US20200063212A1

US20200063212A1 - An inhibitor of atr kinase for use in a method of treating a hyper-proliferative disease

Info

Publication number: US20200063212A1
Application number: US16/488,033
Authority: US
Inventors: Antje Margret Wengner; Gerhard Siemeister; Bernard HÄNDLER; Sven Golfier; Andreas SCHLICKER; Li Liu
Original assignee: Bayer AG; Bayer Pharma AG
Current assignee: Bayer AG; Bayer Pharma AG
Priority date: 2017-02-24
Filing date: 2018-02-22
Publication date: 2020-02-27
Also published as: NZ755553A; CA3054246A1; AU2018223879A1; EP3585908A1; IL268307A; KR20190120204A; US20210404012A1; JOP20190197A1; WO2018153968A1; BR112019017612A2; CN110582581A; AU2018223879B2; IL268307B1; SG11201907119XA; MX2019010095A; IL268307B2; SG10202109220TA; CL2019002431A1; US11976334B2; CL2021000862A1

Abstract

The present invention covers 2-[(3R)-3-methylmorpholin-4-yl]-4-(1-methyl-1H-pyrazol-5-yl)-8-(1H-pyra-zol-5-yl)-1,7-naphthyridine (in the following called “Compound A”), an inhibitor of ATR kinase, for use in a method of treating a hyper-proliferative disease in a subject. Preferably the hyper-proliferative disease or the subject is characterized by one or more biomarker(s) selected from a) one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ATG5, ARID1A, ATM, ATR, ATRIP, ATRX, BAP1, BARD1, BLM, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CCNE1, CCNE2, CDC7, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1B, DCLRE1C, DYRK1A, EGFR, ERBB2, ERBB3, ERCC2, ERCC3, ERCC4, ER-CC5, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXO18, FBXW7, FEN1, GEN1, HDAC2, H2AFX, HRAS, KRAS, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MYC, NBN, NRAS, PALB2, PARP1, PARP2, PARP3, PARP4, PCNA, PIK3CA, PMS2, POLA1, POLB, POLH, POLL, POLN, POLQ, PRKDC, PTEN, RAD9A, RAD17, RAD18, RAD50, RAD51, RAD52, RAD54B, RAD54L, RB1, REV3L, RPA1, RPA2, SLX4, TDP1, TDP2, TM-PRSS2, TMPRSS2-ERG, TOPBP1, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XR-CC1 XRCC2, XRCC3, XRCC4 and/or XRCC6 gene/protein; and/or b) the activation of the ALT pathway; and/or c) microsatellite instability. The present invention also covers a kit comprising Compound A together with means to detect one or more of the afore-biomarker(s) and a method for identifying a subject having a hyper-proliferative disease disposed to respond favorably to Compound A, wherein the method comprises the detection of one or more of the aforementioned biomarker(s). Further, the invention covers a method of determining whether a subject having a hyper-proliferative disease will respond to the treatment with Compound A, wherein the method comprises the detection of one or more of the aforementioned biomarker(s) in a sample of the subject.

Description

The present invention covers an inhibitor of ATR kinase, particularly of 2-[(3R)-3-methylmorpholin-4-yl]-4-(1-methyl-1H-pyrazol-5-yl)-8-(1H-pyrazol-5-yl)-1,7-naphthyridine (in the following called “Compound A”), for use in a method of treating a hyper-proliferative disease in a subject. Preferably the hyper-proliferative disease or the subject is characterized by one or more biomarker(s) selected from

a) one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ATG5, ARID1A, ATM, ATR, ATRIP, ATRX, BAP1, BARD1, BLM, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CCNE1, CCNE2, CDC7, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1B, DCLRE1C, DYRK1A, EGFR, ERBB2, ERBB3, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXO18, FBXW7, FEN1, GEN1, HDAC2, H2AFX, HRAS, KRAS, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MYC, NBN, NRAS, PALB2, PARP1, PARP2, PARP3, PARP4, PCNA, PIK3CA, PMS2, POLA1, POLB, POLH, POLL, POLN, POLQ, PRKDC, PTEN, RAD9A, RAD17, RAD18, RAD50, RAD51, RAD52, RAD54B, RAD54L, RB1, REV3L, RPA1, RPA2, SLX4, TDP1, TDP2, TMPRSS2, TMPRSS2-ERG, TOPBP1, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XRCC1, XRCC2, XRCC3, XRCC4 and/or XRCC6 gene/protein; and/or
b) the activation of the ALT pathway; and/or
c) microsatellite instability.

BACKGROUND

The integrity of the genome of eukaryotic cells is secured by complex signalling pathways, known as DNA damage response (DDR). Recognition of DNA damage activates DDR pathways resulting in cell cycle arrest, suppression of general translation, induction of DNA repair, and, finally, in cell survival or cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR pathway, such as ATR. ATR responds to a broad spectrum of DNA damage, including double-strand breaks and lesions derived from interference with DNA replication as well as increased replication stress that is observed in oncogene-driven tumor cells (e.g. Ras mutation/upregulation, Myc upregulation, CyclinE overexpression).
ATR kinase inhibitors are specifically or generically disclosed in the following publications: J. Med. Chem. 2013, 56, 2125-2138; Exp. Rev. Mol. Med. 16, e10, 2014; WO2010054398A1; WO2010071837A1; WO2010073034A1; WO2011143399A1; WO2011143419A1; WO2011143422A1; WO2011143423A2; WO2011143425A2; WO2011143426A1; WO2011154737A1; WO2011163527A1; WO2012138938A1; WO2012178123A1; WO2012178124A1; WO2012178125A1; WO2013049719A1; WO2013049720A1; WO2013049722A1; WO2013049859A1; WO2013071085A1; WO2013071088A1; WO2013071090A1; WO2013071093A1; WO2013071094A1; WO2013152298A1; WO2014062604A1; WO2014089379A1; WO2014143240; WO 2014143241; WO 2014143242; ACS Med. Chem. Lett. 2015. 6, 37-41; ACS Med. Chem. Lett. 2015. 6, 42-46, WO 2015085132, WO 2015187451.
2-[(3R)-3-methylmorpholin-4-yl]-4-(1-methyl-1H-pyrazol-5-yl)-8-(1H-pyrazol-5-yl)-1,7-naphthyridine (in the following called “Compound A”) is a new ATR kinase inhibitor, which together with more than 400 other ATR kinase inhibitors was described in International Patent Application WO2016020320. Identification of one or more biomarkers that predict sensitivity to Compound A could result in more effective biomarker-driven targeted therapy for hyper-proliferative diseases.
No predictive markers for ATR kinase inhibitors have been identified yet in the clinical setting. However, preclinical evidence suggests a number of candidate predictive biomarkers for ATR kinase inhibitors VE-821, VX-970 and AZD6738: Williamson et al. suggest that ATR kinase inhibitors could have potential as single-agent treatments for ARID1A defective cancers (Nature Communications 7:13837|DOI: 10.1038/ncomms13837, (2016)). According to Mohni et al. ATR pathway inhibition is synthetically lethal in VE-821 treated cancer cells with ERCC1 deficiency and loss of the structure-specific endonuclease ERCC1-XPF (ERCC4) is synthetic lethal with ATR pathway inhibitors (Cancer Res. 74, (2014), 2835-2845). Strong synthetic lethal relationships with ATR inhibition was also shown for the following genes: ATRIP, RPA, CHEK1, CLSPN, HUS1, RAD1, RAD17, TIMELESS, and TIPIN (Mohni et al., Cancer Res. 74, (2014), 2835-2845). ATR inhibition by VE-821 also seems to synergize with loss of ERCC1, ATM and XRCC1 (Mohni et al., PLOS ONE|DOI:10.1371/journal.pone.0125482 May 12, 2015; Sultana et al, PLoS One, 8(2). (2013), e57098. doi: 10.1371/journal.pone.0057098). According to Hocke et al. (Oncotarget Vol. 7, No. 6, (2016), 7080-7095) POLD1 deficiency might represent a predictive marker for treatment response towards ATR inhibitors. Flynn et al. (Science 347, (2015), 273-277) suggest that ATR kinase inhibitors may be useful for treatment of ALT-positive cancers. According to the data described by Menezes et al. (Mol. Cancer. Res. 13(1), (2015), 120-129) single-agent ATR inhibitors may have therapeutic utility in the treatment of mantle cell lymphoma with ATM loss-of-function. Middleton et al. (Oncotarget, Vol. 6, No. 32, (2015), 32396-32409) suggest that defects in ATM, BRCA2, XRCC3 and XRCC1 and high DNA-PKcs expression conferred sensitivity to VE-821 monotherapy.
According to Jones et al. (Cancer Research (2017), Author Manuscript Published OnlineFirst on Oct. 16, 2017; DOI: 10.1158/0008-5472.CAN-17-2056) in Synovial sarcoma SS18-SSX1 or SS18-SSX2 fusion proteins induce ATR kinase inhibitor sensitivity. Nieto-Soler et al. (Oncotarget. 2016; 7:58759-58767) suggest that expression of EWS-FLI1 (also called EWSR1-FLI1) or EWS-ERG (also called EWSR1-ERG oncogenic translocations sensitizes non-ES cells to ATR inhibitors.
Remi-Buisson et al. (Cancer Res 77(17), (2017), 4567-4578) describe that APOBEC3A and APOBEC3B overexpression confers susceptibility to ATR kinase inhibitors.
Kwok et al (Lancet 26, 385, Suppl 1, (2015), S58. doi: 10.1016/S0140-6736(15)60373-7; Blood 4; 127(5), (2016), 582-595. doi: 10.1182/blood-2015-05-644872) showed that AZD6738 sensitized TP53- or ATM-defective primary chronic lymphocytic leukemia (CLL) cells to chemotherapy and ibrutinib. Ruiz et al (Mol Cell 62(2), (2016), 307-313, DOI: 10.1016/j.molce1.2016.03.006) reported that deficiency in cdc25A confers resistance to ATR inhibitors.
The object of the present invention is to provide one or more biomarker(s) for the treatment of one or more hyper-proliferative disease(s) with an ATR kinase inhibitor, particularly with Compound A as described herein, in a subject.

DETAILED DESCRIPTION OF THE INVENTION

Definitions of Terms Used in the Context of the Present Invention

The term “inhibitor of ATR kinase” or the term “ATR kinase inhibitor” as used herein means any compound that inhibits ATR kinase. Examples of ATR kinase inhibitors which may be used in context with the present invention include VX-803, VX-970, AZD-6738 and preferably Compound A (described infra).
In context with the present invention the term “VX-803” means 2-amino-6-fluoro-N-[5-fluoro-4-(4-{[4-(oxetan-3-yl)piperazin-1-yl]carbonyl}piperidin-1-yl)pyridin-3-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide. VX-803 has the following structure
In context with the present invention the term “VX-970” means 3-(3-{4-[(methylamino)methyl]phenyl}-1,2-oxazol-5-yl)-5-[4-(propan-2-ylsulfonyl)phenyl]pyrazin-2-amine. VX-970 has the structure
In context with the present invention the term “AZD-6738” means 4-{4-[(3R)-3-methylmorpholin-4-yl]-6-[1-(S-methylsulfonimidoyl)cyclopropyl]pyrimidin-2-yl}-1H-pyrrolo[2,3-b]pyridine. AZD-6738 has the structure
The term “Compound A” as used herein means 2-[(3R)-3-methylmorpholin-4-yl]-4-(1-methyl-1H-pyrazol-5-yl)-8-(1H-pyrazol-5-yl)-1,7-naphthyridine of structure:
In particular, the term Compound A refers to 2-[(3R)-3-methylmorpholin-4-yl]-4-(1-methyl-1H-pyrazol-5-yl)-8-(1H-pyrazol-5-yl)-1,7-naphthyridine.
The expression “gene/protein” means one gene or one protein. The expression “gene(s)/protein(s) means one or more gene(s) or one or more protein(s). The expression “gene(s)” means one gene or more genes. The expression “protein(s)” means one protein or more proteins.
The term “hyper-proliferative disease” includes but is not limited, e.g., psoriasis, keloids, and other hyperplasias affecting the skin, benign prostate hyperplasia (BPH), as well as malignant neoplasia. Examples of malignant neoplasia treatable with Compound A according to the present invention include solid and hematological tumors. Solid tumors can be exemplified by tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, anum, endocrine glands (e.g. thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, renal, small intestine, soft tissue, testis, stomach, skin, ureter, vagina and vulva. Malignant neoplasias include inherited cancers exemplified by Retinoblastoma and Wilms tumor. In addition, malignant neoplasias include primary tumors in said organs and corresponding secondary tumors in distant organs (“tumor metastases”). Hematological tumors can be exemplified by aggressive and indolent forms of leukemia and lymphoma, namely non-Hodgkins disease, chronic and acute myeloid leukemia (CML/AML), acute lymphoblastic leukemia (ALL), Hodgkins disease, multiple myeloma and T-cell lymphoma. Also included are myelodysplastic syndrome, plasma cell neoplasia, paraneoplastic syndromes, and cancers of unknown primary site as well as AIDS related malignancies.
Examples of breast cancer include, but are not limited to invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ, particularly with bone metastases.
Examples of cancers of the respiratory tract include, but are not limited to small-cell and non-small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma. Examples of brain cancers include, but are not limited to brain stem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumor. Tumors of the male reproductive organs include, but are not limited to prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus. Tumors of the digestive tract include, but are not limited to anal, colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers. Tumors of the urinary tract include, but are not limited to bladder, penile, kidney, renal pelvis, ureter, urethral and human papillary renal cancers. Eye cancers include, but are not limited to intraocular melanoma and retinoblastoma. Examples of liver cancers include, but are not limited to hepatocellular carcinoma (liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma. Skin cancers include, but are not limited to squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head-and-neck cancers include, but are not limited to laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancer, lip and oral cavity cancer and squamous cell. Lymphomas include, but are not limited to AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt lymphoma, Hodgkin's disease, and lymphoma of the central nervous system. Sarcomas include, but are not limited to sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.
In particular, the present invention covers the treatment of lung cancer, colorectal cancer, cervical cancer, bladder cancer, breast cancer, melanoma, B-cell lymphoma, particularly diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, prostate cancer, gliomas, ovarian cancer, glioblastoma, neuroblastoma, chronic lymphocytic leukemia (CLL), fibrosarcoma, gastric cancer, esophageal cancer, pancreatic cancer, chronic and acute myeloid leukemia (CML/AML), acute lymphoblastic leukemia (ALL), Hodgkins disease, multiple myeloma (MM) and T-cell lymphoma, endometrial cancer, vaginal cancer, and vulvar cancer, as well as sarcoma of the uterus.
Preferably, the present invention covers the treatment of prostate cancer, B-cell lymphoma, particularly diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, melanoma, particularly malignant melanoma, ovarian, particularly, ovarian adenocarcinoma, colorectal cancer, lung, particularly non-small cell lung carcinoma, cervical cancer, and breast cancer, particularly triple-negative mammary carcinoma, pancreatic cancer, fibrosarcoma.
The term “functional mutation” as used herein means a mutation of a gene which results in an altered function of the gene, its corresponding RNA or its corresponding protein compared to the function of the respective wildtype gene, corresponding wildtype RNA or corresponding wildtype protein. The term “altered function” as used herein means either reduced or increased function of the gene, its corresponding RNA or its corresponding protein compared to the function of the respective wildtype gene, corresponding wildtype RNA or corresponding wildtype protein. The term “altered function” also includes the complete loss of the function or the gain of a new function of the gene, its corresponding RNA or its corresponding protein compared to the function of the respective wildtype gene, corresponding wildtype RNA or corresponding wildtype protein.
The reference nucleotide sequences of the cDNA's of the respective wildtype genes are described in the attached sequence protocol (SEQ ID Nos 1 to 111). The reference amino acid sequences of the respective wildtype proteins are described in the attached sequence protocol (SEQ ID Nos 112 to 222).
The functional mutation can be a “deleterious mutation” or an “activating mutation”.
The term “deleterious mutation” as used herein means a mutation of a gene which has a deleterious effect on the function of said gene or on the function of its corresponding RNA or its corresponding protein. For example, the deleterious mutation of the gene may result in a reduced gene expression level of said gene, a reduced amount or a reduced activity of the protein corresponding to said gene, or it may result in a nonfunctional gene/protein (“loss-of-function”) compared to the respective wildtype gene/protein. Examples of a deleterious mutation include but are not limited to the following:
The deleterious mutation can be a nonsense mutation, which is a point mutation in the respective gene, resulting in a premature stop codon, or a nonsense codon in the transcribed mRNA, and in a truncated, incomplete, and nonfunctional protein corresponding to the respective gene.
The deleterious mutation can be a missense mutation, which is a point mutation in the respective gene, resulting in the production either of a nonfunctional protein (complete loss of function) or in a protein with partial loss of function compared to the respective wildtype protein.
The deleterious mutation can also result in a frameshift mutation, which is a genetic mutation in the respective gene caused by insertions or deletions of one or more nucleotides in such gene, wherein the number of nucleotides is not divisible by three, and resulting in a (sometimes truncated) nonfunctional protein corresponding to the respective gene.
The deleterious mutation can also be a large rearrangement mutation, for example a deletion of one or more exons disrupting the reading frame or a critical functional domain of the corresponding protein. Another example for a large rearrangement mutation is a duplication of one or more non-terminal exons disrupting the reading frame or a critical functional domain of the corresponding protein.
The deleterious mutation can also be a splice site mutation, which is a genetic mutation that inserts, deletes or changes a number of nucleotides in the specific site at which splicing takes place during the processing of precursor messenger RNA into mature messenger RNA. Splice site consensus sequences that drive exon recognition are located at the very termini of introns. The deletion of the splicing site results in one or more introns remaining in mature mRNA thereby resulting in the production of a nonfunctional protein corresponding to the respective gene.
The deleterious mutation can also be a copy number variant (CNV), particularly a decrease of the gene copy number (e.g. a homozygous or heterozygous deletion) compared to the normal gene copy number of the respective gene.
The term “activating mutation” as used herein means a mutation of a gene which changes said gene, its corresponding RNA and/or its corresponding protein in such a way, that its effects (e.g. the amount of corresponding RNA/protein, or the protein activity) get stronger compared to the respective wildtype gene/RNA/protein. The term “activating mutation” also includes a mutation of a gene, in which the protein corresponding to said gene gets a new function compared to the function of the corresponding wildtype protein. Examples of activating mutations include but are not limited to the following:
The activating mutation can be a substitution of one amino acid residue by another that confers a new or higher activity upon the protein.
The activating mutation can be a copy number variant (CNV), particularly an increase of the gene copy number compared to the normal gene copy number of the respective gene.
The activating mutation can also be a fusion gene or fusion protein, e.g. occurring as a result of translocation, interstitial deletion or chromosomal inversion.
The term “stratification method” as used herein means the method by which one or more of the functional mutation(s) as defined herein, particularly of the deleterious mutations and the activating mutations, the activation of the ALT pathway and/or the microsatellite instability is (are) determined.
Preferably, the stratification method is an in-vitro method. Examples of stratification methods, which can be used in context with the present inventions, are described infra.
The term “activation of the ALT pathway” as used herein refers to cancer cells which overcome replicative senescence by activating the Alternative Lengthening of Telomeres (ALT) pathway.
The term “microsatellite instability” (“MSI”) as used herein is the expansion or reduction in the length of repetitive DNA sequences (known as microsatellites) in the DNA of a sample, e.g. a tumor sample, compared to normal cells.
MSI testing can detect an abnormal number of microsatellite repeats, which indicates that the cancer may arose from cells with defect mismatch repair genes. A microsatellite is a tract of tandemly repeated (i.e. adjacent) DNA motifs that range in length from one to six nucleotides, and are typically repeated 5-50 times. For example, the sequence TATATATATA is a dinucleotide microsatellite, and GTCGTCGTCGTCGTC is a trinucleotide microsatellite (with A being Adenine, G Guanine, C Cytosine, and T Thymine). Repeat units of four and five nucleotides are referred to as tetra- and pentanucleotide motifs, respectively. Microsatellites are distributed throughout the genome. Many are located in non-coding parts of the human genome, however they can also be located in regulatory regions and within the coding region. MSI tumors may result from inactivating germline mutations in one or more genes, including MLH1, MSH2, MSH6 and PMS2, and epithelial cell adhesion molecule (EPCAM), such as occurs in patients with Lynch syndrome, for whom more than 90% of colon cancers test MSI positive. MSI also occurs sporadically in several cancer types, including colorectal, endometrial, ovarian, and gastric cancers. In contrast to Lynch syndrome, sporadic MSI is often due to somatic promoter hypermethylation of MLH1 in the absence of gene sequence mutations.
The term “sample” as used herein means the sample from the subject, preferably an in vitro sample, which is used in the stratification method (as defined herein), e.g. a sample of tumor cells or of tumor tissue, a blood sample, particularly a sample of tumor tissue containing tumor cells.
Aspects of the Present Invention:
Use(s) of the Present Invention
The present invention covers an inhibitor of ATR kinase, particularly of 2-[(3R)-3-methylmorpholin-4-yl]-4-(1-methyl-1H-pyrazol-5-yl)-8-(1H-pyrazol-5-yl)-1,7-naphthyridine (in the following called “Compound A”) or a tautomer, an N-oxide, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof, particularly Compound A, for use in a method of treating a hyper-proliferative disease in a subject.
Particularly, the present invention covers an inhibitor of ATR kinase, particularly of Compound A, or a tautomer, an N-oxide, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof, particularly Compound A, for use in a method of treating a hyper-proliferative disease in a subject, wherein said subject or the hyper-proliferative disease is characterized by one or more biomarker(s) defined herein.
Particularly, the present invention covers an inhibitor of ATR kinase, particularly of Compound A or a tautomer, an N-oxide, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof, particularly Compound A, for use in the treatment of a hyper-proliferative disease in a subject, wherein said subject or the hyper-proliferative disease is characterized by one or more biomarker(s) defined herein.
In one embodiment of the invention said one or more biomarker(s) is (are) selected from

a) one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ATG5, ARID1A, ATM, ATR, ATRIP, ATRX, BAP1, BARD1, BLM, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CCNE1, CCNE2, CDC7, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1B, DCLRE1C, DYRK1A, EGFR, ERBB2, ERBB3, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXO18, FBXW7, FEN1, GEN1, HDAC2, H2AFX, HRAS, KRAS, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MYC, NBN, NRAS, PALB2, PARP1, PARP2, PARP3, PARP4, PCNA, PIK3CA, PMS2, POLA1, POLB, POLH, POLL, POLN, POLQ, PRKDC, PTEN, RAD9A, RAD17, RAD18, RAD50, RAD51, RAD52, RAD54B, RAD54L, RB1, REV3L, RPA1, RPA2, SLX4, TDP1, TDP2, TMPRSS2, TMPRSS2-ERG, TOPBP1, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XRCC1, XRCC2, XRCC3, XRCC4 and/or XRCC6 gene/protein; and/or
b) the activation of the ALT pathway; and/or
c) microsatellite instability, particularly high microsatellite instability.

In another embodiment of the invention said one or more biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BRCA1, ATM, BLM, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
In another embodiment of the invention said one or more biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
The term “POLB/POLL” as used herein means a double mutation comprising one or more deleterious mutation(s) in POLB gene/protein and one or more deleterious mutation(s) in POLL gene/protein.
In another embodiment of the present invention said one or more biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
In a preferred embodiment of the present invention said one or more biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, RAD9A, RAD17, REV3L, TP53BP1, UBE2N.
In another embodiment of the present invention said one or more biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from selected from BRCA1, ATM, FANCD2, H2AFX, RAD17, UBE2N.
In another preferred embodiment of the present invention said one or more biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from selected from BRCA1, ATM, BLM, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, REV3L, TP53BP1, UBE2N.
In another preferred embodiment of the present invention said one or more biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, REV3L, TP53BP1, UBE2N.
In another preferred embodiment of the present invention said one or more biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BRCA1, FEN1, H2AFX, PCNA.
In another embodiment of the present invention said one or more biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
In another embodiment of the present invention said one or more biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
In another embodiment of the present invention said one or more biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, RAD9A, RAD17, REV3L, TP53BP1, UBE2N.
In another embodiment of the present invention said one or more biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, REV3L, TP53BP1, UBE2N.
In another embodiment of the present invention said one or more biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BRCA1, FEN1, H2AFX, PCNA.
Particularly, the present invention covers an inhibitor of ATR kinase, particularly of Compound A, or a tautomer, an N-oxide, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof, particularly Compound A, for use in a method of treating a hyper-proliferative disease in a subject, wherein said subject or the hyper-proliferative disease is characterized by

In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s) selected from one or more functional mutation(s) in one or more of the gene(s)/protein(s) defined herein.
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s) selected from the activation of the ALT pathway.
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s) selected from microsatellite instability, particularly high microsatellite instability (herein also referred to as “MSI-high”).
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by

a) one or more biomarker(s) selected from one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ARID1A, ATG5, ATM, ATR, ATRX, BAP1, BARD1, BLM, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CCNE1, CCNE2, CDC7, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1C, DYRK1A, EGFR, ERBB2, ERBB3, ERCC2, ERCC3, ERCC5, FANCA, FANCB, FANCD2, FANCE, FANCI, FANCM, FBXO18, FBXW7, FEN1, GEN1, HDAC2, H2AFX, KRAS, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MYC, NBN, NRAS, PALB2, PARP4, PIK3CA, PMS2, POLA1, POLB, POLH, POLL, POLN, POLQ, PRKDC, PTEN, RAD17, RAD18, RAD50, RAD51, RAD54B, RAD54L, RB1, REV3L, SLX4, TDP1, TDP2, TMPRSS2, TMPRSS2-ERG, TOP2A, TOP2B, TOPBP1, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XRCC1, XRCC2 and/or XRCC3 gene/protein; and/or
b) microsatellite instability, particularly high microsatellite instability.

In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by

a) one or more biomarker(s) selected from one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ARID1A, ATG5, ATM, ATR, ATRX, BARD1, BLM, BRAF, BRCA1, BRCA2, CCND1, CCNE1, CCNE2, CDC7, CHEK1, CHEK2, DCLRE1C, DYRK1A, EGFR, ERBB2, ERBB3, ERCC2, ERCC3, ERCC5, FANCA, FANCD2, FANCI, FANCM, HDAC2, KRAS, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MYC, NBN, NRAS, PALB2, PIK3CA, POLA1, POLN, POLQ, PRKDC, PTEN, RAD17, RAD18, RAD50, RAD51, RB1, REV3L, SLX4, TDP2, TMPRSS2, TMPRSS2-ERG, TOP2A, TOP2B, TOPBP1, TP53, TP53BP1, TRRAP, UBE2N, USP1, WDR48, WRN, XPA, XRCC1, XRCC2 and/or XRCC3 gene/protein; and/or
b) microsatellite instability, particularly high microsatellite instability.

In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s) selected from one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ARID1A, ATG5, ATM, ATR, ATRIP, ATRX, BAP1, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1B, DCLRE1C, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXO18, FBXW7, FEN1, GEN1, H2AFX, HDAC2, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, NBN, PALB2, PARP1, PARP2, PARP3, PARP4, PMS2, POLA1, POLB, POLH, POLN, POLN, POLQ, PRKDC, PTEN, RAD17, RAD18, RAD50, RAD51, RAD52, RAD54B, RAD54L, RAD9A, RB1, REV3L, RPA1, RPA2, SLX4, TDP1, TDP2, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XRCC1, XRCC2, XRCC3, XRCC4 and/or XRCC6 gene/protein, wherein the functional mutation is (are) a deleterious mutation(s).
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s) selected from one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ARID1A, ATG5, ATM, ATR, ATRX, BARD1, BLM, BRCA1, BRCA2, CHEK1, CHEK2, DCLRE1C, ERCC2, ERCC3, ERCC5, FANCA, FANCD2, FANCI, FANCM, HDAC2, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, NBN, PALB2, POLA1, POLN, POLQ, PRKDC, PTEN, RAD17, RAD18, RAD50, RAD51, RB1, REV3L, SLX4, TDP2, TP53, TP53BP1, TRRAP, UBE2N, USP1, WDR48, WRN, XPA, XRCC1, XRCC2 and/or XRCC3 gene/protein, wherein the functional mutation is (are) a deleterious mutation(s).
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s) selected from one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATR, ATRIP, BRAF, CCND1, CCNE1, CCNE2, CDC7, DYRK1A, EGFR, ERBB2, ERBB3, HRAS, KRAS, MYC, NRAS, PCNA, PIK3CA, TMPRSS2, TOP2A, TOP2B, TOPBP1 and/or TP53 gene/protein, wherein the functional mutation is (are) an activating mutation(s).
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s) selected from one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATR, BRAF, CCND1, CCNE1, CCNE2, CDC7, DYRK1A, EGFR, ERBB2, ERBB3, KRAS, MYC, NRAS, PIK3CA, TMPRSS2, TOP2A, TOP2B, TOPBP1 and/or TP53 gene/protein, wherein the functional mutation is (are) an activating mutation(s).
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from ATM, BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
In a preferred embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, RAD9A, RAD17, REV3L, TP53BP1, UBE2N.
In a preferred embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, REV3L, TP53BP1, UBE2N.
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BRCA1, ATM, FANCD2, H2AFX, RAD17, UBE2N.
In a preferred embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BRCA1, FEN1, H2AFX, PCNA.
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, RAD9A, RAD17, REV3L, TP53BP1, UBE2N.
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, REV3L, TP53BP1, UBE2N.
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BRCA1, FEN1, H2AFX, PCNA.
In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s) selected from one or more functional mutation(s) of the gene(s)/protein(s), particularly deleterious and/or activating mutations, as described in Table 1 and/or in Table 2 infra:

TABLE 1

Deleterious mutations - examples

	Short insertions/deletions
Gene	(INDELs)	Substitution-Nonsense	Substitution-Missense

APC	p.T1556fs*3/c.4666_4667insA	p.R24*/c.70C > T	p.A2D/c.5C > A
ATG5	p.K235fs*4/c.704delA/	p.R9*/c.25C > T	p.K58M/c.173A > T
ARID1A	p.S186fs*209/c.557_570del14	p.Q605*/c.1813C > T	p.Q561H/c.1683G > C
ATM	p.E26fs*7/c.73_76delAAAG	p.R250*/c.748C > T	p.R2832C/c.8494C > T
ATR	p.I774fs*5/c.2320delA	p.E91*/c.271G > T	p.R1015Q/c.3044G > A
ATRIP	p.L63fs*8/c.186_189delGCTT	p.E338*/c.1012G > T	p.S310F/c.929C > T
ATRX	p.D275fs*13/c.824delA	p.G1304*/c.3910G > T	p.L192S/c.575T > C
BAP1	p.K3fs*1/c.6_7insT	p.R60*/c.178C > T	p.G185R/c.553G > A
BARD1	p.D172fs*40/c.513delA	p.S142*/c.425C > A	p.E268K/c.802G > A
BLM	p.N92fs*37/c.271delA	p.W934*/c.2801G > A	p.P30L/c.89C > T
BRCA1	p.E23fs*17/c.66_67delAG	p.Q94*/c.2801G > A/	p.M1V/c.1A > G
BRCA2	p.K437fs*22/c.1301_1304delAAAG	p. E97*/c.289G > T	p.M1I/c.3G > A
BRIP1	p.Y313fs*25/c.937delT	p.R261*/c.781A > T	p.D184Y/c.550G > T
CDK12	p.L21fs*10/c.60_61delTT	p.K172*/c.514A > T	p.R890H/c.2669G > A
CHEK1	p.L355fs*1/c.1061delT	p.R453*/c.1357A > T	p.G361D/c.1082G > A
CHEK2	p.R132fs*29/c.394delA	p.L303*/c.908T > A	p.S428F/c.1283C > T
DCLRE1A	p.K346fs*7/c.1038delA	p.S45*/c.134C > A	p.P137S/c.409C > T
DCLRE1B	C149fs*28/c.443_444insT	p.W44*/c.132G > A	p.D96N/c.286G > A
DCLRE1C	p.Y99fs*7/c.295_296insT	p.G70*/c.208G > T	p.Q137H/c.411G > C
ERCC2	p.E294fs*40/c.880delG	p.S74*/c.221C > G	p.V231M/c.691G > A
ERCC3	p.W493fs*7/c.1475_1476insT	p.R452*/c.1354C > T	p.D60N/c.178G > A
ERCC4	p.K916fs?/c.2743delA	p.Q5*/c.13C > T	p.T809M/c.2426C > T
ERCC5	p.E164fs*6/c.485delA	p.C12*/c.36C > A	p.M222I/c.666G > A
FAM175A	p.E204fs*1/c.609_610insT	p.E142*/c.424G > T	p.Y219C/c.656A > G
FANCA	p.L72fs*6/c.215delT	p.Q1389*/c.4165C > T	p.M415I/c.1245G > A
FANCB	p.F25fs*43/c.74delT	p.Q512*/c.1534C > T	p.L27F/c.81G > T
FANCC	p.N152fs*6/c.455delA	p.R174*/c.520C > T	p.R245W/c.733C > T
FANCD2	p.L446fs*17/	p.R408*/c.1222C > T	p.R1299H/c.3896G > A
	c.1332_1333delCT
FANCE	p.L173fs*15/c.515_516insC	p.E235*/c.703G > T	p.Q285H/c.855G > T
FANCF	p.D27fs*54/c.79delG	p.S18*/c.53C > G	p.R10C/c.28C > T
FANCG	p.S387fs*16/c.1158delC	p.R102*/c.304A > T	p.L589P/c.1766T > C
FANCI	p.H1218fs*2/c.3654delC	p.Q208*/c.622C > T	p.G119V/c.356G > T
FANCL	p.S351fs*2/c.1051_1052delAG	p.W57*/c.170G > A	p.M74I/c.222G > A
FANCM	p.L57fs*9/c.166_167insT	p.S1618*/c.4853C > G	p.S1665F/c.4994C > T
FBXO18	p.L116fs*1/c.345delC	p.Q643*/c.1927C > T	p.R754Q/c.2261G > A
FBXW7	p.T165fs*4/c.493delA	p.S294*/c.881C > G	p.R465C/c.1393C > T
FEN1		p.Q54*/c.160C > T	p.P89L/c.266C > T
GEN1	p.M44fs*1/c.124delA	p.C117*/c.351C > A	p.R93W/c.277C > T
H2AFX	p.P49fs*13/c.146_147delCA		p.E42K/c.124G > A
HDAC2	p.T459fs* > 30/c.1375delA	p.E185*/c.553C > T	p.V154A/c.461T > C
LIG4	p.K424fs*20/c.1271_1275delAAAGA	p.R37*/c.109A > T	p.D165G/c.494A > G
MDC1	p.D580fs*36/c.1738_1739delGA	p.E14*/c.40G > T	p.E149A/c.446A > C
MLH1	p.K196fs*6/c.583delA	p.R226*/c.676C > T	p.E172K/c.514G > A
MLH3	p.N434fs*4/c.1295_1296insA	p.Q173*/c.517C > T	p.M181I/c.543G > A
MRE11A	p.G114fs*31/c.341delG	p.Q97*/c.289C > T	p.D86N/c.256G > A
MSH2	p.F85fs*1/c.252_253delTT	p.Q215*/c.643C > T	p.G221V/c.662G > T
MSH3	p.D190fs*1/c.562_563insT	p.Y227*/c.681C > G	p.N365H/c.1093A > C
MSH6	p.L290fs*1/c.867delC	p.Q4*/c.10C > T	p.V474A/c.1421T > C
NBN	p.R466fs*18/c.1396delA	p.R43*/c.127C > T	p.I35T/c.104T > C
PALB2	p.N186fs*4/c.552_553insA	p.Q552*/c.1654C > T	p.Q479H/c.1437G > C
PARP1	p.P359fs*22/c.1076delC	p.E297*/c.889G > T	p.K59N/c.177G > T
PARP2	p.R13fs*10/c.36_37ins14	p.R395*/c.1183C > T	p.R241W/c.721C > T
PARP3	p.A300fs*29/c.894_897delGCAG	pQ340*/c.1018C > T	p.R524H/c.1571G > A
PARP4	p.K629fs*19/c.1885_1888AAAG > GA	p.E83*/c.247G > T	p.G1003S/c.3007G > A
PMS2	p.E109fs*3/c.325delG	p.K647*/c.1939A > T	p.H24Y/c.70C > T
POLA1	p.Q32fs*4/c.93delC	p.E276*/c.826G > T	p.E89V/c.266A > T
POLB	p.N128fs*5/c.378delA	p.Q159*/c.475C > T	p.P251S/c.751C > T
POLH	p.F18fs*12/c.48delT	p.Q543*/c.1627C > T	p.M14V/c.40A > G
POLL	p.C198fs*2/c.587_588insT	p.R549*/c.1645C > T	p.A285T/c.853G > A
POLN	p.F332fs*14/c.996delT	p.E599*/c.1795G > T	p.G419D/c.1256G > A
POLQ	p.K1068fs*2/c.3204delA	p.R602*/c.1804C > T	p.R375W/c.1123C > T
PRKDC	p.L65fs*13/c.194_195insT	p.E84*/c.250G > T	p.Q16K/c.46C > A
PTEN	p.K6fs*4/c.16_17delAA	p.L25*/c.74T > A	p.I101T/c.302T > C
RAD17	p.N51fs*6/c.147delA	p.K107*/c.319A > T	p.K370N/c.1110A > C
RAD18	p.K345fs*28/c.1035delA	p.E152*/c.454G > T	p.K52T/c.155A > C
RAD50	p.N320fs*5/c.954_955insA	p.W25*/c.75G > A	p.E387D/c.1161G > T
RAD51	p.Y54fs*11/c.159_160insG	p.Q30*/c.88C > T	p.E258D/c.774G > T
RAD52	p.V105fs*7/c.313delG	p.Q221*/c.661C > T	p.R46K/c.137G > A
RAD54B	p.P18fs*10/c.51_52insA	p.E75*/c.223G > T	p.L528F/c.1582C > T
RAD54L	p.L113fs*10/c.336_337insT	p.R75*/c.223C > T	p.F163L/c.489C > A
RAD9A	p.K96fs*6/c.284delA	p.Q205*/c.613C > T	p.R150W/c.448C > T
RB1	p.I124fs*6/c.370_371delAT	p.E54*/c.160G > T	p.V654M/c.1960G > A
REV3L	p.N639fs*16/c.1916delA	p.E1707*/c.5119G > T	p.K1512N/c.4536A > C
RPA1	p.F222fs*3/c.662delT	p.R586*/c.1756C > T	p.V27F/c.79G > T
RPA2	p.V207fs*26/c.620_621delTG	p.Y97*/c.291T > A	p.G204D/c.611G > A
SLX4	p.L470fs*8/c.1406_1407insC	p.E53*/c.157G > T	p.K301N/c.903G > T
TDP1	p.P359fs*21/c.1073delC	p.K177*/c.529A > T	p.K292E/c.874A > G
TDP2	p.K24fs*35/c.71delA	p.W52*/c.156G > A	p.E176D/c.528A > C
TP53	p.L35fs*8/c.102_103insT	p.R213*/c.637C > T	p.R175G/c.523C > G (Ref 1)
TP53BP1	p.N419fs*67/c.1256delA	p.Q106*/c.316C > T	p.F307L/c.919T > C
TRRAP	p.F468fs*52/c.1400delT	p.R1650*/c.4948C > T	p.S722F/c.2165C > T
UBE2N	p.I75fs*6/c.223delA	p.Q100*/c.298C > T	p.R7S/c.21G > T
UIMC1	p.T189fs*2/c.565_566insA	p.W183*/c.549G > A	p.S44F/c.131C > T
USP1	p.N21fs*14/c.57_58insA	p.R180*/c.538C > T	p.E250G/c.749A > G
WDR48	p.W195fs*13/c.580_581insT	p.G107*/c.319G > T	p.R235C/c.703C > T
WRN	p.M497fs*60/c.1485delA	p.E48*/c.142G > T	p.W85L/c.254G > T
XPA	p.C153fs*1/c.459_460delTG	p.E84*/c.250G > T	p.E106K/c.316G > A
XRCC1	p.G61fs*3/c.180_181insT	p.Q134*/c.400C > T	p.R350W/c.1048C > T
XRCC2	p.K267fs* > 14/c.801delA		p.R91Q/c.272G > A
XRCC3	p.T77fs*28/c.228_229insC		p.S23L/c.68C > T
XRCC4	p.C128fs*25/c.380delT	p.E295*/c.883G > T	p.P14A/c.40C > G
XRCC6	p.L41fs*17/c.116delT	p.R80*/c.238C > T	p.G28E/c.83G > A

Ref 1: Xu Y, Induction of genetic instability by gain-of-function p53 cancer mutants. Oncogene. 2008 27(25): 3501-7.

TABLE 2

Activating mutations - examples

Gene	Missense alteration	Fusion

BRAF	p.V600E/c.1799T > A	AKAP9{ENST00000356239}:
		r.1_3551_BRAF{ENST00000288602}:r.1202_2480
EGFR	.L858R/c.2573T > G
ERBB2	p.S1050L/c.3149C > T
ERBB3	p.Q1239H/c.3717G > C
PIK3CA	p.H1047R/c.3140A > G
TMPRSS2		TMPRSS2{ENST00000332149}:
		r.1_79_ERG{ENST00000442448}:r.312_5034
DYRK1A	p.R559C/c.1675C > T
PCNA	p.I88V/c.262A > G
NRAS	p.Q61L/c.182A > T
MYC	p.P57S/c.169C > T
KRAS	p.G12D/c.35G > A
HRAS	p.Q61L/c.182A > T
CDC7	p.E25K/c.73G > A
CCNE2	p.W327R/c.979T > C
CCNE1	p.R240C/c.718C > T
CCND1	p.D240H/c.718G > C
TOP2A	p.R268H/c.803G > A
TOP2B	p.H977Y/c.2929C > T
TOPBP1	P.F699C/c.2096T > G
TP53	p.R273H/c.818G > A (Ref 1)

Further examples of deleterious/activating mutations of the gene(s) mentioned herein are described in publically available databases, such as e.g. ClinVar (Landrum M J, Lee J M, Riley G R, et al., “ClinVar: public archive of relationships among sequence variation and human phenotype”, Nucleic Acids Res. 2014; 42:D980-5; https://www.ncbi.nlm.nih.gov/clinvar), HGMD (the Human Gene Mutation Database, http://www.hgmd.cf.ac.uk/ac/index.php; Stenson P D, Mort M, Ball E V, et al., “The human gene mutation database: 2008 update.”, Genome Med. 2009; 1:13) or in “The Human Variome Project” (http://www.humanvariomeproject.org; Timothy D Smith and Mauno Vihinen, “Standard development at the Human Variome Project”, Database 2015, 2015), which has curated a gene-/disease-specific databases to collect the sequence variants and genes associated with diseases.
Further examples of deleterious/activating mutations of the gene(s), which may be used in context with the method(s)/use(s)/kit(s)/pharmaceutical composition(s) of the present invention, are described in COSMIC database (www.cancer.sanger.ac.uk; “COSMIC: exploring the world's knowledge of somatic mutations in human cancer”, Forbes et al., Nucleic Acids Res. 2015, January; 43 (Database issue):D805-11. doi: 10.1093/nar/gku1075. Epub 2014 Oct. 29), particularly in release 79 of COSMIC (COSMIC v79), which was released on 14 Nov. 2016.
Examples of relevant functional mutations of the TMPRSS2-ERG fusion gene/protein are described for example in Tomlins et al. (Science (New York, N.Y.) 2005; 310(5748):644-648); Soller et al. (Genes, chromosomes & cancer 2006; 45(7):717-719); Clark et al. (Oncogene 2007; 26(18):2667-2673); Wang et al. (Cancer research 2006; 66(17):8347-8351); or in Tu et al. (Modern pathology: an official journal of the United States and Canadian Academy of Pathology, Inc 2007, 20(9):921-928), In another embodiment of the present invention the subject or the hyper-proliferative disease is characterized by one or more biomarker(s) selected from one or more functional mutation(s) of the gene(s)/protein(s) which are described in the Experimental Section infra.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the APC gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the ATG5 gene.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the ARID1A gene.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the ATM gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker invention comprise(s) one or more functional mutation(s), particularly deleterious or activating mutation(s), of the ATR gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious or activating mutation(s), of the ATRIP gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the ATRX gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the BAP1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the BARD1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the BLM gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the BRAF gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the BRCA1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the BRCA2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the BRIP1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the CCND1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the CCNE1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the CCNE2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the CDC7 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the CDK12 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the CHEK1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the CHEK2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the DCLRE1A gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the DCLRE1B gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the DCLRE1C gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the DYRK1A gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the EGFR gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the ERBB2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the ERBB3 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the ERCC2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the ERCC3 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the ERCC4 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the ERCC5 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the FAM175A gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the FANCA gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the FANCB gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the FANCC gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the FANCD2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the FANCE gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the FANCF gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the FANCG gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the FANCI gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the FANCL gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the FANCM gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the FBXO18 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the FBXW7 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the FEN1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the GEN1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the HDAC2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the H2AFX gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the HRAS gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the KRAS gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the LIG4 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the MDC1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the MLH1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the MLH3 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the MRE11A gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the MSH2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the MSH3 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the MSH6 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the MYC gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the NBN gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the NRAS gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the PALB2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the PARP1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the PARP2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the PARP3 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the PARP4 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the PCNA gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the PIK3CA gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the PMS2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the POLA1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the POLB gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the POLH gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the POLL gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the POLN gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the POLQ gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the PRKDC gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the PTEN gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the RAD9A gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the RAD17 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the RAD18 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the RAD50 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the RAD51 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the RAD52 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the RAD54B gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the RAD54L gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the RB1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the REV3L gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the RPA1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the RPA2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the SLX4 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the TDP1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the TDP2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the TMPRSS2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious or activating mutation(s), of the TMPRSS2-ERG gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the TOPBP1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the TOP2A gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly activating mutation(s), of the TOP2B gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious or activating mutation(s), of the TP53 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the TP53BP1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the TRRAP gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the UBE2N gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the UIMC1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the USP1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the WDR48 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the WRN gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the XPA gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the XRCC1 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the XRCC2 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the XRCC3 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the XRCC4 gene/protein.
In another embodiment the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), wherein the biomarker comprise(s) one or more functional mutation(s), particularly deleterious mutation(s), of the XRCC6 gene/protein.
In another embodiment of the present invention, the subject is chemotherapy-naïve.
The term “chemotherapy-naïve” as used herein means that the subject, prior to the treatment with Compound A according to the present invention, has not received a chemotherapy.
In another embodiment of the present invention, the subject has received a chemotherapy prior to the treatment with Compound A. The term “chemotherapy” as used herein means a category of cancer treatment that uses one or more chemotherapeutic agents as part of a standardized chemotherapy regimen. Chemotherapeutic agents are rather non-specific agents including but not limited to alkylating agents, anthracyclines, taxanes, epothilones, histone deacetylase inhibitors, inhibitors of topoisomerase I, inhibitors of topoisomerase II, nucleotide analogues, platinum-based agents, vinca alkaloids.
The present invention also covers an inhibitor of ATR kinase, particularly Compound A, for use in a method of treating a hyper-proliferative disease in a subject, said method comprising the steps:

a) determining if one or more of the biomarker(s) defined herein are present in a sample, preferably in an in vitro sample, of the subject;
b) administering a therapeutically effective amount of the inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by step a) is (are) determined positively.

The present invention also covers an inhibitor of ATR kinase, particularly Compound A, for use in a method of treating a hyper-proliferative disease in a subject said method comprising the steps:

a) determining if one or more of the biomarker(s) selected from
- (i) one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ATG5, ARID1A, ATM, ATR, ATRIP, ATRX, BAP1, BARD1, BLM, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CCNE1, CCNE2, CDC7, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1B, DCLRE1C, DYRK1A, EGFR, ERBB2, ERBB3, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXO18, FBXW7, FEN1, GEN1, HDAC2, H2AFX, HRAS, KRAS, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MYC, NBN, NRAS, PALB2, PARP1, PARP2, PARP3, PARP4, PCNA, PIK3CA, PMS2, POLA1, POLB, POLH, POLL, POLN, POLQ, PRKDC, PTEN, RAD9A, RAD17, RAD18, RAD50, RAD51, RAD52, RAD54B, RAD54L, RB1, REV3L, RPA1, RPA2, SLX4, TDP1, TDP2, TMPRSS2, TMPRSS2-ERG, TOPBP1, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XRCC1, XRCC2, XRCC3, XRCC4 and/or XRCC6 gene/protein; and/or
- (ii) the activation of the ALT pathway; and/or
- (iii) microsatellite instability, particularly high microsatellite instability; are present in a sample, preferably in an in vitro sample, of the subject;
b) administering a therapeutically effective amount of the inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by any one of steps a)(i), a)(ii) and/or a)(iii) is (are) determined positively.

The present invention also covers an inhibitor of ATR kinase, particularly Compound A for use in a method of treating a hyper-proliferative disease in a subject said method comprising the steps:

a) determining if one or more of the biomarker(s) comprising one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N and/or XPA gene/protein are present in a sample, preferably in an in vitro sample, of the subject;
b) administering a therapeutically effective amount of the inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by step a) is (are) determined positively.

In another embodiment of the use of an inhibitor of ATR kinase, particularly of Compound A, in a method of treating a hyper-proliferative disease in a subject according to the present invention said method comprises the steps:

a) assaying a sample, preferably an in vitro sample, from the subject, particularly by one or more of the stratification method(s) described herein;
b) determining if one or more of the biomarker(s) defined herein are present in the sample;
c) administering a therapeutically effective amount of the inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by step (b) is (are) determined positively.

Particularly, the present invention covers the use of an inhibitor of ATR kinase, particularly of Compound A, in a method of treating a hyper-proliferative disease in a subject according to the present invention said method comprises the steps:

a) assaying a sample, preferably an in vitro sample, from the subject, particularly by one or more of the stratification method(s) described herein;
b) determining if one or more of the biomarker(s) defined in (i), (ii) and/or (iii) are present in the sample:
- (i) the one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ATG5, ARID1A, ATM, ATR, ATRIP, ATRX, BAP1, BARD1, BLM, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CCNE1, CCNE2, CDC7, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1B, DCLRE1C, DYRK1A, EGFR, ERBB2, ERBB3, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXO18, FBXW7, FEN1, GEN1, HDAC2, H2AFX, HRAS, KRAS, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MYC, NBN, NRAS, PALB2, PARP1, PARP2, PARP3, PARP4, PCNA, PIK3CA, PMS2, POLA1, POLB, POLH, POLL, POLN, POLQ, PRKDC, PTEN, RAD9A, RAD17, RAD18, RAD50, RAD51, RAD52, RAD54B, RAD54L, RB1, REV3L, RPA1, RPA2, SLX4, TDP1, TDP2, TMPRSS2, TMPRSS2-ERG, TOPBP1, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XRCC1, XRCC2, XRCC3, XRCC4 and/or XRCC6 gene/protein; and/or
- (ii) the activation of the ALT pathway; and/or
- (iii) microsatellite instability, particularly high microsatellite instability;
c) administering a therapeutically effective amount of the inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by any one of steps (b)(i), (b)(ii) and/or (b)(iii) is (are) determined positively.

a) assaying a sample, preferably an in vitro sample, from the subject, particularly by one or more of the stratification method(s) described herein;
b) determining if one or more of the biomarker(s) comprising one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N and/or XPA gene/protein are present in the sample;
c) administering a therapeutically effective amount of the inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by step (b) is (are) determined positively.

In context with the present invention the term “determined positively” means that the presence of said functional mutation, said activation of the ALT pathway and/or microsatellite instability, particularly high microsatellite instability, in the sample, preferably in samples of tumor cells or tumor tissue, was confirmed, particularly by one or more of the stratification method(s) described herein.
In another embodiment the present invention covers an inhibitor of ATR kinase, particularly of Compound A, for use in a method of treating a hyper-proliferative disease in a subject, wherein said subject is selected by having one or more biomarker(s) defined herein.
In another embodiment the present invention covers an inhibitor of ATR kinase, particularly of Compound A, for use in a method of treating a hyper-proliferative disease in a subject, wherein said subject is selected by having one or more of the biomarker(s) selected from
a) one or more functional mutation(s) in one or more gene(s)/protein(s) as defined herein;
b) the activation of the ALT pathway; and/or
c) microsatellite instability, particularly high microsatellite instability.
In another embodiment the present invention covers an inhibitor of ATR kinase, particularly of Compound A, for use in a method of treating a hyper-proliferative disease in a subject, wherein said subject is selected by having one or more of the biomarker(s) selected from

In another embodiment the present invention covers an inhibitor of ATR kinase, particularly of Compound A, for use in a method of treating a hyper-proliferative disease in a subject, wherein said subject is selected by having one or more of the biomarker(s) comprising one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N and/or XPA gene/protein.
The present invention also covers an inhibitor of ATR kinase, particularly of Compound A, for use in a method of treating a hyper-proliferative disease in a subject, wherein said hyper-proliferative disease is characterized by
a) one or more functional mutation(s) in one or more gene(s)/protein(s) as defined herein;
b) the activation of the ALT pathway; and/or
c) microsatellite instability, particularly high microsatellite instability.
In another embodiment the present invention also covers an inhibitor of ATR kinase, particularly of Compound A, for the use in a method of treating a subject diagnosed with a hyper-proliferative disease, said method comprising the steps:

a) assaying a sample, preferably an in vitro sample, from the subject, particularly by one or more of the stratification method(s) described herein;
b) determining if one or more of the biomarker(s) defined herein are present in the sample;
c) administering a therapeutically effective amount of the inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by step b) is (are) determined positively.

In another embodiment the present invention covers an inhibitor of ATR kinase, particularly of Compound A, for the use in a method of treating a subject diagnosed with a hyper-proliferative disease, said method comprising the steps:

a) assaying a sample, preferably an in vitro sample, from the subject, particularly by one or more of the stratification method(s) described herein;
b) determining if one or more of the biomarker(s) defined in (i), (ii) and/or (iii) are present in the sample:
- (i) one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ATG5, ARID1A, ATM, ATR, ATRIP, ATRX, BAP1, BARD1, BLM, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CCNE1, CCNE2, CDC7, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1B, DCLRE1C, DYRK1A, EGFR, ERBB2, ERBB3, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXO18, FBXW7, FEN1, GEN1, HDAC2, H2AFX, HRAS, KRAS, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MYC, NBN, NRAS, PALB2, PARP1, PARP2, PARP3, PARP4, PCNA, PIK3CA, PMS2, POLA1, POLB, POLH, POLL, POLN, POLQ, PRKDC, PTEN, RAD9A, RAD17, RAD18, RAD50, RAD51, RAD52, RAD54B, RAD54L, RB1, REV3L, RPA1, RPA2, SLX4, TDP1, TDP2, TMPRSS2, TMPRSS2-ERG, TOPBP1, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XRCC1, XRCC2, XRCC3, XRCC4 and/or XRCC6 gene/protein; and/or
- (ii) the activation of the ALT pathway; and/or
- (iii) microsatellite instability, particularly high microsatellite instability;
c) administering a therapeutically effective amount of the inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by any one of steps (b)(i), (b)(ii) and/or (b)(iii) is (are) determined positively.

In another embodiment the present invention also covers an inhibitor of ATR kinase, particularly of Compound A, for the use in a method of treating a subject diagnosed with a hyper-proliferative disease, said method comprising the steps:

a) assaying a sample, preferably an in vitro sample, from the subject, particularly by one or more of the stratification method(s) described herein;
b) determining if one or more of the biomarker(s) comprising one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N and/or XPA gene/protein are present in the sample;
c) administering a therapeutically effective amount of the inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by step b) is (are) present in the sample.

In another embodiment the present invention covers the use of an inhibitor of ATR kinase, particularly of Compound A, for the preparation of a medicament for treating a hyper-proliferative disease in a subject, wherein said subject is characterized by one or more biomarker(s) defined herein.
In another embodiment the present invention covers the use of an inhibitor of ATR kinase, particularly of Compound A, for the preparation of a medicament for treating a hyper-proliferative disease in a subject, wherein said subject is characterized by
a) one or more functional mutation(s) in one or more gene(s)/protein(s) as defined herein;
b) the activation of the ALT pathway; and/or
c) microsatellite instability, particularly high microsatellite instability.
In another embodiment the present invention covers the use of an inhibitor of ATR kinase, particularly of Compound A, for the preparation of a medicament for treating a hyper-proliferative disease in a subject, wherein said subject is characterized by

In another embodiment the present invention covers the use of an inhibitor of ATR kinase, particularly of Compound A, in the manufacture of a medicament for treating a hyper-proliferative disease in a subject, wherein said hyper-proliferative disease is characterized by one or more biomarker(s) defined herein.
In another embodiment the present invention covers the use of an inhibitor of ATR kinase, particularly of Compound A, in the manufacture of a medicament for treating a hyper-proliferative disease in a subject, wherein said hyper-proliferative disease is characterized by
a) one or more functional mutation(s) in one or more gene(s)/protein(s) as defined herein; and/or
b) the activation of the ALT pathway; and/or
c) microsatellite instability, particularly high microsatellite instability.
In another embodiment the present invention covers the use of an inhibitor of ATR kinase, particularly of Compound A, in the manufacture of a medicament for treating a hyper-proliferative disease in a subject, wherein said hyper-proliferative disease is characterized by

In another embodiment the present invention covers the use of an inhibitor of ATR kinase, particularly of Compound A, in the manufacture of a medicament for treating a hyper-proliferative disease in a subject, wherein said hyper-proliferative disease or said subject is characterized by one or more biomarker(s) comprising one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N and/or XPA gene/protein.
In another embodiment of the use of an inhibitor of ATR kinase, particularly of Compound A, in the manufacture of a medicament for treating a hyper-proliferative disease in a subject according to the invention the one or more functional mutation(s), the activation of the ALT pathway and/or the microsatellite instability is (are) determined by one or more of the stratification method(s) described herein.
In another embodiment the present invention covers the use of an inhibitor of ATR kinase, particularly of Compound A, in the manufacture of a medicament for a method of treating a hyper-proliferative disease in a subject, said method comprising the steps:

a) assaying a sample from the subject, particularly by one or more of the stratification method(s) described herein;
b) determining if one or more of the biomarker(s) defined herein are present in the sample;
c) administering a therapeutically effective amount of the inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by step (b) is (are) determined positively.

In another embodiment the present invention covers the use of an inhibitor of ATR kinase, particularly of Compound A, in the manufacture of a medicament for a method of treating a hyper-proliferative disease in a subject, said method comprising the steps:

a) assaying a sample from the subject, particularly by one or more of the stratification method(s) described herein;
b) determining if one or more of the biomarker(s) defined in (i), (ii) and/or (iii) are present in the sample:
- (i) one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ATG5, ARID1A, ATM, ATR, ATRIP, ATRX, BAP1, BARD1, BLM, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CCNE1, CCNE2, CDC7, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1B, DCLRE1C, DYRK1A, EGFR, ERBB2, ERBB3, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXO18, FBXW7, FEN1, GEN1, HDAC2, H2AFX, HRAS, KRAS, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MYC, NBN, NRAS, PALB2, PARP1, PARP2, PARP3, PARP4, PCNA, PIK3CA, PMS2, POLA1, POLB, POLH, POLL, POLN, POLQ, PRKDC, PTEN, RAD9A, RAD17, RAD18, RAD50, RAD51, RAD52, RAD54B, RAD54L, RB1, REV3L, RPA1, RPA2, SLX4, TDP1, TDP2, TMPRSS2, TMPRSS2-ERG, TOPBP1, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XRCC1, XRCC2, XRCC3, XRCC4 and/or XRCC6 gene/protein; and/or
- (ii) the activation of the ALT pathway; and/or
- (iii) microsatellite instability, particularly high microsatellite instability;
c) administering a therapeutically effective amount of the inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by any one of steps (b)(i), (b)(ii) and/or (b)(iii) is (are) determined positively.

a) determining if one or more of the biomarker(s) defined herein are present in a sample of said subject;
b) administering a therapeutically effective amount of the inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by step a) is (are) determined positively.

a) determining if one or more of the biomarker(s) selected from
- (i) one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ATG5, ARID1A, ATM, ATR, ATRIP, ATRX, BAP1, BARD1, BLM, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CCNE1, CCNE2, CDC7, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1B, DCLRE1C, DYRK1A, EGFR, ERBB2, ERBB3, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXO18, FBXW7, FEN1, GEN1, HDAC2, H2AFX, HRAS, KRAS, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MYC, NBN, NRAS, PALB2, PARP1, PARP2, PARP3, PARP4, PCNA, PIK3CA, PMS2, POLA1, POLB, POLH, POLL, POLN, POLQ, PRKDC, PTEN, RAD9A, RAD17, RAD18, RAD50, RAD51, RAD52, RAD54B, RAD54L, RB1, REV3L, RPA1, RPA2, SLX4, TDP1, TDP2, TMPRSS2, TMPRSS2-ERG, TOPBP1, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XRCC1, XRCC2, XRCC3, XRCC4 and/or XRCC6 gene/protein; and/or
- (ii) the activation of the ALT pathway; and/or
- (iii) microsatellite instability, particularly high microsatellite instability;
- are present in a sample of said subject;
b) administering a therapeutically effective amount of the inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by any one of steps a)(i), a)(ii) and/or a)(iii) is (are) determined positively.

Method(s) of the Present Invention
In another embodiment the present invention covers a method for the treatment of a hyper-proliferative disease in a subject using an effective amount of an inhibitor of ATR kinase, particularly of Compound A, wherein said subject or said hyper-proliferative disease is characterized by one or more biomarker(s) defined herein.
In another embodiment the present invention covers a method for the treatment of a hyper-proliferative disease in a subject using an effective amount of an inhibitor of ATR kinase, particularly of Compound A, wherein said subject is characterized by
a) one or more functional mutation(s) in one or more gene(s)/protein(s) as defined herein; and/or
b) the activation of the ALT pathway; and/or
c) microsatellite instability, particularly high microsatellite instability.
In another embodiment the present invention covers a method for the treatment of a hyper-proliferative disease in a subject using an effective amount of an inhibitor of ATR kinase, particularly of Compound A, wherein said subject is characterized by

In another embodiment the present invention covers a method for the treatment of a hyper-proliferative disease in a subject using an effective amount of an inhibitor of ATR kinase, particularly of Compound A, wherein said hyper-proliferative disease or said subject is characterized by one or more biomarker(s) comprising one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N and/or XPA gene/protein.
In another embodiment of the method for the treatment of a hyper-proliferative disease in a subject using an effective amount of an inhibitor of ATR kinase, particularly of Compound A, the one or more functional mutation(s), the activation of the ALT pathway and/or the microsatellite instability is (are) determined by one or more of the stratification method(s) described herein.
The present invention also covers a method of treatment of a subject diagnosed with a hyper-proliferative disease comprising the steps

a) assaying a sample from the subject, preferably an in vitro sample from the subject, particularly by one or more of the stratification method(s) described herein;
b) determining if one or more of the biomarker(s) defined herein are present in the sample;
c) administering a therapeutically effective amount of an inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by step b) is (are) determined positively.

The present invention also covers a method of treatment of a subject diagnosed with a hyper-proliferative disease comprising the steps

a) assaying a sample from the subject, particularly by one or more of the stratification method(s) described herein;
b) determining if one or more of the biomarker(s) defined in (i), (ii) and/or (iii) are present in the sample:
- (i) one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ATG5, ARID1A, ATM, ATR, ATRIP, ATRX, BAP1, BARD1, BLM, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CCNE1, CCNE2, CDC7, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1B, DCLRE1C, DYRK1A, EGFR, ERBB2, ERBB3, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXO18, FBXW7, FEN1, GEN1, HDAC2, H2AFX, HRAS, KRAS, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MYC, NBN, NRAS, PALB2, PARP1, PARP2, PARP3, PARP4, PCNA, PIK3CA, PMS2, POLA1, POLB, POLH, POLL, POLN, POLQ, PRKDC, PTEN, RAD9A, RAD17, RAD18, RAD50, RAD51, RAD52, RAD54B, RAD54L, RB1, REV3L, RPA1, RPA2, SLX4, TDP1, TDP2, TMPRSS2, TMPRSS2-ERG, TOPBP1, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XRCC1, XRCC2, XRCC3, XRCC4 and/or XRCC6 gene/protein; and/or
- (ii) the activation of the ALT pathway; and/or
- (iii) microsatellite instability, particularly high microsatellite instability;
c) administering a therapeutically effective amount of an inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by any one of steps (b)(i), (b)(ii) and/or (b)(iii) is (are) determined positively.

a) assaying a sample from the subject, preferably an in vitro sample from the subject, particularly by one or more of the stratification method(s) described herein;
b) determining if one or more of the biomarker(s) comprising one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N and/or XPA gene/protein are present in the sample;
c) administering a therapeutically effective amount of an inhibitor of ATR kinase, particularly of Compound A, to the subject, if one or more of the biomarker(s) determined by step b) is (are) determined positively.

The present invention also concerns a method for identifying a subject having a hyper-proliferative disease disposed to respond favorably to an inhibitor of ATR kinase, particularly of Compound A, wherein the method comprises the detection of one or more of the biomarker(s) defined herein in a sample of said subject, preferably in an in vitro sample of tumor cells or of tumor tissue.
The present invention also concerns a method for identifying a subject having a hyper-proliferative disease disposed to respond favorably to an inhibitor of ATR kinase, particularly of Compound A, wherein the method comprises the detection of one or more of the biomarker(s) selected from:

- (ii) one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ATG5, ARID1A, ATM, ATR, ATRIP, ATRX, BAP1, BARD1, BLM, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CCNE1, CCNE2, CDC7, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1B, DCLRE1C, DYRK1A, EGFR, ERBB2, ERBB3, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXO18, FBXW7, FEN1, GEN1, HDAC2, H2AFX, HRAS, KRAS, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MYC, NBN, NRAS, PALB2, PARP1, PARP2, PARP3, PARP4, PCNA, PIK3CA, PMS2, POLA1, POLB, POLH, POLL, POLN, POLQ, PRKDC, PTEN, RAD9A, RAD17, RAD18, RAD50, RAD51, RAD52, RAD54B, RAD54L, RB1, REV3L, RPA1, RPA2, SLX4, TDP1, TDP2, TMPRSS2, TMPRSS2-ERG, TOPBP1, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XRCC1, XRCC2, XRCC3, XRCC4 and/or XRCC6 gene/protein; and/or
- (ii) the activation of the ALT pathway; and/or
- (iii) microsatellite instability, particularly high microsatellite instability;

in a sample of said subject, preferably in an in vitro sample of tumor cells or of tumor tissue.
The present invention also concerns a method for identifying a subject having a hyper-proliferative disease disposed to respond favorably to an inhibitor of ATR kinase, particularly of Compound A, wherein the method comprises the detection of one or more of the biomarker(s) comprising one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N and/or XPA gene/protein in a sample of said subject, preferably in an in vitro sample of tumor cells or of tumor tissue.
In another embodiment the one or biomarker(s) is (are) determined by one or more of the stratification method(s) described herein.
The present invention also concerns a method for identifying a subject with a hyper-proliferative disease who is more likely to respond to a therapy comprising an inhibitor of ATR kinase, particularly of Compound A, than other subjects, the method comprising

a) determining in a sample from said subject one or more of the biomarker(s) defined herein;
b) identifying those subjects for whom in step a) one or more of the biomarker(s) is (are) determined positively.

The present invention also concerns a method for identifying a subject with a hyper-proliferative disease who is more likely to respond to a therapy comprising an inhibitor of ATR kinase, particularly of Compound A, than other subjects, the method comprising

a) determining in a sample from said subject the biomarker(s) selected from:
- (i) one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ATG5, ARID1A, ATM, ATR, ATRIP, ATRX, BAP1, BARD1, BLM, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CCNE1, CCNE2, CDC7, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1B, DCLRE1C, DYRK1A, EGFR, ERBB2, ERBB3, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXO18, FBXW7, FEN1, GEN1, HDAC2, H2AFX, HRAS, KRAS, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MYC, NBN, NRAS, PALB2, PARP1, PARP2, PARP3, PARP4, PCNA, PIK3CA, PMS2, POLA1, POLB, POLH, POLL, POLN, POLQ, PRKDC, PTEN, RAD9A, RAD17, RAD18, RAD50, RAD51, RAD52, RAD54B, RAD54L, RB1, REV3L, RPA1, RPA2, SLX4, TDP1, TDP2, TMPRSS2, TMPRSS2-ERG, TOPBP1, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XRCC1, XRCC2, XRCC3, XRCC4 and/or XRCC6 gene/protein; and/or
- (ii) the activation of the ALT pathway; and/or
- (iii) microsatellite instability, particularly high microsatellite instability; and
b) identifying those subjects for whom one or more of the biomarker(s) of any one of a)(i), a)(ii) or a)(iii) is (are) determined positively.

The present invention also concerns a method for identifying a subject with a hyper-proliferative disease who is more likely to respond to a therapy comprising an inhibitor of ATR kinase, particularly Compound A, than other subjects, the method comprising
a) determining in a sample from said subject one or more biomarker(s) comprising one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N and/or XPA gene/protein;
b) identifying those subjects for whom in step a) one or more of the biomarker(s) is (are) determined positively.
The present invention also concerns a method of determining whether a subject having a hyper-proliferative disease will respond to the treatment with an inhibitor of ATR kinase, particularly with Compound A, wherein the method comprises the detection of one or more of the biomarker(s) defined herein in a sample of said subject. Preferably the sample is a sample of tumor cells or of tumor tissue of said subject. Particularly, the biomarker(s) is (are) determined by one or more of the stratification method(s) described herein.
The present invention also concerns a method of determining the likelihood that a subject with a hyper-proliferative disease benefits from treatment with an inhibitor of ATR kinase, particularly with Compound A, the method comprising the detection of one or more of the biomarker(s) defined herein in a sample of said subject and identifying the subject being more likely to respond to said treatment with the inhibitor of ATR kinase, particularly with Compound A, when the one or more biomarker(s) is (are) determined positively.
The present invention also covers a method of predicting whether a subject with a hyper-proliferative disease will respond to the treatment with an inhibitor of ATR kinase, particularly with Compound A, wherein the method comprises the detection of one or more of the biomarker(s) defined herein in a sample of said subject.
The present invention also covers the use of one or more of the biomarker(s) defined herein for identifying a subject with a hyper-proliferative disease who is disposed to respond favorably to an inhibitor of ATR kinase, particularly to Compound A.
Kit(s) and Pharmaceutical Composition(s) of the Present Invention
The present invention also covers a kit comprising an inhibitor of ATR kinase, particularly Compound A, together with means, preferably a detecting agent, to detect in a sample from a subject one or more of the biomarker(s) selected from:

The present invention also covers a kit comprising an inhibitor of ATR kinase, particularly Compound A, together with means, preferably a detecting agent, to detect, particularly in a sample from a subject, one or more of the biomarker(s) defined herein.
The present invention also covers a kit comprising an inhibitor of ATR kinase, particularly Compound A, together with means, preferably a detecting agent, to detect in a sample from a subject one or more biomarker(s) comprising one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N and/or XPA gene/protein.
In another embodiment the present invention covers a pharmaceutical composition comprising an inhibitor of ATR kinase, particularly Compound A, together with one or more pharmaceutically acceptable excipients for use in any of the method(s)/use(s) for treating a hyper-proliferative disease in a subject described herein.
The inhibitor of ATR kinase, particularly Compound A, can act systemically and/or locally. For this purpose, it can be administered in a suitable manner, for example by the oral, parenteral, pulmonal, nasal, sublingual, lingual, buccal, rectal, dermal, transdermal, conjunctival, otic route, or as an implant or stent. The inhibitor of ATR kinase, particularly Compound A, can be administered in administration forms suitable for these administration routes.
Suitable administration forms for oral administration are those which deliver Compound A in a rapid and/or modified manner, and contain Compound A in crystalline and/or amorphous and/or dissolved form, for example tablets (uncoated or coated tablets, for example with enteric or retarded-dissolution or insoluble coatings which control the release of Compound A, tablets or films/wafers which disintegrate rapidly in the oral cavity, films/lyophilizates, capsules (for example hard or soft gelatin capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. Parenteral administration can be accomplished with avoidance of an absorption step (for example by an intravenous, intraarterial, intracardial, intraspinal or intralumbal route) or with inclusion of an absorption (for example by an intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal route). Suitable administration forms for parenteral administration include injection and infusion formulations in the form of solutions, suspensions, emulsions, lyophilizates or sterile powders.
For the other administration routes, suitable examples are pharmaceutical forms for inhalation or inhalation medicaments (including powder inhalers, nebulizers), nasal drops, solutions or sprays; tablets, films/wafers or capsules for lingual, sublingual or buccal administration, films/wafers or capsules, suppositories, ear or eye preparations (for example eye baths, ocular insert, ear drops, ear powders, ear-rinses, ear tampons), vaginal capsules, aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, ointments, creams, transdermal therapeutic systems (for example patches), milk, pastes, foams, dusting powders, implants, intrauterine coils, vaginal rings or stents. Compound A can be converted to the administration forms mentioned. This can be done in a manner known per se, by mixing with pharmaceutically suitable excipients.
These excipients include carriers (for example microcrystalline cellulose, lactose, mannitol), solvents (e.g. liquid polyethylene glycols), emulsifiers and dispersing or wetting agents (for example sodium dodecylsulphate, polyoxysorbitan oleate), binders (for example polyvinylpyrrolidone), synthetic and natural polymers (for example albumin), stabilizers (e.g. antioxidants, for example ascorbic acid), dyes (e.g. inorganic pigments, for example iron oxides) and flavour and/or odour correctants.
Pharmaceutically acceptable excipients are non-toxic, preferably they are non-toxic and inert. Pharmaceutically acceptable excipients include, inter alia: fillers and excipients (for example cellulose, microcrystalline cellulose, such as, for example, Avicel®, lactose, mannitol, starch, calcium phosphate such as, for example, Di-Cafos®),

- ointment bases (for example petroleum jelly, paraffins, triglycerides, waxes, wool wax, wool wax alcohols, lanolin, hydrophilic ointment, polyethylene glycols),
- bases for suppositories (for example polyethylene glycols, cacao butter, hard fat)
- solvents (for example water, ethanol, Isopropanol, glycerol, propylene glycol, medium chain-length triglycerides fatty oils, liquid polyethylene glycols, paraffins),
- surfactants, emulsifiers, dispersants or wetters (for example sodium dodecyle sulphate, lecithin, phospholipids, fatty alcohols such as, for example, Lanette®, sorbitan fatty acid esters such as, for example, Span®, polyoxyethylene sorbitan fatty acid esters such as, for example, Tween®, polyoxyethylene fatty acid glycerides such as, for example, Cremophor®, polyoxethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters, poloxamers such as, for example, Pluronic®),
- buffers and also acids and bases (for example phosphates, carbonates, citric acid, acetic acid, hydrochloric acid, sodium hydroxide solution, ammonium carbonate, trometamol, triethanolamine)
- isotonicity agents (for example glucose, sodium chloride),
- adsorbents (for example highly-disperse silicas)
- viscosity-increasing agents, gel formers, thickeners and/or binders (for example polyvinylpyrrolidon, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, carboxymethylcellulose-sodium, starch, carbomers, polyacrylic acids such as, for example, Carbopol®, alginates, gelatine),
- disintegrants (for example modified starch, carboxymethylcellulose-sodium, sodium starch glycolate such as, for example, Explotab®, cross-linked polyvinylpyrrolidon, croscarmellose-sodium such as, for example, AcDiSol®),
- flow regulators, lubricants, glidant and mould release agents (for example magnesium stearate, stearic acid, talc, highly-disperse silicas such as, for example, Aerosil®),
- coating materials (for example sugar, shellac) and film formers for films or diffusion membranes which dissolve rapidly or in a modified manner (for example polyvinylpyrrolidones such as, for example, Kollidon®, polyvinyl alcohol, hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, hydroxypropylmethylcellulose phthalate, cellulose acetate, cellulose acetate phthalate, polyacrylates, polymethacrylates such as, for example, Eudragit®),
- capsule materials (for example gelatine, hydroxypropylmethylcellulose),
- synthetic polymers (for example polylactides, polyglycolides, polyacrylates, polymethacrylates such as, for example, Eudragit®, polyvinylpyrrolidones such as, for example, Kollidon®, polyvinyl alcohols, polyvinyl acetates, polyethylene oxides, polyethylene glycols and their copolymers and blockcopolymers),
- plasticizers (for example polyethylene glycols, propylene glycol, glycerol, triacetine, triacetyl citrate, dibutyl phthalate),
- penetration enhancers,
- stabilisers (for example antioxidants such as, for example, ascorbic acid, ascorbyl palmitate, sodium ascorbate, butylhydroxyanisole, butylhydroxytoluene, propyl gallate),
- preservatives (for example parabens, sorbic acid, thiomersal, benzalkonium chloride, chlorhexidine acetate, sodium benzoate),
- colourants (for example inorganic pigments such as, for example, iron oxides, titanium dioxide),
- flavourings, sweeteners, flavour- and/or odour-masking agents.

The present invention further covers the use of pharmaceutical compositions which comprise the inhibitor of ATR kinase, particularly Compound A, together with one or more, preferably inert, nontoxic, pharmaceutically suitable excipients, for use in any of the method(s)/use(s) for treating a hyper-proliferative disease in a subject described herein.
Based upon standard laboratory techniques known to evaluate compounds useful for the treatment of hyper-proliferative diseases by standard toxicity tests and by standard pharmacological assays for the determination of treatment of the conditions identified above in mammals, and by comparison of these results with the results of known active ingredients or medicaments that are used to treat these conditions, the effective dosage of the compounds of this invention can be determined for treatment of each desired indication. The amount of the active ingredient to be administered in the treatment of one of these conditions can vary widely according to such considerations as the particular compound and dosage unit employed, the mode of administration, the period of treatment, the age and sex of the patient treated, and the nature and extent of the condition treated.
The total amount of the active ingredient to be administered will generally range from about 0.001 mg/kg to about 200 mg/kg body weight per day, and preferably from about 0.01 mg/kg to about 50 mg/kg body weight per day. Clinically useful dosing schedules will range from one to three times a day dosing to once every four weeks dosing. In addition, “drug holidays” in which a patient is not dosed with a drug for a certain period of time, may be beneficial to the overall balance between pharmacological effect and tolerability. A unit dosage may contain from about 0.5 mg to about 1500 mg of active ingredient, and can be administered one or more times per day or less than once a day. The average daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous and parenteral injections, and use of infusion techniques will preferably be from 0.01 to 200 mg/kg of total body weight. The average daily rectal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight. The average daily vaginal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight. The average daily topical dosage regimen will preferably be from 0.1 to 200 mg administered between one to four times daily. The transdermal concentration will preferably be that required to maintain a daily dose of from 0.01 to 200 mg/kg. The average daily inhalation dosage regimen will preferably be from 0.01 to 100 mg/kg of total body weight.
Of course the specific initial and continuing dosage regimen for each patient will vary according to the nature and severity of the condition as determined by the attending diagnostician, the activity of the specific compound employed, the age and general condition of the patient, time of administration, route of administration, rate of excretion of the drug, drug combinations, and the like. The desired mode of treatment and number of doses of a compound of the present invention or a pharmaceutically acceptable salt or ester or composition thereof can be ascertained by those skilled in the art using conventional treatment tests.
In spite of this, it may be necessary to deviate from the amounts specified, specifically depending on body weight, administration route, individual behaviour towards the active ingredient, type of formulation, and time or interval of administration. For instance, less than the aforementioned minimum amount may be sufficient in some cases, while the upper limit mentioned has to be exceeded in other cases. In the case of administration of greater amounts, it may be advisable to divide them into several individual doses over the day.
For example, an inhibitor of ATR kinase, particularly Compound A, may be combined with known antihyperproliferative, cytostatic or cytotoxic substances for treatment of cancers. Examples of suitable antihyperproliferative, cytostatic or cytotoxic combination active ingredients include: 131I-chTNT, abarelix, abiraterone, aclarubicin, adalimumab, ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alectinib, alemtuzumab, alendronic acid, alitretinoin, altretamine, amifostine, aminoglutethimide, hexyl aminolevulinate, amrubicin, amsacrine, anastrozole, ancestim, anethole dithiolethione, anetumab ravtansine, angiotensin II, antithrombin III, aprepitant, arcitumomab, arglabin, arsenic trioxide, asparaginase, atezolizumab axitinib, azacitidine, basiliximab, belotecan, bendamustine, besilesomab, belinostat, bevacizumab, bexarotene, bicalutamide, bisantrene, blinatumomab, bortezomib, buserelin, bosutinib, brentuximab vedotin, busulfan, cabazitaxel, cabozantinib, calcitonine, calcium folinate, calcium levofolinate, capecitabine, capromab, carbamazepine, carboplatin, carboquone, carfilzomib, carmofur, carmustine, catumaxomab, celecoxib, celmoleukin, ceritinib, cetuximab, chlorambucil, chlormadinone, chlormethine, cidofovir, cinacalcet, cladribine, clodronic acid, clofarabine, cobimetinib, copanlisib, crisantaspase, crizotinib, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daratumumab, darbepoetin alfa, dabrafenib, dasatinib, daunorubicin, decitabine, degarelix, denileukin diftitox, denosumab, depreotide, deslorelin, dianhydrogalactitol, dexrazoxane, dibrospidium chloride, dianhydrogalactitol, diclofenac, dinutuximab, docetaxel, dolasetron, doxifluridine, doxorubicin, doxorubicin+estrone, dronabinol, eculizumab, edrecolomab, elliptinium acetate, elotuzumab, eltrombopag, endostatin, enocitabine, epirubicin, epitiostanol, epoetin alfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine, ethinylestradiol, etoposide, everolimus, exemestane, fadrozole, fentanyl, filgrastim, fluoxymesterone, floxuridine, fludarabine, flutamide, folinic acid, formestane, fosaprepitant, fotemustine, fulvestrant, gadobutrol, gadoteridol, gadoteric acid meglumine, gadoversetamide, gadoxetic acid, gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, Glucarpidase, glutoxim, GM-CSF, goserelin, granisetron, granulocyte colony stimulating factor, histamine dihydrochloride, histrelin, hydroxycarbamide, I-125 seeds, lansoprazole, ibandronic acid, ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate, interferon alfa, interferon beta, interferon gamma, iobitridol, iobenguane (123I), iomeprol, ipilimumab, itraconazole, ixabepilone, ixazomib, lanreotide, lansoprazole, lapatinib, Iasocholine, lenalidomide, lenvatinib, lenograstim, lentinan, letrozole, leuprorelin, levamisole, levonorgestrel, levothyroxine sodium, lisuride, lobaplatin, lomustine, lonidamine, masoprocol, medroxyprogesterone, megestrol, melarsoprol, melphalan, mepitiostane, mercaptopurine, mesna, methadone, methotrexate, methoxsalen, methylaminolevulinate, methylprednisolone, methyltestosterone, metirosine, mifamurtide, miltefosine, miriplatin, mitobronitol, mitoguazone, mitolactol, mitomycin, mitotane, mitoxantrone, mogamulizumab, molgramostim, mopidamol, morphine hydrochloride, morphine sulfate, nabilone, nabiximols, nafarelin, naloxone+pentazocine, naltrexone, nartograstim, necitumumab, nedaplatin, nelarabine, neridronic acid, netupitant/palonosetron, nivolumabpentetreotide, nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nintedanib, nitracrine, nivolumab, obinutuzumab, octreotide, ofatumumab, olaratumab, omacetaxine mepesuccinate, omeprazole, ondansetron, oprelvekin, orgotein, orilotimod, osimertinib, oxaliplatin, oxycodone, oxymetholone, ozogamicine, p53 gene therapy, paclitaxel, palbociclib, palifermin, palladium-103 seed, palonosetron, pamidronic acid, panitumumab, panobinostat, pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxy PEG-epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b, pemetrexed, pentazocine, pentostatin, peplomycin, Perflubutane, perfosfamide, Pertuzumab, picibanil, pilocarpine, pirarubicin, pixantrone, plerixafor, plicamycin, poliglusam, polyestradiol phosphate, polyvinylpyrrolidone+sodium hyaluronate, polysaccharide-K, pomalidomide, ponatinib, porfimer sodium, pralatrexate, prednimustine, prednisone, procarbazine, procodazole, propranolol, quinagolide, rabeprazole, racotumomab, radotinib, raloxifene, raltitrexed, ramosetron, ramucirumab, ranimustine, rasburicase, razoxane, refametinib, regorafenib, risedronic acid, rhenium-186 etidronate, rituximab, rolapitant, romidepsin, romiplostim, romurtide, roniciclib, samarium (153Sm) lexidronam, sargramostim, satumomab, secretin, siltuximab, sipuleucel-T, sizofiran, sobuzoxane, sodium glycididazole, sonidegib, sorafenib, stanozolol, streptozocin, sunitinib, talaporfin, talimogene laherparepvec, tamibarotene, tamoxifen, tapentadol, tasonermin, teceleukin, technetium (99mTc) nofetumomab merpentan, 99mTc-HYNIC-[Tyr3]-octreotide, tegafur, tegafur+gimeracil+oteracil, temoporfin, temozolomide, temsirolimus, teniposide, testosterone, tetrofosmin, thalidomide, thiotepa, thymalfasin, thyrotropin alfa, tioguanine, tocilizumab, topotecan, toremifene, tositumomab, trabectedin, trametinib, tramadol, trastuzumab, trastuzumab emtansine, treosulfan, tretinoin, trifluridine+tipiracil, trilostane, triptorelin, trametinib, trofosfamide, thrombopoietin, tryptophan, ubenimex, valatinib, valrubicin, vandetanib, vapreotide, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vismodegib, vorinostat, vorozole, yttrium-90 glass microspheres, zinostatin, zinostatin stimalamer, zoledronic acid, zorubicin.
Biomarker(s) of the Hyper-Proliferative-Disease or Subject
In another embodiment of the use(s)/method(s)/pharmaceutical composition(s)/kit(s) of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BRCA1, ATM, BLM, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
In another embodiment of the use(s)/method(s)/pharmaceutical composition(s)/kit(s) of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BRCA1, ATM, BLM, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
The expression “POLB/POLL” as used herein means a double mutation: one or more deleterious mutation(s) in POLB gene/protein and one or more deleterious mutation(s) in POLL gene/protein.
In a preferred embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BRCA1, ATM, BLM, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, RAD9A, RAD17, REV3L, TP53BP1, UBE2N.
In a preferred embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, RAD9A, RAD17, REV3L, TP53BP1, UBE2N.
In a preferred embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from ATM, BLM, BRCA1, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, REV3L, TP53BP1, UBE2N.
In a preferred embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, REV3L, TP53BP1, UBE2N.
In another preferred embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BRCA1, ATM, FANCD2, H2AFX, RAD17, UBE2N.
In a preferred embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BRCA1, ATM, FEN1, H2AFX, PCNA.
In a preferred embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BRCA1, FEN1, H2AFX, PCNA.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD52, REV3L, TDP2, TP53BP1, UBE2N, XPA.
In a preferred embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, RAD9A, RAD17, REV3L, TP53BP1, UBE2N.
In a preferred embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BLM, BRCA1, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, REV3L, TP53BP1, UBE2N.
In a preferred embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more biomarker(s), wherein the biomarker(s) comprise(s) one or more deleterious mutation(s) in TP53 gene/protein and one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from BRCA1, FEN1, H2AFX, PCNA.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in BLM gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in BRCA1 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in BRCA2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in ERCC5 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in FEN1 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in FANCD2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in FANCG gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in H2AFX gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in PARP1 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in PCNA gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in POLL gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in RAD9A gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in RAD17 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in RAD52 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in REV3L gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in TDP2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in TP53BP1 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in UBE2N gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TP53 gene/protein and by one or more deleterious mutation(s) in XPA gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in ATM gene/protein and/or by one or more deleterious mutation(s) in BRCA2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in POLL gene/protein and by one or more deleterious mutation(s) in POLB gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in POLN gene/protein and by one or more deleterious mutation(s) in POLQ gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in POLH gene/protein and by one or more deleterious mutation(s) in REV3L gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention described herein the hyper-proliferative disease or the subject is characterized by one or more deleterious mutation(s) in TDP1 gene/protein and by one or more deleterious mutation(s) in TDP2 gene/protein.
Prostate Cancer
In another embodiment of the use/methods/pharmaceutical compositions/kits of the present invention the hyper-proliferative disease is prostate cancer.
The term “prostate cancer” as used herein means any histology type of prostate cancer including but not limited to acinar adenocarcinoma, ductal adenocarcinoma, transitional cell (or urothelial) cancer, squamous cell cancer, carcinoid, small cell cancer, sarcomas and sarcomatoid cancers, particularly acinar adenocarcinoma, castration resistant prostate cancer (CRPC), particularly stage M0 castration-resistant prostate cancer (M0 CRPC) or stage M1 castration-resistant prostate cancer (M1 CRPC).
The terms “M0” and “M1” (including M1a, M1b, M1c) are used in accordance with the “TNM staging system” for prostate cancer developed by the American Joint Committee on Cancer as further described in “TNM CLASSIFICATION OF MALIGNANT TUMORS”, 7th edition Edited by James D. Brierley, Mary K. Gospodarowicz, Christian Wittekind, Published by UICC 2011.
According to said TNM classification and as used herein the term “M0 CRPC” means, that there are no distant metastases and that the CRPC has not spread to other parts of the body. The term “M1 CRPC” as used herein means that there are distant metastases and that the CRPC has spread to distant parts of the body.
In another embodiment of the present invention, the castration resistant prostate cancer (CRPC) is stage M0 castration resistant prostate cancer (M0 CRPC) or stage M1 castration-resistant prostate cancer (M1 CRPC).
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is prostate cancer, particularly M0 CRPC or M1 CRPC, and the subject or the prostate cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ATM, ARID1A, ATG5, ATR, ATRX, BARD1, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CDC7, CHEK2, DCLRE1C, DYRK1A, EGFR, ERBB3, ERCC3, ERCC5, FANCA, FANCB, FANCD2, FANCI, GEN1, HDAC, KRAS, LIG4, MLH1, MLH3, MSH2, MSH3, MSH6, MYC, NBN, PALB2, PARP4, PIK3CA, PMS2, POLA1, POLL, PRKDC, PTEN, RAD18, RAD50, RAD51, RB1, REV3L, SLX4, TMPRSS2, TMPRSS2-ERG, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, USP1, WDR48, WRN, XPA, XRCC1 and/or XRCC2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is prostate cancer, particularly M0 CRPC or M1 CRPC, and the subject or the prostate cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATM, ARID1A, ATG5, ATR, ATRX, BARD1, BRAF, BRCA2, CCND1, CDC7, DCLRE1C, DYRK1A, EGFR, ERBB3, FANCA, FANCD2, FANCI, KRAS, MSH2, MSH3, MSH6, MYC, PIK3CA, POLA1, PRKDC, PTEN, RAD50, RAD51, RB1, REV3L, SLX4, TMPRSS2-ERG, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, USP1, WDR48, WRN, XPA, XRCC1 and/or XRCC2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is prostate cancer, particularly M0 CRPC or M1 CRPC, and the subject or the prostate cancer is characterized by microsatellite instability, particularly high microsatellite instability.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is prostate cancer, particularly M0 CRPC or M1 CRPC, and the subject or the prostate cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ARID1A, ATM, ATR, ATRX, BARD1, BRAF, BRCA2, CCND1, CDC7, DCLRE1C, EGFR, ERBB3, FANCA, FANCD2, MSH2, MSH3, MSH6, MYC, PIK3CA, POLA1, PTEN, RAD50, RAD51, REV3L, SLX4, TMPRSS2-ERG, TOP2A, TOP2B, USP1, WDR48, and/or WRN gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is prostate cancer, particularly M0 CRPC or M1 CRPC, and the subject or the prostate cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ARID1A, ATR, ATRX, BARD1, BRAF, BRCA2, CCND1, CDC7, DCLRE1C, EGFR, ERBB3, FANCA, FANCD2, MSH2, MSH3, MSH6, MYC, PIK3CA, POLA1, PTEN, RAD50, RAD51, REV3L, SLX4, TMPRSS2-ERG, TOP2A, TOP2B, USP1, WDR48, and/or WRN gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is prostate cancer, particularly M0 CRPC or M1 CRPC, and the subject or the prostate cancer is characterized by one or more functional mutation(s) of the ATM gene/protein, particularly by a deleterious mutation of the ATM gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is prostate cancer, particularly M0 CRPC or M1 CRPC, and the subject or the prostate cancer is characterized by one or more functional mutation(s) in at least five gene(s)/protein(s) selected from APC, ATM, ARID1A, ATG5, ATR, ATRX, BARD1, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CDC7, CHEK2, DCLRE1C, DYRK1A, EGFR, ERBB3, ERCC3, ERCC5, FANCA, FANCB, FANCD2, FANCI, GEN1, HDAC, KRAS, LIG4, MLH1, MLH3, MSH2, MSH3, MSH6, MYC, NBN, PALB2, PARP4, PIK3CA, PMS2, POLA1, POLL, PRKDC, PTEN, RAD18, RAD50, RAD51, RB1, REV3L, SLX4, TMPRSS2, TMPRSS2-ERG, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, USP1, WDR48, WRN, XPA, XRCC1 and/or XRCC2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is prostate cancer and the subject or the prostate cancer is characterized by one or more functional mutation(s), particularly deleterious mutation(s), in ATM gene/protein and/or in BRCA2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is prostate cancer and the subject or the prostate cancer is characterized by one or more biomarker(s) described in the Experimental Section for one or more of the prostate cancer cell lines, particularly the subject or the prostate cancer is characterized by one or more functional mutation(s) in one or more genes which are described in Table 5.
Ovarian Cancer
In another embodiment of the use/method/pharmaceutical compositions/kits of the present invention the hyper-proliferative disease is ovarian cancer.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is ovarian cancer and the subject or the ovarian cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ARID1A, ATM, ATR, BRAF, BRCA1, BRCA2, CDC7, CHEK1, ERBB2, ERBB3, FANCA, FANCM, FBXW7, KRAS, MLH1, MRE11A, MSH3, MSH6, MYC, PALB2, PARP4, PIK3CA, POLH, POLN, POLQ, PRKDC, PTEN, RAD50, REV3L, TDP1, TP53, TOP2A, TOP2B and/or TOPBP1 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is ovarian cancer and the subject or the ovarian cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ARID1A, ATM, ATR, BRAF, BRCA1, CDC7, CHEK1, ERBB2, ERBB3, FANCM, KRAS, MLH1, MSH6, MYC, PIK3CA, POLN, POLQ, PRKDC, PTEN, RAD50, TP53, TOP2A, TOP2B and/or TOPBP1 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is ovarian cancer and the subject or the ovarian cancer is characterized by one or more functional mutation(s), particularly deleterious mutations, in one or more gene(s)/protein(s) selected from APC, ARID1A, ATR, BRAF, BRCA1, CDC7, CHEK1, ERBB3, FANCM, MLH1, MSH6, PIK3CA, POLN, POLQ, PRKDC, PTEN, RAD50, TOP2A, TOP2B and/or TOPBP1 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is ovarian cancer and the subject or the ovarian cancer is characterized by one or more functional mutation(s), particularly deleterious mutations, in one or more gene(s)/protein(s) selected from APC, ATR, BRAF, CDC7, FANCM, PRKDC, TOP2B and/or TOPBP1 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is ovarian cancer and the subject or the ovarian cancer is characterized by one or more functional mutation(s) of the ATM gene/protein, particularly by a deleterious mutation of the ATM gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is ovarian cancer and the subject or the ovarian cancer is characterized by microsatellite instability, particularly high microsatellite instability.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is ovarian cancer and the subject or the ovarian cancer is characterized by one or more functional mutation(s), particularly deleterious mutation(s), in ATM gene/protein and/or in BRCA2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is ovarian cancer and the subject or the ovarian cancer is characterized by one or more biomarker(s) described in the Experimental Section for one or more of the ovarian cancer cell lines, particularly the subject or the ovarian cancer is characterized by one or more functional mutation(s) in one or more genes which are described in Table 5.
Colorectal Cancer
In another embodiment of the use/method/pharmaceutical compositions/kits of the present invention the hyper-proliferative disease is colorectal cancer.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is colorectal cancer and the subject or the colorectal cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ARID1A, ATM, ATRX, BLM, BRAF, BRCA2, CDK12, CHEK2, ERBB3, ERCC3, ERCC5, FANCA, FANCM, FBXW7, FBX018, GEN1, KRAS, MLH1, MSH2, MSH3, MSH6, MYC, NBN, PIK3CA, POLH, POLN, POLQ, PRKDC, RAD50, REV3L, SLX4, TOP2A, TOP2B, TP53, USP1, WRN and/or XRCC2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is colorectal cancer and the subject or the colorectal cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ARID1A, ATM, ATRX, BLM, BRAF, BRCA2, CHEK2, ERBB3, ERCC5, FANCA, FANCM, KRAS, MLH1, MSH2, MSH3, MSH6, MYC, NBN, PIK3CA, POLN, POLQ, PRKDC, RAD50, REV3L, SLX4, TOP2A, TOP2B, TP53, USP1 and/or XRCC2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is colorectal cancer and the subject or the colorectal cancer is characterized by one or more functional mutation(s) in at least three gene(s)/protein(s) selected from APC, ARID1A, ATM, ATRX, BLM, BRAF, BRCA2, CDK12, CHEK2, ERBB3, ERCC3, ERCC5, FANCA, FANCM, FBXW7, FBXO18, GEN1, KRAS, MLH1, MSH2, MSH3, MSH6, MYC, NBN, PIK3CA, POLH, POLN, POLQ, PRKDC, RAD50, REV3L, SLX4, TOP2A, TOP2B, TP53, USP1, WRN and/or XRCC2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is colorectal cancer and the subject or the colorectal cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ARID1A, ATM, ATRX, BLM, BRAF, BRCA2, CHEK2, ERCC5, FANCA, FANCM, KRAS, MLH1, MSH2, MSH3, MSH6, MYC, NBN, PIK3CA, POLN, POLQ, PRKDC, RAD50, REV3L, SLX4, TOP2A, TOP2B, TP53, USP1 and/or XRCC2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is colorectal cancer and the subject or the colorectal cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ARID1A, ATM, ATRX, BLM, BRAF, BRCA2, CHEK2, ERCC5, FANCA, KRAS, MLH1, MSH2, MSH3, MSH6, MYC, NBN, PIK3CA, PRKDC, RAD50, REV3L, SLX4, TOP2A, TOP2B, TP53, USP1 and/or XRCC2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is colorectal cancer and the subject or the colorectal cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATRX, BRAF, BRCA2, ERCC5, FANCA, MLH1, MSH3, MSH6, MYC, PIK3CA, RAD50, REV3L, SLX4, TOP2A, TOP2B, TP53 and/or USP1 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is colorectal cancer and the subject or the colorectal cancer is characterized by one or more functional mutation(s), particularly deleterious mutation(s), in ATM gene/protein and/or in BRCA2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is colorectal cancer and the subject or the colorectal cancer is characterized by microsatellite instability, particularly high microsatellite instability.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is colorectal cancer and the subject or the colorectal cancer is characterized by one or more biomarker(s) described in the Experimental Section for one or more of the colorectal cancer cell lines, particularly the subject or the colorectal cancer is characterized by one or more functional mutation(s) in one or more genes which are described in Table 5.
Lung Cancer
In another embodiment of the use/method/pharmaceutical compositions/kits of the present invention the hyper-proliferative disease is lung cancer.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is lung cancer and the subject or the lung cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATM, ATR, BARD1, BRCA1, BRIP1, FANCD2, FANCI, CCNE1, CDK12, KRAS, MDC1, MSH3, MYC, NBN, NRAS, PIK3CA, PMS2, PRKDC, RAD5OL, REV3L, SLX4, TOP2B, TP53 and/or XRCC3 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is lung cancer and the subject or the lung cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATM, ATR, CCNE1, KRAS, MSH3, MYC, NRAS, PIK3CA, PRKDC, SLX4, TOP2B, TP53 and/or XRCC3 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is lung cancer and the subject or the lung cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATM, ATR, CCNE1, NRAS, SLX4, TOP2B, TP53 and/or XRCC3 gene/protein
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is lung cancer and the subject or the lung cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATM, CCNE1, KRAS, MSH3, MYC, NRAS, PRKDC, SLX4, TOP2B and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is lung cancer and the subject or the lung cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATR, CCNE1, MSH3, PRKDC and/or KRAS gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is lung cancer and the subject or the lung cancer is characterized by one or more functional mutation(s), particularly deleterious mutation(s), in ATM gene/protein and/or in BRCA2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is lung cancer and the subject or the lung cancer is characterized by one or more biomarker(s) described in the Experimental Section for one or more of the lung cancer cell lines, particularly the subject or the lung cancer is characterized by one or more functional mutation(s) in one or more genes which are described in Table 5.
Melanoma
In another embodiment of the use/method/pharmaceutical compositions/kits of the present invention the hyper-proliferative disease is melanoma.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is melanoma and the subject or the melanoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATM, BRAF, PRKDC and/or XRCC3 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is melanoma and the subject or the melanoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATM, BRAF and/or PRKDC gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is melanoma and the subject or the melanoma is characterized by one or more functional mutation(s), particularly deleterious mutation(s), in ATM gene/protein and/or in BRCA2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is melanoma and the subject or the melanoma is characterized by one or more biomarker(s) described in the Experimental Section for one or more of the melanoma cell lines, particularly the subject or the melanoma is characterized by one or more functional mutation(s) in one or more genes which are described in Table 5.
Cervical Cancer
In another embodiment of the use/method/pharmaceutical compositions/kits of the present invention the hyper-proliferative disease is cervical cancer.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is cervical cancer and the subject or the cervical cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from BRIP1, EGFR, REV3L and/or UIMC1 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is cervical cancer and the subject or the cervical cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from EGFR and/or REV3L gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is cervical cancer and the subject or the cervical cancer is characterized by one or more functional mutation(s), particularly deleterious mutation(s), in ATM gene/protein and/or in BRCA2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is cervical cancer and the subject or the cervical cancer is characterized by one or more biomarker(s) described in the Experimental Section for one or more of the cervical cancer cell lines, particularly the subject or the cervical cancer is characterized by one or more functional mutation(s) in one or more genes which are described in Table 5.
Breast Cancer
In another embodiment of the use/method/pharmaceutical compositions/kits of the present invention the hyper-proliferative disease is breast cancer.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is breast cancer and the subject or the breast cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATR, BLM, BRCA1, BRCA2, ERBB2, FANCA, FANCE, FANCI, FBXO18, MLH3, MSH3, MYC, PRKDC, PTEN, RB1, SLX4, TP53 and/or TMPRSS2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is breast cancer and the subject or the breast cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATR, BRCA1, ERBB2, FANCA, MSH3, MYC, PRKDC, PTEN, RB1, TP53 and/or TMPRSS2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is breast cancer and the subject or the breast cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from BRCA1, MSH3, MYC, PRKDC, RB1, TP53 and/or TMPRSS2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is breast cancer and the subject or the breast cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from MSH3, PTEN, RB1 and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is breast cancer and the subject or the breast cancer is characterized by one or more functional mutation(s), particularly deleterious mutation(s), in ATM gene/protein and/or in BRCA2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is breast cancer and the subject or the breast cancer is characterized by one or more biomarker(s) described in the Experimental Section for one or more of the breast cancer cell lines, particularly the subject or the breast cancer is characterized by one or more functional mutation(s) in one or more genes which are described in Table 5.
Pancreatic Cancer
In another embodiment of the use/method/pharmaceutical compositions/kits of the present invention the hyper-proliferative disease is pancreatic cancer.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is pancreatic cancer and the subject or the pancreatic cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ARID1A, BRAF, DYRK1A, ERCC2, FBXW7, KRAS, MLH1, PALB2, PARP4, PRKDC and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is pancreatic cancer and the subject or the pancreatic cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from BRAF, DYRK1A, KRAS, PRKDC and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is pancreatic cancer and the subject or the pancreatic cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from BRAF, DYRK1A, and/or PRKDC gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is pancreatic cancer and the subject or the pancreatic cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from BRAF, PRKDC and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is pancreatic cancer and the subject or the pancreatic cancer is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from DYRK1A, KRAS, PRKDC and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is pancreatic cancer and the subject or the pancreatic cancer is characterized by one or more functional mutation(s), particularly deleterious mutation(s), in ATM gene/protein and/or in BRCA2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is pancreatic cancer and the subject or the pancreatic cancer is characterized by one or more biomarker(s) described in the Experimental Section for one or more of the pancreatic cancer cell lines, particularly the subject or the pancreatic cancer is characterized by one or more functional mutation(s) in one or more genes which are described in Table 5.
Mantle Cell Lymphoma
In another embodiment of the use/method/pharmaceutical compositions/kits of the present invention the hyper-proliferative disease is mantle cell lymphoma.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is mantle cell lymphoma and the subject or the mantle cell lymphoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATM, ATR, ATRX, BAP1, BRCA1, CHEK2, DCLRE1A, ERCC2, FANCM, KRAS, MLH3, MSH3, POLN, PRKDC, RB1, SLX4, TMPRSS2 and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is mantle cell lymphoma and the subject or the mantle cell lymphoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATM, ATR, FANCM, KRAS, MLH3, MSH3, PRKDC, RB1,TMPRSS2 and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is mantle cell lymphoma and the subject or the mantle cell lymphoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from FANCM, KRAS, MSH3 and/or TMPRSS2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is mantle cell lymphoma and the subject or the mantle cell lymphoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from FANCM, MSH3 and/or PRKDC gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is mantle cell lymphoma and the subject or mantle cell lymphoma is characterized by one or more functional mutation(s), particularly deleterious mutation(s), in ATM gene/protein and/or in BRCA2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is mantle cell lymphoma and the subject or the mantle cell lymphoma is characterized by one or more biomarker(s) described in the Experimental Section for one or more of the mantle cell lymphoma cell lines, particularly the subject or the mantle cell lymphoma is characterized by one or more functional mutation(s) in one or more genes which are described in Table 5.
Diffuse Large B-Cell Lymphoma (DLBCL)
In another embodiment of the use/method/pharmaceutical compositions/kits of the present invention the hyper-proliferative disease is diffuse large B-cell lymphoma.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is diffuse large B-cell lymphoma and the subject or the diffuse large B-cell lymphoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from APC, ATM, BRAF, BRIP1, CDC7, ERCC2, FANCD, FEN1, PRKDC, MLH1, MYC, REV3L, TOP2A and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is diffuse large B-cell lymphoma and the subject or the diffuse large B-cell lymphoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATM, BRAF, CDC7, PRKDC, MYC, TOP2A and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is diffuse large B-cell lymphoma and the subject or the diffuse large B-cell lymphoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATM, BRAF, CDC7, MYC, PRKDC, TOP2A and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is diffuse large B-cell lymphoma and the subject or the diffuse large B-cell lymphoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from MYC gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is diffuse large B-cell lymphoma and the subject or the diffuse large B-cell lymphoma is characterized by one or more functional mutation(s) in at least two, particularly at least three gene(s)/protein(s) selected from APC, ATM, BRAF, BRIP1, CDC7, ERCC2, FANCD, FEN1, PRKDC, MLH1, MYC, REV3L, TOP2A and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is diffuse large B-cell lymphoma and the subject or the diffuse large B-cell lymphoma is characterized by one or more functional mutation(s), particularly deleterious mutation(s), in ATM gene/protein and/or in BRCA2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is diffuse large B-cell lymphoma and the subject or the diffuse large B-cell lymphoma is characterized by one or more biomarker(s) described in the Experimental Section for one or more of the diffuse large B-cell lymphoma cell lines, particularly the subject or the diffuse large B-cell lymphoma is characterized by one or more functional mutation(s) in one or more genes which are described in Table 5.
Glioblastoma
In another embodiment of the use/method/pharmaceutical compositions/kits of the present invention the hyper-proliferative disease is glioblastoma.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is glioblastoma and the subject or the glioblastoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATRX, CCNE1, ERBB2, FANCA, PRKDC, PTEN, RAD50, RAD54, TDP2 and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is glioblastoma and the subject or glioblastoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATRX, CCNE1, ERBB2, PRKDC, PTEN, TDP2 and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is glioblastoma and the subject or glioblastoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from CCNE1, ERBB2, PRKDC, PTEN, TDP2 and/or TP53 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is glioblastoma and the subject or glioblastoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from ATRX and/or PTEN gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is glioblastoma and the subject or the glioblastoma is characterized by one or more functional mutation(s), particularly deleterious mutation(s), in ATM gene/protein and/or in BRCA2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is glioblastoma and the subject or the glioblastoma is characterized by one or more biomarker(s) described in the Experimental Section for one or more of the glioblastoma cell lines, particularly the subject or the glioblastoma is characterized by one or more functional mutation(s) in one or more genes which are described in Table 5.
Neuroblastoma
In another embodiment of the use/method/pharmaceutical compositions/kits of the present invention the hyper-proliferative disease is neuroblastoma.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is neuroblastoma and the subject or neuroblastoma is characterized by one or more functional mutation(s) in one or more gene(s)/protein(s) selected from CHEK2, MSH3 and/or PRKDC gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is neuroblastoma and the subject or the neuroblastoma is characterized by one or more functional mutation(s), particularly deleterious mutation(s), in ATM gene/protein and/or in BRCA2 gene/protein.
In another embodiment of the use/method/pharmaceutical composition/kit of the invention the hyper-proliferative disease is neuroblastoma and the subject or the neuroblastoma is characterized by one or more biomarker(s) described in the Experimental Section for one or more of the neuroblastoma cell lines, particularly the subject or the neuroblastoma is characterized by one or more functional mutation(s) in one or more genes which are described in Table 5.
Stratification Methods
Various stratification methods can be used in context of the present invention to identify one or more functional mutation(s) in one or more gene(s), an activation of the ALT pathway and/or microsatellite instability (MSI) in a sample.
Functional Mutation(s)
The determination of functional mutations, particularly of deleterious and activating mutations, of gene(s)/protein(s) is known to the person skilled in the art. Deleterious mutations and activating mutations can be, for example, determined by one or more of the following stratification methods: Next generation sequencing (NGS) (Metzker M L, “Sequencing technologies—the next generation”, Nat Rev Genet. 2010; 11:31-46); Sanger sequencing and other first generation sequencing methods (Lilian T. C. Franca, Emanuel Carrilho and Tarso B. L. Kist, A review of DNA sequencing techniques, Quarterly Reviews of Biophysics 35, 2 (2002), pp. 169-200); PCR, particularly multiplex PCR; Fluorescence in situ hybridization (FISH); array comparative genomic hybridization (array CGH); single nucleotide polymorphism microarray (SNP microarrays), in particular to determine copy number variants (CNVs); or immunohistochemistry (IHC), in particular to determine the loss or overexpression of the respective protein.
The term “NGS” does not denote a single technique; rather, it refers to a diverse collection of post-Sanger sequencing technologies developed in the last decade. These methods include sequencing-by-synthesis (Ronaghi M et al., “A sequencing method based on real-time pyrophosphate”, Science. 1998; 281:363-365), sequencing-by-ligation (Shendure J et al., “Accurate multiplex polony sequencing of an evolved bacterial genome”, Science. 2005; 309:1728-32.16), ion semiconductor sequencing (Rothberg J M et al., “An integrated semiconductor device enabling non-optical genome sequencing.”, Nature. 2011; 475:348-52.17), and others.
Bioinformatics approaches are used for detecting and analyzing the sequence variants from NGS data (Teng S, “NGS for Sequence Variants.”, Adv Exp Med Biol. 2016; 939:1-20). NGS variant detection consists of quality control (to remove potential artifacts and bias from data), sequence alignment (reads are mapped to positions on a reference genome), and variant calling (which is performed by comparing the aligned reads with known reference sequences to find which segments are different with the reference genomes).
The sequence variants detected from NGS can be classified to single nucleotide variants (SNVs), small insertions and deletions (INDELs), and large structural variants (SVs) based on their sequences in length. SNVs, the most common type of sequence variants, are single DNA basepair differences in individuals. INDELs are defined as small DNA polymorphisms including both insertions and deletions ranging from 1 to 50 bp in length. SVs are large genomic alterations (>50 bp) including unbalanced variants (deletions, insertions, or duplications) and balanced changes (translocations and inversions). Copy number variants (CNVs), a large category of unbalanced SVs, are DNA alterations that result in the abnormal number of copies of particular DNA segments.
Variant analysis includes variant annotation which can be used to determine the effects of sequence variants on genes and proteins and filter the functional important variants from a background of neutral polymorphisms.
Variant association analyses connects the functional important variants with complex diseases or clinical traits. The disease-related casual variants can be identified by combining these approaches. Results of these variant analysis are stored in public databases, such as for example COSMIC (the Catalogue Of Somatic Mutations In Cancer, www.cancer.sanger.ac.uk), ClinVar (Landrum M J, Lee J M, Riley G R, et al., “ClinVar: public archive of relationships among sequence variation and human phenotype.”, Nucleic Acids Res. 2014; 42:D980-5), HGMD (Stenson P D, Mort M, Ball E V, et al., “The human gene mutation database: 2008 update.”, Genome Med. 2009; 1:13) or “The Human Variome Project” (http://www.humanvariomeproject.org/), which has curated the gene-/disease-specific databases to collect the sequence variants and genes associated with diseases.
As described above, public data bases, relevant literatures and ongoing evidences associated with the recurrence and function of the gene are used to determine the reportable status of an alteration found from NGS data for the genes of interest. Functional mutations can be classified by any one of the following reportable status: deleterious mutation(s) and activation mutation(s).
Activation of the ALT Pathway
In normal somatic cells, significant telomere shortening leads to p53-dependent senescence or apoptosis (Heaphy and Meeker, J Cell Mol Med. 15(6): 1227-1238 (2011)). Cancer cells rely on telomerase or the alternative lengthening of telomeres (ALT) pathway to overcome replicative mortality. Most neoplastic cells express telomerase to support immortalization and tumor progression. However, approximately 10% to 15% of cancers achieve immortalization via a telomerase-independent mechanism of telomere lengthening, the alternative lengthening of telomeres (ALT) (Cesare A. J., Reddel R. R. Alternative lengthening of telomeres: Models, mechanisms and implications. Nat. Rev. Genet. 2010; 11:319-330.). ALT is a recombination-based mechanism of telomere maintenance characterized by heterogeneous, fluctuating telomere lengths, high levels of telomere sister chromatid exchanges (t-SCEs), abundant extrachromosomal telomeric repeat DNA (ECTR), and a specialized telomeric DNA nuclear structure termed ALT-associated promyelocytic leukemia (PML) bodies (APBs) (Robert L. Dilley, Roger A. Greenberg, ALTernative Telomere Maintenance and Cancer, Trends Cancer. 2015 Oct. 1; 1(2): 145-156). Specific mutational events including recurrent mutations of the Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) or Death-Domain Associated Protein (DAXX) genes have been reported to influence ALT activation and maintenance (Amorim et al, The Role of ATRX in the Alternative Lengthening of Telomeres (ALT) Phenotype, Genes (Basel). 2016 September; 7(9): 66.). Recent studies have implicated the long-noncoding RNA telomeric repeat-containing RNA (TERRA), nuclear receptors, and RPA in the recombinogenic potential of ALT telomeres. ATR protein kinase, a critical regulator of recombination, recruited by the Replication Protein A might be involved in ALT regulation and become a viable target for treatment of ALT tumors (Flynn, R. L.; Cox, K. E.; Jeitany, M.; Wakimoto, H.; Bryll, A. R.; Ganem, N. J.; Bersani, F.; Pineda, J. R.; Suva, M. L.; Benes, C. H.; et al. Alternative lengthening of telomeres renders cancer cells hypersensitive to ATR inhibitors. Science 2015, 347, 273-277.)
Stratification methods known in the art can be used to identify subjects as having a cancer associated with activation of the ALT pathway (i.e., for identifying a cancer as associated with ALT activation, also referred to herein as an ALT cancer or an ALT+ cancer). For example, detection of maintenance of telomeres in the absence of telomerase activity (Bryan et al., EMBO J., 14:4240-4248 (1995)); detection of a pattern of telomere lengths, e.g., by terminal restriction fragment Southem blots, ranging from very short to extremely long, and with a modal length approximately twice that in comparable telomerase-positive or normal cells (Bryan et al., EMBO J., 14:4240-4248 (1995); Gollahon et al., Oncogene, 17:709-717 (1998)), detection of rapid, unsynchronized changes in telomere length cause telomere length heterogeneity (Mumane et al., EMBO J., 13:4953-4962 (1994)), detection of ALT-associated PML bodies (APBs) (Yeager et al., Cancer Res., 59:4175-4179 (1999)), detection of copying of engineered telomeric tags from one telomere to another (Pickett et al. EMBO J., 28:799-809 (2009)), detection of tandem repeat instability at telomeres and the MS32 minisatellite (Jeyapalan et al., Hum. Mol. Genet., 14: 1785-1794 (2005)), detection of Telomere-sister chromatid exchange (T-SCE) (Fan et al. Nucleic Acids Res., 37:1740-1754 (2009)), detection of an increase in the level of telomeric t-circles (Cesare et al., Mol. Cell. Biol., 24:9948-9957 (2004)), detection of single stranded C-strand telomeric DNA (ss-C-strand) (Grudic et al., Nucleic Acids Res., 35:7267-7278 (2007)), detection of C circles (Henson et al., Nat. Biotechnol., 27:1181-1185 (2009)). See, e.g. Henson and Reddel, FEBS Lett. 584(17):3800-3811 (2010); and US20150247866. In some embodiments, a branched DNA assay in RNA in situ hybridization (RNA-ISH) is used, e.g., as described in WO2015/123565.
The activation of the ALT pathway is preferably determined by one of the stratification methods described above.
Microsatellite Instability (MSI)
MSI analysis involves comparing allelic profiles of microsatellite markers generated by amplification of DNA from matching normal wildtype and test samples, which may be mismatch-repair (MMR) deficient. Alleles that are present in the test sample but not in corresponding normal wildtype samples indicate MSL MSI can be for example analyzed by a MSI-PCR method which includes fluorescently labelled primers for co-amplification of microsatellite markers, by MSI-immunohistochemistry (IHC) staining of four MMR pathway proteins: MLH1, PMS2, MSH2, or MSH6, or by computational methods using next generation DNA sequencing (NGS) data detecting an abnormal number of microsatellite repeats: The term “high microsatellite instability” (herein also called “MSI-high”) means that a significant number, particularly at least one, preferably at least two, of microsatellite markers were found: MSI status can be determined by a MSI-PCR Analysis System (e.g. by Promega Corp, Madison, USA) which is based on the use of five nearly monomorphic mononucleotide microsatellite markers (BAT-25, BAT-26, NR-21, NR-24, and MONO-27). In this system high microsatellite instability (“MSI-high”) is defined as the phenotype, in which at least 2 of the tested microsatellite markers (BAT-25, BAT-26, NR-21, NR-24, and MONO-27) are altered in the sample of the subject compared to a normal reference sample. Low microsatellite instability (“MSI-low”) is defined as the phenotype, in which only one of the tested microsatellite markers is altered in the sample of the subject compared to a normal reference sample. Microsatellite stable (MSS) is defined as the phenotype, in which none of the tested microsatellite markers is altered in the sample of the subject compared to a normal reference sample. MSI status can also be determined by a MSI-PCR method using the five microsatellite loci (BAT-25, BAT-26, D2S123, D5S346, and D175250) recommended by the National Cancer Institute (NCI), which are amplified in a single multiplex PCR reaction. In this system high microsatellite instability (“MSI-high”) is defined as the phenotype, in which at least two of the tested mononucleotide microsatellite markers (BAT-25, BAT-26, D2S123, D5S346, and D175250) are altered in the sample of the subject compared to a normal reference sample. Low microsatellite instability (“MSI-low”) is defined as the phenotype, in which only one of the tested mononucleotide microsatellite markers (BAT-25, BAT-26, D2S123, D5S346, and D175250) is altered in the sample of the subject compared to a normal reference sample. Microsatellite stable (MSS) is defined as the phenotype, in which none of the tested microsatellite markers (BAT-25, BAT-26, D2S123, D5S346, and D175250) is altered in the sample of the subject compared to a normal reference sample (Boland C R, et al, A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res. 1998; 58(22):5248-5257).
In this context a “normal reference sample” used for MSI testing can be for example a genomic DNA template provided by the assay kit, or DNA isolated from blood or from another non-cancerous tissue from the subject to be tested,
MSI status can be assessed by computational methods to using next generation DNA sequencing (NGS) data generated from tumor or other tissues. These computational methods include but are not limited to mSINGS (Salipante, S. J. et al, “Microsatellite instability detection by next generation sequencing”, Clin. Chem. 60, 1192-1199, 2014), MSISensor (Niu B et al., “MSlsensor: microsatellite instability detection using paired tumor-normal sequence data.”, Bioinformatics. 2014; 30(7):1015-1016.), MANTIS (Microsatellite Analysis for Normal Tumor InStability) (Kautto E A et al, “Performance evaluation for rapid detection of pan-cancer microsatellite instability with MANTIS”, Oncotarget. 2016 Dec. 12), MOSAIC (Ronald J Hause et al, “Classification and characterization of microsatellite instability across 18 cancer types”, Nature Medicine 22, 1342-1350, 2016), or the Foundation Medicine NGS-MSI analysis using 114 intronic homopolymer repeat loci (10-20 bp long in the human reference genome) (Michael J. Hall et al, J Clin Oncol 34, 2016 (suppl 4S; abstr 528).
In these computational methods MSI status can be defined by cutoff numbers for MSI-high, MSI-low or MSS based on an index score for each sample determined by using computer algorithm and validated by comparing to the other MSI detection methods.
MSI can also be detected by an immunohistochemistry (IHC) staining of four microsatellite marker proteins: MLH1, PMS2, MSH2, or MSH6. If any of these four proteins are found significantly reduced in quantity by IHC, particularly if at least one of the four proteins cannot be detected by IHC, the sample is labeled as MSI-high.
In another embodiment of the use/method/pharmaceutical composition/kit of the present invention the microsatellite instability is characterized by the alteration of one or more, preferably of two or more, microsatellite markers selected from BAT-25, BAT-26, NR-21, NR-24, MONO-27, D2S123, D5S346, D175250 in the sample of the subject compared to a normal reference sample and/or microsatellite instability is characterized by the absence of one or more proteins selected from MLH1, PMS2, MSH2 and/or MSH 6.
In another embodiment of the use/method/pharmaceutical composition/kit of the present invention the microsatellite instability is characterized by the alteration of one or more, preferably of two or more, microsatellite markers selected from BAT-25, BAT-26, NR-21, NR-24, MONO-27, D2S123, D5S346 and/or D17S250 in the sample of the subject compared to a normal reference sample.
In another embodiment of the use/method/pharmaceutical composition/kit of the present invention the microsatellite instability is characterized by the alteration of one or more, preferably of two or more, microsatellite markers selected from BAT-25, BAT-26, NR-21, NR-24 and/or MONO-27 in the sample of the subject compared to a normal reference sample.
In another embodiment of the use/method/pharmaceutical composition/kit of the present invention the microsatellite instability is characterized by the alteration of at least one, preferably of at least two, microsatellite markers selected from BAT-25, BAT-26, NR-21, NR-24, and MONO-27 in the sample of the subject compared to a normal reference sample.
In another embodiment of the use/method/pharmaceutical composition/kit of the present invention the microsatellite instability is characterized by the alteration of one or more microsatellite markers selected from BAT-25, BAT-26, D2S123, D5S346 and/or D17S250 in the sample of the subject compared to a normal reference sample.
In another embodiment of the use/method/pharmaceutical composition/kit of the present invention the microsatellite instability is characterized by the alteration of at least one, preferably of at least two, microsatellite markers selected from BAT-25, BAT-26, D2S123, D5S346 and/or D17S250 in the sample of the subject compared to a normal reference sample.
In another embodiment of the use/method/pharmaceutical composition/kit of the present invention the microsatellite instability is characterized by the absence of one or more proteins selected from MLH1, PMS2, MSH2 and/or MSH 6.
In another embodiment is determined by immunohistochemistry staining of MMR pathway proteins (MLH1, PMS2, MSH2, or MSH6). In this method the “MSI-high” phenotype is characterized by a significant reduction in quantity of one or more of the proteins selected from MLH1, PMS2, MSH2, and/or MSH6, particularly by a loss of expression of at least one of the proteins selected from MLH1, PMS2, MSH2 and/or MSH6. In this context the term “loss of expression” means no positive nuclear staining in a tumor cell, particularly in a tumor cell, by IHC.
The percentages in the tests and examples which follow are, unless indicated otherwise, percentages by weight; parts are parts by weight. Solvent ratios, dilution ratios and concentration data for liquid/liquid solutions are based in each case on volume.

EXPERIMENTAL SECTION

Preparation of Compound A
Compound A was prepared according to the procedure described in example 111 of International Patent Application WO2016020320.

Example 1

Treatment of Different Prostate Cancer Cell Lines with Compound A

LAPC-4 human prostate cancer cells were obtained from the VTT Technical Resarch Center (Finland). They were plated in RPMI 1640 (RPMI=Roswell Park Memorial Institute) medium without phenol red+10% charcoal-stripped FCS (FCS=Fetal Calf Serum)+2 mM L-Glutamine at 4000 cells/well in a 96-well microtiter plate. After 1 day, the cells were treated with R1881 (1 nM) and Compound A (day 0). Cell number was determined by Alamar Blue staining (2 h) at day 7. Fluorescence was determined in a Victor X3 device (excitation 530 nm; emission 590 nm). The inhibition of cell growth was calculated by normalization with respect to the fluorescence reading (cell number) measured at the end of the experiment for cells treated with R1881 alone compared to the fluorescence reading (cell number) measured at the end of the experiment for DMSO-treated cells.
VCaP human prostate cancer cells were obtained from the VTT Technical Research Center (Finland). They were plated in DMEM medium (DMEM=Dulbecco's Modified Eagle Medium) with stable glutamine+10% FCS at 16000 cells/well in a 96-well microtiter plate. Compound A was added at day 0. Cell number was determined by Alamar Blue staining (2 h) at day 0 and day 7. Fluorescence was determined in a Victor X3 device (excitation 530 nm; emission 590 nm). The inhibition of cell growth was calculated by normalization with respect to the fluorescence reading (cell number) measured at the end of the experiment for cells treated with R1881 alone compared to the fluorescence reading (cell number) measured at the start of the experiment for DMSO-treated cells.
LNCaP human prostate cancer cells were obtained from the DSMZ-German Collection of Microorganisms and Cell Cultures (Germany) (DSMZ ACC-256). They were plated in RPMI1640 medium without phenol red+10% charcoal-stripped FCS at 600 cells/well in a 384-well white plate. R1881 (1 nM) and Compound A were added at day 0. Cell number was determined by CellTiter-Glow (Promega) at day 0 and day 6. Luminescence was determined in Victor X3. The inhibition of cell growth was calculated by normalization with respect to the fluorescence reading (cell number) measured at the end of the experiment for cells treated with R1881 alone compared to the fluorescence reading (cell number) measured at the start of the experiment for DMSO-treated cells. 22RV1 human prostate cancer cells were obtained from the American Type Culture Collection (ATCC CRL-2505). They were plated in RPMI1640 medium supplemented with 10% FCS at 5000 cells/well in a 96-well microtiter plate. After 24 h, the cells from one microtiter plate were stained with crystal violet (==>0 plate), whereas the cells in the test plates were exposed continuously for 4 days to test substances. Cell proliferation was determined by staining with crystal violet. The absorbance was determined photometrically at 595 nm using a Tecan Sunrise instrument. The percentage change of cell growth was calculated by normalization with respect to the absorbance reading (cell number) at the beginning of treatment of cells (0 plate) and the absorbance reading (cell number) of the untreated control group. DU-145 human prostate cancer cells were obtained from the DSMZ-German Collection of Microorganisms and Cell Cultures (Germany) (DSMZ ACC-261). They were plated in DMEM/Ham's F12 medium at 5000 cells/well in a 96-well microtiter plate. After 24 h, the cells from one microtiter plate were stained with crystal violet (==>0 plate), whereas the cells in the test plates were exposed continuously for 4 days to test substances. Cell proliferation was determined by staining with crystal violet. The absorbance was determined photometrically at 595 nm using a Tecan Sunrise instrument. The percentage change of cell growth was calculated by normalization with respect to the absorbance reading (cell number) at the beginning of treatment of cells (0 plate) and the absorbance reading (cell number) of the untreated control group.
PC-3 human prostate cancer cells were obtained from the DSMZ-German Collection of Microorganisms and Cell Cultures (Germany) (DSMZ ACC-465). They were plated in DMEM/Ham's F12 medium with stable Glutamine+10% FCS at 5000 cells/well in a 96-well microtiter plate. After 24 h, the cells from one microtiter plate were stained with crystal violet (==>0 plate), whereas the cells in the test plates were exposed continuously for 4 days to test substances. Cell proliferation was determined by staining with crystal violet. The absorbance was determined photometrically at 595 nm using a Tecan Sunrise instrument. The percentage change of cell growth was calculated by normalization with respect to the absorbance reading (cell number) at the beginning of treatment of cells (0 plate) and the absorbance reading (cell number) of the untreated control group.
Treatment of Further Cancer Cell Lines with Compound A
The cells (See Table 3: Test systems) were seeded in their appropriate medium supplemented with 10% FCS at 1,250-5,000 cells/well (depending on their proliferation rate) in 96-well microtiter plates. Cells were allowed to adhere for 24 h, and then the compound was added using a digital dispenser. The final concentration of Compound A was between 1E-09 mol/L and 3E-06 mol/L, and the final concentration of the solvent DMSO was 0.03%. After 4 days of continuous incubation, the cells were fixed with glutaraldehyde, stained with crystal violet, and the absorbance was recorded at 595 nm. All measurements were done in quadruplicates. The values were normalized to the absorbance of solvent treated cells (=100%) and the absorbance of a reference plate which was fixed at the time point of compound application (=0%). Half-maximal growth inhibition (IC₅₀) was determined as compound concentration, which was required to achieve 50% inhibition of cellular growth using a 4-parameter fit. Non-adherent growing GRANTA-519, Jeko-1, JVM-2, NCI-H929, Rec-1 and SU-DHL-8 cells were seeded in 150 μl of growth medium at 4000 cells/well (NCI-H929, 5000 cells/well) in 96-well microtiter plates and incubated for 24 h at 37° C. Compound A was added using a digital dispenser to the cells in the test plates and incubated continuously for 4 days at 37° C. To determine cell viability (corresponding to cell number) CTG solution (Promega Cell Titer Glo solution, # G755B and G756B) was added. After incubation for further 10 min luminescence was measured using Perkin Elmer Victor V equipment. All measurements were done in quadruplicates. The percentage change of cell viability was calculated by normalization with respect to the luminescence reading (cell number) at the beginning of treatment of cells (a reference plate was measured at the time point of compound application to the measurement plates) and the luminescence reading (cell number) of the untreated control group. Half-maximal growth inhibition (IC₅₀) was determined as compound concentration, which was required to achieve 50% inhibition of cellular growth using a 4-parameter fit.
Treatment of Isogenic Cancer Cell Lines with Compound A
The isogenic DLD-1 cell lines DLD-1 parental, DLD-1 BRCA2 (−/−) and DLD-1 ATM (−/−) (see Table 3: Test systems) were seeded in RPMI 1640 (RPMI=Roswell Park Memorial Institute) medium without phenol red+10% charcoal-stripped FCS (FCS=Fetal Calf Serum)+2 mM L-Glutamine+25 mM Sodium Bicarbonate at 2,500 cells/well in a 96-well microtiter plate. Cells were allowed to adhere for 24 h, and then the compound was added using a digital dispenser. The final concentration of Compound A was between 7E-10 mol/L and 5E-06 mol/L, and the final concentration of the solvent DMSO was 0.03%. After 7 days of continuous incubation at 37° C. cell viability was determined (corresponding to cell number) using CTG solution (Promega Cell Titer Glo solution, # G755B and G756B) was added. After incubation for further 10 min luminescence was measured using Perkin Elmer Victor V equipment. All measurements were done in quadruplicates. The percentage change of cell viability was calculated by normalization with respect to the luminescence reading (cell number) at the beginning of treatment of cells (a reference plate was measured at the time point of compound application to the measurement plates) and the luminescence reading (cell number) of the untreated control group. Half-maximal growth inhibition (IC50) was determined as compound concentration, which was required to achieve 50% inhibition of cellular growth using a 4-parameter fit.

TABLE 3

Test systems

Cell line	Tumor entity	Source

A2780	ovarian carcinoma	ECACC-93112519
AsPC1	pancreatic carcinoma	ATCC CRL-1682
BxPC3	pancreatic carcinoma	ATCC CRL-1687
Caco2	colorectal carcinoma	DSMZ ACC-169
GRANTA-519	mantle cell lymphoma	DSMZ ACC-342
DLD-1 (parental)	colorectal carcinoma	HD PAR-008
DLD-1 BRCA2 (−/−)	colorectal carcinoma	HD 105-007
DLD-1 ATM (−/−)	colorectal carcinoma	HD 105-061, clone 11517
HeLa	human cervical adenocarcinoma	ATCC CCL-2
HT-144	malignant melanoma	ATCC HTB-63
HT-29	colorectal carcinoma	DSMZ ACC-299
Jeko-1	mantle cell lymphoma	DSMZ ACC-553
LOVO	colorectal carcinoma	DSMZ ACC-350
MDA-MB-436	mammary carcinoma	CLS 300278
MDA-MB-468	mammary carcinoma	ATCC HTB-132
MIAPaca-2	pancreatic carcinoma	ATCC CRL-1420
NCI-H460	non-small cell lung carcinoma	ATCC HTB-177
NCI-H929	multiple myeloma	ATCC CRL-9068
OVCAR-8	ovarian carcinoma	NCI-60 panel; Sample ID No. 25
REC-1	mantle cell lymphoma	ATCC CRL-3004
SK-OV-3	ovarian carcinoma	ATCC HTB-77
SU-DHL-8	germinal center B cell DLBCL	DSMZ ACC-573
JVM-2	mantle cell lymphoma	ATCC CRL-3002
TMD-8	activated B cell DLBCL	Charite, Berlin, Germany
C4-2B	prostate cancer	MD Anderson Cancer Center
HCT116	colorectal carcinoma	DSMZ ACC-581
IGR-OV-1	ovarian carcinoma	NCI-60 panel; Sample ID No. 26
NCI-H23	non-small cell lung carcinoma	ATCC CRL-5800
NCI-H1838	non-small cell lung carcinoma	ATCC CRL-5899
NCI-H1703	non-small cell lung carcinoma	ATCC CRL-5889
A549	non-small cell lung carcinoma	DSMZ ACC-107
NCI-H2030	non-small cell lung carcinoma	ATCC CRL-5914
HCC70	mammary carcinoma	ATCC CRL-2315
M059J	glioblastoma	ATCC CRL-2366
U-87MG	glioblastoma	ATCC HTB-14
SH-SY5Y	neuroblastoma	ATCC CRL-2266

ATCC=American Type Culture Collection; NCI=National Cancer Institute; CLS=Cell Line Service GmbH, Germany; DSMZ=Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Germany; MD Anderson Cancer Center, Houston, USA; HD=Horizon Discovery Ltd
Results:
The genetic mutations and DNA copy number alterations of the above-mentioned cancer cell lines were determined by targeted whole exome sequencing testing and/or acquired from the public databases of Cancer Cell Line Encyclopedia (CCLE, Barretina, Caponigro, Stransky et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 2012 28; 483(7390:603-7.), Genentech Panel (Klijn C, Durinck S, Stawiski E W, Haverty P M, Jiang Z, et al, A comprehensive transcriptional portrait of human cancer cell lines. Nat Biotechnol. 2015 March; 33(3):306-12.) and Sanger Cell Line Panel in COSMIC database (www.cancer.sanger.ac.uk; “COSMIC: exploring the world's knowledge of somatic mutations in human cancer”, Forbes et al., Nucleic Acids Res. 2015, January; 43 (Database issue):D805-11. doi: 10.1093/nar/gku1075. Epub 2014 Oct. 29).
Mutations in DNA damage (DDR) or mismatch repair (MMR) genes as well as mutations in genes inducing oncogenic replication stress or in TP53/tumor suppressor genes of these cancer cell lines (Table 3) are listed in Table 4, selected functional mutations of these cell lines are described in Table 5.
The activity of Compound A in these cell lines was tested. As shown in Table 4, Compound A inhibited the proliferation of the tumor cell lines tested. These results demonstrate that Compound A potently inhibits the proliferation of human tumor cell lines when tested as single agent and that the genetic background of the cells impacts their sensitivity to ATR inhibition.
The activity of Compound A was also tested in isogenic cell lines, in the parental DLD-1 cells and in two DLD-1 cell clones that are deficient for BRCA2 or ATM: DLD-1 BRCA2 (−/−) and DLD-1 ATM (−/−). As shown in Table 6, Compound A inhibited the proliferation of the parental DLD-1 to a lesser extent than the mutant cell lines. The strongest effect was detectable in ATM deficient DLD-1 cells. These results demonstrate that mutations in the genes BRCA2 and ATM sensitize tumor cells to treatment with Compound A.

TABLE 4

Inhibition of tumor cell proliferation by Compound A

			Oncogenic
			replication stress or
			TP53/tumor	in vitro
Indication	Cell line	DDR/MMR defects	suppressors	(IC50, nM)

Prostate	LNCaP	ARID1Afs, ATG5fs,	APCR2714C,	18
		ATMA1119V/K1572N, ATRXEE2264-	ATRK1379N,
		2265E, BRCA2fs, CHEK2T430N,	ERBB3K177N,
		ERCC3A740T; R391W, ERCC5L1023I,	MYCN45S,
		FANCAE369D, Q652*, HDAC2A62V,	PTENfs,
		MLH3I541V, MSH3PPA66-68-; fs,	TOP2Afs,
		POLBfs, POLHD631G, PRKDCfs,	TOP2BG323*/V889A
		RAD50fs, RAD54LL532M,
		RB1splice_acceptor, SLX4S605N,
		TDP2T308S, TP53BP1R639Q; Q111*,
		TRRAPR2665W; P3554L, WDR48G107*,
		XRCC3P87L, XRCC4L70M
Prostate	22Rv1	ATMK1101E, ARID1Afs, BARD1fs,	PIK3CAQ546R,	36
		BRCA2V1810I, fs, DCLRE1Cfs,	ATRfs, BRAFL597R,
		FANCAfs, MSH3fs, NBNR43Q,	ERBB3R683Q,
		PALB2V1123M, PARP4R970W,	TP53Q331R
		PRKDCfs, RAD18L314V,
		RAD50T532I, SLX4fs, TP53BP1fs,
		USP1fs, WRNfs, XRCC2fs
Prostate	VCaP	MSH3PPA66-68-, MSH6fs	CCND1S219N,	51
			TMPRSS2-ERG,
			MYCamp,
			TP53R248W
Prostate	LapC4	ARID1A-2138-2139X, BAP1P723L,	ATRR1951*stop/amp,	55
		BRCA2fs, CDK12W1459X,	CDC7A342V/D571G,
		ERCC2E313K,	EGFRV980D,
		ERCC3R642Q/V443A/E259D,	TP53H178PX, R175H
		ERCC5G1080R,
		FANCD2N405S, P714L, P1081X,
		splice_donor, GEN1K42E, H2AFXN95S,
		LIG4T219A, MLH3K585X,
		MSH2M300R, splice_donor,
		MSH3K381X, PALB2V398A,
		PARP1W589C, PARP3H441R,
		PARP4T1170I, V1065A, Q1059R, I1039T,
		V626D, POLHD140N,
		POLQS1797I, M587I, PRKDCQ4041H,
		RAD17R49X, RAD51C320Y,
		RAD54BI164T, REV3LS2862T, P2172L,
		TRRAPD394G, M1087I, P2026L, H3174Y,
		A3655V, WDR48S611P,
		WRNE3X, K1126X
Prostate	DU-145	ATG5splice_donor, BRCA1E962K,	KRASamp,	110
		BRCA2S2284L, BRIP1T132N,	DYRK1R226H,
		DYRK1AR226H, FANCBG702W,	TMPRSS2splice_acceptor,
		FANCIfs, GEN1Q554H, LIG4R32C,	TP53V274F; P223L
		MLH1A586V, splice, MSH2L736I,
		MSH6S1067I, PMS2H189Y,
		POLA1A550S, POLLA285T,
		POLQV124M, PRKDCfs, RAD50N509K,
		RB1K715*, REV3LR2523C,
		RPA2E252D, TP53BP1splice_acceptor,
		TRRAPfs; A1389T, UIMC1A96D,
		UBE2Nfs, USP1fs, XPAfs,
		XRCC1splice_acceptor, XRCC2fs
Prostate	PC3	MSH3AAAAAAAAPP55-64A; PPA66-68,	MYCamp, TP53fs	490
		PRKDCfs
Prostate	C4-2B	MSI-H,	PTENdel	50
		ARID1A_c.854delG_p.G285fs*78,
		ATRX-E2265del, MSH2loss, TRRAP-
		Q1984*
Breast	HCC70	MSH3PPA66-68-, RB1DN479-480D	PTENfs, TP53R248Q	27
Breast	MDA-MB-436	BRCA1splice_donor, FANCIS812G,	TMPRSS2V101F,	120
		MLH3F92L, PRKDCL1824F; fs,	MYCamp+, TP53fs
Breast	MDA-MB-468	BLMD554V, BRCA2M965I,	ERBB2fs, ATRP?	130
		FANCAQ869*, FANCEG245-,	(2633 + 5A > G,
		FBXO18G193A, SLX4E1784Q	Substitution − intronic)
			PTENsplice_donor,
			TP53R273H
Cervix	HeLa	BRIP1R855H, REV3LQ2891*,	EGFRI646L	150
		UIMC1R536W
Colorectal	LOVO	MSI-H, ARID1A-F2141fs*59, ATM-	APCR1114*; R2816Q;	71
		splice site 1236-2_1237delAGGC,	fs,
		CHEK2-T389fs*25, ERCC3Q711R,	KRASG13D
		FANCAR350W, FBXW7R505C, MSH2-
		G71del, MSH3L795H, NBNfs,
		POLHT477I, BLMfs, POLQfs, PRKDCfs,
		RAD50fs, XRCC2-L117fs*17
Colorectal	HT29	FANCMfs, POLNR761, POLQS1819,	APCE853*, fs,	160
		PRKDCfs, WRNL1255V	MYCamp,
			BRAFV600E, T119S,
			PIK3CAP449T,
			TP53R273H
Colorectal	Caco2	none	APCQ1367*,	240
			ERBB3D857N
Colorectal	HCT-116	ATMA1127V, ATRXTK1529-1530K,	ERBB3Q261*,	25
		BRCA2fs, CDK12P250H, CHEK2L398P,	TOP2Afs,
		ERCC5splice_donor, FANCAfs,	TOP2BR651H,
		FBXO18A1062V, GEN1R401Q,	KRAS G13D,
		MLHlS252*, MSH3fs, MSH6fs,	PIK3CA H1047R
		POLHA112T, POLQK2571N,
		PRKDCY2964C, RAD50fs, REV3Lfs,
		SLX4A1461fs*2,
		TRRAPH3023Y; T3663A, USP1R180*,
		WRNE480V
Glioblastoma	U-87MG	RAD50D515G, RAD54LR691Q	ATRXN564S	64
			PTENsplice_donor,
Glioblastoma	M059J	FANCAR1409W, PRKDCfs,	CCNE1R95L,	80
		RAD54BP98L, TDP2R317*	ERBB2W452S
			PTENfs, TP53E286K
Lung	NCI-H1838	ATMW1279*, BRCA1C328Y,	ATRP1991S,	24
		CDK12R1473Q, RAD50L347P,	TP53R273L
		MSH3AAAAAAAAPP55-64A; PPA66-68-,
		PRKDCfs
Lung	NCI-H1703	ATMV1521L; G1998E, BRCV1G890V,	TP53splice_donor	46
		FANCD2V97I, MSH3AAAAAAAAPP55-
		64A; PPA66-68-, PRKDCTs
Lung	NCI-H460	NBNG224A, REV3LQ1367L,	KRASQ61H,	65
		MSH3AAAAAAAAPP55-64A, PRKDCfs	PIK3CAE545K,
			MYCamp
Lung	NCI-H2030	MSH3AAAAAAAAPP55-64A; PPA66-68-,	KRASG12C,	160
		PRKDCfs	TP53G262V
Lung	NCI-H23	ATMQ1919P, BARD1A168T,	CCNE1D83N,	18
		BRIP1E1054A, FANCID1048Y,	KRASG12C/amp,
		MSH3E251*, MDC1N1233S, NBNV153I,	TOP2BH977Y,
		PMS2E491K, PRKDCfs, SLX4ED1148-	MYCamp,
		1149D	NRASamp,
			TP53M246I
Lung	A549	ATRsplice_acceptor, MSH3PPA66-68-,	KRASG12S,	29
		PRKDCfs	ATRsplice,
			CCNE1amp
Lymphoma, B	SU-DHL-8	ATMK1964E, FANCD2R1165Q,	MYCp72S, Q10H,	9
cell		FEN1L190V, REV3LV1004E, PRKDCfs	BRAFT599TT,
			CDC7K42N,
			TOP2AG1197E,
			TP53R249G; Y234N
Lymphoma, B	TMD-8	BRIP1S59P, ERCC2H148R, MLH1V16L	APCK1170E,	179
cell			MYCF3L
Lymphoma,	REC-1	ATMS707P/amp, PRKDCK3872R; fs,	KRASamp,	10
mantle cell		POLNN382S	TP53Q317*; G245D
Lymphoma,	Jeko-1	ATMamp, ATRXR246C, BAP1S609G,	ATRT1751A,	18
mantle cell		BRCA1N742S, CHEK2V218A,	TMPRSS2Y82D,
		DCLRE1AR1002C, ERCC2V231M,	TP53fs
		MLH3I397-, PRKDCfs, RB1R621S,
		SLX4G395C
Lymphoma,	GRANT	ATMR2832C	none	30
mantle cell	A-519
Lymphoma,	JVM-2	FANCMQ1701*,	none	32
mantle cell		MSH3AAAAAAAAPP55-64A, PRKDCfs
Melanoma	HT-144	ATMW2845*, PRKDCfs, XRCC3E278K	BRAFV600E	40
Neuroblastoma	SH-SY5Y	CHEK2fs, MSH3PPA66-68-, PRKDCfs	none	13
Ovarian	A2780	ARID1AQ1430, R1721fs4, ATMP604S,	ATRI123V,	21
		FANCMfs, PARP4G630E, POLHR356Q,	ERBB3V1082I,
		PRKDCfs	PTENK128_R130del,
			TOP2BV530I,
			BRAFV226M,
			PIK3CAE365K
Ovarian	SK-OV-3	ARID1AQ586*,	APCfs,	33
		ATMsplice_acceptor, FANCMA205V,	TOPBP1N295S,
		FBXW7R505L, TDP1Y46C	PIK3CAH1047R,
			KRASamp,
			CDC7del, TP53fs
Ovarian	IGROV-1	MSI-H, ARID1AD1850fs4, G276fs87,	ERBB3K742,	96
		ATMR248Q, BRCA1K654fs*47,	PIK3CAR38C; *1069W,
		BRCA2P3150T, CHEK1fs,	PTENfs,
		FANCA3prime_UTR, MLH1S505fs*3,	TOP2AH605Q,
		MRE11AR525K,	TOPBP1D395G,
		MSH3G539V; F780L; D943N, MSH6fs,	TP53Y126C
		PALB2T787I, POLQfs; L45I, POLNfs,
		PRKDCC1454Y, Y155C, RAD50fs,
		RAD52E130K, RB1fs, TDP1N179S,
		TRRAPS2051F, USP1V636I,
		UIMC1A418T
Ovarian	OVCAR8	ATMV613L, MSH6T727S,	APCA1225S,	110
		REV3LL3040V	ERBB2G776V,
			KRASP121H,
			MYCamp,
			TP53splice_acceptor
Pancreas	BxPC3	ERCC2R156Q, PRKDCfs	BRAFVTAPTP487-	44
			492A, TP53Y220C
Pancreas	AsPc-1	FBXW7R465C, PARP4M1110L,	DYRK1AS14C,	49
		PRKDCfs	KRASG12D, TP53fs
Pancreas	MIAPaCa2	ARID1AP1940L, MLH1T270I,	KRASG12C,	380
		PALB2S64L	TP53R248W

TABLE 5

Functional mutations of genes of tested cell lines

Functional Mutation

			TP53/tumor	oncogenic replication
Indication	Cell line	DDR/MMR deleterious	suppressors	stress

Prostate	LNCaP	ARID1Afs, ATGfs,	TP53BP1R639Q; Q111*	ATRK1379N,
		ATRXEE2264-2265E,		ERBB3K177N,
		BRCA2fs,		MYCN45S, TOP2Afs,
		FANCAE369D, Q652*,		TOP2BG323*/V889A
		MSH3PPA66-68-; fs,
		PRKDCfs, RAD50fs,
		RB1splice_acceptor,
		WDR48G107*
Prostate	22Rv1	ARID1Afs, BARD1fs,	TP53Q331R,	PIK3CAQ546R, ATRfs,
		BRCA2V1810I, fs,	TP53BP1fs	BRAFL597R,
		DCLRE1Cfs, FANCAfs,		ERBB3R683Q
		MSH3fs, PRKDCfs,
		SLX4fs, USP1fs, WRNfs,
		XRCC2fs
Prostate	VCaP	MSH3PPA66-68-,	TP53R248W	CCND1S219N,
		MSH6fs		TMPRSS2-ERG,
				MYCamp
Prostate	LapC4	ATM splice donor,	TP53H178PX,	ATRR1951*stop/amp,
		BRCA2fs, FANCD2splice_donor,	R175H	CDC7A342V/D571G,
		MSH2splice_dono		EGFRV980D
Prostate	DU-145	ATG5splice_donor,	TP53V274F;	KRASamp,
		FANCIfs, PRKDCfs,	P223L,	DYRK1R226H,
		RB1K715*, TRRAPfs;	TP53BP1splice_acceptor	TMPRSS2splice_acceptor
		UBE2Nfs, USP1fs, XPAfs,
		XRCC1splice_acceptor,
		XRCC2fs
Prostate	PC3	MSH3AAAAAAAAPP55-	TP53fs	MYCamp
		64A; PPA66-68-,
		PRKDCfs
Prostate	C4-2B	MSI-H,	PTENdel	none
		ARID1A_c.854delG_p.G285fs*78,
		MSH2loss, TRRAP-
		Q1984*, ATRX-E2265del
Breast	HCC70	MSH3PPA66-68-,	PTENfs,	none
		RB1DN479-480D	TP53R248Q
Breast	MDA-MB-436	BRCA1splice_donor,	TP53fs	TMPRSS2V101F,
		PRKDCL1824F; fs		MYCamp
Breast	MDA-MB-468	FANCAQ869*	PTENsplice_donor,	ERBB2fs, ATRP?
			TP53R273H	(2633 + 5A > G,
				Substitution − intronic)
Cervix	HeLa	REV3LQ2891*	none	EGFRI646L
Colon	LOVO	MSI-H, ARID1A-	APCR1114*	KRASG13D
		F2141fs*59, ATM-splice
		site 1236-
		2_1237delAGGC,
		CHEK2-T389fs*25,
		MSH2-G71del, NBNfs,
		BLMfs, POLQfs,
		PRKDCfs, RAD50fs,
		XRCC2-L117fs*17
Colon	HT29	FANCMfs, POLNR761*,	APCE853*, fs,	MYCamp,
		POLQS1819*, PRKDCfs	TP53R273H	BRAFV600E, T119S,
				PIK3CAP449T
Colon	Caco2	none	APCQ1367*	ERBB3D857N
Colon	HCT-116	ATMA1127V,	none	ERBB3Q261*, TOP2Afs,
		ATRXTK1529-1530K,		TOP2BR651H, KRASG13D,
		BRCA2fs,		PIK3CA H1047R
		ERCC5splice_donor,
		FANCAfs,
		MLH1S252*, MSH3fs,
		MSH6fs, RAD50fs,
		REV3Lfs,
		SLX4A1461fs*2,
		USP1R180*
Glioblastoma	U-87MG	none	PTENsplice_donor	ATRXN564S
Glioblastoma	M059J	PRKDCfs, TDP2R317*	PTENfs,	CCNE1R95L,
			TP53E286K	ERBB2W452S
Lung	NCI-H1838	ATMW1279*,	TP53R273L	ATRP1991S
		MSH3AAAAAAAAPP55-
		64A; PPA66-68-,
		PRKDCfs
Lung	NCI-H1703	ATMG1998E,	TP53splice_donor,	none
		MSH3AAAAAAAAPP55-
		64A; PPA66-68-,
		PRKDCfs
Lung	NCI-H460	MSH3AAAAAAAAPP55-	none	KRASQ61H,
		64A, PRKDCfs		PIK3CAE545K, MYCamp
Lung	NCI-H2030	MSH3AAAAAAAAPP55-	TP53G262V	KRASG12C
		64A; PPA66-68-,
		PRKDCfs
Lung	NCI-H23	ATMQ1919P,	TP53M246I	CCNE1D83N,
		MSH3E251*,		KRASG12C/amp,
		PRKDCfs, SLX4ED1148-		TOP2BH977Y, MYCamp,
		1149D		NRASamp
Lung	A549	MSH3PPA66-68-,	none	KRASG12S, ATRsplice,
		PRKDCfs		CCNEamp
Lymphoma, B	SU-DHL-8	ATMK1964E, PRKDCfs,	TP53R249G; Y234N,	MYCp72S, Q10H,
cell				BRAFT599TT,
				CDC7K42N,
				TOP2AG1197E
Lymphoma, B	TMD-8	none	none	MYCF3L
cell
Lymphoma,	REC-1	PRKDCfs	TP53Q317*;	KRASamp
mantle cell			G245D,
Lymphoma,	Jeko-1	MLH3I397-, PRKDCfs,	TP53fs	ATRT1751A,
mantle cell		RB1R621S		TMPRSS2Y82D
Lymphoma,	GRANTA-519	ATMR2832C	none	none
mantle cell
Lymphoma,	JVM-2	FANCMQ1701*,	none	none
mantle cell		MSH3AAAAAAAAPP55-
		64A, PRKDCfs
Melanoma	HT-144	ATMW2845*, PRKDCfs	none	BRAFV600E
Neuroblastoma	SH-SY5Y	CHEK2fs, MSH3PPA66-	none	none
		68-, PRKDCfs,
Ovarian	A2780	ARID1AQ1430, R1721fs4,	PTEN	ATRI123V, ERBB3V1082I,
		ATMP604S, FANCMfs,	K128_R130del	TOP2BV530I,
		PRKDCfs,		BRAFV226M,
				PIK3CAE365K
Ovarian	SK-OV-3	ARID1AQ586*,	APCfs, TP53fs	TOPBP1N295S,
		ATMsplice_acceptor		PIK3CAH1047R,
				KRASamp, CDC7del
Ovarian	IGROV-1	MSI-H,	PTENfs,	ERBB3K742,
		ARID1AD1850fs*4,	TP53Y126C	PIK3CAR38C; *1069W,
		G276fs*87, ATMR248Q,		TOP2AH605Q,
		BRCA1K654fs*47,		TOPBP1D395G
		CHEK1fs,
		MLH1S505fs*3, MSH6fs,
		POLQfs; POLNfs,
		RAD50fs
Ovarian	OVCAR8	ATMV613L	TP53splice_acceptor	ERBB2G776V,
				KRASP121H, MYCamp
Pancreas	BxPC3	PRKDCfs	TP53Y220C	BRAFVTAPTP487-492A,
Pancreas	AsPc-1	PRKDCfs	TP53fs	DYRK1AS14C,
				KRASG12D
Pancreas	MIAPaCa2	none	TP53R248W	KRASG12C

Abbreviations used in Tables 4 and 5:
DDR: DNA damage repair;
MMR: Mismatch repair;
fs: frame shift;
del: deletion;
*stop codon;
amp: gene amplification;
MSI-H: Microsatellite Instability High
IC50: compound concentration required to achieve 50% inhibition of the maximal cell growth.

TABLE 6

Inhibition of isogenic tumor cell line proliferation by Compound A

			in vitro
Indication	Cell line	Defect	(IC50, nM)

Colorectal	DLD-1 parental		50
Colorectal	DLD-1 BRCA2	BRCA2 deficiency	27
	(−/−)	(deleterious mutation
		of BRCA2)
Colorectal	DLD-1 ATM	ATM deficiency	1.5
	(−/−)	(deleterious mutation
		of ATM)

Abbreviations used in Table 6:
IC50: compound concentration required to achieve 50% inhibition of the maximal cell growth.

Example 2

In Vivo Xenotransplantation Models

The anti-tumor activity of of Compound A was examined in murine xenotransplantation models of human cancer. For this purpose, mice were implanted subcutaneously with tumor cells. At a mean tumor size of 20-30 mm²animals were randomized into treatment and control groups (n=10 animals/group) and treatment started with vehicle only or Compound A (formulation: 60% PEG400/10% Ethanol/30% Water; application route: p.o./per os, orally; dose/schedule: 50 mg/kg twice daily for 3 days on/4 days off). The oral application volume was 10 ml/kg. The time interval between two applications per day was 6-7h. The experiment was ended when the untreated control group had tumors of area ≤225 mm². The tumor size and the body weight were determined three times weekly. Changes in the body weight were a measure of treatment-related toxicity (>10%=critical, stop of treatment until recovery, >20%=toxic, termination). The tumor area was detected by means of an electronic caliper gauge [length (mm)×width (mm)]. In vivo anti-tumor efficacy is presented as T/C ratio (Treatment/Control) calculated with tumor areas at study end by the formula [(tumor area of treatment group at day x)−(tumor area of treatment group at day before first treatment)]/[(tumor area of control group at day x)−(tumor area of control group at day before first treatment)]. A compound having a T/C below 0.5 is defined as active (effective). Statistical analysis was assessed using SigmaStat software. A one-way analysis of variance was performed and differences to the control were compared by a pair-wise comparison procedure (Dunn's method).
Results (Table 7):
Compound A showed potent anti-tumor efficacy in different xenograft models of human tumors upon monotherapy treatment inducing stable disease in ovarian (A2780), prostate (PC3), colorectal cancer (LOVO) and complete tumor remission in mantle cell lymphoma (REC-1) at good tolerability.

TABLE 7

Anti-tumor activity of Compound A in different
human cancer xenograft models in mice.

	Xenograft Model	T/C^a	Max. weight loss^b(%)

REC-1	−0.13*	−10
PC3	−0.02*	−7
LOVO	0.13*	−8
A2780	0.13*	−6

*P < 0.05 (compared to vehicle treated control)
^aT/C = ratio of the tumor area of treatment versus [(tumor area of treatment group at day x) − (tumor area of treatment group at day before first treatment)]/[(tumor area of control group at day x) − (tumor area of control group at day before first treatment)].
^bLoss of body weight: Changes in body weight compared to the initial body weight at the start of treatment (>10% = critical, stop of treatment until recovery, >20% = toxic, termination).
The abbreviation 2QD means twice per day, po means peroral

Example 3

Treatment of Isogenic DT40 Chicken Lymphoma Cell Lines with Compound A

DT40 cells from isogenic cell lines (see Table 8) were seeded in 40 μl of growth medium (RPMI 1640 medium containing stabilized glutamine (# FG1215, Merck/Biochrom), supplemented with 10% fetal calf serum, 1% chicken serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 5E-05M ß-mercaptoethanol) at 200 cells/well in 384-well white microtiter plates ((#6007680; Perkin Elmer Life Sciences) and incubated for 24 h at 37° C. Compound A was added using a digital dispenser (Tecan) to the cells in the test plates and incubated continuously for 3 days at 37° C. To determine cell viability (corresponding to cell number) 10 μl/well of CTG solution (Promega Cell Titer Glo solution, # G755B and G756B) was added. After incubation for further 10 min luminescence was measured using a PHERAstar FSX (BMG Labtech) equipment. All measurements were done in quadruplicates. The percentage change of cell viability was calculated by normalization with respect to the luminescence reading (cell number) at the beginning of treatment of cells (a reference plate was measured at the time point of compound application to the measurement plates) and the luminescence reading (cell number) of the untreated control group. Half-maximal growth inhibition (IC₅₀) was determined as compound concentration, which was required to achieve 50% inhibition of cellular growth using a 4-parameter fit.
To evaluate the relative cellular sensitivity of the isogenic DT40 cell lines towards Compound A the mean IC₅₀of each mutant cell line was divided by the mean IC₅₀of wild-type cells, and then the quotient was converted into logarithmic scale (base 2). Loge ratios of ≤−1 or ≥+1, corresponding to a 2-fold change in sensitivity relative to wild-type cells, were considered as particularly relevant.
Results
The activity of Compound A was tested in a panel of 46 isogenic cell lines derived from DT40 chicken lymphoma cells, which do not express TP53 (Takao et al., Oncogene 1999; 18: 7002-7009), covering inactivation of various genes involved in DNA damage signaling and DNA repair. Relative sensitivities against Compound A were calculated for the mutant cell lines versus the parental wild-type cell line (Table 9). The results indicate that cells deficient in the genes TP53BP1, RAD9A, RAD17, H2AFX, RAD52, BRCA1, BRCA2. UBE2N, PCNA, PARP1, TDP2, FANCD2, FANCG, POLL, POLL/POLB double mutated, REV3L, FEN1, XPA, ERCC5, or BLM are 2-fold or more than 2-fold more sensitive towards Compound A as compared to wild-type cells. Strong sensitizations (>4-fold) were observed with RAD17, PARP1, FANCD2, UBE2N, RAD9A, REV3L, TP53BP1, ERCC5, and BLM deficient DT40 cells, whereas the strongest effects (>8-fold) were detectable in PCNA, FEN1, H2AFX, BRCA1 deficient DT40 cells. These results demonstrate that deleterious mutations in the genes TP53BP1, RAD9A, RAD17, H2AFX, RAD52, BRCA1, BRCA2. UBE2N, PCNA, PARP1, TDP2, FANCD2, FANCG, POLL, POLL/POLB double mutated, REV3L, FEN1, XPA, ERCC5, or BLM sensitize tumor cells to treatment with Compound A.

TABLE 8

DT40 isogenic mutant cell lines. All cell lines were obtained from Kyoto University, Japan.

Cell line	Gene	Function of deleted (mutated) gene(s), and annotation	Ref.

KU70	XRCC6	Non-homologous end joining	1
LIGASE IV	LIG4	Non-homologous end joining	2
DNA-PKcs	PRKDC	Non-homologous end joining	3
RAP80	UIMC1	Functional interaction with Top2, Component of BRCA1-A complex,	4
		K63 poly-ubiquitin binding protein
53BP1	TP53BP1	Inhibition of homologous recombination (Homologous recombination)	5
ATM	ATM	Damage check point control	6
RAD9	RAD9A	Damage check point control	7
RAD17	RAD17	Damage check point control	7
H2AX	H2AFX	Homologous recombination	8
RAD52	RAD52	Homologous recombination, Rad51 like protein, Homologous	9
		recombination, single-strand DNA annealing
NBS1p70	NBN	Homologous recombination	10
BRCA1	BRCA1	Homologous recombination	11
BRCA2	BRCA2	Homologous recombination	12
UBC13	UBE2N	E2 ligase, post-replication repair, Homologous recombination	13
RAD18	RAD18	E3 ligase of PCNA, Post replication repair	14
PCNAK164R	PCNA	Post-replication repair	15
PARP1	PARP1	DNA damage sensing, poly(ADP-rybosyl)ation, SSB and DSB repair	16
TDP1	TDP1	Removal of Top1 cleavage complex (Top1cc)	17
TDP2	TDP2	Removal of Top2 cleavage complex (Top2cc)	18
TDP1/TDP2	TDP1/TDP2	(See above)	19
FANCC	FANCC	Interstrand crossslink repair, Homologous recombination	20
FANCD2	FANCD2	Interstrand crossslink repair, Homologous recombination	21
FANCG	FANCG	Interstrand crossslink repair, Homologous recombination	22
USP1	USP1	Interstrand crossslink repair, Homologous recombination	23
UAF1	WDR48	Interstrand crossslink repair, Homologous recombination, USP1	23
		association factor
SNM1A/1B	DCLRE1A/	Interstrand crossslink repair	24
	DCLRE 1B
ARTEMIS	DCLRE1C	5′-3′ exonuclease, non-homologous end joining	24
POLB	POLB	Base excision repair	25
POLL	POLL	DNA polymerase, Base excision repair	25
POLB/POLL	POLB/POLL	(See above)	25
POLN	POLN	Translesion synthesis DNA polymerase	26
POLQ	POLQ	Translesion synthesis DNA polymerase, Base excision repair, Helicase	26
		domain
POLN/POLQ	POLN-POLQ	(See above)	26
POLH	POLH	Translesion synthesis DNA polymerase	27
POLZ	REV3L	Translesion synthesis DNA polymerase	28
POLH/POLZ	POLH-REV3L	(See above)	29
FEN1	FEN1	5′ flap endonuclease, base excision repair, Homologous recombination	30
XPA	XPA	Nuclear excision repair	31
XPG	ERCC5	Nuclear excision repair	32
FBH1	FBXO18	DNA helicase, Similar phenotype of BLM	33
BLM	BLM	RecQ helicase responsible for Bloom syndrome	34
WRN	WRN	RecQ helicase responsible for Werner syndrome	35
MSH3	MSH3	Mismatch repair	36
ATG5	ATG5	Autophagy related 5 homolog, autophagy, negative regulation of	37
		apoptosis

TABLE 9

Inhibition of proliferation of isogenic DT40 cells by
Compound A and relative sensitivities (log₂ratios).

Cell line	Gene	IC₅₀(M)	log₂(ratio)

Wild-type		1.3E−07	0.00
KU70	XRCC6	1.2E−07	−0.12
LIGASE IV	LIG4	1.0E−07	−0.38
DNA-PKcs	PRKDC	8.5E−08	−0.61
RAP80	UIMC1	1.0E−07	−0.38
53BP1	TP53BP1	3.7E−08	−1.81
ATM	ATM	1.1E−07	−0.24
RAD9	RAD9A	2.5E−08	−2.38
RAD17	RAD17	2.1E−08	−2.63
H2AX	H2AFX	1.2E−08	−3.44
RAD52	RAD52	5.4E−08	−1.27
NBS1p70	NBN	1.2E−07	−0.12
BRCA1	BRCA1	1.4E−08	−3.22
BRCA2	BRCA2	4.7E−08	−1.47
UBC13	UBE2N	2.4E−08	−2.44
RAD18	RAD18	6.7E−08	−0.96
PCNAK164R	PCNA	8.5E−09	−3.93
PARP1	PARP1	2.2E−08	−2.56
TDP1	TDP1	9.9E−08	−0.39
TDP2	TDP2	5.6E−08	−1.22
TDP1/TDP2	TDP1/TDP2	6.6E−08	−0.98
FANCC	FANCC	8.1E−08	−0.68
FANCD2	FANCD2	2.3E−08	−2.50
FANCG	FANCG	5.2E−08	−1.32
USP1	USP1	1.3E−07	0.00
UAF1	WDR48	7.0E−08	−0.89
SNM1A/1B	DCLRE1A/DCLRE 1B	9.9E−08	−0.39
ARTEMIS	DCLRE1C	2.0E−07	0.62
POLB	POLB	1.3E−07	0.00
POLL	POLL	4.7E−08	−1.47
POLB/POLL	POLB/POLL	5.6E−08	−1.22
POLN	POLN	1.3E−07	0.00
POLQ	POLQ	7.4E−08	−0.81
POLN/POLQ	POLN-POLQ	9.2E−08	−0.50
POLH	POLH	7.7E−08	−0.76
POLZ	REV3L	3.1E−08	−2.07
POLH/POLZ	POLH-REV3L	1.2E−07	−0.12
FEN1	FEN1	1.1E−08	−3.56
XPA	XPA	4.6E−08	−1.50
XPG	ERCC5	4.1E−08	−1.66
FBH1	FBXO18	1.5E−07	0.21
BLM	BLM	4.3E−08	−1.60
WRN	WRN	7.1E−08	−0.87
MSH3	MSH3	7.1E−08	−0.87
ATG5	ATG5	1.5E−07	0.21

REFERENCES

1. Takata M, Sasaki M S, Sonoda E, Morrison C, Hashimoto M, Utsumi H, et al. Homologous recombination and non-homologous end-joining pathways of DNA doublestrand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. Embo J 1998; 17:5497-508.
2. Adachi N, Ishino T, Ishii Y, Takeda S, Koyama H. DNA ligase IV-deficient cells are more resistant to ionizing radiation in the absence of Ku70: Implications for DNA double-strand break repair. Proceedings of the National Academy of Sciences of the United States of America 2001; 98:12109-13.
3. Fukushima T, Takata M, Morrison C, Araki R, Fujimori A, Abe M, et al. Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late SG2 phase DNA double-strand break repair. J Biol Chem 2001; 276:44413-8.
4. Iijima J, Zeng Z, Takeda S, Taniguchi Y. RAP80 Acts Independently of BRCA1 in Repair of Topoisomerase H Poison-Induced DNA Damage Cancer Res. 2010; 70:8467-8474
5. Nakamura K, Sakai W, Kawamoto T, Bree R T, Lowndes N F, Takeda S, et al. Genetic dissection of vertebrate 53BP1: a major role in non-homologous end joining of DNA double strand breaks. DNA Repair (Amst) 2006; 5:741-9.
6. Takao N, Kato H, Mori R, Morrison C, Sonada E, Sun X, et al. Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation. Oncogene 1999; 18:7002-9.
7. Kobayashi M, Hirano A, Kumano T, Xiang S L, Mihara K, Haseda Y, Matsui O, Shimizu H, Yamamoto K. Critical role for chicken Rad17 and Rad9 in the cellular response to DNA damage and stalled DNA replication. Genes Cells 2004; 9:291-303
8. Sonoda E, Zhao G Y, Kohzaki M, Dhar P K, Kikuchi K, Redon C, et al. Collaborative roles of gammaH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair. DNA Repair (Amst) 2007; 6:280-92.
9. Yamaguchi-Iwai Y, Sonoda E, Buerstedde J M, Bezzubova O, Morrison C, Takata M, et al. Homologous recombination, but not DNA repair, is reduced in vertebrate cells deficient in RAD52. Mol Cell Biol 1998; 18:6430-5.
10. Nakahara M, Sonoda E, Nojima K, Sale J E, Takenaka K, Kikuchi K, et al. Genetic evidence for single-strand lesions initiating Nbsl-dependent homologous recombination in diversification of Ig v in chicken B lymphocytes. PLoS genetics 2009; 5:e1000356.
11. Martin R W, Orelli B J, Yamazoe M, Minn A J, Takeda S, Bishop D K. RAD51 upregulation bypasses BRCA1 function and is a common feature of BRCA1-deficient breast tumors. Cancer Res 2007; 67:9658-65.
12. Hatanaka A, Yamazoe M, Sale J E, Takata M, Yamamoto K, Kitao H, et al. Similar effects of Brca2 truncation and Rad51 paralog deficiency on immunoglobulin V gene diversification in DT40 cells support an early role for Rad51 paralogs in homologous recombination. Molecular and cellular biology 2005; 25:1124-34.
13. Zhao G Y, Sonoda E, Barber L J, Oka H, Murakawa Y, Yamada K et al. A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination. Mol Cell. 2007; 25:663-75.
14. Yamashita Y M, Okada T, Matsusaka T, Sonoda E, Zhao G Y, Araki K, et al. RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells. Embo J 2002; 21:5558-66.
15. Arakawa H, Moldovan G L, Saribasak H, Saribasak N N, Jentsch S, Buerstedde J M. A role for PCNA ubiquitination in immunoglobulin hypermutation. PLoS biology 2006; 4:e366.
16. Hochegger H, Dejsuphong D, Fukushima T, Morrison C, Sonoda E, Schreiber V, et al. Parp-1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells. Embo J. 2006; 25:1305-14.
17. Murai J, Huang S Y, Das B B, Dexheimer T S, Takeda S, Pommier Y. Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs DNA damage induced by topoisomerases I and II and base alkylation in vertebrate cells. J Biol Chem 2012; 287:12848-57.
18. Zeng Z, Cortes-Ledesma F, El Khamisy S F, Caldecott K W. TDP2/TTRAP is the major 5′-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular resistance to topoisomerase II-induced DNA damage. J Biol Chem 2011; 286:403-9.
19. Zeng Z, Sharma A, Ju L, Murai J, Umans L, Vermeire L, et al. TDP2 promotes repair of topoisomerase I-mediated DNA damage in the absence of TDP1. Nucleic Acids Res 2012.
20. Hirano S, Yamamoto K, Ishiai M, Yamazoe M, Seki M, Matsushita N, et al. Functional relationships of FANCC to homologous recombination, translesion synthesis, and BLM. Embo J 2005; 24:418-27.
21. Yamamoto K, Hirano S, Ishiai M, Morishima K, Kitao H, Namikoshi K, et al. Fanconi anemia protein FANCD2 promotes immunoglobulin gene conversion and DNA repair through a mechanism related to homologous recombination. Molecular and cellular biology 2005; 25:34-43.
22. Yamamoto K, Ishiai M, Matsushita N, Arakawa H, Lamerdin J E, Buerstedde J M, et al. Fanconi anemia FANCG protein in mitigating radiation- and enzyme-induced DNA double-strand breaks by homologous recombination in vertebrate cells. Molecular and cellular biology 2003; 23:5421-30.
23. Murai J, Yang K, Dejsuphong D, Hirota K, Takeda S, D′Andrea A D. The USP1/UAF1 Complex Promotes Double-Strand Break Repair through Homologous Recombination. Mol Cell Biol 2011; 31:2462-9.
24. Ishiai M, Kimura M, Namikoshi K, Yamazoe M, Yamamoto K, Arakawa H, et al. DNA cross-link repair protein SNM1A interacts with PIAS1 in nuclear focus formation. Molecular and cellular biology 2004; 24:10733-41.
25. Tano K, Nakamura J, Asagoshi K, Arakawa H, Sonoda E, Braithwaite E K, et al. Interplay between DNA polymerases beta and lambda in repair of oxidation DNA damage in chicken DT40 cells. DNA Repair (Amst) 2007; 6:869-75.
26. Yoshimura M, Kohzaki M, Nakamura J, Asagoshi K, Sonoda E, Hou E, et al. Vertebrate POLQ and POLbeta cooperate in base excision repair of oxidative DNA damage. Molecular cell 2006; 24:115-25.
27. Kawamoto T, Araki K, Sonoda E, Yamashita Y M, Harada K, Kikuchi K, et al. Dual roles for DNA polymerase eta in homologous DNA recombination and translesion DNA synthesis. Molecular cell 2005; 20:793-9.
28. Sonoda E, Okada T, Zhao G Y, Tateishi S, Araki K, Yamaizumi M, et al. Multiple roles of Rev3, the catalytic subunit of polzeta in maintaining genome stability in vertebrates. Embo J 2003; 22:3188-97.
29. Hirota K, Sonoda E, Kawamoto T, Motegi A, Masutani C, Hanaoka F, et al. Simultaneous disruption of two DNA polymerases, Poleta and Polzeta, in Avian DT40 cells unmasks the role of Poleta in cellular response to various DNA lesions. PLoS genetics 2010; 6.
30. Matsuzaki Y, Adachi N, Koyama H. Vertebrate cells lacking FEN-1 endonuclease are viable but hypersensitive to methylating agents and H2O2. Nucleic Acids Res 2002; 30:3273-7.
31. Okada T, Sonoda E, Yamashita Y M, Koyoshi S, Tateishi S, Yamaizumi M, et al. Involvement of vertebrate polkappa in Rad18-independent postreplication repair of U V damage. J Biol Chem 2002; 277:48690-5.
32. Kikuchi K, Taniguchi Y, Hatanaka A, Sonoda E, Hochegger H, Adachi N, et al. Fen-1 facilitates homologous recombination by removing divergent sequences at DNA break ends. Molecular and cellular biology 2005; 25:6948-55.
33. Kohzaki M, Hatanaka A, Sonoda E, Yamazoe M, Kikuchi K, Vu Trung N, et al. Cooperative roles of vertebrate Fbh1 and Blm DNA helicases in avoidance of crossovers during recombination initiated by replication fork collapse. Mol Cell Biol 2007; 27:2812-20.
34. Imamura O, Fujita K, Shimamoto A, Tanabe H, Takeda S, Furuichi Y, et al. Bloom helicase is involved in DNA surveillance in early S phase in vertebrate cells. Oncogene 2001; 20:1143-51.
35. Imamura O, Fujita K, Itoh C, Takeda S, Furuichi Y, Matsumoto T. Werner and Bloom helicases are involved in DNA repair in a complementary fashion. Oncogene 2002; 21:954-63.
36. Nojima K, Hochegger H, Saberi A, Fukushima T, Kikuchi K, Yoshimura M, Orelli B J, Bishop D K, Hirano S, Ohzeki M, Ishiai M, Yamamoto K, Takata M, Arakawa H, Buerstedde J M, Yamazoe M, Kawamoto T, Araki K, Takahashi J A, Hashimoto N, Takeda S, Sonoda E. Multiple repair pathways mediate tolerance to chemotherapeutic cross-linking agents in vertebrate cells. Cancer Res. 2005; 65:11704-11711.
37. Maede Y, Shimizu H, Fukushima T, Kogame T et al. Differential and common DNA repair pathways for topoisomerase I- and II-targeted drugs in a genetic DT40 repair cell screen panel. Mol Cancer Ther 2013; 13; 214-20.

Claims

1. A method of treating a hyper-proliferative disease in a subject, comprising administering a therapeutically effective amount of an inhibitor of ATR kinase to the subject, wherein said subject or the hyper-proliferative disease is characterized by one or more biomarker(s) selected from the group consisting of:

a) one or more functional mutation(s) in one or more gene(s)/protein(s) selected from the group consisting of BRCA1, APC, ATG5, ARID1A, ATM, ATR, ATRIP, ATRX, BAP1, BARD1, BLM, BRAF, BRCA2, BRIP1, CCND1, CCNE1, CCNE2, CDC7, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1B, DCLRE1C, DYRK1A, EGFR, ERBB2, ERBB3, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXO18, FBXW7, FEN1, GEN1, HDAC2, H2AFX, HRAS, KRAS, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, MYC, NBN, NRAS, PALB2, PARP1, PARP2, PARP3, PARP4, PCNA, PIK3CA, PMS2, POLA1, POLB, POLH, POLL, POLN, POLQ, PRKDC, PTEN, RAD9A, RAD17, RAD18, RAD50, RAD51, RAD52, RAD54B, RAD54L, RB1, REV3L, RPA1, RPA2, SLX4, TDP1, TDP2, TMPRSS2, TMPRSS2-ERG, TOPBP1, TOP2A, TOP2B, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XRCC1, XRCC2, XRCC3, XRCC4 and XRCC6 gene/protein;

b) the activation of the ALT pathway; and

c) microsatellite instability.

2. The method of claim 1, wherein the inhibitor of ATR kinase is 2-[(3R)-3-methylmorpholin-4-yl]-4-(1-methyl-1H-pyrazol-5-yl)-8-(1H-pyrazol-5-yl)-1,7-naphthyridine or a tautomer, an N-oxide, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof.

3. The method of claim 1, wherein the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), which comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from the group consisting of BRCA1, APC, ARID1A, ATG5, ATM, ATR, ATRIP, ATRX, BAP1, BARD1, BLM, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, DCLRE1A, DCLRE1B, DCLRE1C, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXO18, FBXW7, FEN1, GEN1, H2AFX, HDAC2, LIG4, MDC1, MLH1, MLH3, MRE11A, MSH2, MSH3, MSH6, NBN, PALB2, PARP1, PARP2, PARP3, PARP4, PMS2, POLA1, POLB, POLH, POLN, POLN, POLQ, PRKDC, PTEN, RAD17, RAD18, RAD50, RAD51, RAD52, RAD54B, RAD54L, RAD9A, RB1, REV3L, RPA1, RPA2, SLX4, TDP1, TDP2, TP53, TP53BP1, TRRAP, UBE2N, UIMC1, USP1, WDR48, WRN, XPA, XRCC1, XRCC2, XRCC3, XRCC4 and XRCC6.

4. The method of claim 1, wherein the subject or the hyper-proliferative disease is characterized by one or more biomarker(s), which comprise(s) one or more deleterious mutation(s) in one or more gene(s)/protein(s) selected from the group consisting of BRCA1, ATM, BLM, BRCA2, ERCC5, FEN1, FANCD2, FANCG, H2AFX, PARP1, PCNA, POLL, POLB/POLL, RAD9A, RAD17, RAD52, REV3L, TDP2, TP53BP1, UBE2N, and XPA.

5. The method of claim 4, wherein the one or more gene(s)/protein(s) is (are) selected from the group consisting of BRCA1, ATM, BLM, ERCC5, FEN1, FANCD2, H2AFX, PARP1, PCNA, RAD9A, RAD17, REV3L, TP53BP1, and UBE2N.

6. The method of claim 4, wherein the one or more gene(s)/protein(s) is (are) selected from the group consisting of BRCA1, ATM, FANCD2, H2AFX, RAD17, and UBE2N.

7. The method of claim 4, wherein the one or more gene(s)/protein(s) is (are) selected from the group consisting of BRCA1, ATM, FEN1, H2AFX, and PCNA.

8. The method of claim 4, wherein the one or more gene(s)/protein(s) is BRCA1.

9. The method of claim 4, wherein the one or more gene(s)/protein(s) is ATM.

10. The method of claim 4, wherein the one or more gene(s)/protein(s) is FANCD2.

11. The method of claim 4, wherein the one or more gene(s)/protein(s) is H2AFX.

12. The method of claim 4, wherein the one or more gene(s)/protein(s) is RAD17.

13. The method of claim 4, wherein the one or more gene(s)/protein(s) is UBE2N.

14. The method of claim 1, further comprising:

a) determining if one or more of the biomarker(s) as defined in claim 1 are present in a sample of the subject;

b) administering a therapeutically effective amount of the inhibitor of ATR kinase, to the subject, if one or more of the biomarker(s) determined by step (a) is (are) determined positively.

15. The method of claim 14, wherein the sample of the subject is an in vitro sample.

16. (canceled)

17. A kit comprising 2-[(3R)-3-methylmorpholin-4-yl]-4-(1-methyl-1H-pyrazol-5-yl)-8-(1H-pyrazol-5-yl)-1,7-naphthyridine or a tautomer, an N-oxide, a hydrate, a solvate, or a pharmaceutically acceptable salt together with means to detect one or more of the biomarker(s) defined in selected from the group consisting of:

b) the activation of the ALT pathway; and

c) microsatellite instability.

18. A method for identifying a subject having a hyper-proliferative disease disposed to respond favorably to an inhibitor of ATR kinase wherein the method comprises the detection of one or more of the biomarker(s) selected from the group consisting of:

b) the activation of the ALT pathway; and

c) microsatellite instability,

in a sample of the subject.

19. A method of determining whether a subject having a hyper-proliferative disease will respond to the treatment with an inhibitor of ATR kinase, wherein the method comprises the detection of one or more of the biomarker(s) selected from the group consisting of:

b) the activation of the ALT pathway; and

c) microsatellite instability,

in a sample of the subject.

20. The method of claim 14, wherein the inhibitor of ATR kinase is 2-[(3R)-3-methylmorpholin-4-yl]-4-(1-methyl-1H-pyrazol-5-yl)-8-(1H-pyrazol-5-yl)-1,7-naphthyridine or a tautomer, an N-oxide, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof.