Targeting Homologous Recombination Increases Cytotoxicity While Reducing Genomic Instability Induced By Melphalan in Myeloma

Abstract

Currently the greatest problem in successfully treating cancer is the genomic adaptability of the tumor cells leading to their continual survival and progression including development of resistance to treatment. Using genome sequencing, we have previously demonstrated that multiple myeloma (MM) cells derived from patients display heterogeneity of mutational spectrum and diverse patterns of clonal evolution, including linear evolution, differential clonal response, or branching evolution. This clonal evolution could be driven by pressures from inherent or acquired genomic and epigenomic changes, microenvironment and/or therapy itself, leading to both clonal selection and formation of new clones. A fundamental problem is that most chemotherapeutics agents are DNA damaging agents which kill cancer cells by increasing damage to their DNA. Although such a treatment can kill a large fraction of cancer cells, it is also likely to increase genomic instability in surviving cancer cells, possibly resulting in emergence of drug-resistant clones and subsequent relapse. A similar effect may emerge in normal cells of body.The purpose of this study was to investigate the impact of a chemotherapeutic agent melphalan on genome stability in the presence or absence of inhibitors of homologous recombination (HR) . MM cells were cultured with or without melphalan, RI-1 (which inhibits HR through inhibition of RAD51), nilotinib (which inhibits HR through inhibition of ABL-kinase) and combination of melphalan with these HR inhibitors. The cells were evaluated for viability using CellTiter-Glo luminescent c ell viability assay and apoptosis (using flow cytometry) at different time points. Cells were also evaluated for HR activity using a plasmid based assay as well as DNA breaks which were assessed from levels of gH2AX (detected by Western blotting and immunofluorescence). To investigate the impact of these treatments on genomic instability, dead cells were removed and live cells were examined for micronuclei, a marker of genomic instability. Treatment of MM cells with HR inhibitors was associated with both inhibition of HR activity and near complete loss of gH2AX signal indicating strong inhibition of spontaneous DNA breaks, whereas treatment with melphalan caused an increase in both the HR activity (by ~ 2.4-fold) and levels of gH2AX (by > 3-fold). Interestingly, although both HR inhibitors could completely reverse melphalan-induced HR, the levels of gH2AX were in fact increased ~ ≥ 2-fold. The increased levels of gH2AX by melphalan and combination treatments were also associated with a proportionate increase in cell death and apoptosis in these cells. These data indicate that inhibition of HR-mediated repair increases cytotoxicity of melphalan by increasing DNA breaks. Although the melphalan treatment increased the genomic instability (as assessed from micronuclei) in MM cells by ~80%, both the HR inhibitors could completely reverse melphalan-induced genomic instability.Taken together these data suggest that dysregulated HR in MM cells not only contributes to increased DNA breaks but is also involved in recombinational repair/utilization of DNA damaging agent-induced breaks. Thus providing a mechanism for both the genomic adaptability as well as survival against genotoxic insults. Inhibitors of HR, therefore, have potential to inhibit/reduce intrinsic as well as chemotherapy-induced genomic instability in myeloma cells as well as can increase in the efficacy of chemotherapy. DisclosuresNo relevant conflicts of interest to declare.