The Simultaneous Accumulation of the Extremely Cytotoxic Interstrand Cross-Links and Double-Strand Breaks Contributes to the Successful Anti-Myeloma Therapy; The Effect of DNA Repair Inhibitors

Several anticancer drugs exert their antitumor effects through the production of DNA interstrand cross-links (ICLs). 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). Herein, using the ICL-inducing drug melphalan, we investigated the underlying mechanisms in processing and repair of pharmaceutical induced DNA damage and their contribution to the successful outcome of melphalan therapy.

We studied two human multiple myeloma (MM) cell lines (RPMI8226 and LR5, melphalan-sensitive and -resistant, respectively) as well as CD138+ bone marrow plasma cells (BMPCs) isolated from 15 newly diagnosed MM patients (8M/7F; median age 61 years) who underwent high-dose melphalan with autologous stem cell transplantation as first line therapy. Response assessment was based on the International Myeloma Working Group (IMWG) criteria. The patients categorized based on their outcome to responders (≥PR, n =9) and non-responders (<PR, n =6) to subsequent melphalan therapy. MM cell lines and BMPCs were ex vivo treated with melphalan alone or in combination with selective inhibitors of homologous recombination (RI-1) or non-homologous end joining (SCR7, NU7026) and the extent of the DNA damage formation/repair (monoadducts using Southern blot analysis; ICL using quantitative-PCR; DSBs using immunofluorescence quantification of γH2AX foci by confocal microscopy) as well as the induction of the apoptotic pathway (using a photometric enzyme-immunoassay) were evaluated.

In all MM patients, following ex vivo treatment of BMPCs with melphalan, monoadducts were repaired over two sharply demarcated phases: a first rapid phase (0-2h), and a delayed second phase (2-48h). Interestingly, responders showed higher accumulation of monoadducts due to the significantly slower first-phase repair observed in compare to non-responders (P <0.001). Similar rates of monoadducts removal were observed in all MM patients during the second phase of repair. To note, ICL "unhooking" rates were found to be similar in all MM patients. However, accumulation of ICLs was significantly higher in responders' BMPCs compared to non-responder's (P<0.05), due to higher levels of monoadducts (precursors of ICLs) left unrepaired in responders' cells, resulting in higher formation of ICLs. Moreover, DSBs burden was significantly higher in responders compared to non-responders, due to higher accumulation of ICLs (precursors of DSBs) and lower rates of DSBs repair in responders (P <0.001). More importantly, an inverse correlation was found between the apoptotic rates and the first-phase monoadducts repair capacity of the BMPCs as well as with the DSBs repair efficiency of these cells, with the apoptotic rates being significantly higher in responders compared to non-responders (all P <0.05). In line with the BMPCs data, significantly higher accumulation of melphalan-induced monoadducts, ICLs and DSBs were found in RPMI8226 compared to LR5 cells (P <0.01).

To further elucidate the mechanisms of drug-induced DSBs repair and their contribution to cell sensitivity, MM cell lines and BMPCs were treated with melphalan in combination with nontoxic doses of DSBs repair inhibitors. We found that the combined treatment of melphalan with an inhibitor significantly increased the melphalan-induced phosphorylation of H2AX and strongly enhanced the cytotoxic activity of melphalan (all P< 0.01). As expected, co-treatment with a DSB repair inhibitor had no effect on the levels of melphalan-induced monoadducts or ICLs.

We conclude that responders' BMPCs are characterized by slower first-phase repair of DNA damage resulting in higher levels of monoadducts and greater accumulation of ICLs, which in combination with the deficient DSBs repair efficiency leads to much higher DSBs burden in these cells. The simultaneous accumulation of high levels of the extremely cytotoxic ICLs and DSBs in responders' cells triggers the induction of the apoptotic pathway, a priority for successful clinical outcome. Finally, the enhancement of melphalan cytotoxicity in BMPCs by DSBs repair inhibitors offers a novel therapeutic strategy toward the treatment of MM eligible for ASCT patients.