The Checkpoint Kinase Inhibitor AZD7762 Potentiates Chemotherapy-Induced Apoptosis of p53-Mutated Multiple Myeloma Cells

DNA cross-linking agents are frequently used in the treatment of multiple myeloma–generating lesions, which activate checkpoint kinase 1 (Chk1), a critical transducer of the DNA damage response. Chk1 activation promotes cell survival by regulating cell-cycle arrest and DNA repair following genotoxic stress. The ability of AZD7762, an ATP-competitive Chk1/2 inhibitor to increase the efficacy of the DNA-damaging agents bendamustine, melphalan, and doxorubicin was examined using four human myeloma cell lines, KMS-12-BM, KMS-12-PE, RPMI-8226, and U266B1. The in vitro activity of AZD7762 as monotherapy and combined with alkylating agents and the “novel” drug bortezomib was evaluated by studying its effects on cytotoxicity, signaling, and apoptotic pathways. The Chk1/2 inhibitor AZD7762 potentiated the antiproliferative effects of bendamustine, melphalan, and doxorubicin but not bortezomib in multiple myeloma cell lines that were p53-deficient. Increased γH2AX staining in cells treated with bendamustine or melphalan plus AZD7762 indicates a greater degree of DNA damage with combined therapy. Abrogation of the G2–M checkpoint by AZD7762 resulted in mitotic catastrophe with ensuing apoptosis evidenced by PARP and caspase-3 cleavage. In summary, the cytotoxic effects of bendamustine, melphalan and doxorubicin on p53-deficient multiple myeloma cell lines were enhanced by the coadministration of AZD7762. These data provide a rationale for testing these combinations in patients with relapsed and/or refractory multiple myeloma. Mol Cancer Ther; 11(8); 1781–8. ©2012 AACR.

[1]  K. Anderson,et al.  The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications. , 2003, Blood.

[2]  S. Boehrer,et al.  In vitro induction of apoptosis of neoplastic cells in low-grade non-Hodgkin's lymphomas using combinations of established cytotoxic drugs with bendamustine. , 2001, Haematologica.

[3]  E. Moler,et al.  CHIR-124, a Novel Potent Inhibitor of Chk1, Potentiates the Cytotoxicity of Topoisomerase I Poisons In vitro and In vivo , 2007, Clinical Cancer Research.

[4]  G. Schwartz,et al.  Sequential dependent enhancement of caspase activation and apoptosis by flavopiridol on paclitaxel-treated human gastric and breast cancer cells. , 1999, Clinical cancer research : an official journal of the American Association for Cancer Research.

[5]  G. Zachos,et al.  Chk1-Dependent S-M Checkpoint Delay in Vertebrate Cells Is Linked to Maintenance of Viable Replication Structures , 2005, Molecular and Cellular Biology.

[6]  James B. Mitchell,et al.  In vitro and In vivo Radiation Sensitization of Human Tumor Cells by a Novel Checkpoint Kinase Inhibitor, AZD7762 , 2010, Clinical Cancer Research.

[7]  T. Therneau,et al.  Clinical course of patients with relapsed multiple myeloma. , 2004, Mayo Clinic proceedings.

[8]  E. Sausville,et al.  UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53. , 1996, Journal of the National Cancer Institute.

[9]  D. Conrad,et al.  Interruption of the NF-kappaB pathway by Bay 11-7082 promotes UCN-01-mediated mitochondrial dysfunction and apoptosis in human multiple myeloma cells. , 2004, Blood.

[10]  D. Dingli,et al.  Improved survival in multiple myeloma and the impact of novel therapies. , 2008, Blood.

[11]  P. Hasty,et al.  The checkpoint kinases Chk1 and Chk2 regulate the functional associations between hBRCA2 and Rad51 in response to DNA damage , 2008, Oncogene.

[12]  A. D’Andrea,et al.  Chk1-Mediated Phosphorylation of FANCE Is Required for the Fanconi Anemia/BRCA Pathway , 2007, Molecular and Cellular Biology.

[13]  A. Gunasekera,et al.  Chk1 Mediates S and G2 Arrests through Cdc25A Degradation in Response to DNA-damaging Agents* , 2003, Journal of Biological Chemistry.

[14]  G. Morgan,et al.  High-dose chemotherapy with hematopoietic stem-cell rescue for multiple myeloma. , 2003, The New England journal of medicine.

[15]  G. Schwartz,et al.  Potentiation of Cytotoxicity of Topoisomerase I Poison by Concurrent and Sequential Treatment with the Checkpoint Inhibitor UCN-01 Involves Disparate Mechanisms Resulting in Either p53-Independent Clonogenic Suppression or p53-Dependent Mitotic Catastrophe , 2004, Cancer Research.

[16]  P. Moreau,et al.  Bortezomib plus dexamethasone induction improves outcome of patients with t(4;14) myeloma but not outcome of patients with del(17p). , 2010, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[17]  B. Zhivotovsky,et al.  Death through a tragedy: mitotic catastrophe , 2008, Cell Death and Differentiation.

[18]  K. Yamashita,et al.  Cleavage-mediated Activation of Chk1 during Apoptosis* , 2008, Journal of Biological Chemistry.

[19]  Stephen Green,et al.  AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies , 2008, Molecular Cancer Therapeutics.

[20]  L. Cro,et al.  p53 gene mutations in multiple myeloma are associated with advanced forms of malignancy. , 1993, Blood.

[21]  James B. Mitchell,et al.  In Vitro and In Vivo Radiation Sensitization of Human Tumor Cells by a Novel Checkpoint Kinase Inhibitor , 2009 .

[22]  Y. Pommier,et al.  The Intra-S-Phase Checkpoint Affects both DNA Replication Initiation and Elongation: Single-Cell and -DNA Fiber Analyses , 2007, Molecular and Cellular Biology.

[23]  H. Einsele,et al.  International Myeloma Working Group consensus statement regarding the current status of allogeneic stem-cell transplantation for multiple myeloma. , 2010, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[24]  Jacques Corbeil,et al.  Bendamustine (Treanda) Displays a Distinct Pattern of Cytotoxicity and Unique Mechanistic Features Compared with Other Alkylating Agents , 2008, Clinical Cancer Research.

[25]  Xin Lu,et al.  Live or let die: the cell's response to p53 , 2002, Nature Reviews Cancer.

[26]  O. Cope,et al.  Multiple myeloma. , 1948, The New England journal of medicine.

[27]  P. Richardson,et al.  Proteasome inhibition as a novel therapeutic target in human cancer. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[28]  K. Anderson,et al.  Interleukin-6 Overcomes p21WAF1 Upregulation and G1 Growth Arrest Induced by Dexamethasone and Interferon-γ in Multiple Myeloma Cells , 1997 .

[29]  A. Almasan,et al.  Differential upregulation of p53-responsive genes by genotoxic stress in hematopoietic cells containing wild-type and mutant p53. , 1999, Gene expression.

[30]  Y. Matsuo,et al.  Guidelines for the characterization and publication of human malignant hematopoietic cell lines , 1999, Leukemia.

[31]  I. Roninson,et al.  If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells. , 2001, Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy.

[32]  P. O'Connor,et al.  Breaching the DNA damage checkpoint via PF-00477736, a novel small-molecule inhibitor of checkpoint kinase 1 , 2008, Molecular Cancer Therapeutics.

[33]  Jiri Bartek,et al.  Checking on DNA damage in S phase , 2004, Nature Reviews Molecular Cell Biology.

[34]  Y. Pommier,et al.  Chk1 inhibition after replicative stress activates a double strand break response mediated by ATM and DNA-dependent protein kinase , 2010, Cell cycle.