Bortezomib Induced BRCAness Sensitizes Multiple Myeloma Cells to PARP Inhibitors
Authors: Neri Paola , Ren Li , Gratton Kathy , Stebner Erin , Owen Carolyn J , Duggan Peter , Mansoor Adnan , Stewart Douglas A. , Bahlis Nizar J 
Published: October 2019

Abstract
Abstract 789

Background:
Poly-ADP-ribose-polymerase (PARP) inhibitors are cytotoxic to tumor cells with impaired DNA damage repair machinery (DRR), in particular those with a deficient homology directed repair (HR) of DNA double stranded breaks (DSB). Multiple Myeloma (MM) cells are characterized by a highly unstable genome and while the exact mechanisms for this karyotypic instability is largely unknown, their DDR machinery is thought to be highly stressed. The ubiquitin-proteasome system (UPS) is involved in the regulation of several cellular functions including DDR and in particular HR. In addition proteasome inhibitors are reported to induce an unfolded protein response (UPR) in MM cells resulting in their apoptotic death. We have postulated that inhibition of the 26S proteasome also alters the DNA-DSB repair machinery leading to a BRCAness state in MM cells, sensitizing them to PARP inhibitors.

Methods and results:
In order to biochemically inhibit PARP in MM cells, we used a novel selective inhibitor of PARP1 and PARP2, 2-(R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide or ABT-888. We first demonstrated inhibition of PARP activity as measured by a reduction in poly-ADP-ribose (PAR) polymer levels (western blotting) in human MM cell lines (MM1S, U266, H929, RPMI8226, KMS-11, OPM2, INA-6) treated with ABT-888 (5 μM). PARP inhibition and the reduction of PAR levels resulted in DNA damage as evidenced by ATM phosphorylation and induced DNA-DSBs with increased γH2AX (phospho-Ser139-H2AX) levels within 6–12 hours of MM cells treatment with ABT-888. Increased γH2AX foci formation was also detected by immunofluorescent staining within 6–12 hours of ABT-888 treatment and nearly fully resolved by 24 hours, consistent with repair of resultant DNA-DSBs. As expected treatment with ABT-888 alone had no effect on the viability of MM cells consistent with their ability to repair DNA-DSBs resulting from PARP inhibition.
We then examined the effect of bortezomib on HR-mediated repair of DNA-DSBs, in particular on the BRCA/FA pathway. A significant reduction of MM cells' FANCD2, BRCA1, BRCA2 and RAD51 mRNA levels (qRT-PCR) was observed within 6–12 hours of bortezomib treatment (10 nM). Similar results were observed at the protein level indicating that bortezomib impedes homology-directed DNA-DSBs repair and results in an operational BRCAness state in MM cells. Therefore, we next tested whether this bortezomib-induced BRCAness was sufficient to sensitize MM cells to PARP inhibition with ABT-888. Consistent with our hypothesis, we observed that co-treatment of MM cell lines with bortezomib and ABT-888 lead to persistent and increased γH2AX foci at 24 hours compared to treatment with ABT-888 alone. Co-treatment also significantly potentiated cell death (Annexin V/PI staining) compared to treatment with bortezomib alone. Similar results were observed in CD138+ primary MM cells (n=8) with strong synergistic effect (CI < 1) between bortezomib and ABT-888. Importantly, no impaired viability (Annexin/PI staining) or function (colony forming unit assay) was noted for CD138− cells or CD34+ peripheral blood stem cells after bortezomib and ABT-888 co-treatment. Mechanistic studies have also shown that apoptotic events (caspase 3, caspase 8 and PARP cleavage) are markedly enhanced by this combination.
Based on our in vitro data, we evaluated in vivo the activity of ABT-888 in combination with bortezomib in a Scid murine xenograft model of human MM. Significant inhibition of tumour growth (p<0.005) was noted in mice treated with the combination of bortezomib and ABT-888 compared to bortezomib alone or control-treated mice. This tumour growth inhibition also resulted in a significant increase in survival (p<0.05) of the animals. No toxicity (e.g. weight loss, ruffled coats, paralysis, etc.) was observed in mice treated with the combination. Induction of DNA-DSBs was also confirmed in vivo as shown by an increase in 53BP1 and γH2AX foci formation in tumors of mice treated with the combination compared to bortezomib alone.

Conclusion:
Our studies indicate that bortezomib induces a BRCAness state in MM cells by impairing HR-mediated repair of DNA-DSBs and results in a contextual synthetic lethality when combined with the PARP inhibitor ABT-888. These data provide the scientific basis for the future clinical testing of PARP inhibitors in combination with proteasome inhibitors for the treatment of MM.

Disclosures:
No relevant conflicts of interest to declare.