Ongoing Spontaneous DNA Damage Creates Synthetic Lethality Targeted By Novel RAD51 Inhibitors in Multiple Myeloma

Unrepaired DNA double-strand breaks (DSBs) pose a serious threat to genomic stability, potentially leading to the formation of oncogenic mutations, as well as translocations, deletions and amplifications. Almost all multiple myeloma (MM) patients are characterized by such copy number alterations and structural variations. Therefore, we evaluated and report that MM cells have increased ongoing spontaneous DNA damage which could be exploited to create synthetic lethality in combination with inhibitors of homologous recombination (HR). We evaluated various types of DNA damage in peripheral blood mononuclear cells (PBMCs)from 15healthy controls, 4 MM cell lines and CD138+ bone marrow plasma cells (BMPCs) from 16 patients with monoclonal gammopathy of undetermined significance, 18 with smoldering myeloma (SMM) and 15 with MM. Relative to normal PBMCs, MGUS and MM cells showed significantly higher levels of endogenous DNA breaks (measured as olive tail moments with single-cell gel electrophoresis), abasic sites (Elisa-based detection) as well as DNA break repair efficiency (all P<0.001). Interestingly, a gradual significant increase of all the DNA damage parameters was observed in BMPCs duringthe progression from MGUS to MM, with PBMCs from healthy controls showing the least levels (all P<0.05).Thus, MM cells have both the increased levels of endogenously generated DNA damage as well as DNA repair. A functioning HR is thus a dependency for MM cell survival. Our data confirms elevated HR activity in MM contributing to both genomic evolution as well as tumor growth. We have further exploited this molecular and functional dependency by targeting RAD51, a recombinase involved in the recognition of sequence homology and exchange of DNA strands between homologous sequences, playing a key role in the HR. We evaluated antitumor activity of novel RAD51 inhibitors (CYT-0178 and CYT-0851) in MM. CYT-0851 is currently in clinical testing in a range of cancers. First, we evaluated HR using 3 different functional HR assays in MM as well as other cancer cell lines. Treatment of MM1S and FLO-1 cancer cell lines with CYT-0178 at 5 mM demonstrated > 70% inhibition of HR activity measured using plasmid-based assay. Inhibition of HR by CYT-0178 was also demonstrated in U2OS cell using DRGFP (Addgene) assay. Moreover, treatment of RPMI-8226, MM1S, H929 MM cell lines with CYT-0851caused a dose-dependent inhibition of HR activity as assessed by homologous strand exchange assay. To evaluate the impact of these drugs on cell viability, MM cell lines (U266, RPMI8226 and MM1S) and normal cells were incubated with CYT-0851 and CYT-0178 at various concentrations and durations. Treatment with CYT-0851 induced dose- and time-dependent cytotoxicity in all myeloma cell lines tested with minimal impact on normal cells. For CYT-0178, the treatment at 10 𝛍M for seven days resulted in 50% to ~80% cell death in MM cell lines tested, although lower (5 𝛍M) dose had no significant impact. We further evaluated combination of these inhibitors with conventional MM drugs melphalan, cyclophosphamide and lenalidomide,for impact on cytotoxicity. Treatment with CYT-0851 led to a synergistic anticancer effect when combined with these chemotherapeutic agents in both the MM cell lines tested. Increased cytotoxicity of lenalidomide and melphalan was also observed by CYT-0178 in MM1S cells. In summary, we demonstrate that MM cells have ongoing endogenous DNA damage which provides basis for synthetic lethality in combination with HR inhibitors while sparing normal cells. Moreover, novel RAD51 inhibitors CYT-0178 and CYT-0851 inhibit HR activity, impair cell growth and increase efficacy of existing drugs in multiple myeloma.