Inhibition of Homologous Recombination Pathway Promotes Telomere Shortening and Cell Survival without Affecting Telomerase Activity In Myeloma

Abstract 786

Recombinase (RAD51) expression and homologous recombination (HR) activity are low in normal human cells including plasma cells. It is significantly induced following exposure of normal human cells to carcinogen, and is constitutively elevated in cancer cells including multiple myeloma (MM) cells. Besides its effect on genomic stability, elevated or dysregulated HR has also been implicated in telomere maintenance in tumor and immortalized cells. These cells usually lack telomerase activity and maintain telomere length by ALT mechanism (alternate lengthening of telomeres). Inhibitors of homologous recombination, therefore, have potential not only to prevent/reduce genomic instability, but also inhibit telomere maintenance, and cancer survival. We have here investigated the effect of inhibitor of HR on telomere maintenance mechanism in MM. We have evaluated effect of Nilotinib, a tyrosine kinase inhibitor and RAD51 shRNA on HR in MM. First we observed that nilotinib inhibits and RAD51 phosphorylation in MM. Nilotinib at both 5 and 10 mM concentration also led to dose-dependent inhibition of recombinase expression in MM cells. Importantly, Nilotinib also inhibited HR activity in MM cells as well as other cancer cell lines, as measured by a plasmid based assay in which leuciferase activity is generated following homologous recombination. We next evaluated effect of nilotinib on telomere maintenance alone as well as in combination with agents inhibiting telomere maintenance. The MM cells were treated for 48 hrs, either with nilotinib, telomerase inhibitor, or both nilotinib and telomerase inhibitor and evaluated for telomerase activity as well as effect on telomere length. As expected, the treatment of myeloma cells with telomerase inhibitor at 1 mM led to 88% inhibition of telomerase activity relative to control cells. Nilotinib, either alone or in the presence of telomerase inhibitor, did not have any major effect on telomerase activity in these cells. The cells were cultured in the presence of these agents for 2 weeks and evaluated for telomere length, using telomere specific real time PCR. Cells in presence of Telomerase inhibitor at 1 mM in fact had slightly increased telomere length (9%), probably due to presence or activation of ALT mechanism, following loss of telomerase activity. Importantly, nilotinib alone at 10 mM led to 20% reduction in telomere length and when combined with telomerease inhibitor at 1 mM concentrations led to reduction in the telomere length in MM cells by 52%. Moreover we have observed that transduction of MM cells with shRNA targeting RAD51 combined with telomerase inhibitor induced greater and quicker MM cell kill compared to either of these treatments alone. These data indicate that elevated HR pathway contributes to telomere maintenance in MM and combining inhibitors of HR with telomerase would expedite telomere shortening and cell death providing more effective therapeutic strategy.