US008.999632B2 (12) United States Patent (10) Patent No.: US 8,999,632 B2 Falcon et al. (45) Date of Patent: Apr. 7, 2015 (54) METHOD FOR MEASURING ATR (52) U.S. Cl. INHIBITION MEDIATED INCREASES IN DNA CPC ............ G0IN33/5047 (2013.01); G0IN33/68 DAMAGE (2013.01) (58) Field of Classification Search (71) Applicants: Vertex Pharmaceuticals Incorporated, USPC ........................................................ 435/2, 15 Cambridge, MA (US); University of See application file for complete search history. Newcastle Upon Tyne, Newcastle Upon Tyne (GB) (56) References Cited (72) Inventors: Susanna Falcon, Newbury (GB); Philip PUBLICATIONS Reaper, Shillingford (GB); John Kao et al. Inhibition of Gamma-H2AXAfter Ionizing Radiation. As a Pollard, Abingdon (GB); Nicola Curtin, Biological Surrogate of Impaired Upstream DNA Damage Signaling Newcastle upon Tyne (GB); Fiona and Radiosensitivity; Journal of Cancer Molecules, vol. 5, No. 2 Middleton, South Gosforth (GB); Tao (2010) pp. 49-54.* Chen, Suzhou (CN) Buscemi, G., et al., “DNA Damage-Induced Cell Cycle Regulation of Novel Chk2 Phosphoresidues'. Molecular and Cellular Biology, (73) Assignee: Vertex Pharmaceuticals Incorporated, 26(21), (2006), pp. 7832-7845 (DOI: 10.1128/MCB.00534-06). Boston, MA (US) Chen, T., et al., “Targeting the S and G2 checkpoint to treat cancer. s Drug Discovery Today, 17(5/6), (2012), pp. 194-202, (DOI: 10.1016/ - r - j.drudis.2011, 12.009). (*) Notice: Subject to any disclaimer, the term of this Chen, T., et al., “Development of a biomarker of ATR activity in patent is extended or adjusted under 35 surrogate human tissues”, Newcastle University, Poster, Nov. 2012. U.S.C. 154(b) by 0 days. Middleton, F.K., and Curtin, N.J., “ATR as a Therapeutic Target'. Cancer Drug Discovery and Development, 2013, Author's Proof. (21) Appl. No.: 14/045,373 Ward, I.M. and Chen, J., “Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress'. The (22) Filed: Oct. 3, 2013 Journal of Biological Chemistry, 276(51), (2001), pp. 47759-47762. (65) Prior Publication Data * cited by examiner US 2014/0134596 A1 May 15, 2014 Primary Examiner — Susan Hanley Assistant Examiner — Paul Martin (74) Attorney, Agent, or Firm — Rory C. Stewart Related U.S. Application Data (60) Provisional application No. 61/709,384, filed on Oct. (57) ABSTRACT 4, 2012. The present relates to methods for detecting DNA damage in subjects treated with an ATR inhibitor. More specifically, this (51) Int. Cl. invention relates to a method for measuring changes in levels AOIN I/02 (2006.01) of YH2AX and/or pChk1''' in, e.g., surrogate tissue cells, CI2O I/48 (2006.01) following ex vivo stimulation with a DNA damaging agent. GOIN33/50 (2006.01) GOIN33/68 (2006.01) 5 Claims, 11 Drawing Sheets U.S. Patent Apr. 7, 2015 Sheet 1 of 11 US 8,999,632 B2 40 30 20 10 O 0.001 0.01 0.1 1 10 VE-822 uM) Figure 1 80 70 60 50 40 30 20 1O O 0.001 0.01 0.1 1 1O 100 VE-822 uM Figure 2 U.S. Patent Apr. 7, 2015 Sheet 2 of 11 US 8,999,632 B2 60 50 40 30 20 1O 0.001 0.01 0.1 1 1O 100 VE-822 uM) Figure 3 60 40 O.OO1 O.O1 O.1 1 10 100 VE-822 uM) Figure 4 U.S. Patent Apr. 7, 2015 Sheet 3 of 11 US 8,999,632 B2 C W 2hr 2hr 24hr ATR pchkiser383 Attir Figure 5 U.S. Patent Apr. 7, 2015 Sheet 5 of 11 US 8,999,632 B2 or less & s 38 Š :8 88 Figure 7 U.S. Patent Apr. 7, 2015 Sheet 6 of 11 US 8,999,632 B2 s & S&S w S. & Šiše:SS a .S. r r s y s Figure 8 U.S. Patent Apr. 7, 2015 Sheet 7 of 11 US 8,999,632 B2 AP pChkii" WYYY DAP - YEA : s & & s SS SRst : - s: 3 & & 8. 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CROSS-REFERENCE TO RELATED FIG.5 depicts a western blot of ATR, PARP, pChk1’, APPLICATIONS and Actin in PBMCs following exposure to UV. The present application claims the benefit of under 35 FIG. 6 depicts the levels of YH2AX and pChk1''' in U.S.C. S 119 U.S. Provisional Application No. 60/709,384, purified PBMCs, after treatment with DNA damaging agents filed Oct. 4, 2012. 10 and VE-821 using immunofluorescence. FIG. 7 depicts the levels of YH2AX and pChk1''' in PBMCs after treatment of human whole blood with a DNA BACKGROUND OF THE INVENTION damaging agents and VE-821 using immunofluorescence. 15 FIG. 8 depicts the levels of YH2AX and pChk1''' in PBMCs after treatment of human whole blood with 4-nitro Ataxia talangiectasia mutated and Rad-3 related (ATR) quinoline and VE-822 using immunofluorescence. kinase is an enzyme involved in the DNA damage response FIG. 9 depicts the levels of YH2AX and pChk1''' in (DDR). This signaling network acts to detect and orchestrate PBMCs after treatment of human whole blood with 4-nitro a cell's response to certain forms of DNA damage, most quinoline at varying doses. notably double strand breaks and replication stress. Follow ing treatment with many types of DNA damaging drugs and FIG. 10 depicts pChk1 and YH2AX levels in MCF7 ionizing radiation, cells are reliant on the DDR for survival. It cells treated with varying concentrations of 4-nitroquinoline has been shown that disruption of the DDR can increase using immunofluorescence. cancer cell sensitivity to these DNA damaging agents and 25 FIG. 11 depicts the levels of pChk1''' and YH2AX in thus may improve patient responses to such therapies. Inhi MCF7 cells treated with 4-nitroquinoline and VE-821 (ATR bition of ATR is one approach that can be taken to disrupt the inhibitor) or KU55933 (ATM inhibitor) and NU7441 (DNA DDR and it has been shown that inhibition of ATR can mark PK inhibitor) for varying time intervals using immunofluo edly increase cancer cell sensitivity to DNA damaging agents. CSCCC. To support the clinical progression of ATR inhibitors it is 30 FIG. 12 depicts the levels of pChk1''' and YH2AX in necessary to develop biomarkers that can measure the degree MCF7 cells treated with UV and VE-821 or KU55933 and of ATR inhibition or the impact ATR inhibition has on cellular NU7441 for varying time intervals using immunofluores DNA damage. CCCC. FIG. 13 depicts immunofluorescence of 4-nitroquinoline SUMMARY OF INVENTION 35 induced pChk1''' in MCF7 cells at varying dilutions of the anti-pChk1''' antibody. This invention relates to methods for detecting DNA dam age in subjects administered an ATR inhibitor. More specifi DESCRIPTION OF THE INVENTION cally, this invention relates to a method for measuring changes in levels of phosphorylated H2AX (YH2AX) and/or 40 phosphorylated Chk1 (pChk1''') in, e.g., surrogate tissue This invention provides a method for monitoring DNA cells, following ex vivo stimulation with a DNA damaging damage in a Subject by measuring changes in YH2AX and/or agent. pChk1, the method comprising: Moreover, this invention relates to methods for detecting 45 a) administering an ATR inhibitor to a subject; DNA damage in the blood of subjects treated with an ATR b) taking Surrogate tissue cell samples from the Subject at inhibitor. More specifically, this invention relates to a method various intervals; of measuring increases in levels of YH2AX in CD3+ white c) treating the Surrogate tissue cell samples with a DNA blood cells from blood, stimulated ex vivo with a DNA dam damaging agent; aging agent, by flow cytometry. 50 d) measuringyH2AX and/orpChk1''' levels in the cells by using antibodies specific for phospho-H2AX and/or BRIEF DESCRIPTION OF THE DRAWINGS pChk1 Se345 The invention is herein described, by way of example only, It shall be understood that “subject' includes patients, with reference to the accompanying drawings. 55 humans, and other animals, such as mice. In one embodiment, FIG. 1 depicts the level of YH2AX in purified human the Subject is a non-human animal such as a mouse, rat, or peripheral blood mononuclear cells (PBMCs) at varying dog. In a preferred embodiment, the Subject is a human. doses of compound VE-822 using flow cytometry, following Any compound that inhibits ATR may be utilized when exposure to 4-nitroquinoline. administering a compound to the Subject. This may include FIG.2 depicts the level of YH2AX in lymphocytes in whole 60 those compounds that indirectly inhibit ATR via inhibition of human blood at varying doses of compound VE-822 using an upstream or downstream target in the same biological flow cytometry (scatterplot gating), following exposure to pathway.