WO2019057852A1

WO2019057852A1 - Use of kap1 as a biomarker for detecting or monitoring atr inhibition in a subject

Info

Publication number: WO2019057852A1
Application number: PCT/EP2018/075535
Authority: WO
Inventors: Sven Golfier; Antje Margret Wengner; Anna BEHNKE; Sabine SCHUBERT
Original assignee: Bayer AG; Bayer Pharma AG
Current assignee: Bayer AG; Bayer Pharma AG
Priority date: 2017-09-22
Filing date: 2018-09-20
Publication date: 2019-03-28
Anticipated expiration: 2020-03-22

Abstract

The present invention covers a method for detecting or monitoring ATR inhibition in a subject, the method comprising quantifying the amount of pKAP1 protein phosphorylated at serine 824 in a sample from said subject. It also covers the use of pKAP1 protein phosphorylated at serine 824 as a biomarker for detecting or monitoring ATR inhibition in a subject, the use comprising quantifying the amount of pKAP1 protein phosphorylated at serine 824 in a sample obtained from said subject.

Description

USE OF PKAP1 AS A BIOMARKER FOR DETECTING OR MONITORING ATR INHIBITION

IN A SUBJECT

BACKGROUND

In the early stages of clinical development of ATR kinase inhibitors it is important to demonstrate that the new drug inhibits its target sufficiently to modulate the relevant pathways to elicit a biological response.

For ATM inhibitors a number of potential biomarkers have been identified to serve this purpose, such as for example phosphorylation of the ATM downstream targets p53, KAP1 , SMC1 , CHK2 and H2AX (Weber and Ryan, ATM and ATR as therapeutic targets in cancer, Pharmacology & Therapeutics 149 (2015), 124-138). However, several of these markers are thought to be not specific measures of ATM kinase activity as they may be also targets of other kinases, such as for example ATR kinase.

Awasthi et al. (Journal of Cell Science 128, (2015), 4255-4262, see ) also describe KAP1 as a downstream target of ATM kinase.

According to Weber and Ryan (Pharmacology & Therapeutics 149 (2015), 124-138) for ATR kinase activity CHK1 phosphorylation is the most widely used preclinical biomarker. However, ATM can also phosphorylate CHK1 albeit to a lesser extent and a recent study in ovarian cancer has suggested that CHK1 phosphorylation status may not be a reliable marker for inhibition of the ATR-CHK1 pathway. Less direct measures of ATR inhibitor activity could include increased DNA replication stress markers such as phosphorylation of Histone H2AX (gammaH2AX), but this marker is unlikely to be specific for ATR inhibition because it has been shown that H2AX is a common target for other kinases such as ATM kinase or DNA-PKcs (Weber and Ryan, ATM and ATR as therapeutic targets in cancer, Pharmacology & Therapeutics 149 (2015), 124-138).

Hall et al. (Oncotarget. 5, (2014), 5674-5685) show an increase in gammaH2AX and pKAP1 induction in combination treatment of cisplatin with increasing ATR kinase inhibitor VX-970 concentrations in H2009 lung cancer cells (see Figure 1 b of Hall et al.). VX-970 alone shows only weak induction of gammaH2AX and, according to Hall et al., led to mild induction of P-H2AX and P-KAP 1 . However, Figure 1 b of Hall et al. does not show an ATR kinase inhibitor concentration- dependent induction of pKAP1 in H2009 lung cancer cells (Figure 1 b of Hall et al.). At least 4 out of 5 ATR kinase inhibitor concentrations tested by Hall et al. in a VX-970 monotherapy treatment, show no induction of pKAP1 (see Figure 1 b, lanes 2 to 5), This is in line with the fact that VX-970 has only weak anti-tumor activity in monotherapy treatment in contrast to cisplatin monotherapy and cisplatin/VX-970 combination treatment (see Figure 4 of Hall et al.). Based upon the data of Hall et al. the person skilled in the art would have refrained from further evaluating the use of pKAP1 as a potential biomarker for ATR inhibition in tumors or surrogate tissue samples after ATR kinase inhibitor treatment, in particular in context with ATR kinase inhibitor monotherapy.

International Patent Application WO2014055756 describes a method for monitoring DNA damage in a subject treated with an ATR kinase inhibitor by measuring changes in gammaH2AX and/or pCHK1 . This method is based on the use of surrogate tissue samples. Due to the relatively low expression levels of gammaH2AX and/or pCHK1 in surrogate tissues, the method requires an ex vivo stimulation step with a DNA damaging agent to induce an increased gammaH2AX and/or pCHK1 level. The performance of this ex vivo stimulation step can be time and cost consuming. Therefore, there is a need for identifying methods for detecting or monitoring ATR kinase activity which can use surrogate tissue samples without requiring an ex vivo stimulation step with a DNA damaging agent.

There also is a need for identifying other biomarkers which specifically reflect ATR kinase inhibition to be used in clinical development of ATR kinase inhibitors, in particular in clinical development of ATR kinase inhibitor monotherapy, to specifically demonstrate ATR kinase inhibitor activity on the ATR kinase target and/or to identify suitable ATR kinase inhibitor treatment regimens.

Blasius et al (Genome Biol. 201 1 ;12(8):R78) showed that DNA-damage-induced KAP1 Ser824 phosphorlyation was mainly ATM dependent (Figure 4a of Blasius et al). This ATM dependency of KAP1 Ser824 phosphorlyation has also been shown in ATM knockout cells by Tomimatsu et al. (EMBO Rep. 2009; 10:629-635, see Figure 1 a) or in ATM knock down cells by Ziv et al (Nat Cell Biol. 2006;8(8):870-6, see Figure 4f).

Pires et al. (British Journal of Cancer (2012) 107, 291 -299) describe the cellular effects of pharmacological inhibition of ATR kinase with VE-821 in severe hypoxic conditions and its potential as a radiosensitiser. According to Pires et al. VE-821 inhibits ATR-mediated signalling in response to replication arrest induced by severe hypoxia. Under these severe hypoxia conditions (<0.02% O2) , VE-821 induced DNA damage and increased ATM-mediated phosphorylation of H2AX and KAP1 . In contrast, Pires et al. (Figure 3A) show no induction of pKAP1 by VE-821 alone under non-hypoxic conditions. Further, according to Pires et al. phosphorylation of KAP1 is considered to be ATM-dependent. According to Wagner et al. (Proteomics 2016, 16, 402-416, in particular page 412, Figure 4 E) KAP1 phosphorylation is functionally associated with ATM kinase and no upregulated phosphorylation of Ser824 KAP1 is shown after treatment with ATR kinase inhibitor VE-821.

It has now surprisingly been found, and this constitutes the basis of the present invention, that phosphorylation of Ser824 KAP1 (pKAP1 ) can be used as a biomarker for detecting or monitoring the activity of a ATR kinase inhibitor on the ATR kinase target in ATR kinase monotherapy and/or in ATR kinase combination therapy.

DESCRIPTION of the INVENTION

METHODS OF THE PRESENT INVENTION

The present invention covers a method of detecting or monitoring ATR inhibition in a subject, the method comprising

a) quantifying the amount of pKAP1 protein in a first sample from said subject prior to the administration of the ATR kinase inhibitor to the subject;

b) quantifying the amount of pKAP1 protein in a second sample from the same subject after the administration of the ATR kinase inhibitor to the subject; and

c) comparing the amounts of pKAP1 protein from the first and second sample.

As further described in the "DEFINITIONS" section below the term "pKAP1 protein" as used herein means human KAP1 -protein, which is phosphorylated at serine 824.

The present invention also covers a method of detecting or monitoring ATR inhibition in a subject, the method comprising

a) providing a first sample of said subject prior to the administration of the ATR kinase inhibitor to the subject;

b) quantifying the amount of pKAP1 protein in said first sample from said subject;

c) providing a second sample of the same subject after the administration of the ATR kinase inhibitor to the subject;

d) quantifying the amount of pKAP1 protein in said second sample from said subject; and e) comparing the amounts of pKAP1 protein from the first and second sample.

The method(s) of the present invention are performed outside the human body, they are in vitro method(s).

In another embodiment of the method(s) of the present invention an elevation of the amount of pKAP1 protein measured in the second sample compared to the amount of pKAP1 protein measured in the first sample indicates that the ATR kinase inhibitor has bound to the ATR kinase target and/or that the ATR kinase target has been inhibited by the ATR kinase inhibitor or by the ATR kinase inhibitor treatment regimen in the subject. In another embodiment of the method(s) of the present invention of detecting or monitoring ATR inhibition in a subject, the target engagement of an ATR kinase inhibitor to the ATR kinase target is detected or monitored.

In another embodiment of the method(s) of the present invention the administration of the ATR kinase inhibitor to the subject is a ATR kinase inhibitor monotherapy treatment.

In another embodiment of the method(s) of the present invention the second sample is from the same subject after single dose treatment with the ATR kinase inhibitor.

In another embodiment of the method(s) of the present invention the second sample is from the same subject after or during multiple dose treatment with the ATR kinase inhibitor.

In another embodiment of the method(s) of the present invention the subject is characterized by one or more deleterious mutation(s) of the ATM gene/protein.

In another embodiment of the method(s) of the present invention the first and the second sample are surrogate tissue samples from the same surrogate tissue.

In another embodiment of the method(s) of the present invention the surrogate tissue sample is a blood sample, a skin sample or a hair follicle sample, particulartly a hair follicle or skin sample.

In another embodiment of the method(s) of the present invention the first and the second sample are blood samples from the subject enriched in circulating tumor cells.

In another embodiment of the method(s) of the present invention the first and the second sample each is a blood sample, in which circulating tumor cells are enriched in the first and in the second blood sample, and the quantification of the amount of pKAP1 protein is performed in said circulating tumor cell enriched blood samples.

In another embodiment of the method(s) of the present invention the first and the second sample are circulating tumor cells, particularly circulating tumor cells isolated from a blood sample, from said subject.

In another embodiment of the method(s) of the present invention the first and the second sample each is a blood sample, from which circulating tumor cells are isolated in the first and in the second sample, and the quantification of the amount of pKAP1 protein is performed in said isolated circulating tumor cells.

Methods for enriching or isolating circulating tumor cells in blood samples are known to the person skilled in the art (see for example Alix-Panabieres C, Pantel K., Clin Chem. 59(1 ), (2013), 1 10-1 18; Alix-Panabieres C, Pantel K., Nature Reviews Cancer 14, (2014), 623-631 ; Kallergi G et al., Cell Physiol Biochem. 40 (3-4), (2016), 41 1 -419; Millner et al., Ann Clin Lab Sci. 43(3), (2013), 295-304). In another embodiment of the method(s) of the present invention the first and the second sample are tumor tissue samples from the same tumor tissue.

In another embodiment of the method(s) of the present invention the first and the second sample is a tumor tissue sample and the first and the second sample are obtained from the same tumor tissue.

In another embodiment of the method(s) of the present invention the first and the second sample each is a tumor tissue sample; the first and the second sample each is obtained from the same tumor tissue; and the first and the second tumor tissue sample each does not comprise severe hypoxic tumor tissue.

In another embodiment of the method(s) of the present invention the first and the second sample each is a tumor tissue sample; the first and the second sample each is obtained from the same tumor tissue; and the first and the second tumor tissue sample each does not comprise severe hypoxic tumor tissue and/or each does not comprise moderate hypoxic tumor tissue.

In another embodiment of the method(s) of the present invention the first and the second sample each is a tumor tissue sample; the first and the second sample each is obtained from the same tumor tissue; and the first and the second tumor tissue sample each consists of non- hypoxic tumor tissue.

The terms "severe hypoxic tumor tissue", "moderate hypoxic tumor tissue" and "non-hypoxic tumor tissue" are further defined below ( see "DEFINITIONS"). In another embodiment of the method(s) of the present invention the first and the second sample was obtained within 4 to 36 hours, particularly within 6 to 30 hours, preferably within 8 to 24 hours, preferably (about) 8 or (about) 24 hours after the administration of the ATR kinase inhibitor to the subject.

In another embodiment of the method(s) of the present invention the first and the second sample is a surrogate tissue sample from the same surrogate tissue and the second surrogate tissue sample was obtained within 4 to 36 hours, particularly within 6 to 30 hours, preferably within 8 to 24 hours, preferably (about) 8 or (about) 24 hours after the administration of the ATR kinase inhibitor to the subject.

In another embodiment of the method(s) of the present invention the first and the second sample is a blood sample enriched in circulating tumor cells and the second blood sample enriched in circulating tumor cells was obtained within 4 to 36 hours, particularly within 6 to 30 hours, preferably within 8 to 24 hours, preferably (about) 8 or (about) 24 hours after the administration of the ATR kinase inhibitor to the subject.

In another embodiment of the method(s) of the present invention the first and the second sample is a circulating tumor cell sample and the second circulating tumor cell sample was obtained within 4 to 36 hours, particularly within 6 to 30 hours, preferably within 8 to 24 hours, preferably (about) 8 or (about) 24 hours after the administration of the ATR kinase inhibitor to the subject.

In another embodiment of the method(s) of the present invention the first and the second sample is a tumor tissue sample from the same tumor tissue and the second tumor tissue sample was obtained within 4 to 36 hours, particularly within 6 to 30 hours, preferably within 8 to 24 hours, preferably (about) 8 or (about) 24 hours after the administration of the ATR kinase inhibitor to the subject.

In another embodiment of the method(s) of the present invention the amount of the pKAP1 protein is determined by using an antibody specific for pKAP1 protein, preferably an antibody specific for pKAP1 , which is phosphorylated at serine 824.

In another embodiment of the method(s) of the present invention the antibody specific for pKAP1 protein is selected from Phospho-TIF1 β (Ser824) Antibody and Phospho-TRIM28 (Ser824) Antibody.

The term "Phospho-TIF1 β (Ser824) Antibody" as used herein refers to a polyclonal antibody specific for the detection of Ser824 phosphorylation of KAP1 protein, which can be purchased from Cell Signaling Technology, USA, Product No. 4127.

The term "Phospho-TRIM28 (Ser824) Antibody" as used herein refers to a recombinant rabbit monoclonal antibody specific for the detection of Ser824 phosphorylation of KAP1 protein, which can be purchased for example from ThermoFisher Scientific, USA, Product No. 702084. In another embodiment of the method(s) of the present invention the amount of the pKAP1 protein is determined by IHC (= immunohistochemistry), Western Blot, ELISA (= enzyme- linked immunosorbent assay), Collaborative Enzyme Enhanced Reactive-immunoassay (CEER), LC-MS/MS, proximity extension assay (PEA) technology, or proximity ligation assay (PLA) technology, particularly the amount of the pKAP1 protein is determined by IHC (= immunohistochemistry) or Western Blot.

Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) is a method known to the person skilled in the art, which is for example described in Kim et al., Proteome Sci. 201 1 ; 9: 75.

Proximity extension assay (PEA) and proximity ligation assay (PLA) technology are methods which are known to the person skilled in the art and which are for example described in Greenwood et al., Biomolecular Detection and Quantification, Volume 4, June 2015, Pages 10- 16.

In another embodiment of the method(s) of the present invention the ATR kinase inhibitor is selected from VX-803, VX-970, AZD-6738 and 2-[(3R)-3-methylmorpholin-4-yl]-4-(1 -methyl- 1 H-pyrazol-5-yl)-8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridine, preferably the ATR kinase inhibitor is 2- [(3R)-3-methylmorpholin-4-yl]-4-(1 -methyl-1 H-pyrazol-5-yl)-8-(1 H-pyrazol-5-yl)-1 ,7- naphthyridine.

In another embodiment of the method(s) of the present invention the second sample is from the same subject after the administration of the ATR kinase inhibitor to the subject and after the administration to the subject of one or more active ingredient(s) selected from antihyperproliferative, cytostatic or cytotoxic substances for the treatment of the hyper- proliferative disease.

In another embodiment of the method(s) of the present invention - prior to quantifying the amount of pKAP1 protein in the second sample - the second sample is treated in vitro with a DNA damaging agent.

In another embodiment of the method(s) of the present invention the DNA damaging agent is selected from ionizing radiation, UV radiation, 4-nitroquinoline, a platinating agent, an inhibitor of topoisomerase I, an inhibitor of topoisomerase II, an antimetabolite, an alkylating agent and a cytotoxic antibiotic.

USES OF THE PRESENT INVENTION

The present invention covers the use of pKAP1 protein for detecting or monitoring ATR inhibition in a subject, the use comprising quantifying the amount of pKAP1 protein in a sample obtained from said subject.

In another embodiment of the use of the present invention the use comprises: a) quantifying the amount of pKAP1 protein in a first sample obtained from the subject prior to the administration of the ATR kinase inhibitor to the subject;

b) quantifying the amount of pKAP1 protein in a second sample obtained from the same subject after the administration of the ATR kinase inhibitor to the subject; and

c) comparing the amounts of pKAP1 protein from the first and second sample.

The use of the present invention is performed in vitro.

In another embodiment of the use of the present invention an elevation of the amount of pKAP1 protein measured in the second sample compared to the amount of pKAP1 protein measured in the first sample indicates that the ATR kinase inhibitor has bound to the ATR kinase target and/or that the ATR kinase target has been inhibited by the ATR kinase inhibitor or by the ATR kinase inhibitor treatment regimen in the subject. In another embodiment of the use of pKAP1 protein for detecting or monitoring ATR inhibition in a subject, the target engagement of an ATR kinase inhibitor to the ATR kinase target is detected or monitored.

In another embodiment of the use of the present invention the second sample was obtained from the same subject after single dose treatment with the ATR kinase inhibitor.

In another embodiment of the use of the present invention the second sample is obtained from the same subject after or during multiple dose treatment with the ATR kinase inhibitor.

In another embodiment of the use of the present invention the subject is characterized by one or more deleterious mutation(s) of the ATM gene/protein.

In another embodiment of the use of the present invention the second sample is obtained from the same subject after the administration of the ATR kinase inhibitor to the subject and after the administration of one or more active ingredient(s) selected from antihyperproliferative, cytostatic or cytotoxic substances for the treatment of the hyper-proliferative disease to the subject. In this embodiment of the invention the second sample is obtained from a subject, which has received a combination therapy, which comprises a treatment of the subject with the ATR kinase inhibitor in combination with one or more active ingredient(s) selected from antihyperproliferative, cytostatic or cytotoxic substances.

In another embodiment of the use of the present invention - prior to quantifying the amount of pKAP1 protein in the second sample - the second sample is treated in vitro with a DNA damaging agent.

In another embodiment of the use of the present invention the DNA damaging agent is selected from ionizing radiation, UV radiation, 4-nitroquinoline, a platinating agent, an inhibitor of topoisomerase I, an inhibitor of topoisomerase II, an antimetabolite, an alkylating agent and a cytotoxic antibiotic.

In another embodiment of the use of the present invention the first and the second sample is a surrogate tissue sample and the first and the second sample are obtained from the same surrogate tissue.

In another preferred embodiment of the of the use of the present invention the surrogate tissue sample is a blood sample, a skin sample or a hair follicle sample, particularly a skin sample or hair follicle sample.

In another embodiment of the use of the present invention the first and the second sample each is a blood sample, in which circulating tumor cells are enriched in the first and in the second blood sample, and the quantification of the amount of pKAP1 protein is performed in said circulating tumor cell enriched blood samples. In another embodiment of the use of the present invention the first and the second sample each is a blood sample, from which circulating tumor cells are isolated in the first and in the second sample, and the quantification of the amount of pKAP1 protein is performed in said isolated circulating tumor cells.

Methods for enriching or isolating circulating tumor cells in blood samples are known to the person skilled in the art (see for example Alix-Panabieres C, Pantel K., Clin Chem. 59(1 ), (2013), 1 10-1 18; Alix-Panabieres C, Pantel K., Nature Reviews Cancer 14, (2014), 623-631 ; Kallergi G et al., Cell Physiol Biochem. 40 (3-4), (2016), 41 1 -419; Millner et al., Ann Clin Lab Sci. 43(3), (2013), 295-304)

In another embodiment of the use of the present invention the first and the second sample is a tumor tissue sample and the first and the second sample are obtained from the same tumor tissue.

In another embodiment of the use of the present invention the first and the second sample each is a tumor tissue sample; the first and the second sample each is obtained from the same tumor tissue; and the first and the second tumor tissue sample each does not comprise severe hypoxic tumor tissue.

In another embodiment of the use of the present invention the first and the second sample each is a tumor tissue sample; the first and the second sample each is obtained from the same tumor tissue; and the first and the second tumor tissue sample each does not comprise severe hypoxic tumor tissue and/or each does not comprise moderate hypoxic tumor tissue.

In another embodiment of the use of the present invention the first and the second sample each is a tumor tissue sample; the first and the second sample each is obtained from the same tumor tissue; and the first and the second tumor tissue sample each consists of non-hypoxic tumor tissue.

The terms "severe hypoxic tumor tissue", "moderate hypoxic tumor tissue" and "non-hypoxic tumor tissue" are further defined below ( see "DEFINITIONS").

In another embodiment of the use of the present invention the amount of the pKAP1 protein is determined by using an antibody specific for pKAP1 protein, preferably an antibody specific for pKAP1 , which is phosphorylated at serine 824.

In another embodiment of the use of the present invention the antibody specific for pKAP1 protein is selected from Phospho-TIF1 β (Ser824) Antibody and Phospho-TRIM28 (Ser824) Antibody.

In another embodiment of the use of the present invention the amount of the pKAP1 protein is determined by IHC (=immunohistochemistry), Western Blot, ELISA (=enzyme-linked immunosorbent assay), Collaborative Enzyme Enhanced Reactive-immunoassay (CEER), LC- MS/MS, proximity extension assay (PEA) technology, or proximity ligation assay (PLA) technology, particularly the amount of the pKAP1 protein is determined by IHC (= immunohistochemistry) or Western Blot.

In another embodiment of the use of the present invention the ATR kinase inhibitor is selected from VX-803, VX-970, AZD-6738 and 2-[(3R)-3-methylmorpholin-4-yl]-4-(1 -methyl-1 H-pyrazol- 5-yl)-8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridine, preferably the ATR kinase inhibitor is 2-[(3R)-3- methylmorpholin-4-yl]-4-(1 -methyl-1 H-pyrazol-5-yl)-8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridine.

In a preferred embodiment of the use of the present invention the use is an in vitro use.

DEFINITIONS

The terms as mentioned in the present text have the following meanings:

The term "comprising" when used in the specification includes "consisting of".

If within the present text any item is referred to as "as mentioned herein", it means that it may be mentioned anywhere in the present text.

The term "detecting" means a singular measurement of a relevant parameter, while the term "monitoring" refers to multiple measurements of a relevant parameter within a certain time period.

The term "pKAP1 protein" means human KAP1 -protein, which is phosphorylated at serine 824.

The term "KAP1 protein", which is also called "KRAB-associated protein 1 ", "E3 SUMO-protein ligase TRIM28" or "Transcription intermediary factor 1 -beta (TIF1 β)", means a human protein which amino acid sequence is described under SEQ ID NO: 1 .

KAP1 protein was initially identified as a universal transcriptional co-repressor because it interacts with a large KRAB-containing zinc finger protein (KRAB-ZFP) transcription factor family. KAP1 structure, post-translational modifications and interacting proteins are described in Figure 2 of Chun-Ting Cheng, Ching-Ying Kuo, David K Ann, KAPtain in charge of multiple missions: Emerging roles of KAP1 , World J Biol Chem. Aug 26, 2014; 5(3): 308-320. Many studies demonstrate that KAP1 affects gene expression by regulating the transcription of KRAB-ZFP-specific loci, trans-repressing as a transcriptional co-repressor or epigenetically modulating chromatin structure. Emerging evidence suggests that KAP1 also functions independent of gene regulation by serving as a SUMO/ubiquitin E3 ligase or signaling scaffold protein to mediate signal transduction. KAP1 is subjected to multiple post-translational modifications (PTMs), including serine/tyrosine phosphorylation, SUMOylation, and acetylation, which coordinately regulate KAP1 function and its protein abundance. KAP1 is involved in multiple aspects of cellular activities, including DNA damage response, virus replication, cytokine production and stem cell pluripotency. Moreover, knockout of KAP1 results in embryonic lethality, indicating that KAP1 is crucial for embryonic development and possibly impacts a wide-range of (patho)physiological manifestations. Indeed, studies from conditional knockout mouse models reveal that KAP1 -deficiency significantly impairs vital physiological processes, such as immune maturation, stress vulnerability, hepatic metabolism, gamete development and erythropoiesis (Cheng et al, World J Biol Chem. Aug 26, 2014; 5(3): 308-320).

Upon DNA damage induced by double-strand break (DSB) inducing agents the KAP1 serine- 824 phosphorylation is responsible for ATM-mediated chromatin relaxation, a crucial step for DNA double-strand break (DSB) repair (Ziv et al., Nat Cell Biol. 2006 Aug;8(8):870-876).

The term "treatment regimen" means a treatment plan that specifies the dosage and the schedule of treatment.

The term "ATR kinase inhibitor treatment regimen" means a treatment plan that specifies the dosage and the schedule of treatment with an ATR kinase inhibitor, particularly in ATR kinase inhibitor monotherapy or in combination therapy with one or more active ingredient(s) selected from antihyperproliferative, cytostatic or cytotoxic substances for the treatment of the hyper- proliferative disease.

The term "ATR kinase inhibitor monotherapy treatment" means a treatment of the hyper- proliferative disease solely with an ATR kinase inhibitor but without the additional administration of one or more active ingredient(s) selected from antihyperproliferative, cytostatic or cytotoxic substances for the treatment of the hyper-proliferative disease to the subject.

The term ATR kinase inhibitor refers to any inhibitor of ATR kinase, particularly an inhibitor of ATR kinase selected from VX-803, VX-970, AZD-6738 and 2-[(3R)-3-methylmorpholin-4-yl]-4- (1 -methyl-1 H-pyrazol-5-yl)-8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridine (Compound A) as described infra.

In context with the present invention the term "VX-803" is understood as meaning 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 is of structure

In context with the present invention the term "VX-970" is understood as meaning 3-(3-{4- [(methylamino)methyl]phenyl}-1 ,2-oxazol-5-yl)-5-[4-(propan-2-ylsulfonyl)phenyl]pyrazin-2- amine. VX-970 is of structure

In context with the present invention the term "AZD-6738" is understood as meaning 4-{4- [(3R)-3-methylmorpholin-4-yl]-6-[1 -(S-methylsulfonimidoyl)cyclopropyl]pyrimidin-2-yl}-1 H- pyrrolo[2,3-b]pyridine. AZD-6738 is of structure

In a preferred embodiment of the present invention the ATR kinase inhibitor is 2-[(3R)-3- methylmorpholin-4-yl]-4-(1 -methyl-1 H-pyrazol-5-yl)-8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridine, or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment of the present invention the ATR kinase inhibitor is Compound A of structure

Compound A.

The synthesis of Compound A is described in Example 1 1 1 of WO2016020320 (A1 ).

The term "target engagement" refers to the binding of a ligand, e.g. the ATR kinase inhibitor, to the target of interest, e.g. the ATR kinase, thereby inhibiting the target^'s activitiy, e.g. ATR kinase activity. The term "treating" or "treatment" as used herein is used conventionally, for example the management or care of a subject for the purpose of combating, alleviating, reducing, relieving, improving the condition of a disease or disorder, such as a carcinoma.

The term "hyper-proliferative disease" as used herein includes but is not limited to, for example: psoriasis, keloids, and other hyperplasias affecting the skin, benign prostate hyperplasia (BPH), as well as malignant neoplasia. Examples of malignant neoplasia 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.

These diseases have been well characterized in humans, but also exist with a similar etiology in other mammals, and can be treated by administering pharmaceutical compositions of the present invention.

The term "subject" means a human or an animal, such as for example mice, rat, rabbit, dog and monkey. In a preferred embodiment the subject is a human, particularly a human having a hyper-proliferative disease.

The term "sample" refers to biological material obtained from the subject. The sample assayed by the method/use of the present invention is not limited to any particular type. Samples include, as non-limiting examples, single cells, circulating tumor cells, multiple cells, tissues, tumor tissue, biological fluids, biological molecules, or supernatants or extracts of any of the foregoing. Examples include tissue obtained for biopsy, tissue obtained during resection, blood, urine, skin tissue, hair follicles, lymph tissue, lymph fluid, cerebrospinal fluid, mucous, and stool samples. The sample used will vary based on the assay format, the detection method and the nature of the tumors, tissues, cells or extracts to be assayed. Methods for preparing samples are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method/use of the present invention.

The term "surrogate tissue sample" means a sample obtained from a surrogate tissue.

The term "surrogate tissue" means any tissue, cell or body fluid of the subject having the hyper-proliferative disease, excluding the primary tumor tissue. A surrogate tissue responds to the pharmacological intervention of the ATR kinase inhibitor treatment and reflects its impact on the subject^'s organism, especially on the primary site of the disease. Measurable effects in surrogate tissue can therefore indicate ATR inhibition, particularly they can indicate target engagement of the ATR kinase inhibitor to the ATR kinase target. Surrogate tissue includes for example hair follicle tissue/cells, skin tissue/cells, peripheral blood mononuclear cells (PBMCs), blood, plasma, serum, lymph, urine, tears, synovial fluid, wound fluid and/or cerebrospinal fluid.

Surrogate tissue samples may be gathered using a variety of methods known in the art, e.g., via a skin swab, hair pluck, skin puncture or intravenous bleed.

The term "severe hypoxic tumor tissue" means a tumor tissue which shows an oxygen concentration of equal or less than 0.02% 0₂.

The term "moderate hypoxic tumor tissue" means a tumor tissue which shows an oxygen concentration in the range of 0.02% to 2,00% 0₂.

The term "non-hypoxic tumor tissue" means a tumor tissue which shows an oxygen concentration of more than 2,00%, particularly an oxygen concentration in the range of 2% to 8%, preferably an oxygen concentration in the range of 2.5% to 5%. The oxygen concentration of the first and the second tumor tissue sample can be determined by methods known to the person skilled in the art. Preferably, it is determined by using a two-channel fiberoptic oxygen- sensing device, such as for example Oxylite 2000 (Oxford Optronix, Oxford, UK), particularly it is determined by using the method described by Brurberg et al. (Int. J. Radiation Oncology Biol. Phys., Vol. 58, No. 2, (2004), pp. 403-409).

Preferably the oxygen concentration of the tumor tissue is determined in vivo to identify tumor tissue of the patient, which does not comprise severe hypoxic tumor tissue and/or which does not comprise moderate hypoxic tumor tissue. After such identification, the first and the second tumor tissue sample, which each does not comprise severe hypoxic tumor tissue and/or which each does not comprise moderate hypoxic tumor tissue, is then isolated from the subject and is then further tested according to the method(s) of the present invention.

In context with the method/use of the present invention the term "in vitro" means that said method/use is performed in a controlled environment (e.g. test tube, reaction vessel) outside of a living subject (e.g. human, animal).

The term "deleterious mutation of the ATM gene/protein" as used herein means a mutation of the ATM 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 ATM gene may result in a reduced gene expression level of said gene, a reduced amount or a reduced activity of the ATM protein, or it may result in a nonfunctional ATM gene/protein ("loss-of-function") compared to the respective wildtype ATM 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 ATM gene, resulting in a premature stop codon, or a nonsense codon in the transcribed mRNA, and in a truncated, incomplete, and nonfunctional ATM protein.

The deleterious mutation can be a missense mutation, which is a point mutation in the ATM gene, resulting in the production either of a nonfunctional ATM protein (complete loss of function) or in a ATM protein with partial loss of function compared to the respective wildtype ATM protein.

The deleterious mutation can also result in a frameshift mutation, which is a genetic mutation in the ATM 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 ATM protein.

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 ATM protein.

Another example for a large rearrangement mutation is a duplication of one or more nonterminal exons disrupting the reading frame or a critical functional domain of the ATM 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 ATM protein.

The deleterious mutation can also be a copy number variant (CNV), particularly a decrease of the ATM gene copy number (e.g. a homozygous or heterozygous deletion) compared to the normal gene copy number of the ATM gene.

Particularly, the deleterious mutation(s) of the ATM gene/protein result(s) in the loss of the ATM protein in the sample.

The loss of the ATM protein in the sample can be determined by methods such as IHC (=immunohistochemistry), Western Blot, ELISA (=enzyme-linked immunosorbent assay), Collaborative Enzyme Enhanced Reactive-immunoassay (CEER), LC-MS/MS, proximity extension assay (PEA) technology, or proximity ligation assay (PLA) technology, particularly the amount of the ATM protein is determined by IHC (= immunohistochemistry) by Western Blot, preferably by Western Blot as described in the Experimental Section, Example 4. The term "antihyperproliferative, cytostatic or cytotoxic substances for treatment of cancers" as used herein includes, for example, the following active ingredients:

131 1-chTNT, abarelix, abiraterone, aclarubicin, ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alemtuzumab, Alendronic acid, alitretinoin, altretamine, amifostine, aminoglutethimide, Hexyl aminolevulinate,amrubicin, amsacrine, anastrozole, ancestim, anethole dithiolethione, angiotensin II, antithrombin III, aprepitant, arcitumomab, arglabin, arsenic trioxide, asparaginase, axitinib, azacitidine, basiliximab, belotecan, bendamustine, belinostat, bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin, bortezomib, buserelin, bosutinib, brentuximab vedotin, busulfan, cabazitaxel, cabozantinib, calcium folinate, calcium levofolinate, capecitabine, capromab, carboplatin, carfilzomib, carmofur, carmustine, catumaxomab, celecoxib, celmoleukin, ceritinib, cetuximab, chlorambucil, chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin, cladribine, clodronic acid, clofarabine, copanlisib , crisantaspase, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, darbepoetin alfa, dabrafenib, dasatinib, daunorubicin, decitabine, degarelix, denileukin diftitox, denosumab, depreotide, deslorelin, dexrazoxane, dibrospidium chloride, dianhydrogalactitol, diclofenac, docetaxel, dolasetron, doxifluridine, doxorubicin, doxorubicin + estrone, dronabinol, eculizumab, edrecolomab, elliptinium acetate, eltrombopag, endostatin, enocitabine, enzalutamide, epirubicin, epitiostanol, epoetin alfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine, etoposide, everolimus, exemestane, fadrozole, fentanyl, filgrastim, fluoxymesterone, floxuridine, fludarabine, fluorouracil, 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 (1231), iomeprol, ipilimumab, irinotecan, Itraconazole, ixabepilone, lanreotide, lapatinib, lasocholine, lenalidomide, 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, nedaplatin, nelarabine, neridronic acid, nivolumabpentetreotide, nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nitracrine, nivolumab, obinutuzumab, octreotide, ofatumumab, omacetaxine mepesuccinate, omeprazole, ondansetron, oprelvekin, orgotein, orilotimod, oxaliplatin, oxycodone, oxymetholone, ozogamicine, p53 gene therapy, paclitaxel, palifermin, palladium-103 seed, palonosetron, pamidronic acid, panitumumab, pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxy PEG-epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b, pemetrexed, pentazocine, pentostatin, peplomycin, Perflubutane, perfosf amide, 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, radium-223 chloride, radotinib, raloxifene, raltitrexed, ramosetron, ramucirumab, ranimustine, rasburicase, razoxane, refametinib, regorafenib, risedronic acid, rhenium-186 etidronate, rituximab, romidepsin, romiplostim, romurtide, roniciclib, samarium (153Sm) lexidronam, sargramostim, satumomab, secretin, sipuleucel-T, sizofiran, sobuzoxane, sodium glycididazole, sorafenib, stanozolol, streptozocin, sunitinib, talaporfin, 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, 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.

The term "DNA damaging agent" means any agent which can cause damage either directly or indirectly to the nucleotides in the genome, particularly the DNA damaging agent is selected from ionizing radiation, UV radiation, 4-nitroquinoline, a platinating agent, an inhibitor of topoisomerase I, an inhibitor of topoisomerase II, an antimetabolite, an alkylating agent and a cytotoxic antibiotic.

The term "platinating agent" as used herein covers any platinum-based antineoplastic drug which is used to treat hyper-proliferative a hyper-proliferative disease. It includes, for example, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, lobaplatin, triplatin tetranitrate and picoplatin. In a preferred embodiment the DNA damaging agent is a platinating agent, particularly cisplatin or carboplatin.

The term "inhibitor of topoisomerase I" as used herein covers any drug that inhibits topoisomerase I. It includes, for example, agents such as camptothecin, topotecan, irinotecan, SN38, rubitecan and belotecan. The term "inhibitor of topoisomerase II" as used herein covers any drug that inhibits topoisomerase II. It includes, for example, agents such as etoposide, daunorubicin, doxorubicin, aclarubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin and teniposide.

The term "antimetabolite" as used herein covers any drug that inhibits the use of a metabolite and that can be used in the treatment of a hyper-proliferative disease. It includes, for example, purine antagonists or pyrimidine antagonists, such as methotrexate, pemetrexed, thioguanine, fludarabine, cladribine, 6-mercaptopurine, cytarabine, gemcitabine, 5-fluorouracil (5FU), aminopterin, raltitrexed, pentostatin, clofarabine, capecitabine, tegafur, carmofur, floxuridine, azacitidine and hydroxyurea.

The term "alkylating agent" as used herein covers any drug used in in the treatment of a hyper- proliferative disease that attaches an alkyl group to DNA. It includes, for example, agents such as nitrogen mustards, triazenes, alkyl sulphonates, procarbazines and aziridines, in particular cyclophosphamide, melphalan, chlorambucil, carmustine, dacarbazine, temozolomide, busulfan, mechlorethamine, ifosfamide, trofosfamide, prednimustine, bendamustine, uramustine, estramustine, carmustine, lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocin, mannosulfan, treosulfan, triaziquone, triethylenemelamine, altretamine, mitobronitol.

The term "cytotoxic antibiotic" as used herein covers any drug used in in the treatment of a hyper-proliferative disease that interrupts cell division. The most important subgroup is the anthracyclines and the bleomycins; other examples include mitomycin C, plicamycin, mitoxantrone, and actinomycin. The term also includes, for example, agents such as doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin and mitoxantrone.

EXPERIMENTAL SECTION

The various aspects of the invention described in this application are illustrated by the following examples which are not meant to limit the invention in any way.

The example testing experiments described herein serve to illustrate the present invention and the invention is not limited to the examples given.

EXAMPLES Example 1 :

Quantification of pKAP1 (Ser824) and pCHK1 (Ser317) of in vitro treated isogenic cancer cell lines

The isogenic cell lines DLD-1 parental and DLD-1 ATM knockout (=DLD1 ATM KO) 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 24h (h=hours), and then the ATR inhibitor, Compound A, was added using a digital dispenser. The final concentration of Compound A was 1 μΜ and the final concentration of the solvent DMSO was 0.01 %. After 1 h of incubation at 37Ό cisp latin was added to untreated as well to Compound A pretreated cells at a final concentration of 10μΜ in PBS (=phosphate buffered saline). The cells were lysed after 21 h of continuous incubation at 37Ό and 12^g protein were subjected to capillar electrophoresis. Subsequently pKAP1 and pCHK1 were detected by Phospho-TIF13 (Ser824) Antibody (Cell Signaling Technology, USA, Product No. 4127) and Phospho-CHK1 (Ser317) Antibody (Cell Signaling Technology, USA, Product No. 12302) and immunoprobed using an HRP-(=horseradish peroxidase)-conjugated secondary antibody and chemiluminescent substrate. The resulting chemiluminescent signal is quantitated.

In parental as well as in ATM-deleted DLD-1 cells cisplatin treatment alone led to an increase of phosphorylated Ser824 of KAP1 (Figure 1 a) and Ser318 of CHK1 (Figure 1 b).

Pretreatment of the cells by Compound A, followed by cisplatin treatment led to an inhibition of the cisplatin induced phosphorylation of Ser317 of CHK1 thereby demonstrating the effective inhibition of ATR (Figure 1 b). In contrast, in such Compound A/cisplatin combination treatment Ser824 phosphorylation of KAP1 exceeded the Ser824 phosphorlyation level observed for treatment with cisplatin or Compound A alone (Figure 1 a).

Independently of the genetic deletion of ATM, inhibition of ATR resulted in an increase of phosphorylated Ser824 of KAP1 (Figure 1 a) but in no change in phosphorylation of Ser317 of CHK1 (Figure1 b). The observed phosphorylation of Ser824 of KAP1 is independent of ATM. It was observed in both, in DLD-1 parental and in DLD-1 ATM knockout (DLD1 ATM KO) cells. Measurement of Ser824 of KAP1 is suitable to monitor ATR inhibition in vitro.

Example 2:

Quantification of pKAP1 (Ser824) in vivo in GRANTA-519 xenografts after treatment

Target engagement after treatment with Compound A was examined in the cell line derived human mantle cell lymphoma xenograft model GRANTA-519. For that purpose, GRANTA-519 [DSMZ (=Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) Accession No. 342] tumor cells were transplanted subcutaneously into female SCID (=severe combined immunodeficiency) beige mice. The tumor area was detected by means of an electronic caliper gauge [length (mm) x width (mm)]. At a mean tumor size of 78 mm² animals were randomized into treatment and control groups (n=21 animals/group) and treatment started with vehicle (60% PEG400/ 10% Ethanol/ 30% Water; application route: p.o./per os/orally, QD/quaque die/every day x1 ) or Compound A (formulation: 60% PEG400/ 10% Ethanol/ 30% Water; application route: p.o./per os/orally) upon different doses and schedules: once on one day (QD x1 ) upon 10 mg/kg or 30 mg/kg; on 3 consecutive days: twice daily for two days and once on the third day (2QDx2 + QDx1 ) upon 3 mg/kg, 10 mg/kg or 30 mg/kg. The oral application volume was 10 ml/kg. The time interval between two applications per day was 6-7h. After the last treatment blood and tumors were sampled at 0.5h, 1 h, 3h, 8h, 24h, 48h and 72h post treatment. Matched samples were frozen and formalin fixed respectively and used for further analysis.

Immune detection of pKAP1 by capillary electrophoresis

Frozen tumors were subjected to protein extraction and 12.5 μg protein were separated by capillar electrophoresis. Subsequently pKAP1 was detected by Phospho-TIF13 (Ser824) Antibody (Cell Signaling Technology, USA, Product No. 4127) and immunoprobed using an HRP-conjugated secondary antibody and chemiluminescent substrate. The resulting chemiluminescent signal is quantitated.

Treatment of mice bearing GRANTA-519 tumors by Compound A led to a time, dose and dose schedule dependent induction of Ser824 of KAP1 . Single dose application (QDx1 ) of 30mg/kg Compound A resulted in a pharmacodynamic change in Ser824 KAP1 phosphorylation, cumulating between 8 and 24 hours for the maximal phosphorylation (Figure 2). Induction of the phosphorylation was not detected for 10 mg/kg Compound A applied as single dose. In contrast subsequent analysis of tumors of mice having been treated twice daily for two days and once on the third day (2QDx2 + QDx1 ) by 10 mg/kg of Compound A resulted in a increased and prolonged phosphorylation of Ser824 of KAP1 compared to a single dose of 30 mg/kg. An additional increased phosphorylation of Ser824 KAP1 was observed for 30 mg/kg applied 2QDx2 + QDx1 . In vivo inhibition of ATR translates into pharmacodynamic changes of Ser824 phosphorylation of KAP1 (Figure 2).

Immune detection of pKAP1 Immunohistochemistry

Formalin fixed and Paraffin embedded (FFPE) 5μιη tissue section of tumor samples were subjected to immunohistochemical analysis of Ser824 KAP1 phosphorylation. Therefore sections were incubated for 1 hour at room temperature with Phospho-TIF1 β (Ser824) Antibody (Cell Signaling Technology, USA, Product No. 4127) at 0.35 \g/m\ final concentration in TBS-T. Bound antibody was detected by rabbit EnVision System (Agilent K4010) and quantitated by automated image analysis (3DHistech; DensitoQuant).

24 hours after the last dose application (2QDx2 + QDx1 ) of 30 mg/kg Compound A in mice bearing GRANTA-519 tumors a high amount of Ser824 phosphorylated KAP1 positive cells was detected (Figure 3). Tumors of untreated animals displayed barely positive cells (Figure 3).

Figure 4 shows an exemplary staining of untreated and treated tumors [24 hours after the last dose application (2QDx2 + QDx1 ) of 30 mg/kg Compound A] in mice.

Immunohistochemical analysis of tumor tissue is suitable for monitoring ATR activity after ATR inhibitor treatment.

Example 3:

Quantification of pKAP1 (Ser824), pKAP1 (Ser473), pCHK1 (Ser317) and pH2AX (Ser139) of in vitro treated cancer cells by different DNA repair inhibitors in monotherapy and in combination with cisplatin.

The DLD-1 cell lines described in Example 1 above 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 24h (h=hours), and then the ATR inhibitors (Compound A, AZD6738, VX970, VX803) or the PARP inhibitor (Olaparib) was added using a digital dispenser. The final concentration was 1 μΜ and the final concentration of the solvent DMSO was 0.01 %. After 1 h of incubation at 37Ό cisplatin was added to untreated as well pretreated cells at a final concentration of 10μΜ in PBS (=phosphate buffered saline). The cells were lysed after 21 h of continuous incubation at 37Ό and 12^g protein was subjected to capillar electrophoresis. Subsequently pKAP1 and pCHK1 were detected by Phospho-TIF1 β (Ser824) Antibody (Cell Signaling Technology, USA, Product No. 4127), Phospho-TIF1 β (Ser473) Antibody (BioLegend, USA, Product No. 644602), Phospho-CHK1 (Ser317) Antibody (Cell Signaling Technology, USA, Product No. 12302) and pH2AX (Ser139) Antibody (Merck Millipore, Germany, Product No. 05-636) and immunoprobed using an HRP-(=horseradish peroxidase)-conjugated secondary antibody and chemiluminescent substrate. The resulting chemiluminescent signal is quantitated.

Cisplatin treatment alone led to an increase of phosphorylated Ser824 of KAP1 (Figure 5) and Ser318 of CHK1 . Ser473 of KAP1 or Ser139 of pH2AX were not induced.

Pretreatment of the cells by ATR inhibitors but not by Olaparib, followed by cisplatin treatment led to an inhibition of the cisplatin induced phosphorylation of Ser317 of CHK1 demonstrating the specific inhibition of the ATR pathway. At equimolar concentration this effect was the most pronounced for Compound A.

In addition ATR inhibitor/cisplatin combination treatment induces a strong phosphorylation of KAP1 at Ser824 and to a less extend at Ser473, as well of H2AX at Ser139.

In contrast, in monotherapy differences in phosphorylation levels upon ATR inhibition could only be detected for Ser824 of KAP1 but not for the distinct Ser473 and moreover neither for the commonly analysed DNA repair markers pCHK1 (Ser317) and pH2AX (S139) demonstrating the specific induction of Ser824 upon ATR inhibition, which is also not observed by Olaparib treatment inhibiting a different DNA repair pathway. Among the tested ATR inhibitors Compound A shows the strongest KAP1 (Ser824) phosphorylation in ATR inhibitor monotherapy (Figure 5).

Example 4:

Verification of ATM loss by Western-Blot Immunodetection

2x10E7 in vitro cultured cells were subjected to protein extraction and 10 μg protein were separated by Western Blot. Subsequently ATM was detected by monoclonal ATM Antibody (Abeam, USA, Product No. ab59541 ) and immunoprobed using an infrared dye-conjugated secondary antibody (LiCor Goat anti Mouse IRDye 800C (LiCor, USA, Product No. 926- 32210). The fluorescence signal was detected on a LiCor ODYSSEY CLx scanner.

ATM protein could be detected in NCI-H460 postive control cells (pos. Ctr.) as well in DLD1 parental cells. In contrast the lack of protein signal in DLD1 ATM -/- cells confirmed the loss of ATM protein. The detection of control protein GAPDH confirmed the protein loading of all samples (Figure 6). Example 5:

Quantification of pKAP1 (Ser824) of in vitro treated cancer cells by ATR inhibitor in monotherapy.

The DLD-1 cell lines 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 24h (h=hours), and then the ATR inhibitor, Compound A, was added using a digital dispenser. The final concentration was 1 μΜ and the final concentration of the solvent DMSO was 0.01 %. The cells were lysed after 21 h of continuous incubation at 37Ό and 7,5μΙ were subjected to capillar electrophoresis at different protein concentrations. Subsequently pKAP1 was detected by the monoclonal antibody Phospho-TIF13 (Ser824) Antibody (Thermo Fischer, USA, Product Cat# 702084) at different antibody dilutions and immunoprobed using an HRP-(=horseradish peroxidase)-conjugated secondary antibody and chemiluminescent substrate. The resulting chemiluminescent signal is quantitated.

The induction of KAP1 phosphorylation at Ser824 upon ATR inhibition by Compound A could be detected by the monoclonal murine antibody used (Figure 7).

Example 6:

Immune detection of pKAP1 and pH2AX Immunohistochemistry

Formalin fixed and Paraffin embedded (FFPE) 5μιη tissue section of tumor samples were subjected to immunohistochemical analysis of Ser824 KAP1 phosphorylaton and compared to respective H2Ax Ser139 phosphorylation. Therefore sections were incubated for 1 hour at room temperature with Phospho-TIF1 β (Ser824) Antibody (Cell Signaling Technology, USA, Product No. 4127) or Phospho Histone H2AX (Millipore, USA, Product No. 05-636) at 1 μς/ηπΙ final concentration in TBS-T. Bound antibody was detected by rabbit EnVision System (Agilent K4010) and quantitated by automated image analysis (3DHistech; DensitoQuant).

3h post final treatment a clear induction of phosphorylation of Ser814 of KAP1 and Ser139 of H2AX became detectable for doses starting from 10mg/kg given 2QDx2 +QDx1 , but not for 3 mg/kg at the same dose schedule, nor for 10 mg/kg or 30 mg/kg given QDx1 . The observed phosphorylation was more prominent for Ser814 of KAP1 than for Ser139 of H2AX. This difference in induction of phosphorylation became much more pronounced 24h post last treatment for all doses tested except for the lowest dose of 3mg/kg given 2QDx2 + QDx1 . For all dosing regimens tested the induction of Ser824 phosphorylation of pKAP1 was equal or higher than the induction of Ser139 phosphorylation of H2AX (Figure 8). The dynamic range of Ser824 phosphorylation of KAP1 upon ATR inhibition is superior to that of Ser139 phosphorylation of H2AX.

Claims

1 . A method of detecting or monitoring ATR inhibition in a subject, the method comprising a) quantifying the amount of pKAP1 protein phosphorylated at serine 824 in a first sample from said subject prior to the administration of the ATR kinase inhibitor to the subject;

b) quantifying the amount of pKAP1 protein phosphorylated at serine 824 in a second sample from the same subject after the administration of the ATR kinase inhibitor to the subject; and c) comparing the amounts of pKAP1 protein phosphorylated at serine 824 from the first and second sample,

2. The method of claim 1 , wherein an elevation of the amount of pKAP1 protein

phosphorylated at serine 824 measured in the second sample compared to the amount of pKAP1 protein phosphorylated at serine 824 measured in the first sample indicates that the ATR kinase target has been inhibited by the ATR kinase inhibitor in the subject.

3. The method of any one of claims 1 to 2, wherein the amount of the pKAP1 protein phosphorylated at serine 824 is determined by using an antibody specific for pKAP1 protein phosphorylated at serine 824.

4. The method of any one of claims 1 to 3, wherein the subject is characterized by one or more deleterious mutation(s) of the ATM gene or ATM protein.

5. The method of claim 4, wherein the deleterious mutation(s) of the ATM gene or ATM protein result(s) in a loss of the ATM protein.

6. The method of any one of claims 1 to 5, wherein the administration of the ATR kinase inhibitor to the subject is a ATR kinase inhibitor monotherapy treatment.

7. The method of any one of claims 1 to 6, wherein the second sample is from the same subject after the administration of the ATR kinase inhibitor to the subject and after the administration of one or more active ingredient(s) selected from antihyperproliferative, cytostatic or cytotoxic substances for the treatment of the hyper-proliferative disease to the subject.

8. The method of any one of claims 1 to 7, wherein the first and the second sample are surrogate tissue samples from the same surrogate tissue.

9. The method of claim 8, wherein the surrogate tissue sample is a blood sample, a skin sample or a hair follicle sample.

10. The method of any one of claims 1 to 8, wherein the first and the second sample are blood samples from said subject enriched in circulating tumor cells.

1 1 . The method of any one of claims 1 to 8, wherein the first and the second sample are circulating tumor cells from said subject.

12. The method of any one of claims 1 to 7, wherein the first and the second sample are tumor tissue samples from the same tumor tissue.

13. The method of any one of claims 1 to 12, wherein the amount of the pKAP1 protein is determined by by IHC (= immunohistochemistry), Western Blot, ELISA (= enzyme-linked immunosorbent assay), Collaborative Enzyme Enhanced Reactive-immunoassay (CEER), LC- MS/MS, proximity extension assay (PEA) technology, or proximity ligation assay (PLA) technology.

14. The method of any one of claims 1 to 13, wherein the ATR kinase inhibitor is selected from VX-803, VX-970, AZD-6738 and 2-[(3R)-3-methylmorpholin-4-yl]-4-(1 -methyl-1 H-pyrazol-5-yl)- 8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridine.

15. The method of any one of claims 1 to 14, wherein the ATR kinase inhibitor is 2-[(3R)-3- methylmorpholin-4-yl]-4-(1 -methyl-1 H-pyrazol-5-yl)-8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridine.