Abstract 2827: Alternative non-homologous end joining DNA repair as therapeutic target in multiple myeloma

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Abstract Alternative Non Homologous End Joining DNA repair as therapeutic target in Multiple MyelomaDaniele Caracciolo, Martina Montesano, Emanuela Altomare, Grazia Consolo, Nicola Amodio, Maria Teresa Di Martino, Marco Rossi, Cirino Botta, Eugenio Morelli, Giada Juli, Pierosandro Tagliaferri, Pierfrancesco Tassone. UMG of Catanzaro, Catanzaro, ItalyBackground- Multiple Myeloma (MM) is a hematologic malignancy strongly characterized by genomic instability. We previously demonstrated that LIG3-dependent Alt-NHEJ repair is significantly involved in the genomic instability which promote survival and drug resistance of MM cells. Based on this rationale, we aimed to elucidate the efficacy of available Alt-NHEJ pathway inhibitors as new therapeutic agents 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 and EJ5-GFP plasmids. LIG3, LIG1, PARP1, Caspase 3 and p-H2AX, levels were analyzed by Western blot of whole protein extracts. In vivo anti-MM activity was evaluated in NOD-SCID mice bearing subcutaneous AMO-1 Bortezomib resistant (ABZB) xenografts, daily treated with Olaparib via oral gavage. Results- By interrogating public available datasets, we found that higher mRNA expression of Alt-NHEJ core components was correlated with shorter survival of MM patients. Indeed, plasmid-based reporter assays confirmed a prevalent activation of Alt-NHEJ repair in MM cells as compared to normal lymphoblastoid cell lines, except for U266 cells which instead displayed a prevalence of Canonical-NHEJ repair activity. Next, we attempted to evaluate the effects of available Alt-NHEJ inhibitors on MM cell survival. Consistently, L67 (LIG3-LIG1 inhibitor) or Olaparib (PARP-inhibitor) induced a significant reduction of proliferation and clonogenic cell growth of different MM cell lines at low micromolar concentrations, while no significant effects on survival were observed in U226 as well as in normal lymphoblastoid cells. Importantly L67 or Olaparib impaired viability of MM cell lines or primary plasmacells co-cultured on stromal cells, thus overcoming the supporting role played by MM-microenvironment on plasma-cells survival. As result of Alt-NHEJ repair inhibition, anti-proliferative effects were correlated to increase of DNA double-strand breaks (DSBs), cell cycle arrest and finally apoptosis. Notably, we observed that Bortezomib resistant AMO-1 derivative cells (ABZB) exhibited significantly increased sensitivity to a combination of L67 and Olaparib that increased the number of DSBs. Moreover, Alt-NHEJ inhibitors synergistically sensitized primary cells as well as MM cell lines to Bortezomib. Finally, to demonstrate the in vivo relevance of our findings, we showed that clinically available Parp-inhibitor Olaparib, exerted a significant anti-MM activity on ABZB cells injected in immunocompromised mice. Conclusion- Taken together, our findings indicate that MM cells are addicted to Alt-NHEJ repair parthway, which therefore represent a novel druggable target pathway in MM. Citation Format: Daniele Caracciolo, Martina Montesano, Emanuela Altomare, Grazia Consolo, Nicola Amodio, Maria Teresa Di Martino, Marco Rossi, Cirino Botta, Eugenio Morelli, Giada Juli, Pierosandro Tagliaferri, Pierfrancesco Tassone. Alternative non-homologous end joining DNA repair as therapeutic target in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2827.