Dinaciclib, a CDK Inhibitor, Impairs Homologous Recombination and Sensitizes Multiple Myeloma Cells to PARP Inhibition

Multiple myeloma (MM) cells are characterized by genomic instability, implicating aberrant DNA damage repair. They exhibit pervasive double strand breaks (DSBs), the most lethal DNA lesions, as indicated by the constitutive abundance of γH2AX foci. DSBs can be repaired through homologous recombination (HR) or nonhomologous end joining (NHEJ). We previously showed elevated HR in MM cells, which were reported to have impaired NHEJ function; they may thus depend on elevated HR for survival. Poly (ADP-ribose) polymerase 1 (PARP1), hyperactivated in MM cells and required for single-strand break (SSB) repair, when inhibited results in accumulation of SSBs that degenerate into lethal DSBs during replication. HR occurs predominantly in the S and G2 cell-cycle phases, and is the preferred route of DSB repair. Should HR be unavailable, as is the case in BRCA-deficient tumor cells, these DSBs persist and/or are repaired by the more error-prone NHEJ, ultimately leading to cell death. Cyclin-dependent kinases (CDKs), upregulated in MM and previously targeted in chemo­therapy, are known to modulate HR. CDK1 and 2 phosphorylate BRCA1, a modification considered essential for BRCA1 to function in checkpoint activation and DSB repair. CDK5 activates the intra-S and G2 checkpoints via ATM phosphorylation and mediates cell sensitivity to PARP inhibition. Previous studies have shown that a proteasome inhibitor, bortezomib, induces a state of “BRCAness” in MM cells and sensitizes the cells to PARP inhibition. We hypothesized that targeting CDKs with dinaciclib (which inhibits CDKs 1, 2, 5 and 9, and is being evaluated for MM treatment), will disrupt HR repair and create a “contextual synthetic lethality” in MM cells when combined with PARP inhibition. We investigated the effects on human MM cell lines of dinaciclib, doxorubicin (a topoisomerase-II inhibitor that induces DSBs), and veliparib (an inhibitor of PARP1/2). Confocal immuno­fluores­cence microscopy was used to quantify γH2AX, BRCA1 and RAD51 foci in order to assess dinaciclib ± veliparib effects on recruitment of HR proteins to DSB sites. PCR arrays and qRT-PCR were used to examine the effects of dinaciclib on mRNA levels of DNA-repair genes, and western blotting to measure levels of phospho-BRCA1 (at Ser1497, a CDK target site) and RAD51 proteins. Dinaciclib effects on HR were measured in a MM cell line (MM.1S) bearing a chromosomally integrated DR-GFP reporter. Cytotoxicity was determined by WST-1 and colony-formation assays. In vivo consequences of dinaciclib ± veliparib were examined in a myeloma xenograft model, CB.17/SCID mice with MM cells injected subcutaneously. Total RNA was extracted from those tumors and analyzed by qRT-PCR to determine in vivoeffects of dinaciclib on HR-repair genes. We observed significant decreases in the percentage of cells with ≥5 BRCA1 foci (47% to 6%, p In conclusion, CDK inhibition via dinaciclib inhibits HR in MM cells, and creates a contextual synthetic lethality when combined with PARP inhibition. This combination represents an effective treatment strategy for MM. Disclosures No relevant conflicts of interest to declare.