Dinaciclib, a CDK Inhibitor, Impairs Homologous Recombination and Sensitizes Multiple Myeloma Cells to PARP Inhibition
Authors: Alagpulinsa David , Ayyadevara Srinivas , Yaccoby Shmuel , shmookler Reis Robert 
Published: October 2019

Abstract
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<0.0001) or ≥5 RAD51 foci (26% to 4%, p<0.0001), in cells treated with veliparib plus dinaciclib, relative to veliparib alone. On the other hand, 19% of veliparib-treated cells showed ≥5 γH2AX foci compared to 52% for cells treated with veliparib plus dinaciclib (p<0.0001). Dinaciclib significantly reduced the transcript levels of genes involved in HR, including BRCA1 and RAD51 both in vitro and invivo, and elicited significant decreases (p≤0.05) in RAD51 protein levels and doxorubicin-induced phosphorylation of BRCA1 on S1497. In a DR-GFP reporter of HR events, 24% of vehicle-treated cells but only 4% of dinaciclib-treated cells were GFP+ (p<0.0001). Dinaciclib + veliparib treatment produced synergistic cytotoxicity (p≤0.05 for synergy) compared to either agent alone. MM cells were implanted subcutaneously, and tumor volume monitored in vivo over a 3-week period. Tumor volume increased 20- fold in mice exposed only to vehicle, but 9-fold with dinaciclib, 19-fold with veliparib, and 1.7-fold with the combination (p≤0.012 compared to vehicle or either agent alone). Log-rank tests for Kaplan-Meier survival curves showed a significant increase in survival (p≤0.004) for the dinaciclib+ veliparib treatment relative to single agent or vehicle treatment.
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.