Abstract 2400: Dinaciclib, an inhibitor of cyclin-dependent kinases (CDKs), impairs homologous recombination, abrogates G2 checkpoint arrest and sensitizes myeloma cells to PARP inhibition
Authors: David A. Alagpulinsa, Shmuel Yaccoby, Srinivas Ayyadevara, Robert J. S. Reis
Journal: Cancer Research (2014)

Abstract Background: Multiple myeloma (MM) is the second most common hematologic malignancy, and yet remains incurable in the majority of patients. We previously observed dysfunctional homologous recombination (HR) to mediate genomic instability and progression of MM.CDKs are key components of HR DNA repair and have been targeted by therapeutic drugs for MM. CDK1 phosphorylates BRCA1, and CDK5 (upregulated in MM and predictive of patient survival) phosphorylates ATM, modifications considered essential for checkpoint activation and HR repair. Poly(ADP-ribose)polymerase (PARP) is required for single-strand break (SSB) repair and when inhibited, SSBs accumulate and degenerate into double-strand breaks (DSBs) during DNA replication, resulting in cell death if HR is deficient. Dinaciclib (DC) is a potent inhibitor of CDK1, 2, 5 and 9, currently in clinical trials for MM. Here, we show that DC disrupts HR repair, abrogates G2 checkpoint arrest, and sensitizes MM cells to PARP inhibition. Methods: We investigated effects of DC, doxorubicin (topoisomerase II inhibitor), and veliparib (PARP inhibitor) on MM cell lines and myeloma cells from MM patients. We used confocal immunofluorescence microscopy to assess formation of γH2AX, BRCA1 and RAD51 foci, and western blotting to measure levels of phospho-BRCA1 (at Ser1497,CDK1 phosphorylation site) in order to assess the effects of DC on recruitment of HR proteins to DSBs. We measured DC effects on HR-mediated DSB repair, following transient infection with adenovirus encoding I-SceI, which forms DSBs within a DR-GFP reporter chromosomally integrated in MM.1S myeloma cells. Cell viability was determined using WST-1 and colony-formation assays. Flow cytometry after dual staining with propidium iodide (PI)/RNaseA and anti-phospho-histone H3 (Ser10) antibody was used to determine G2 checkpoint activation. In vivo consequences of DC treatment are currently being examined in a myeloma-xenograft mouse model. Results: We observed significant decreases in the percentage of cells with ≥5 BRCA1 foci (46.7% to 5.7%, p&amp;lt;0.001) or ≥5 RAD51 foci (26% to 4%, p&amp;lt;0.001), in cells treated with veliparib plus 20 nM DC, relative to veliparib alone. DC elicited ∼8-fold decrease in doxorubicin-induced phosphorylation of BRCA1 on S1497. In the DR-GFP assay of HR, 23.9% of vehicle-treated cells and 4.2% of DC-treated cells were GFP+, indicating that DC disrupts HR repair of DSBs. Cell viability following 72h treatments with veliparib ± DC, or vehicle alone, implied synergistic cytotoxicity between veliparib and DC. DC treatment significantly inhibited doxorubicin- or veliparib-induced G2/M phase cell accumulation and increased the fraction of mitotic cells in G2/M. Conclusion: DC significantly impairs HR DNA repair and sensitizes myeloma cells to PARP inhibition, and this combination may thus offer an effective treatment strategy for MM. Citation Format: David A. Alagpulinsa, Shmuel Yaccoby, Srinivas Ayyadevara, Robert J. S. Reis. Dinaciclib, an inhibitor of cyclin-dependent kinases (CDKs), impairs homologous recombination, abrogates G2 checkpoint arrest and sensitizes myeloma cells to PARP inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2400. doi:10.1158/1538-7445.AM2014-2400