dea¾aguanine and 7-dea¾aademne and 3- deazagua ne and 3~deazaadenine.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance or modify pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance of the oligonucleotide. Conjugate groups are routinely used in the chemical arts and are linked directl or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an

oligomerie compound, such as an oligonucleotide. Conjugate groups include without limitation, mtercaiators, reporter molecules, polyamines,

polyamides, polyethylene glycols, thioetbers, polye there, eholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenantbridine, anthraquinone, adaniantane, aeridine,

fluoresceins, rhodamines, conmarins and dyes. Said conjugate group or groups may be directly attached to oligonucleotides in oligomerie

compounds. It is preferred that the conjugate group or groups are attached to oligonucleotides by a conjugate linking group. In certain sucn

embodiments, conjugate linking groups, including, but not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein. Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilising groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound. In genera! a bifunctional linking moiety comprises a. hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group. In some embodiments, the conjugate linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units, Examples of functional groups that are routinely used in a bifunctional Unking moiety include, but are not limited to, electrophiles for reacting with nucleophilie groups and nueleophiles for reacting with

electrophilie groups, in some embodiments, bifunctional linking moieties include amino, hydroxy!, earboxyiic acid, thiol, unsaiurations (e.g., double or triple bonds), and the like.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. The term "chimeric" antisense compound", as used herein refers to an antisense compound, particularly an oligonucleotide, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA: DMA or ENA: RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand 18 of an RNA: DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression, Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate

deoxyoligomicleotides hybridizing to the same target region. Cleavage of the ENA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified

oligonucleotides, oligemic! eosides and/or oligonucleotide niimetics as described, above. Such compounds have also been referred to in the art as hybrids or gapmers.

The antisense compounds need in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.

Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA) . Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule

structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.

The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other

bioequiva!ents. The term "prodrug" indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions, in particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [ (S-acetyl-2- thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 in WO 94/28764.

The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts arc formed with metals or amines, such as alkali and alkaline earth metals or organic amines , Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are Ν,Ν' - dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methyiglucamine. and procaine. The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may he regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar IS solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a "pharmaceutical addition salt" includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic earboxylic, sulfonic, sulfo or phospho acids or N~substituted s ilfamie acids, for example acetic acid, propionic acid, glyeolic acid, succinic acid, maleie acid, hydroxyrnaleic acid, methylmaleic acid, fu marie acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, henmic acid, cinnamie acid, mandelic acid, salicylic acid, 4~ aminosalicylic acid, 2-phenoxyben¾oic acid, 2-acetoxybengoic acid, embonic acid, nicotinic acid or isonicotmic acid; and with amino acids, such as natural alpha-amino acids, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfo ie acid, ethanesulfonic acid, 2- hydroxyethanesulfonic acid, ethane-1, 2-disulfonic acid, bengenesulfbnie acid, 4-methyihenzenesulfonic acid, naphthalene-2~sulfonic acid,

naphthalene-l, 5-disulfonic acid, 2- or S-phosphoglycerate, gIncose~8- phosphate, N-cyclohe^ylsnJfamic acid (with the formation of cyc!amates) , or with other acid organic compounds, such as ascorbic acid, Pharmaceutically acceptable salts of compounds may also he prepared with a

pharmaceutically acceptable cation. Suitable pharmaceutically acce table cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible, For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are sot limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyandries such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromie acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalene sulfonic acid, methanesulfonic acid, p-toluenesulfonie acid, naphthalenedisulfome acid, pol alacturonic acid, and the like; and (d) salts formed from elemental a ions such as chlorine, bromine, and iodine.

A therapeutic amount or dose of an antisense RNA compound of the present invention may range from about 0.1 to 500 mg/kg/day, such, as 0.1, 0.5, 1.0, 1.5, 2.0, 3.0, 5.0, 10, 25, 50, 100, 200, or 500 mg/kg/day. A targeting

compound is preferably provided in 0.9% NaCL The resulting solution can be administered, for example by infusion with a portable volumetric infusion pump.

An antisense RNA compound is preferably provided as an expression

product to a cancer cell. Said antisense RNA compound, or a DNA sequence coding for an antisense RNA compound, is preferably linked to one or more control sequences, i.e. regulatory DNA sequences, in such a manner that said antisense RNA compound is highly expressed in a cancer cell. Control sequences that are operably linked to the sequences encoding the targeting RNA molecule include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in. The term promoter is well-known in the art and encompasses nucleic acid regions ranging in s½e and complexity from minimal promoters to promoters including upstream elements and enhancers. The promoter is typically selected from promoters that are functional in mammalian cells, although promoters functional in other e karyotic cells may he used. The type of promoter is chosen to accomplish a useful expression profile for said targeting RNA molecule in the context of said replication competent

adenovirus. Preferred promoters are ENA polymerase II promoters, such as a viral promoter, for example SV40 early gene promoter and GM^'V promoter and RNA polymerase III promoters, including but not limited to the U8, Hi and tRNA (Val) promoters are especially suitable for the invention, but other promoters are not excluded.

A preferred promoter/enhancer drives specific expression of the antisense RNA compound in target cancer cells. The term "specific expression", as used herein, refers to a high level of expression from the promoter/enhancer in target cells, when compared to other cells or other cell types. As is known to the skilled person, the Tissue-specific Gene Expression and Regulation (TiGER) database developed by the Bioinformati s Lah at Wilmer Eye

Institute of Johns Hopkins University contains eis-re gulatory module (CRM) data. These CRM data allow the expression of an antisense ENA compound in a specific target tissue or cell For example, a preferred

promoter/enhancer for expression in hepatocellular carcinoma cells is the alpha -fetoprotein promoter/enhancer; for expression in breast cancer cells is the alp a-lactalhumin (ALA) promoter/enhancer, for expression in prostate cancer cells is the PSA/FMSA and/or the kallikrein-2 promoter/enhancer, for expression in melanoma cells is the tyrosinase promoter/enhancer

(Papadakis et a! 2004. Current Gene Therapy 4: 89-113).

The antisen.se RNA compound and control sequences are preferably incorporated in a vector. In one embodiment, said vector is a plasmid.

Methods for introducing a plasmid into cells, preferably mammalian cells, are known in art and include nanoparticle -mediated delivery. Preferred vector are viral vectors, including but not limited to an adenoviral vector, an adeno-associated viral vector, and a retroviral vector, A preferred retroviral vector is provided by a replication non-competent murine leukemia virus (MLV) and a replication non-competent human immunodeficiency virus 1 and 2.

Methods for providing an expression vector comprising an antisense SNA compound to cancer cells are known in the art an include ex vivo

administration, where cells are removed from the body, incubated with the vector, and the gene-engineered cells are returned to the body; in situ administration, where the vector is placed directly into the affected tissues; and in vivo administration, where a vector is injected directly into the bloodstream or tissue. A therapeutic amount or dose of an expression vector comprising an antisense ENA compound of the present invention may range from about 1*10E9 to 1*1E13 copies/kg, or from about 1*I0E10 to i*lE12 copies/kg. In general, treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about l*i0E10 to 1* IE.1.2 copies/kg of an expression vector comprising an antisense RNA compound of this invention in single or multiple doses.

Therapeutic amounts or doses will also vary depending on route of administration; as well as the possibility of co-usage with other agents. The term "copies" refers to plasmid numbers for p!asmid vectors, and to particle numbers for viral vectors, as is known to the skilled person.

RNA interference (ENAi) is a conserved cellular surveillance system that recognises double-stranded RNA (dsENA) and activates a sequence-specific degradation of ENA species homologous to the dsR A (Hannon (2002). Nature 418: 244-251). in addition, RNAi can cause transcriptional gene silencing by RNA- directed promoter DNA methyl&tion and or histone methylation (Kawasaki and Taira 2004, Nature 431: 211-217; Morris et al 2004. Science 305: 1239-1292). RNAi -related processes have been described in almost all e karyo ic organisms, including protozoa, flies, nematodes, insects, parasites, and mouse and human cell lines (reviewed in: Zamore 2001. Nat, Struct, Biol. 8: 746-750; Hannon 2002, Nature 418: 244-251; Agrawal et al 2003. Microbiol. MoL Biol, Rev. 67: 657-885), By now, RNA interference is the most widely used method to specifically downregulate genes for functional studies.

RNAi is mediated by a targeting RNA molecule capable of decreasing expression of a target gene through the process of RNA interference. Said targeting RNA molecule preferably comprises a double stranded portion, preferably having a length of at least 19 nucleotides (per strand). The double stranded portion preferably has a len gt of less than 30 nucleotides, and said RNA molecule preferably comprises a short hairpin SNA (shRNA), or comprises a double-stranded structure consisting of two different RNA molecules having complementary regions of preferably the complete length of the RNA molecules. In the latter case, the said RNA molecules can be transcribed from different promoters. Said RNA molecules are preferably susceptible to the action (i.e. the recognition and cleavage of dsRNA) of RNAse III enzymes, resulting in the above-discussed siRNAs, The term "target gene" is understood to indicate that the process of decreasing expression of said gene occurs with specificity towards ARIHl, DUSP15, UBE1X, RPL7L1, USp8, 4EH.P and/or PRK 1.

The targeting RNA molecule, or a DNA sequence coding for a targeting RNA molecule, can be provided to cancer ceils by any method known in the art. For example, the nncleic acids can be coupled to a nanoparticle of an inert solid (commonly gold) which is then shot directly into a target cell's nucleus. As an alternative, magnet-assisted transfeetion uses magnetic force to deliver nucleic acids into target cells after associating said nucleic acids with magnetic nanopartieles. Furthermore, inipalefection is carried out by impaling cells by elongated anostruetures and arrays of such

nanostrueture s such as carbon nanofibers or silicon nanowires which ha e been inaction aliped with nucleic acid molecules.

A therapeutic amount or dose of a targeting RNA molecule of the present invention may range from about 0.1 to 500 mg kg day, such as 0.1, (1,5, 1.0, 1,5, 2.0, 3.0, 5.0, 10, 25, 50, 100, 200, or 500 nig/kg/day. A targeting compound is preferably provided in 0,9% NaCL The resulting solution can he administered, for example by infusion with a portable volumetric infusion pump.

Said targeting ENA molecule, or a DNA sequence codi g for a targeting RNA molecule, is preferably linked to one or more control sequences, i.e. regulatory DNA sequences, in such a manner that said targeting RNA molecule is highly expressed in a cancer cell. Control sequences that are operahly linked to the sequences encoding the targeting RNA molecule include promoters/enhancers and other expression regulation signals. These control sequences may be selected to he compatible with the host cell for which the expression vector is designed to be used in. The term promoter is well-known in the art and encompasses nucleic acid regions ranging in sise and complexity from minimal promoters to promoters including upstream elements and enhancers. The promoter is typically selected from promoters that are functional in mammalian cells, although promoters functional in other eukaryotic cells may be used. The type of promoter is chosen to accomplish a useful expression profile for said targeting ENA molecule in the context of said replication competent adenovirus. RNA polymerase III promoters, including but not limited to the U6, HI and tENA(¥al)

promoters are especially suitable for the invention, but other promoters are not excluded, In another variation of the invention, said DNA sequence coding for a targeting RNA molecule is functionally linked to one or more control sequences, i.e. regulatory DNA sequences, in such a manner that said targeting RNA molecule is only expressed or is expressed at a higher level in a cell into which the targeting RNA molecule is introduced tinder certain conditions that can be modulated by an external signal, where the term "external" means having its origin outside of the DNA fragment

encompassing said DNA sequence coding for a targeting ENA molecule and said regulatory DNA sequences. In this aspect of the invention, the

expression of the targeting RNA molecule is driven by a so-called

regulatahie or inducible promoter.

Preferred targeting ENA molecules are directed to the target sequences depicted in Table 1. It is further preferred that the targeting RNA molecules is or comprises one or more targeting siRNA/shENA molecules that are depicted in Table 1. These targeting siRNA/shRNA molecules are

preferably, selected rom the human targeting si RNA/ shRNA molecules having identification numbers M-019984-00; M-019984-00, M-019984-00, M- 019984-00, TRCN0000007501, TRCN0000007502, TRCN0000007503, M- 008484-02, M-008484-02, M-008484-02, M-008484-02, MU-019870-01, MU- 019870-01, MU-019870-01, MU-019870-01, M-005028-02, M-QG5G28-02, M- 005028-02, M-005028-02, M-027238-01, M-027238-01, M-027238-01, M- 027238-01, M.-004509-Ol , M-,004509-01, M-004509-01, M-004509-01, M- 005203-01, M-0Q5203 01, M-005203-01, and/or M-005203-01, as depicted in Table 1.

The targeting ENA molecule and control sequences are preferably

incorporated in a vector. In one embodiment, said vector is a plasmid.

Methods for introdncing a plasmid into cells, preferably mammalian ceils, are known in art and inclnde nanop article -mediated delivery as is detailed herein above. A preferred vector is a viral vector, including but not limited to n adenoviral vector, an adeno-assoeiated viral vector, and a retroviral vector. A preferred retroviral vector is provided by a replication non- competent murine leukemia virus (MLV) and a replication non-competent human immunodeficiency virus 1 and 2.

The invention also provides methods and means for formulating the replication non-competent viruses according to the invention that can be used to preserve said replication competent viruses and to administer said replication non-competent viruses to cells. In one variation, the formulations are used to administer said replication non-competent viruses to cells in vitro, in another variation the formulations are used to administer said replication non-competent viruses to cells in vivo.

The invention furthermore provides methods for administering the

formulations according to the invention to cells, leading to infection of said cells with the replication non-competent viruses of the invention. In one variation, the methods are used to administer said formulations to cells in vitro, in another variation the methods are used to administer said formulations to cells in vivo.

Methods for providing an expression vector comprising a targeting RNA molecule to cancer cells are known in the art an include ex vivo

administration, where cells are removed from the body, incubated with the vector, and the gene-engineered cells are returned to the body; in situ administration, where the vector is placed directly into the affected tissues; and in vivo administration, where a vector is injected directly into the bloodstream or tissue. A therapeutic amount or dose of an expression vector comprising a targeting RNA molecule of the present invention may range from about 1*10E9 to 1*1E13 copies/kg, or from about 1*10E10 to 1*1E12 28 copies/kg. In general, treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 1*10E1G to 1*1E12 copies/kg of an expression vector comprising a targeting RNA molecule of this invention in single or multiple doses. Therapeutic amounts or doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents. The term "copies" refers to plasmid numbers for plasmid vectors, arid to particle numbers for viral vectors, as is known to the skilled person.

A further preferred susceptibility agent according to the invention is an inhibitor molecule, such as a large or a small inhibitor molecule.

Known large inhibitor molecules include, for example, antibodies that are directed to a gene product of ARM 1, DUSF1.5, UBE1X, EFL7L1, USp8, 4EHF and/or PRKDl. The term antibody includes conventional antibodies such as an IgG which comprises two heavy and two light chain .molecules. Further included are dimers, diabodies, triabodies, bispecific, trispeeific, tetraspecifie antibody formats, monovalent, divalent, trivalent, tetravalent or other multivalent antibody formats, or any portion or fragment thereof such as, hut not limited to, single domain antibodies, F(ab').sub.2, Fab, Fab", Facb, Fc, and scFv,

The delivery of antibodies into cells is currently of great commercial and scientific interest. For example, a method for the delivery of antibodies into cells using the TAT-fused protein has been reported (Lee et ai 2005.

Biocbem Biophys Ees Comms 337: 701). A further example is provided by the use of liposomal systems. A number of relatively inexpensive lipid-based delivery systems are commercially available. For example, Lipodin~Ab®

(Abb io tec, LLC, San Diego) and Ab-DeliverIN® (OZ Biosciences, Marseille, France) are antibody delivery systems that are able to deliver functional antibodies to their intracellular targets. In addition, a composition comprising encapsulated antibody vesicles have been described

(IJS20110111038) wherein tbe vesicles comprise an amphophilic block copolymer having a hydropbilie and a hydrophobic block.

As an alternative, nucleic acid encoding one or more antibodies against a gene product of ARIH1, BUSP15, UBE1X, RPL7L1, USp8, 4EHP and/or FRKD1 can be incorporated into a gene delivery vector which further comprises control sequences that are operah!y linked to tbe sequences encoding the antibody or antibodies. These control sequences include promoters/enhancers and other exp ession regulation signals. These control sequences ma be selected to be compatible with the host cell for which the expression vector is designed to be used in. The term promoter is well- known in the art and encompasses nucleic acid regions ranging in :m and complexity from minimal promoters to promoters including upstream elements and enhancers. The promoter is typically selected from promoters that are functional in mammalian cells, although promoters functional in other eukaryo ie cells may be used. The type of promoter is chosen to accomplish a useful expression profile for said targeting RNA molecule in the context of said replication competent adenovirus. Preferred promoters are ENA polymerase II promoters, as are indicated hereinabove. Preferred vectors are plasmids and viral vectors, preferably adenoviral, retroviral or adeno-assoeiated viral vectors.

Antibodies against a gene product of AR!H!, DUSP15, UBElX, RPL7L1, USpS, 4EHP and/or PRKD1 are known in the art. For example, Abeam (Cambridge, USA) provides an anti-AEIHl antibody (ab3891); an anti- DU3F15 antibody (ab .123267); an anti-El Ubiqnitin Activating Enzyme antibody (ab31S38); an anti-RPL7Ll antibody (abl28581), anti-UBPY/USP8 antibody (ab38365, ah59949, ab 108524 and ah 108102); an anti-EIF4EL8 23 antibody (ab63Q62; ab50934 and ab56637) and an anti-PRKDl antibody (ab59413)„ In addition, other companies such as Nevus Bioiogicals

(Littleton, USA), USCN Life Sciences, INC. (Houston, USA) and Origene (Rockville, USA) provide one or more antibodies against ARIH1, DUSPI5, UBE1X, RPL7L1, USp8, 4EHP and/or PRKD1 that can be used for the present invention,

Known small inhibitor molecules of PRKDI include CID 2011758 (5~(3- c orophenyl)~N 4-(morphoIin-4-ylmethyI)phenyI]foran-2"Carboxamide; Tocris Bioscience), CID 755873 (2, 3_;4_; 5-tetrab dro-7-bydroxy- 1 H~

benzofuro [2, 3 -c] azepin- 1 -one ; Tocris Bioscience), and NB 142-70 (9~hydroxy- 3_}4~dihyd?o-2H~[ll-ben¾;otb.ioIo[2_?3"fl [l,4]thiazepin-5~one (Tocris Bioscience), A therapeutic amount or dose of a small inhibitor molecule of the present invention may range from about 0.1 nag/kg to about 500 mg kg, alternatively from about 1 to about 50 mg kg. Irs general, treatment regimens accordmg to the present invention comprise administration to a patient in need of such treatment from about 10 mg to about 1000 mg of a small inhibitor molecule of this invention per day in single or multiple doses. Therapeutic amounts or doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents.

The invention also provides compositions of the replication non-competent viruses according to the invention and cells in which the replicatio non- competent viruses according to the invention induce expression of a susceptibility agent.

The invention further provides a susceptibility agent for use according to the invention, whereby the susceptibility agent is administered before, during or after treatment an individual suffering from cancer with a genotoxic agent. It is preferred that a susceptibility agent for use according to the invention is provided prior or adjacent to treatment of an individual suffering from cancer with a genotoxic agent.

Said individual suffering from cancer is preferably a mammal, more preferably a human.

Figure legends

Figure 1. RNAi screen for ubiqidtination / sumoylation enzymes identifies cisplatin response modulators, (A) Hits identified in primary screens;

protecting siRNA Smartpools (IS) on the left, sensitizing siENA Smartpools (32) on the right, (B) Results of de convolution screen for 50 Smartpools identified in primary screen, (O) -scores obtained for 27 confirmed hits in deconvokition screen. Asterisk marks ARIHl results. (D) Distribution of kits over different gene families as indicated, (E) Me tacore - rediete d network derived form screen, hits; interactions with p53 are indicated.

Figure 2, Silencing ARIHl sensitises to genotoxie stress. (A) ES cell viability after treatment with 10 microM cisplatin (CP), 10 mg/ml mitomycin C (MMC), 150nm etoposide (ETO), 250nM doxorubicin (DOX), 250 microM diethylmaleathe (DSM)_S 1 microM thapsigargm (THAPS), or ΙΟηΜ

Vincristine (Vine), (B) ES cell viability in presence of control or ARIHl siENA after treatment with CP, Etc, or MMC. (C) ES cell viability in presence of Kifll -siENA (only for PBS), GFP-siR A, p53-8_KNA, or 3 individual siENA sequences targeting AEIH1 after treatment with vehicle control, CP, DOX, DEM, THAPS or VINC (normalized to siGFF). (D) ASIHl protein levels and H2B loading control in U20S expressing shcontrol or 3 individual sbRNAs targeting ARIHl, followed by bulk puromycin selection. Percentages indicate remaining ARIHl expression. Asterisks indicate shARIHl #2 and #3 used in all further experiments. (E) U20S cell viability in shcontrol or 2 individual sh ARIHl cell lines after treatment with vehicle (PBS) or 0 or 25 microM CP for 48k (F, G, H) Colony formation capacity in shcontrol or shARIHl-2 and -3 IJ20S cell lines after treatment with gamma irradiation (XR), CP, or UV-radiation at indicated exposure conditions.

*p<0.G5; **p<0.0L Figure 3. Silencing AEIH1 enhances cancer cell death in response to ge no toxic stress in a p53 & caspa.se -3 independent manner. (A) i)

Immunoetaining for gammaH2AX, 53BP1, and DAPI in U20S cells expressing indicated shRNAs u de BOB -irradiated conditions (NO) or after treatment with gamma-irradiation for indicated times, ii) Quantification of remaining ganimaH2AX foci at indicated tixnepoints derived from 3 independent experiments. (B) i) p53 and tubulin control protein levels in ES cells in presence of indicated siSNAs treated with PBS control or 5 mieroM CP for 8!L ii) Quantification of Western blot data from i) (B™4). (C) i) p53 and tubulin control protein levels in U20S cells in presence of control or ARJ l 11 shRNAs treated with PBS control or 10 microM CP for 16h. ii) Quantification of Western blot data from i) (η·"·· 3), (B) 53 and tubulin control protein levels in p53-deflcient H1299 cells treated with indicated concentrations of CP for 2 h. (E) H i 299 cell viability under control or siAMHl conditions after treatment with vehicle control PBS or 25 microM CP for 24h. (F) WB for p-p53 and p53 in p53-mutant 4T1 cells treated with 5 or 25 microM CP for 12h. (G) 4T1 cell viahility under control siARIHl, or sip 53 conditions after treatment with PBS or 12.5 microM CP for 24b. (H) i) FACS profiles for HM1 cell cycle content under control, siGFP, or siARIHl conditions after treatment with control or 1 microM CP. ii) Cell cycle distribution derived from profiles from i) (n~8). (I) Sub-GI/GO apoptotic fraction of control or siARIHl ES cells treated with 7.5 microM CP for 24h. (J) MCF7 cell viability under control or siARIHl conditions after treatment with PBS or 25 microM CP for 4Sh. (K) AEIH1 and tubulin control protein levels in MCF7 cells hulk puromyein-sorted for expression of control shR A or different shRNAs targeting ARII l. (L) Cell viahility for shcontroi and shARIHl-2 and -3 MCF7 cell lines treated for 48h with PBS or 10 or 25 microM CP. (M) FACS analysis for cell cycle content in shcontroi and shAEIHl-2 MCF7 cell lines treated for 2 h with FBS or 10 microM CP. *p<0.05; **p<0.01. Figure 4. ARIHl mediate® DNA damage-induced Cap binding of 4EHP. (A) ARIHl protein levels in U20S cells treated for 4h with PBS or 5 microM CP. (B) qPCR analysis of ARIHl RNA levels, normalized to GAPDH in U20S cells treated for 4 with PBS or 5 microM CP, (C) FLAG pulldown from sheontrol and shARIHl U20S cells transfecte with GFP control or FLAG-4ehp plasniid and treated with vehicle control or 5 microM CP followed by Western blot for FLAG or ARIHl, (D-F) m?G~Cap pulldown from control and ARIHl -silenced U20S (D), MCF7 (E) or ES cells (F) treated with vehicle control or indicated concentrations CP followed by Western blot for 4ehp or tubulin control. Numbers at the bottom indicate Cap-associated 4ehp levels relative to total 4ehp, (G) Quantification of m7GCap-bound 4ehp in control (n=4) and siARIHl (n=2) ES cells, (H) ES cell viability under sicontrol or si4ehp conditions after treatment with PBS, 10 microM CP, 150nm Eto, 10 mg/ml MMC for 24b. (I) H1299 cell viability under sicontrol or si4elip conditions after treatment with PBS or 25 microM CP. *p<0.05; **p<0,QL

Figure 5. ARIHl mediates eisplatin-induced mRNA translation arrest. (A) Methionine incorporation in U20S cells after treatment with 15 microM CP for 2h, 4h or 8h or cyclohexamide (CHX) for lb. Alexa546 signal (reflecting amount of newly synthesized proteins) / number of nuclei (DAPI) is shown. (B) Methionine incorporation in sheontrol and two different sbARIHl U20S cell lines after treatment with CP for indicated times. Effect of co-treatment with 15 microM CP and 2.5 microM salubrinal (SAL) for 2b. is shown and CHX is used as positive control. Normalized to Alexa546 fluorescence / nucleus value in PBS for each cell line, (C-E) Cell survival in cells

expressing indicated siRNAs or shBNAs after treatment with indicated concentrations of CP in absence or presence of 2.5 microM SAL. 0, U20S 48h treatment; D, ES cells 24h treatment; E, MCF7 48b treatment, (E) Cartoon depicting the role for AEIH1 in regulating sensitivity to genotoxie stress: i) Under non-stressed conditions e!F4E binds to the mRNA Cap and recrnits eiF4G and—A driving mRNA translation; ii) upon genotoxic stress ARIHl accumulates and associates with 4ehp. resulting in recruitment of 4ehp to the Cap where it replaces elF4E resulting in translation arrest that is ey top rotecti ve ; iii) in the absence of ARIHl (or 4ehp) DNA damage- induced translation arrest does not occur and cell survival is compromised; iv) co-treatment with salnbrinai inhibits translation under stressed conditions through inhibition of eiF2alpha dephosphorylatiori causing restored translation arrest and s urvival in the absence of ARIHl under genotoxic stress.

Figure 6 RNAi screen for CP response modifiers in ESC. (A) siRNA

SMARTpools from indicated gene families affecting cell viability under control (PBS) condition (pie diagram) with examples of known survival genes for each family. B) Graphs show -seore ranking in primary screen of SMARTpools after exclusion of those affecting general viability. Pie diagrams show number of SMARTpools reducing (left) or enhancing (right) CP-induced loss of viability [absolute Z~Score>L5; p<0.05|. (C) Confirmation of hits from primary screen by deeon volution using 4 individual siRNAs against each target gene. (D) Number of primary hits confirmed (dark lb medium dark) and rejected (light).

Examples

Example 1

SNAi screen for (de)ubiquitinases and sumoylases

HMl mouse ES ceils derived from OLA/129 genetic background (provided by Dr. Klaus Wl!lecke, University of Bonn GE) were maintained under feeder free conditions in GMEM medium containing 5x105U mouse recombinant leukemia inhibitory factor (LIF; PAA). All other cell lines were purchased from ATCC. MCF7 hurnan breast cancer cells, 4T1 mouse breast cancer cells and H1299 human non-small cell lung cancer ceils were maintained in EPMX medium. U20S human sarcoma cells were kept in DMEM. All media contained 10% FBS and 25U/ml penicillin, and 25 g/r J streptomycin. All cell lines, including stable sbJEiNA expressing derivatives, were confirmed to be mycoplasuia-free using the Mycosensor kit from Stratagene. For stable gene silencing, cells were transduced nsing lentiviral TRC shRNA vectors at MOI 1 (LentiExpressTM; Sig na-Aldrieh: Dr. Rob Hoeben and Mr Martijn Rabelink, University Hospital, Leidnn NL) according to the manufacturers^* procedures and bulk selected in medium containing 2.5pg/mi puromycin. Control vector expressed shRNA targeting TurboGFP,

Genotoxieants included the DNA cross-linker cispiati (Cis-PtCl2(NH3)2) (provided by the Pharmacy unit of University Hospital, Leiden NL) and the inhibitors of topoisomera.se IJ-mediated DNA unwinding, doxorubicin

(Sigma.) and etoposide (Sigma). Oxidative stressor diethyl maleate (DEM), microtubule poison Vincristine, and ES stressor Tbapsigaragin were from Sigma, The pan~caspase inhibitor gA^abAla-DL-Asp-fiuerometbylketone (so VAD-fink) was purchased from Bachem, the eif2alpha phosphorylation inhibitor sa!nbrina! was from Ca!biochem. Antibodies against ad and p p!hioosspphboo--'ppo6S8 wweerree p puurrcchhaasseedd fr froomm NNoovvaaccoossttrraa aannda CCeellll s siiggnnaalliinngg,,

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RRNNAAii.. eexxppeerriimmeennttss

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As a qaalit control Z' -factors were determined for each plate, using La in A/C as a negative control and p53 as a positive control. To rank the results, Z-seores were calculated using as a. reference i) the mean of ail test samples in the primary screen and ii) the mean of the negative control samples in the secondary deeonvolution screen (in order to prevent bias due to pre- enrichment of hits) (Birmingham et a!. 2009. Nat Methods 6: 569-575). Hit determination was done using Z-seores with a cut off value of 1.5 below or above the reference and -value lower than. 0.05.

Enrichment of canonical pathways and formation of p53/ ubiqnitination signaling network was performed using MetaCore™ data-mining software. Apoptosis and cell cycle analysis ES cells wore exposed to vehicle or cisplatin for 8h for ceil cycle analysis or 24b. for apoptosis analysis. MCF7 cells were exposed, for 24h for cell cycle analysis. Floating and attached cells were pooled and fixed in 80% ethane! overnight. Cells were stained usi g PBS EDTA containing 7.5rnM propidium iodine and 40mg/ml RNAseA and measured by flow cytometry (FACSCanto XI; Becton Dickinson), The amount of cells in the different ceil cycle fractions or in sub G0/G1 for apoptotie cells was calculated using BD FACSDiva software. Alternatively, apoptosis was determined using live imaging of Annexin V labeling, as described

previously (Puigyert et aL 2010. Cure Protoc Cell. Bio! Chapter 18, Unit 18A0 11-13).

Clonogeme survival assay

IJ20S cells (250 cells plate) expressing different shRMAs were seeded in triplicate in 9 cm plates. The following day, cells were treated with a dose range of genotoxie agents (γ-irradiation, cisplatin. UVC). After a recovery period of 10 days, surviving ceils were fixed, stained and colonies were counted to assay each cell-line's clonogenlc potential.

Western blot analysis Extracts were prepared in TSE containing protein inhibitor cocktail and separated by S DSP AGE on poly aery iaraide gels, transferred to PVDF membranes, and membranes were blocked using d% BSA. Following incubation with primary and secondary antibodies signal was detected using a Typhoon™ 9400 from GE Healthcare.

Imm nofluoresc^^

U20S cells were seeded on glass coverslips and allowed to grow for two days. Subsequently, they were irradiated with O.oGy and fixed^■using 2% formaldehyde at the indicated tiniepo nts. Alter washing with PBS, post- fixation extraction took place by incubating with 0.25% Triton-X. Cells were extensively washed with PBS to remove detergent and then blocked in S% BSA, Finally eoverslips were ixnnxunostained with rabbit 53BP1 and mo se γίΙ2ΑΧ antibodies, followed by eounterstaining with DAPI and appropriate secondary fluorescent antibodies.

Cap binding assay

HMI ES cells, U20S. and MCF7 breast cancer ceils were seeded in 8-well plates at a density of 0,5 million cells/ well. Cells were treated with different concentrations of cisplatin for 4 h (U20S, MCF7) or 8h (ES) and proteins were harvested in lysis buffer containing liaM phenyirnethylsulfonyl fluoride (Cell Signalling), Cap binding proteins were precipitated using 7- raethyl-GTP-Sepharose 4B beads (Amersha n) as described previously (Moody et al. 2005, J Virol 79; 5499-5506). Precipitated proteins were separated on 12% SDS-PAGE gels and analyzed by immunoblotting for 4ehp (E1F4E2).

Metabolic labeling for detection of translational. changes after cisplatin treatment

Cliek-iT® Metabolic Labeling Reagents for Proteins was purchased from n vitrogen and used according to manufacturers Instructions. In short U20S cells were seeded to 80% confluence in 96 well rnicroclear plates and subsequently treated with 15 microM cisplatin for 2-8h or with 2mg/ml eydohex&mi e (CHX) for lb, or for 2b with a combination of 15 microM ciBplatin and 2.5 micreM sai brmal. During the last .hour of treatment medium was replaced with m.ethiomne-free medium. Subsequently, cells were incubated with aside-labeled methionine analogue for in and fixed for 15 min in 4% formaldehyde and stained according to manufacturers protocol.

DAP! was used as coanterstain and images were acquired using a BD- pathway imaging system. Image analysis was performed using BD

Atto ision software.

I.I20S cells, expressing different shENAs, were transiently transfeeted with FLAG-tagged ehp cD A (provided by Dong-Ex Zhang, Scripps Research Institute, La Jolia CA. - through Addgene; plasnnd 17342) (Okumura et al. 2007. Genes Dev 21: 255-260) or GFP control plasraid in OptiMEM

(Invitrogen), using JeiPEI (Poly las Transfection, The following day, medium was refreshed and 72br post transfection cells were lysed in FLAG- lysisbuffer (50mM Tris-HCl pH 7.4, ISGmM NaCl, ImM^' EDTA, 0,5% P-40, 0,5% Triton-X, ImM^" PMSF, supplemented with complete protease inhibitor cocktail (Roche)), After 30^s incubation on ice, lysates were diluted 5 times with FLAG-dilntion buffer (50mM Tris-HCl pH7,4, ISOmM NaCl, ImM EDTA_S ImM PMSF, supplemented with complete protease inhibitor cocktail) and incubated, with prewashed M2-FLAG magnetic beads (Sigma) for 3h. Subsequently, beads were washed 3 times for amin with FLAG- dilution h after and !ysed in Laemmh-SDS-sample buffer. aPCOE

ENA was e tracted using ENeasy Plus Mini Kit from Qiagen. cDNA was made from 50 ng total RNA with EevertAid H minus First strand cDNA synthesis kit (Fermentas) and real-time qPCS was subsequently performed in triplicate using SYBR green PGR (Applied Brosystems) on a 7900HT last real-time PGR system (Applied Biosystems). The following qPC.E primer sets were used:

GAPDH, forward (rw) AGCCACATCGCTCAGACACC

reverse (rev) ACCCGTTGACTCCGACCTT;

ARIHl (fw) TCATGCCTCTACCCAAGCCTT

(rev) ACCAAACCCACAGCAACACA.

Data were collected and analyzed -using SDS2.3 software (Applied

Biosystems). Relative nsRNA levels after correction for GAPDH control mRNA were expressed nsing 2^Λ(-ΔΔ(¾) method.

Results

We performed an siENA-based. screen using the Dharmacon nbiquitination Smartpool library and custom made Srnartpooi libraries targeting all known cellular deublquitinases (DUBs). sum ylases, and desumoylases. Mouse embryonic stem (ES) cells that display a robust apoptotic response to genotoxic compounds, including cispiatin, were treated with 10 microM cisplatin or vehicle and cell viability was monitored after 24h. 50

Smartpools were identified that met selection criteria [Z-score +/- 1.5; p- value > 0.05] (Fig IA). As controls, we included siSNA Smartpools targeting Kifll, expected to induce cell killing due to mitotic spindle defects and targeting p53_s expected to protect ES cells against cispla tin-induced killing. In ail experimental plates, si ifll resulted in --90% reduction in viability and sipSd protected against cisplatindnduced loss of viability. As a quality measurement Z^s-factors were calculated based on siLamin (negative control) and srp53. The average of calculated Z'-feetors was 0.45, indicating a good signal to noise ratio and reproducibility of the screens. To exclude off target effects, selected Smartpools entered a deeonvointion screen where 27/50 bits could be confirmed with at least 2/4 sequences reproducing the effect of the Smartpool (Fig 1B,C).

The 27 confirmed Mts included six deubiquitinating enzymes (DUBs), one El ubiqu tin -activating enzyme, Ubelx, one E2 iibiqu tin -con j ugating enzyme, UBE2D3, as well as 12 siRNAs targeting E3 ubiquitin ligases, Moreover, we identified seven siRNAs targeting proteins with no described ubiquitinase function that were included in the ThermoFischer

"ubiquitination library* presumably based on the presence of predicted domains associated with ubiquitinase function, including RING, SOCS, or SPRY. The knockdown of the El ubiquitin enzyme Ubelx (Uba), which has recently been shown to he a crucial El enzyme in the DDR following ionizing radiation and replication stress (Moudry et aL. 2012. Cell Cycle 11: 1573-1582), resulted in a particularly strong reduction of viability (Fig 1C).

A large proportion of the identified hits have been previously established to regulate the transcription factor 53, which acts as a master regulator of the outcome of the DDR i various cell types including ES cells (Fig IE). Three of the identified DUBs, USP7 (HAUSP), USP4, and USP5 can directly or indirectly influence p53 protein levels (Brooks and Gu 2006. Mol Cell 21: 307-315; Dayal et aL 2069, J Biol Chem 284: 5030-5041; Meuimeester et al, 2005. Mol Cell 18: 585-576; Zhang et al. 2011. EMBO J 30: 2177-2189). In addition, the E3 ligases Rfwd3, Pirh2 and TOPORS were previously shown to affect p53 stability (Brooks and Gu, 2008. Mol Cell 21; 307-315; Fu et aL 2010. Free Natl Acad Sci U S A 107: 4579-4584; Yang et al. 2009. J Biol Chem 284: 18588-13592) (Fig IE; Table S3). Resides p53 regulators we identified several other ubiquitin ligases implicated in DNA repair processes, such as postreplieation repair (SHPEH (Lin et aL 2011. Mol Cell 42; 237-249), translesion synthesis (Pirh2; Jung et aL 2011. Mol Cell Riol 31; 3997-4006), DSB repair (BRCAl; Roy et al. 2012, Nat Eev Cancer 12: 68- 78), and the RPA-mediated repair of single strand breaks (RfwdS; Liu et al. 2011. J Biol Cheni 288: 22314-22322). Accordingly, Metacore-baeed pathway analysis of the 27 confirmed ubiquitination-screen hits identified DNA damage signaling and repair processes to be prominently enriched.

One of the strongest hits in the screen was the Parkin family ubiquitln ligase Ariadne homologue 1 (ARIHI) (Tan et al. 2000; Cytogenet Cell Genet 90: 242-245). The ARIHI Smartpool and all four of the individual sequences tested in the deeonvobation experiments, significantly sensitised ES cells to cisplatin-induced oss-of- viability (Fig 1A,C). In order to examine if the effect of silencing ARIHI was specific for the type of induced (genotoxic) stress, the effect of ARIHI knockdown in ES cells was examined after treatment with various genotoxic and nongenotoxic compounds. Compounds were used at a dose, which induces comparable loss-of viability at 24h (Fig 2A). Notably, knockdown of ARIHI using the Smartpool or either of three individual siRNAs did not significantly affect ES cell viability under control conditions (Fig 2B,C). Similar to its effect on cisp!atin-sensitivity, silencing ARIHI significantly sensitised ES cells to all tested genotoxic drags, including the topoisomerase Inhibitors etoposide and doxorubicin and the DNA crosslinking compound mitomycin C (Fig 2B,C). In contrast,

knockdown of ARIHI did not sensitize ES cells to non- enotoxic agents such as the ER stressor thapsigargin, the oxidative stressor diethyl maleate (DEM), or the microtubule poison Vincristine (Fig 2A,C).

We also tested the effect of silencing ARIHI in U20S p53 wild type human sarcoma cells, which are relatively resistant to eisplatin compared to ES cells. We introduced lentiviral shRNAs targeting ARIHI and following bulk puromycin selection identified two short hairpins providing ~90% reduction in ARIHI protein levels (Fig 2D). Basal cell survival was reduced in these knockdown cells when compared to a lentiviral control cell line (Fig 2E) and, analogous to its role in ES cells, silencing ARIHl significantly sensitized IJ20S cells to treatment with 10 or 25 mieroM cisplatin for 48k (Fig 2E).

Finally, we tested whether ARIHl plays a similar role in controlling sensitivity to radiation-induced DNA damage. Control and ARIHl -depleted U20S cells were treated with increasing dosages of cisplatin, ionizing radiation or UV light and colony-forming ability was assessed. Silencing ARIHl sensitised U20S cells to cisplatin and γ -irradiation hut did not affect the response to UV (Fig 2F-H),

Since silencing ARIHl sensitized cells to several genotoxie stressors that lead to the formation of primary and secondary DSBs. we asked if ARIHl affects the dynamics of DSB repair foci. Formation of gammaH2AX and 53BP1 foci after treatment with gamma-irradiation was similar for sheontrol and shA Hl IJ20S cells. S bsequent foci disappearance, which is associated with repair, was slightly delayed hut was similar in control and ARIHl-dep!eted cells at 24 hours after gamma irradiation (Fig 3A), ARIHl, in contrast to many of the other identified hits (Fig IS), also did net control basal or genotoxic stress-induced p53 stability in ES cells or U2QS cells (Fig 3B,C)_' In agreement, silencing ARIHl sensitised the p53-defieient non- sxn ll-cell lung cancer cell line H1.299 (Li et al. 2011. Int J Bioehem Mol Biol 2: 138-145) and the p53 mntant mouse breast cancer cell line 4T1, to cisplatin (Fig 3D-G). DNA damage can trigger a p53~depen.dent or

independent cell cycle arrest (Medema and, Macixrek, 2012. Oncogene 31: 2801-13). In ES cells, ARIEL 1 knockdown did not significantly alter cell cycle distribution or cis latin-induced S/G2 phase arrest (Fig 311).

These data suggested that AEIH1 is mainly implicated in maintaining viability while the cell cycle is arrested and. repair is ongoing. Indeed, silencing ARIHl increased the si bGl/GO fraction after treatment with cisplatin (Fig 31). This effect of AEIH1 was not restricted to easpase 3- mediated apoptosis, since transient or stable silencing of ARIHl also sensitized the easpase -3 deficient hum n breast cancer ceil line MCF7 (Fig 3J-L), Notably, as observed in ES cells ARIHl knockdown did not affect basal eel! cycle distribution or eisplatin-indnced cell cycle arrest in MCF7 (Fig 3M).

In summary, AR H 1 -depleted cells respond to DSBs by initiating DDE foci and arresting the cell cycle. However, in the absence of ARIHl cell survival is more severely compromised following DNA damage, which does not depend on p53 or cas ase - 3 ~ me di ie d apoptosis.

In response to DNA damage_s ongoing cellular activities are suppressed while stress programs and DNA repair processes are activated. One typical response is the acute inhibition of protein synthesis through alterations of the cap-dependent translation Initiation complex (Kumar et al. 2000. Biol Chem 275: 10779-10787), This can be achieved in several ways, including recruitment of 4ehp (eIF4E2), a competitive inhibitor of the canonical cap- binding translation initiation factor, eIF4E (Kong and Lasko 2012. Nat Rev Genet 13; 388-394). In contrast to eIF4E₅ 4ehp cannot bind the structural component eiF4G that is required for ribosome recruitment and subsequent inRNA translation. Interestingly, although ARIHl can act as an E3 ubiquitin ligase for 4ehp (Tan et al. 2003. FEES Lett 554; 501-504.) more recently it has been established that ARIHl can ISGylate 4ehp thus enhancing its affinity for the mRNA cap structure and replacing eIF4E

(Okumura et ai 2007. Genes Dev 21: 255-260),

We tested if DNA damage may trigger the interaction between AEIHl and 4ehp. AEIHl protein levels were enhanced following cisplatin treatment in U20S cells (Fig 4A). This conld not be explained by enhanced mRNA levels, indicating that genotoxie stress triggers enhanced synthesis or stability of the ARIH1 protein (Fig 4B). Co-immuno precipitations in U20S cells showed that the increased levels of AEIHl in cisplatin treated cells led to ARIHl association with 4ehp (Fig 4C), We used 5' cap-pulldown assays to investigate whether cisplatin treatment caused 4ehp translocation to the mRNA cap. Indeed, 4ebp binding to the mRNA cap was induced in response to cisplatin in U20S, MCF7, as well as ES cells (Fig 4D-F). Importantly, this response was disturbed in the absence of ARIHl. While basal 4ehp cap- binding was somewhat enhanced especially in Mcf? and ES cells; cisplatin- induced 4ehp:cap association was abrogated in U20S, MCF7, and ES cells where ARIHl was depleted (Fig 4D~G). Subsequently, to test if 4ehp:cap association represents an ARIHI-regulated pathway that could explain the protective role of ARIHl, 4ehp itself was silenced. In line with such a mechanism, ES cells as well as p53 deficient HI 299 cells were strongly sensitized to genotoxie compounds following 4ehp silencing (Fig 4¾ Ϊ). We next investigated whether the identification of ARIHl as a mediator of DNA damage- induced 4ehp association with the mRNA cap pointed to a role for ARIHl in DNA damage-induced translation arrest. CHck-iT® metabolic labeling showed that cisplatin treatment caused a strong translation block in U20S cells at 2h post-treatment while at later timepoints (4, 8h) a more modest suppression of translation was maintained (Fig 5 A). In line with a critical role for ARIHl in mediating this arrest, two independent sh ARIHl hues failed to arrest translation in response to cisplatin while a

cyeiohexamide -induced translation block was intact (Fig 5B). Co-treatment with salubrinai a.s inhibitor of eiF2aipha dephosphorylation that inhibits translation under stressed conditions, could restore the translation block at 2b cisplacin treatment in ΑΚΓΗ1 knockdown cells (Fig 5B). Finally, i good agreement with a protective function of the AEIHLcontroled translation arrest_; saliibrxnal also restored, cell viability in cispiatin-treated ARIH1- silenced U20S cells, ES cells and MCF7 cells (Fig 5CE).

Altogether, these findings indicate that DNA damage-induced increase in ARIH1 protein levels lead to association of ARiHl with 4ehp. This causes 4ehp recruitment to the mRNA cap where it replaces eIF4E, The resulting niRNA translation arrest acts eytoproteeiive: AR.IH1 or 4ehp depletion sensitizes cells to geaotoxic stress while reestablishing the translation block with sal brinal alleviates this effect (Fig 5F),

Example 2

RNAi screen identifies modulators of chemosensjtivity

HMl mouse ESC derived from OLA/129 genetic background la in et ah 1992, Nucleic Acids Research 20: 3795-3796) were maintained as described in Example 1. B4418 mouse ESC derived from C57/B16 genetic background (provided by Dr. Monique de Waard, Erasmus Medical Center, Rotterdam ML (Kruse et ah 2007. uta Res. 617: 58--70 (2007) and wild type and pS3- /- D3 mouse ESC (do Fries et al 2002, Free. Natl. Acad. Sci. U.S.A. 99:

2948— 2953) (provided by Dr, Anneroieke do Vries_s National Institute of Public Health and the Environment, Biithoven NL) were cultured in KO- DhiEM medium (Invitrogen) with 10% FBS, 5x105 U F, and 25 micro /mi streptomycin on feeders. These cells were transferred to gelatinized plates and ES BRL medium (1:1 KO-DME and ES BEL conditioned medium) two passages before starting experiments. For RNAi screens and micro-arrays ESC were used at passage 22 and for ail other experiments ESC were used between passage 20 and 27,

AM cell lines, including stable shENA expressing derivatives, were

confirmed to be myeoplasma-free using the Myeosensor kit from Stra agene. Genotoxieants included the DNA cross-linker oisplaiin (CP; Cis- PtC12(NH3)2) (provided by the Pharmacy unit of University Hospital, Leiden NL) and the inhibitors of topoisonierase Il-mediated DNA

unwinding, doxorubicin (Sigma) and etoposide (Sigma), Oxidative stressors included menadione (Sigma), diethyl roa!eate (DEM; Sigma), and 11202 (Merck). The pan-easpase inhibitor z-Val-Ala-DL-Agpfluorometbylketone (z» VAD-fmk) was purchased from. Bacheni. Retinoie acid (RA) and LiC!2 were obtained from Sigma. SB-481542 TGFbeta receptor inhibitor was obtained from Tocris Bioscience. Antibodies against p58 and pbospbo-po3 wore purchased from Novaeostra and Cell signaling, respectively. Antibody against 53.BP1 was from BD Biosciences, antibody against Tubulin was obtained from Sigma, Antibody against active beta-catenin. (anti-ABC; clone 8E7) was from Millipore and antibody against p--Ser45 beta-eatenin was from Cell Signaling,

RNAi screening

For primary screens SMARTpooi siGENOME libraries targeting all known mouse kinases, phosphatases, and transcription, factors were used

(TbermoFisher Scientific). For deconvolution confirmation screens, customized libraries containing 4 individual siRNAs targeting each selected RNA were used (TbermoFisher Scientific). GFP, Lamin A/C, and RISC free control siRNAs were used according to MIARE guidelines (F!aney 200?. Pharmacogenetics 8: 1037-1049). Kifll siRNA was used as transfectien efficiency control. The siRNA screens were performed on a Biomek FX

(Beckman Coulter) liquid, handling system. 50xiM siRNA was transfected in 96 well plates u ing Dharmafectl transfection. reagent (Therm oFisher Scientific). The medium was refreshed every 24h and cells were exposed to indicated compounds or vehicle controls 64h post-transfection for 24k

Primary screens were done in duplicate and deconvointxon screens were done in quadruplicate. As readout, a ceil viability assay using ATPlite IStep kit (Perkin Elmer) was performed according to the manufacturer's

instructions followed by luminescence measurement using a plate reader. RNAi screen data analysis

As a quality control Z'-foctors were determined for each plate, as decribed in. Example 1, using Lamin A/C as a negative control and p53 as a positive control.

HMI ESC were treated with CP (1 microM, 5 roierofVi_s or 10 ieroM or vehicle control for 8k in 3 independent experiments. B4418 ESC were treated for 8h with the genotoxicants CP„ doxorubicin or etoposide, or the oxidative stressors menadione, DEM or H202. Total SNA was isolated using the RNAeasy kit (Qiagen) according to manufacturer's instructions. SNA quality and integrity was assessed with Agilent 21.00 Bioanalyser system (Agilent technologies). Gene expression was measured using

Atliuietrix MG430 PM Array plates. All raw data passed the Affimetrlx quality criteria.

Normalisation of raw data using the robust multi-array average algorithm and statistical analysis was performed using BRBarray tools

(http: linus.nci, nih.gov/BRB-ArrayTools. html).

The experimental details of global phosphormroteomics in CP-treated ESC are as described in Pines et al₃ 2011. Molecular and. Cellular Biology 31. 4964-4977). In short, SILAC labeling, isolation, and purification of phosphopeptides was performed according to published procedures (Villen ei ai. 2007. Proc. Natl. Acad. Sci. U.S.A. 104: 1488-1493) and analyzed by tandem Mass Spec.

Cells were exposed to vehicle or CP for 8k for cell cycle analysis or 24b. for apoptosis analysis. Floating and attached cells were pooled and fixed in 80% ethano! overnight. Ceils were stained using PBS EDTA containing 7.5mM propidinni iodine and 40mg/mi ENAseA and measured by flow cytometry (EACSCanto II; Beeton Dickinson). The amount of cells in the different ceil, cycle fractions (and in sub GO/Gl for apoptotic cells) was calculated using the BD FAOSDiva software. As an alternative method to determine apoptosis, phosphatidyl serine exposure at the outer membrane leaflet was detected hy Annexin V-PITC in real-time in attached cells as described previously (Puigvert et ai 2010. Curr Protoc Cell Biol Chapter 18, Unit 18,10.1-13).

Wmt LMsLm&km

Total extracts were prepared in SDS protein lysis sample buffer and boiled for 5 mm at 95°C. Extracts were separated by SDS-PAGE on polyacrylami.de gels, transferred to PVDF membranes, and membranes were blocked using 5% BSA.. Following incubation with primary and secondary antibodies signal was detected using a Typhoon™ 9400 from GE Healthcare.

Immunofluorescence

Cells were plated in pCiear 96 weii/piates (G REINER) coated with 1% gelatin and exposed to vehicle (PBS) or 5μΜ CP for indicated times.

Fixation of the samples was done using 4% paraformaldehyde following incubation with primar and. secondary antibodies and images were captured using a Nikon TE2000 EPI microscope. gPCR

RNA was extracted and real-time qPCR was subsequently performed as

Results

KMAi screen identifies modulators of chemose∞itivity m ESC

An RNAi screen targeting ail know kinases, phosphatases, and

transcription factors was performed in mouse ESC, Fluorescence activated ceil sorting (FACS) for DNA content or ATP-based viability measurement showed 60-70% ESC death after 24h lOpM CP treatment, which could be prevented hy the pan-Caspase inhibitor, ZAeADfhxk, pointing to CP-induced apoptosis (Fig, S1A,B).. For the screening protocol, Kit 11 si iNA was used, as transfection efficiency control, and si-GFP and si-Lanrin A C as negative controls. Further, since the role of po3 in the DDR in ESC is debated

(Aladjem et ai. 1998, Current Biology 8: 145--155; Sabapathy et al. 1997, The EMBO Journal 16: 621.7-6229; Solozobova et al, 2009, BiV!C Cell Biol. 10: 46), we tested the effect of si-p58 on CP-induced apoptosis. Silencing p53 mimicked the protective effect of Z-VAD-fmk in. CP-treated cells while none of the negative controls affected viability (data not shown).

In the primary screen, 2,351. individual genes were silenced using siRNA SMARTpools and viability under control and 5 microfVl CP treatment conditions was determined. The average Z'factor (Boutros et aL 2008.

Genome Biol 7: E66) of all CP-treated plates based on si-Larnin A C and si- pod was ~"0,5, indicating a strong signal to noise ratio. For hit selection, we first excluded. siRNAs that significantly reduced viability in. control conditions. This list contained expected survival genes from ail three gene libraries, such as Plkl, Oct-3/4, and Wi.pl (Fig, 6A),

After exclusion of siRNAs affecting general survival, siRNAs were ranked by Z-scores and hits were defined as [absolute Z-Seore>1.5: p<0,06]. Using these criteria, 106 SMARTpools protected against CP and 78 sensitized (Fig, 2B), These hits entered a secondary deconvolution screen where hit confirmation was defined as at least 3 out of 4 individual siRNAs copying the effect of the SMARTpool [absolute Z-Seore>L5; p<0,05, ranked against Lamin Ά C] (19). la this way, about 2.5% of all kinases, phosphatases, and. transcription factors (~32% of the primary screen hits) were confirmed as CP response modifiers (Fig. 6QD).

In parallel to the transcription factor and kinase/phosphatase RNAi screens, micro-array analyses and SILAC were employed to map global changes in mENA expression and protein phosphorylation, respectively in response to CP treatment (Fig. 1). ESC were exposed to vehicle or 1, 5, or 10 mieroM CP for 8 h. followed hy NA isolation. FACS analysis at 24h rom parallel plates of the same experiment confirmed dose-dependent induction of apoptosis (data not shown), A concentration-dependent inductio of differentially expressed gen.es (DEGs; p<0.05) was observed and 2,269 DEGs were identified at 10 microM exposure. 29 of the 47 DEGs already

responding to 1 microM CP, showed a concentration-dependent increase in fold-change including known p63-targets such as Mdm2 and Btg2, in agreement with a pS3~mediated response to CP in ESC, The SILAC experiment was performed as described previously (Pines et al. 2011.

Molecular and Cellular Biology 31: 4964-4977). In short, isotope-iaheled amino acids were used to distinguish hetween proteins isolated from untreated ESC and ESC treated with 5 microM CP for Ah. Isolated peptide mixtures were enriched for phosphopeptides on a titanium column and samples were analyzed by tandem mass spectrometry. Of the 8,251 identified phosphopeptides, 1,612 showed differential phosphorylation

[ratio<0.8? or ratio>l,5; p<0.05] representing 1,025 different proteins. These included several known ΑΤΜ/ΆΤΚ-targets, such as ATM-Serl987, BRCAl-

Exam le 3

All 72 hits, which were shown to modulate cispiatin sensitivity in mouse embryonic stem cells and which show rohust apoptosis after DNA damage, were tested in different cancer cell lines, including the human non-small cell lung cancer cell lines HI 299, the human breast cancer cell line Mcf?, the mouse breast cancer cell line 4T1 and the human liver cancer cell line Hepg2.

All tested cancer cell lines were found to be more resistant to eisplatin induced killing, than mouse ES cells. Furthermore, the cell lines differ in their genetic backgrounds, H1299 are p53 deficient, while 4T1 cells have mutant pS3 and Mcf7 cells are deficient for Caspase-3. p53 knockdown failed to rescue eisplatin induced killing in HI 299. Mcf?, and 4T1 cell lines, while it could protect Hepg2 cells from eisplatin induced loss of viability. Furthermore, only 4T1 and He g2 cells could be rescued hy addition of the Pan-Caspase inhibitor Z-VAD (data not shown). Using siR A screens we were able to identify a panel of genes, which were shown to sensitize all four cell lines. Screening conditions were optimized based on the sensitivity of the cancer cell lines, and concentrations that did not induce complete killing were used for the screens. In 4T1 ceil, screens were carried out with a 24b treatment and a CP concentration of 5 microM and 12.5 microM. In the Mcf/ cells, incubation with CP was prolonged until 48h using a concentration of 25 microM and H1299 and Hepg2 cells were treated for 24b with 25 microM CP, Hits were ranked based on survival as well as en the observed p- value for both control and CP treated conditions. Groups of common hits were identified by overlaying the top 20 sensitizers from all individual cell lines. Hits that enhanced sensitivity of at least three of the four cell lines for CP included AriHl, PE D1, DUSP15, RpMl, USPS, Uba. FEKDl and RpITll enhanced sensitivity of all four cell lines for CP (data not shown).

Claims

Claims

1. A susceptibility agent for use in a method for treatment of an individual suffering from cancer, whereby said susceptibility agent is combined w th a genotoxic agent,

2. The susceptibility agent for use according to claim 1, whereby the susceptibility agent improves treatment of the individual by the genotoxic agent,

3. The susceptibility agent for use according to any of claims 1-2, whereby the genotoxic agent is selected from gamma radiation, a platinum- based compound and/or an anthracyelin,

4. The susceptibility agent for use according to any of claims 1-3, whereby the genotoxic agent is cispiatin.

5. The susceptibility agent for use according to any of claims 1-3, whereby the genotoxic agent is doxorubicin.

6. The susceptibility agent for use according to any of claims 1-5, whereby said individual suffers from breast cancer, ovarian cancer, lung cancer, liver cancer, head and neck cancer, squamous cell carcinoma, bladder cancer, colorectal cancer, cervical cancer, renal cell carcinoma, stomach cancer, prostate cancer, melanoma, brain cancer, and/or esophageal cancer,

7. The susceptibility agent for use according to any of claims 1-6, whereby the susceptibility agent inhibits expression and/or activity of a product of a gene selected from the group consisting of ARIHI, DUSP15, UBE1X, RP.L7L1, USp8, 4EHF and PRKDl.

8. A method for treatment of an individual suffering from cancer, comprising

(a) providing said individual with a genotoxic agent; and

(h) providing said individual with a susceptibility agent.

9. The method of claim 8, whereby treatment is improved compared to treatment in the absence of said susceptibility agent,

10. Use of a susceptibility agent, in the preparation of a medicament for treatment of an individual suffering from cancer, whereby said medicament is combined with a genotoxic agent,

11. Use according to claim 10, whereby treatment of the individual is improved compared to treatment in the absence of said susceptibility agent.