Deficient double strand breaks repair of bone marrow plasma cells correlates with better clinical outcome of multiple myeloma patients

Abstract

DNA Interstrand Crosslinks (ICLs) are complex DNA lesions generated by bi-functional alkylating agents, a class of compounds extensively used in cancer chemotherapy. During ICL repair, replication forks stall at the ICL, inducing the formation of a lethal form of DNA damage termed DNA Double-strand Breaks (DSBs), which are repaired mainly by homologous recombination (HR) and Non-homologous End Joining (NHEJ). In this study we applied the properties of the ICL-inducing agent melphalan as a model, to elucidate the underlying mechanisms in processing and repair of ICLs. We studied two Human Multiple Myeloma (HMCLs) cell lines (melphalan-sensitive RPMI-8226 and -resistant LR5) and CD138+ Bone Marrow Plasma Cells (BMPCs) from 15 newly diagnosed MM patients (8M/7F; median age 61 years) before they receive high-dose melphalan therapy supported by ASCT. HMCLs and BMPCs were treated with melphalan alone or in combination with RI-1 (selective inhibitor of HR) or SCR7 (selective inhibitor of NHEJ) and the extent of the N-ras-specific ICLs using a quantitative PCR assay and DSBs (intermediates of ICL repair) using γH2Ax foci measurements by confocal microscopy were evaluated. The induction of the melphalan-induced apoptosis using a photometric enzyme-assay was also evaluated.The ICLs repair efficiency correlated with response to treatment and could identify 2 groups of patients: non-responders (<PR, n=6), with higher ICLs repair efficiency (t_1/2 23h) and responders (≥PR, n=9) with lower efficiency (t_1/2 48h). Moreover, using γ-H2AX foci formation/removal measurement, we found that the repair efficiency of DSBs in BMPCs was significantly higher in non-responders (t_1/2 9h) than in responders (t_1/2 12h) (all p<0.001). Also, the melphalan-induced apoptosis in BMPCs inversely correlated with the repair efficiencies of ICLs and DSBs, with the toxicity being higher in responders than in non-responders. Furthermore, LR5 cells showed higher repair efficiency of both ICLs and DSBs and lower toxicity than RPMI-8226 cells. Interestingly, in all cellular populations analysed, significant correlation between ICL and DSBs levels was observed.To further elucidate the mechanism of drug-induced DSBs repair, HMCLs and BMPCs were treated with melphalan in combination with nontoxic doses of RI-1 or SCR7. We found that the combined treatment of melphalan with RI-1 or SCR7 significantly increased the induction of the melphalan-only phosphorylation of H2AX, delayed the repair of ICLs and strongly enhanced the cytotoxic activity of melphalan (all p<0.01). Collectively, our study demonstrates that significant changes in the repair efficiency of DSBs occur in MM. These changes affect the repair of ICLs, modify drug sensitivity of malignant BMPCs, and correlate with clinical outcome. Specific inhibition of HR and/or NHEJ might be an effective strategy to enhance sensitivity of cancer cells in MM.