Quantitative measurement of alterations in DNA damage repair (DDR) pathways using single cell network profiling (SCNP)

Journal of Translational Medicine

Rachael E Hawtin¹  Alessandra Cesano³  Scott Z Fields²  Iuliana Shapira²  Andrew Conroy¹  James A Cordeiro¹  Brent Louie¹  Ling Y Leung¹  David B Rosen¹ 

[1] Research, Nodality Inc., 170 Harbor Way, Suite 200, South San Francisco, CA 94080, USA;Monter Cancer Center, North Shore Long Island Jewish Medical School, 450 Lakeville Road Lake Success, New York, NY 11042, USA;Clinical Affairs, Nodality Inc., South San Francisco, CA, USA

关键词: FACS;    Multiparameter flow cytometry;    PARP;    ATM;    BRCA;    Non-homologous end joining;    Homologous recombination;    Cell cycle;    DNA damage repair;    Genomic instability;   

Others  :  1148787
DOI  :  10.1186/1479-5876-12-184

 received in 2014-02-24, accepted in 2014-06-18,  发布年份 2014

【 摘 要 】

Background

Homologous recombination repair (HRR) pathway deficiencies have significant implications for cancer predisposition and treatment strategies. Improved quantitative methods for functionally characterizing these deficiencies are required to accurately identify patients at risk of developing cancer and to identify mechanisms of drug resistance or sensitivity.

Methods

Flow cytometry-based single cell network profiling (SCNP) was used to measure drug-induced activation of DNA damage response (DDR) proteins in cell lines with defined HRR pathway mutations (including ATM-/-, ATM+/-, BRCA1+/-, BRCA2-/-) and in primary acute myeloid leukemia (AML) samples. Both non-homologous end joining (NHEJ) and HRR pathways were examined by measuring changes in intracellular readouts (including p-H2AX, p-ATM, p-DNA-PKcs, p-53BP1, p-RPA2/32, p-BRCA1, p-p53, and p21) in response to exposure to mechanistically distinct genotoxins. The cell cycle S/G2/M phase CyclinA2 marker was used to normalize for proliferation rates.

Results

Etoposide induced proliferation-independent DNA damage and activation of multiple DDR proteins in primary AML cells and ATM +/+but not ATM -/- cell lines. Treatment with the PARPi AZD2281 +/- temozolomide induced DNA damage in CyclinA2+ cells in both primary AML cells and cell lines and distngiushed cell lines deficient (BRCA2-/-) or impaired (BRCA1+/-) in HRR activity from BRCA1+/+ cell lines based on p-H2AX induction. Application of this assay to primary AML samples identified heterogeneous patterns of repair activity including muted or proficient activation of NHEJ and HRR pathways and predominant activation of NHEJ in a subset of samples.

Conclusions

SCNP identified functional DDR readouts in both NHEJ and HRR pathways, which can be applied to identify cells with BRCA1+/- haploinsuffiency and characterize differential DDR pathway functionality in primary clinical samples.

【 授权许可】

   
2014 Rosen et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 6.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

【 参考文献 】

• [1]Harper JW, Elledge SJ: The DNA damage response: ten years after. Mol Cell 2007, 28:739-745.
• [2]Kass EM, Jasin M: Collaboration and competition between DNA double-strand break repair pathways. FEBS Lett 2010, 584:3703-3708.
• [3]Jalal S, Earley JN, Turchi JJ: DNA repair: from genome maintenance to biomarker and therapeutic target. Clin Cancer Res 2011, 17:6973-6984.
• [4]Mladenov E, Iliakis G: Induction and repair of DNA double strand breaks: the increasing spectrum of non-homologous end joining pathways. Mutat Res 2011, 711:61-72.
• [5]Irish JM, Hovland R, Krutzik PO, Perez OD, Bruserud O, Gjertsen BT, Nolan GP: Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell 2004, 118:217-228.
• [6]Sachs K, Perez O, Pe’er D, Lauffenburger DA, Nolan GP: Causal protein-signaling networks derived from multiparameter single-cell data. Science 2005, 308:523-529.
• [7]Rosen DB, Cordeiro JA, Cohen A, Lacayo N, Hogge D, Hawtin RE, Cesano A: Assessing signaling pathways associated with in vitro resistance to cytotoxic agents in AML. Leuk Res 2012, 36:900-904.
• [8]Rosen DB, Putta S, Covey T, Huang YW, Nolan GP, Cesano A, Minden MD, Fantl WJ: Distinct patterns of DNA damage response and apoptosis correlate with Jak/Stat and PI3kinase response profiles in human acute myelogenous leukemia. PLoS One 2010, 5:e12405.
• [9]Brooks DJ, Srinivas NR, Alberts DS, Thomas T, Igwemzie LM, McKinney LM, Randolph J, Schacter L, Kaul S, Barbhaiya RH: Phase I and pharmacokinetic study of etoposide phosphate. Anticancer Drugs 1995, 6:637-644.
• [10]Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, Mortimer P, Swaisland H, Lau A, O’Connor MJ, Ashworth A, Carmichael J, Kaye SB, Schellens JH, de Bono JS: Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med 2009, 361:123-134.
• [11]Britten CD, Rowinsky EK, Baker SD, Agarwala SS, Eckardt JR, Barrington R, Diab SG, Hammond LA, Johnson T, Villalona-Calero M, Fraass U, Statkevich P, Von Hoff DD, Eckhardt SG: A Phase I and pharmacokinetic study of temozolomide and cisplatin in patients with advanced solid malignancies. Clin Cancer Res 1999, 5:1629-1637.
• [12]Purvis N, Stelzer G: Multi-platform, multi-site instrumentation and reagent standardization. Cytometry 1998, 33:156-165.
• [13]Stelzer GT, Goodpasture L: Use of multiparameter flow cytometry and immunophenotyping for the diagnosis and classification of acute myeloid leukemia. In Immunophenotyping. Edited by Stewart CC, Nicholson JKA. Wilmington DE: Wiley-Liss; 2000:215-238.
• [14]Cesano A, Rosen DB, O’Meara P, Putta S, Gayko U, Spellmeyer DC, Cripe LD, Sun Z, Uno H, Litzow MR, Tallman MS, Paietta E: Functional pathway analysis in acute myeloid leukemia using single cell network profiling assay: effect of specimen source (bone marrow or peripheral blood) on assay readouts. Cytometry B Clin Cytom 2012, 82B:158-172.
• [15]Ashworth A: A synthetic lethal therapeutic approach: poly(ADP) ribose polymerase inhibitors for the treatment of cancers deficient in DNA double-strand break repair. J Clin Oncol 2008, 26:3785-3790.
• [16]De Soto JA, Wang X, Tominaga Y, Wang RH, Cao L, Qiao W, Li C, Xu X, Skoumbourdis AP, Prindiville SA, Thomas CJ, Deng CX: The inhibition and treatment of breast cancer with poly (ADP-ribose) polymerase (PARP-1) inhibitors. Int J Biol Sci 2006, 2:179-185.
• [17]Maecker H, Rinfret A, D’Souza P, Darden J, Roig E, Landry C, Hayes P, Birungi J, Anzala O, Garcia M, Harari A, Frank I, Baydo R, Baker M, Holbrook J, Ottinger J, Lamoreaux L, Epling CL, Sinclair E, Suni MA, Punt K, Calarota S, El-Bahi S, Alter G, Maila H, Kuta E, Cox J, Gray C, Altfeld M, Nougarede N, et al.: Standardization of cytokine flow cytometry assays. BMC Immunol 2005, 6:13. BioMed Central Full Text
• [18]Kolitz JE, George SL, Dodge RK, Hurd DD, Powell BL, Allen SL, Velez-Garcia E, Moore JO, Shea TC, Hoke E, Caligiuri MA, Vardiman JW, Bloomfield CD, Larson RA: Dose escalation studies of cytarabine, daunorubicin, and etoposide with and without multidrug resistance modulation with PSC-833 in untreated adults with acute myeloid leukemia younger than 60 years: final induction results of Cancer and Leukemia Group B Study 9621. J Clin Oncol 2004, 22:4290-4301.
• [19]Abrahamsson J, Forestier E, Heldrup J, Jahnukainen K, Jónsson ÓG, Lausen B, Palle J, Zeller B, Hasle H: Response-guided Induction therapy in pediatric acute myeloid leukemia with excellent remission rate. J Clin Oncol 2011, 29:310-315.
• [20]Plummer R: Perspective on the pipeline of drugs being developed with modulation of DNA damage as a target. Clin Cancer Res 2010, 16:4527-4531.
• [21]Ledford H: Drug candidates derailed in case of mistaken identity. Nature 2012, 483:519.
• [22]Borg A, Haile RW, Malone KE, Capanu M, Diep A, Torngren T, Teraoka S, Begg CB, Thomas DC, Concannon P, Mellemkjaer L, Bernstein L, Tellhed L, Xue S, Olson ER, Liang X, Dolle J, Borrersen-Dale AL, Bernstein JL: Characterization of BRCA1 and BRCA2 deleterious mutations and variants of unknown clinical significance in unilateral and bilateral breast cancer: the WECARE study. Hum Mutat 2010, 31:E1200-E1240.
• [23]Darzynkiewicz Z, Traganos F, Zhao H, Halicka HD, Skommer J, Wlodkowic D: Analysis of individual molecular events of DNA damage response by flow- and image-assisted cytometry. Methods Cell Biol 2011, 103:115-147.
• [24]Amrein L, Loignon M, Goulet AC, Dunn M, Jean-Claude B, Aloyz R, Panasci L: Chlorambucil cytotoxicity in malignant B lymphocytes is synergistically increased by 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026)-mediated inhibition of DNA double-strand break repair via inhibition of DNA-dependent protein kinase. J Pharmacol Exp Ther 2007, 321:848-855.
• [25]Zhao H, Traganos F, Albino AP, Darzynkiewicz Z: Oxidative stress induces cell cycle-dependent Mre11 recruitment, ATM and Chk2 activation and histone H2AX phosphorylation. Cell Cycle 2008, 7:1490-1495.
• [26]Zhao H, Traganos F, Darzynkiewicz Z: Phosphorylation of p53 on Ser15 during cell cycle and caused by Topo I and Topo II inhibitors in relation to ATM and Chk2 activation. Cell Cycle 2008, 7:3048-3055.
• [27]Tanaka T, Huang X, Halicka HD, Zhao H, Traganos F, Albino AP, Dai W, Darzynkiewicz Z: Cytometry of ATM activation and histone H2AX phosphorylation to estimate extent of DNA damage induced by exogenous agents. Cytometry A 2007, 71:648-661.
• [28]Zhao H, Traganos F, Darzynkiewicz Z: Kinetics of histone H2AX phosphorylation and Chk2 activation in A549 cells treated with topotecan and mitoxantrone in relation to the cell cycle phase. Cytometry A 2008, 73:480-489.
• [29]Cesano A, Gotlib JR, Lacayo NJ, Putta S, Lackey A, Gayko U, Kornblau SM: Sample cryopreservation does not affect functional read outs in SCNP assays: implications for biomarker development. ASH Annual Meeting Abstracts 2010, 116:4843.
• [30]Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, Santarosa M, Dillon KJ, Hickson I, Knights C, Martin NM, Jackson SP, Smith GC, Ashworth A: Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 2005, 434:917-921.
• [31]Rottenberg S, Jaspers JE, Kersbergen A, van der Burg E, Nygren AO, Zander SA, Derksen PW, de Bruin M, Zevenhoven J, Lau A, Boulter R, Cranston A, O’Connor MJ, Martin NM, Borst P, Jonkers J: High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs. Proc Natl Acad Sci USA 2008, 105:17079-17084.
• [32]Strom CE, Johansson F, Uhlen M, Szigyarto CA, Erixon K, Helleday T: Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate. Nucleic Acids Res 2011, 39:3166-3175.
• [33]Patel AG, Sarkaria JN, Kaufmann SH: Nonhomologous end joining drives poly(ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells. Proc Natl Acad Sci USA 2011, 108:3406-3411.
• [34]Daemen A, Wolf DM, Korkola JE, Griffith OL, Frankum JR, Brough R, Jakkula LR, Wang NJ, Natrajan R, Reis-Filho JS, Lord CJ, Ashworth A, Spellman PT, Gray JW, van’t Veer LJ: Cross-platform pathway-based analysis identifies markers of response to the PARP inhibitor olaparib. Breast Cancer Res Treat 2012, 135:505-517.
• [35]Nakanishi K, Cavallo F, Brunet E, Jasin M: Homologous recombination assay for interstrand cross-link repair. Methods Mol Biol 2011, 745:283-291.
• [36]Salmon AY, Salmon-Divon M, Zahavi T, Barash Y, Levy-Drummer RS, Jacob-Hirsch J, Peretz T: Determination of molecular markers for BRCA1 and BRCA2 heterozygosity using gene expression profiling. Cancer Prev Res (Phila) 2013, 6:82-90.
• [37]Kornblau SM, Minden MD, Rosen DB, Putta S, Cohen A, Covey T, Spellmeyer DC, Fantl WJ, Gayko U, Cesano A: Dynamic single-cell network profiles in acute myelogenous leukemia are associated with patient response to standard induction therapy. Clin Cancer Res 2010, 16:3721-3733.
• [38]Cesano A, Evensen E, Ptacek J, Cordeiro J, Hawtin RE, Ware JR, Nichele I, Scupoli MT: BCR responsiveness is associated with Time to First Treatment (TTFT) in B-Cell Chronic Lymphocytic Leukemia (B-CLL): results from a Single Cell Network Profiling (SCNP) verification study. ASH Annual Meeting Abstracts 2011, 118:2834.