PARP1-Mediated Alt-NHEJ Repair Is a Therapeutic Target in Multiple Myeloma

Background- Multiple Myeloma (MM) is a hematologic malignancy strongly characterized by genomic instability, which promotes disease progression and drug resistance. We previously demonstrated that LIG3-dependent Alt-NHEJ repair is involved in genomic instability, drug resistance and survival of MM cells. On these premises, we investigated PARP1 as driver component of Alt-NHEJ pathway and new therapeutic target in MM.

Materials and methods- Cell proliferation and apoptosis were evaluated with CellTiter-Glo assay and Annexin V staining. Alt-NHEJ repair was evaluated using EJ2-GFP. PARP1, Caspase 3, MYC and DNA Damage Response protein levels were analyzed by Western blot of whole protein extracts. In vivo anti-MM activity was evaluated in NOD-SCID mice bearing subcutaneous H929 and AMO-1 Bortezomib resistant (ABZB) xenografts, daily treated with Olaparib (Selleckchem) via oral gavage.

Results- By interrogating public available datasets, we found significant correlation between higher mRNA expression of PARP1 and shorter survival of MM patients. On these findings, we investigated the effect of available PARP inhibitors (PARPi) on MM cell survival. We found that Olaparib, a clinically available PARPi induced a significant reduction of proliferation and clonogenic growth of MM cell lines at low micromolar concentrations. Importantly, Olaparib impaired viability of MM cell lines or primary malignant plasmacells co-cultured with stromal cells, thus overcoming the bone marrow microenvironment supportive effect for MM survival. As result of PARP-mediated Alt-NHEJ repair inhibition, anti-proliferative effects were associated to increase of DNA double-strand breaks (DSBs), activation of DNA damage response, cell cycle arrest and finally apoptosis. To identify predictive biomarkers for PARPi in MM, a published sensitivity gene expression signature was applied to our MM gene expression profiling (GEP) data. Interestingly, this signature was particularly enriched in TC2 MM and secondary plasma cell leukemia (PCL). Therefore, in order to evaluate concordantly modulated sets of genes that were possibly associated to PARPi signature in MM, PARPi-positive and PARPi-negative MM-TC2 cases were compared by GSEA analysis. Interestingly, groups of genes regulated by MYC or involved in DNA repair resulted among the most significantly up-regulated in PARPi-positive versus PARPi-negative MM-TC2 cases. Accordingly, MYC transcript reached the highest median expression levels in sPCL and HMCLs across PC dyscrasia groups, and in MM-TC2 class. Consistently U266 cells, which was quiet insensitive to PARP knockdown or PARP inhibitor Olaparib, were null for c-MYC as compared to multiple myeloma cell lines evaluated in this study. Conversely, as formal proof of our hypothesis, over-expression of c-MYC in U266 cells (MYC-OE) induced cell death upon PARP silencing or PARP inhibitor treatment. Notably, we found that c-MYC-PARP1 loop was also hyper-activated in Bortezomib resistant cells, thus confirming pivotal role of Alt-NHEJ repair in drug resistance development.

Remarkably, to demonstrate the in vivo relevance of our findings, we showed that clinically available Parp-inhibitor Olaparib exerted a significant anti-MM activity on both Bortezomib sensitive (H929) and resistant (ABZB) MM cells injected in immunocompromised mice.

Conclusion Taken together, our findings indicate that MM cells are dependent on PARP-mediated Alt-NHEJ repair pathway, which therefore represents a novel druggable target pathway in MYC-driven MM cells.