Treatment options for TP53-mutated lymphoid tumors are very limited. In experimental models, TP53-mutated lymphomas were sensitive to direct inhibition of checkpoint kinase 1 (Chk1), a pivotal regulator of replication. We initially tested the potential of the highly specific Chk1 inhibitor SCH900776 to synergize with nucleoside analogs (NAs) fludarabine, cytarabine and gemcitabine in cell lines derived from B-cell malignancies. In p53-proficient NALM-6 cells, SCH900776 added to NAs enhanced signaling towards Chk1 (pSer317/pSer345), effectively blocked Chk1 activation (Ser296 autophosphorylation), increased replication stress (p53 and γ-H2AX accumulation) and temporarily potentiated apoptosis. In p53-defective MEC-1 cell line representing adverse chronic lymphocytic leukemia (CLL), Chk1 inhibition together with NAs led to enhanced and sustained replication stress and significantly potentiated apoptosis. Altogether, among 17 tested cell lines SCH900776 sensitized four of them to all three NAs. Focusing further on MEC-1 and co-treatment of SCH900776 with fludarabine, we disclosed chromosome pulverization in cells undergoing aberrant mitoses. SCH900776 also increased the effect of fludarabine in a proportion of primary CLL samples treated with pro-proliferative stimuli, including those with TP53 disruption. Finally, we observed a fludarabine potentiation by SCH900776 in a T-cell leukemia 1 (TCL1)-driven mouse model of CLL. Collectively, we have substantiated the significant potential of Chk1 inhibition in B-lymphoid cells.

CEITEC Central European Institute of Technology Brno University of Technology Brno Czech Republic

CEITEC Central European Institute of Technology Masaryk University Brno Czech Republic

Center of Biomolecular and Cellular Engineering International Clinical Research Center St Anne's University Hospital Brno Czech Republic

Department of Chemistry and Toxicology Veterinary Research Institute Brno Czech Republic

Department of Chemistry CZ Openscreen Faculty of Science Masaryk University Brno Czech Republic

Department of Cytokinetics Institute of Biophysics Academy of Sciences of the Czech Republic Brno Czech Republic

Department of Internal Medicine Hematology and Oncology University Hospital Brno and Faculty of Medicine Masaryk University Brno Czech Republic

Institute of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Institute of Physical Engineering Faculty of Mechanical Engineering Brno University of Technology Brno Czech Republic

Zobrazit více v PubMed

Robles AI, Harris CC. Clinical outcomes and correlates of TP53 mutations and cancer. Cold Spring Harb Perspect Biol. 2010;2:a001016. PubMed PMC

Stilgenbauer S, Schnaiter A, Paschka P, Zenz T, Rossi M, Döhner K, Bühler A, Böttcher S, Ritgen M, Kneba M, Winkler D, Tausch E, Hoth P, et al. Gene mutations and treatment outcome in chronic lymphocytic leukemia: results from the CLL8 trial. Blood. 2014;123:3247–54. PubMed

Farooqui MZ, Valdez J, Martyr S, Aue G, Saba N, Niemann CU, Herman SE, Tian X, Marti G, Soto S, Hughes TE, Jones J, Lipsky A, et al. Ibrutinib for previously untreated and relapsed or refractory chronic lymphocytic leukaemia with TP53 aberrations: a phase 2 single-arm trial. Lancet Oncol. 2015;16:169–76. PubMed PMC

Kwok M, Davies N, Agathanggelou A, Smith E, Oldreive C, Petermann E, Stewart G, Brown J, Lau A, Pratt G, Parry H, Taylor M, Moss P, et al. ATR inhibition induces synthetic lethality and overcomes chemoresistance in TP53 or ATM defective chronic lymphocytic leukemia cells. Blood. 2015;127:582–95. PubMed

Zhao H, Piwnica-Worms H. ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol Cell Biol. 2001;21:4129–39. PubMed PMC

Capasso H, Palermo C, Wan S, Rao H, John UP, O'Connell MJ, Walworth NC. Phosphorylation activates Chk1 and is required for checkpoint-mediated cell cycle arrest. J Cell Sci. 2002;115:4555–64. PubMed

Dai Y, Grant S. New insights into checkpoint kinase 1 in the DNA damage response signaling network. Clin Cancer Res. 2010;16:376–83. PubMed PMC

Lane D, Levine A. p53 Research: the past thirty years and the next thirty years. Cold Spring Harb Perspect Biol. 2010;2:a000893. PubMed PMC

Ma CX, Janetka JW, Piwnica-Worms H. Death by releasing the breaks: CHK1 inhibitors as cancer therapeutics. Trends Mol Med. 2011;17:88–96. PubMed PMC

Del Nagro CJ, Choi J, Xiao Y, Rangell L, Mohan S, Pandita A, Zha J, Jackson PK, O'Brien T. Chk1 inhibition in p53-deficient cell lines drives rapid chromosome fragmentation followed by caspase-independent cell death. Cell Cycle. 2014;13:303–14. PubMed PMC

Chen Z, Xiao Z, Gu WZ, Xue J, Bui MH, Kovar P, Li G, Wang G, Tao ZF, Tong Y, Lin NH, Sham HL, Wang JY, et al. Selective Chk1 inhibitors differentially sensitize p53-deficient cancer cells to cancer therapeutics. Int J Cancer. 2006;119:2784–94. PubMed

Zenvirt S, Kravchenko-Balasha N, Levitzki A. Status of p53 in human cancer cells does not predict efficacy of CHK1 kinase inhibitors combined with chemotherapeutic agents. Oncogene. 2010;29:6149–59. PubMed

Xiao Y, Ramiscal J, Kowanetz K, Del Nagro C, Malek S, Evangelista M, Blackwood E, Jackson PK, O'Brien T. Identification of preferred chemotherapeutics for combining with a CHK1 inhibitor. Mol Cancer Ther. 2013;12:2285–95. PubMed

Bryant C, Scriven K, Massey AJ. Inhibition of the checkpoint kinase Chk1 induces DNA damage and cell death in human Leukemia and Lymphoma cells. Mol Cancer. 2014;13:147. PubMed PMC

Höglund A, Nilsson LM, Muralidharan SV, Hasvold LA, Merta P, Rudelius M, Nikolova V, Keller U, Nilsson JA. Therapeutic implications for the induced levels of Chk1 in Myc-expressing cancer cells. Clin Cancer Res. 2011;17:7067–79. PubMed

Ferrao PT, Bukczynska EP, Johnstone RW, McArthur GA. Efficacy of CHK inhibitors as single agents in MYC-driven lymphoma cells. Oncogene. 2012;31:1661–72. PubMed

Guzi TJ, Paruch K, Dwyer MP, Labroli M, Shanahan F, Davis N, Taricani L, Wiswell D, Seghezzi W, Penaflor E, Bhagwat B, Wang W, Gu D, et al. Targeting the replication checkpoint using SCH 900776 a potent and functionally selective CHK1 inhibitor identified via high content screening. Mol Cancer Ther. 2011;10:591–602. PubMed

Montano R, Chung I, Garner KM, Parry D, Eastman A. Preclinical development of the novel Chk1 inhibitor SCH900776 in combination with DNA-damaging agents and antimetabolites. Mol Cancer Ther. 2012;11:427–38. PubMed PMC

Montano R, Thompson R, Chung I, Hou H, Khan N, Eastman A. Sensitization of human cancer cells to gemcitabine by the Chk1 inhibitor MK-8776: cell cycle perturbation and impact of administration schedule in vitro and in vivo. BMC Cancer. 2013;13:604. PubMed PMC

Sampath D, Shi Z, Plunkett W. Inhibition of cyclin-dependent kinase 2 by the Chk1-Cdc25A pathway during the S-phase checkpoint activated by fludarabine: dysregulation by 7-hydroxystaurosporine. Mol Pharmacol. 2002;62:680–8. PubMed

Schenk EL, Koh BD, Flatten KS, Peterson KL, Parry D, Hess AD, Smith BD, Karp JE, Karnitz LM, Kaufmann SH. Effects of selective checkpoint kinase 1 inhibition on cytarabine cytotoxicity in acute myelogenous leukemia cells in vitro. Clin Cancer Res. 2012;18:5364–73. PubMed PMC

Iwasaki H, Huang P, Keating MJ, Plunkett W. Differential incorporation of ara-C gemcitabine and fludarabine into replicating and repairing DNA in proliferating human leukemia cells. Blood. 1997;90:270–8. PubMed

Galmarini CM, Mackey JR, Dumontet C. Nucleoside analogues: mechanisms of drug resistance and reversal strategies. Leukemia. 2001;15:875–90. PubMed

Robertson LE, Chubb S, Meyn RE, Story M, Ford R, Hittelman WN, Plunkett W. Induction of apoptotic cell death in chronic lymphocytic leukemia by 2-chloro-2′-deoxyadenosine and 9-beta-D-arabinosyl-2-fluoroadenine. Blood. 1993;81:143–50. PubMed

Ewald B, Sampath D, Plunkett W. Nucleoside analogs: molecular mechanisms signaling cell death. Oncogene. 2008;27:6522–37. PubMed

Bartek J, Lukas J. Mammalian G1- and S-phase checkpoints in response to DNA damage. Curr Opin Cell Biol. 2001;13:738–47. PubMed

Bastin-Coyette L, Cardoen S, Smal C, de Viron E, Arts A, Amsailale R, Van Den Neste E, Bontemps F. Nucleoside analogs induce proteasomal down-regulation of p21 in chronic lymphocytic leukemia cell lines. Biochem Pharmacol. 2011;81:586–93. PubMed

Rodriguez R, Meuth M. Chk1 and p21 cooperate to prevent apoptosis during DNA replication fork stress. Mol Biol Cell. 2006;17:402–12. PubMed PMC

Thompson R, Eastman A. The cancer therapeutic potential of Chk1 inhibitors: how mechanistic studies impact on clinical trial design. Br J Clin Pharmacol. 2013;76:358–69. PubMed PMC

Tse AN, Rendahl KG, Sheikh T, Cheema H, Aardalen K, Embry M, Ma S, Moler EJ, Ni ZJ, Lopes de Menezes DE, Hibner B, Gesner TG, Schwartz GK. CHIR-124 a novel potent inhibitor of Chk1 potentiates the cytotoxicity of topoisomerase I poisons in vitro and in vivo. Clin Cancer Res. 2007;13:591–602. PubMed

Zachos G, Black EJ, Walker M, Scott MT, Vagnarelli P, Earnshaw WC, Gillespie DA. Chk1 is required for spindle checkpoint function. Dev Cell. 2007;12:247–60. PubMed PMC

Slabý T, Kolman P, Dostál Z, Antoš M, Lošťák M, Chmelík R. Off-axis setup taking full advantage of incoherent illumination in coherence-controlled holographic microscope. Opt Express. 2013;21:14747–62. PubMed

Patten PE, Chu CC, Albesiano E, Damle RN, Yan XJ, Kim D, Zhang L, Magli AR, Barrientos J, Kolitz JE, Allen SL, Rai KR, Roa S, et al. IGHV-unmutated and IGHV-mutated chronic lymphocytic leukemia cells produce activation-induced deaminase protein with a full range of biologic functions. Blood. 2012;120:4802–11. PubMed PMC

Willimott S, Baou M, Naresh K, Wagner SD. CD154 induces a switch in pro-survival Bcl-2 family members in chronic lymphocytic leukaemia. Br J Haematol. 2007;138:721–32. PubMed

Blackwood E, Epler J, Yen I, Flagella M, O'Brien T, Evangelista M, Schmidt S, Xiao Y, Choi J, Kowanetz K, Ramiscal J, Wong K, Jakubiak D, et al. Combination drug scheduling defines a “window of opportunity” for chemopotentiation of gemcitabine by an orally bioavailable selective ChK1 inhibitor GNE-900. Mol Cancer Ther. 2013;12:1968–80. PubMed

Bichi R, Shinton SA, Martin ES, Koval A, Calin GA, Cesari R, Russo G, Hardy RR, Croce CM. Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression. Proc Natl Acad Sci U SA. 2002;99:6955–60. PubMed PMC

Morgan MA, Parsels LA, Zhao L, Parsels JD, Davis MA, Hassan MC, Arumugarajah S, Hylander-Gans L, Morosini D, Simeone DM, Canman CE, Normolle DP, Zabludoff SD, et al. Mechanism of radiosensitization by the Chk1/2 inhibitor AZD7762 involves abrogation of the G2 checkpoint and inhibition of homologous recombinational DNA repair. Cancer Res. 2010;70:4972–81. PubMed PMC

Knapp S, Arruda P, Blagg J, Burley S, Drewry DH, Edwards A, Fabbro D, Gillespie P, Gray NS, Kuster B, Lackey KE, Mazzafera P, Tomkinson NC, et al. A public-private partnership to unlock the untargeted kinome. Nat Chem Biol. 2013;9:3–6. PubMed

Lossaint G, Besnard E, Fisher D, Piette J, Dulić V. Chk1 is dispensable for G2 arrest in response to sustained DNA damage when the ATM/p53/p21 pathway is functional. Oncogene. 2011;30:4261–74. PubMed

Gottifredi V, Karni-Schmidt O, Shieh SS, Prives C. p53 down-regulates CHK1 through p21 and the retinoblastoma protein. Mol Cell Biol. 2001;21:1066–76. PubMed PMC

Ewald B, Sampath D, Plunkett W. H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation. Mol Cancer Ther. 2007;6:1239–48. PubMed

Bartek J, Bartkova J, Lukas J. DNA damage signalling guards against activated oncogenes and tumour progression. Oncogene. 2007;26:7773–9. PubMed

Petermann E, Woodcock M, Helleday T. Chk1 promotes replication fork progression by controlling replication initiation. Proc Natl Acad Sci U SA. 2010;107:16090–5. PubMed PMC

Zegerman P, Diffley JF. DNA replication as a target of the DNA damage checkpoint. DNA Repair (Amst) 2009;8:1077–88. PubMed

Shi Z, Azuma A, Sampath D, Li YX, Huang P, Plunkett W. S-Phase arrest by nucleoside analogues and abrogation of survival without cell cycle progression by 7-hydroxystaurosporine. Cancer Res. 2001;61:1065–72. PubMed

Sampath D, Cortes J, Estrov Z, Du M, Shi Z, Andreeff M, Gandhi V, Plunkett W. Pharmacodynamics of cytarabine alone and in combination with 7-hydroxystaurosporine (UCN-01) in AML blasts in vitro and during a clinical trial. Blood. 2006;107:2517–24. PubMed PMC

Portugal J, Mansilla S, Bataller M. Mechanisms of drug-induced mitotic catastrophe in cancer cells. Curr Pharm Des. 2010;16:69–78. PubMed

Morgan MA, Parsels LA, Parsels JD, Mesiwala AK, Maybaum J, Lawrence TS. Role of checkpoint kinase 1 in preventing premature mitosis in response to gemcitabine. Cancer Res. 2005;65:6835–42. PubMed

Peddibhotla S, Lam MH, Gonzalez-Rimbau M, Rosen JM. The DNA-damage effector checkpoint kinase 1 is essential for chromosome segregation and cytokinesis. Proc Natl Acad Sci U SA. 2009;106:5159–64. PubMed PMC

Oberic L, Vaillant W, Hebraud B, Recher C, Suc E, Houyau P, Laurent G, Ysebaert L. Clinical activity of a new regimen combining gemcitabine and alemtuzumab in high-risk relapsed/refractory chronic lymphocytic leukemia patients. Eur J Haematol. 2015;94:37–42. PubMed

Durot E, Michallet AS, Leprêtre S, Le QH, Leblond V, Delmer A. Platinum and high-dose cytarabine-based regimens are efficient in ultra high/high-risk chronic lymphocytic leukemia and Richter's syndrome: results of a French retrospective multicenter study. Eur J Haematol. 2015;95:160–7. PubMed

Ten Hacken E, Burger JA. Microenvironment interactions and B-cell receptor signaling in Chronic Lymphocytic Leukemia: Implications for disease pathogenesis and treatment. Biochim Biophys Acta. 2016;1863:401–13. PubMed PMC

Johnson AJ, Lucas DM, Muthusamy N, Smith LL, Edwards RB, De Lay MD, Croce CM, Grever MR, Byrd JC. Characterization of the TCL-1 transgenic mouse as a preclinical drug development tool for human chronic lymphocytic leukemia. Blood. 2006;108:1334–8. PubMed PMC

Malcikova J, Smardova J, Rocnova L, Tichy B, Kuglik P, Vranova V, Cejkova S, Svitakova M, Skuhrova Francova H, Brychtova Y, Doubek M, Brejcha M, Klabusay M, et al. Monoallelic and biallelic inactivation of TP53 gene in chronic lymphocytic leukemia: selection impact on survival and response to DNA damage. Blood. 2009;114:5307–14. PubMed

Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P, Olivier M. Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Hum Mutat. 2007;28:622–9. PubMed

Camps J, Salaverria I, Garcia MJ, Prat E, Beà S, Pole JC, Hernández L, Del Rey J, Cigudosa JC, Bernués M, Caldas C, Colomer D, Miró R, Campo E. Genomic imbalances and patterns of karyotypic variability in mantle-cell lymphoma cell lines. Leuk Res. 2006;30:923–34. PubMed

Malcikova J, Stano-Kozubik K, Tichy B, Kantorova B, Pavlova S, Tom N, Radova L, Smardova J, Pardy F, Doubek M, Brychtova Y, Mraz M, Plevova K, et al. Detailed analysis of therapy-driven clonal evolution of TP53 mutations in chronic lymphocytic leukemia. Leukemia. 2015;29:877–85. PubMed PMC

Dwyer MP, Keertikar K, Paruch K, Alvarez C, Labroli M, Poker C, Fischmann TO, Mayer-Ezell R, Bond R, Wang Y, Azevedo R, Guzi TJ. Discovery of pyrazolo[1 5-a]pyrimidine-based Pim inhibitors: a template-based approach. Bioorg Med Chem Lett. 2013;23:6178–82. PubMed

Labroli M, Paruch K, Dwyer MP, Alvarez C, Keertikar K, Poker C, Rossman R, Duca JS, Fischmann TO, Madison V, Parry D, Davis N, Seghezzi W, et al. Discovery of pyrazolo[1 5-a]pyrimidine-based CHK1 inhibitors: a template-based approach--part 2. Bioorg Med Chem Lett. 2011;21:471–4. PubMed

Collakova J, Krizova A, Kollarova V, Dostal Z, Slaba M, Vesely P, Chmelik R. Coherence-controlled holographic microscopy enabled recognition of necrosis as the mechanism of cancer cells death after exposure to cytopathic turbid emulsion. J Biomed Opt. 2015;20:111213. PubMed

Drets ME, Shaw MW. Specific banding patterns of human chromosomes. Proc Natl Acad Sci U SA. 1971;68:2073–7. PubMed PMC

Widhopf GF, Cui B, Ghia EM, Chen L, Messer K, Shen Z, Briggs SP, Croce CM, Kipps TJ. ROR1 can interact with TCL1 and enhance leukemogenesis in Eμ-TCL1 transgenic mice. Proc Natl Acad Sci U SA. 2014;111:793–8. PubMed PMC

The CHK1 inhibitor MU380 significantly increases the sensitivity of human docetaxel-resistant prostate cancer cells to gemcitabine through the induction of mitotic catastrophe

Novel CHK1 inhibitor MU380 exhibits significant single-agent activity in TP53-mutated chronic lymphocytic leukemia cells

Distinct cellular responses to replication stress leading to apoptosis or senescence

Chk1 Inhibitor SCH900776 Effectively Potentiates the Cytotoxic Effects of Platinum-Based Chemotherapeutic Drugs in Human Colon Cancer Cells