Compromised Nuclear Sirtuins Activity Sensitizes BRCA-Proficient multiple Myeloma Cells to DNA Damage Agents

Multiple myeloma (MM) is a clonal malignancy of plasma cells with hallmark genetic instability resulting in large-scale changes at diagnosis, as well as further evolution contributing to disease progression. Inhibition of DNA repair mechanisms leads to significant reduction in acquisition of new genetic changes and associated progression of MM. Mammalian sirtuins are class III NAD+-dependent deacetylases emerging as innovative proteins involved in multiple pathways, including genome maintenance.

A panel of 18 MM cell lines, both sensitive and resistant to conventional and novel anti-MM therapies, was used in the study. The antitumor effect of a pan-sirtuins inhibitor, Nicotinamide (Nam), alone and combined with DNA-damaging agents, was investigated by CTG assay and Annexin-V/propidium iodide staining. Mechanistic studies were performed with thymidine incorporation, Western-blotting, lentivirus-mediated shRNAs and immunofluorescence assay. Analysis of DNA DSB repair was done using chromosomally integrated reporter constructs, followed by cytometer analysis.

We analyzed an Affymetrix GeneChip (GSE6477) array of patient MM cells (n=162) compared with normal plasma cells, and found that transcript levels of two nuclear sirtuins (SIRT6 and SIRT7) were significantly higher in monoclonal gammopathy of undetermined significance (MGUS), smoldering MM, active MM, and relapsed MM compared with normal plasma cells. Importantly, protein analysis confirmed increased nuclear levels of these deacetylases in MM cell lines, including those resistant to DNA-damaging agents (MM.1R, LR-5, Dox40), as well as in patient CD138⁺ MM cells compared to PBMCs from healthy donors. Next we evaluated the functional role of these Sirtuins in MM cells by using loss of function approaches with RNAi. SIRT6 and SIRT7 silencing by knockdown reduced MM cell proliferation compared with control scrambled cells, with only a modest induction of cytotoxicity. We also examined the effects of Nam on DNA-damage response signaling triggered by conventional anti-MM agents melphalan and doxorubicin. Nam treatment did not appreciably affect MM cell viability; however, pretreatment with Nam impaired DNA double-strand breaks (DSBs) repair as well as DNA repair mechanisms triggered by conventional DNA damaging agents, evidenced by γH2AX and RPA phosphorylation, respectively. Consistent with these findings, Nam-pretreated MM cells formed fewer RAD51 foci in response to Doxorubicin and Melphalan, thereby conferring sensitivity to these agents. Importantly, this sensitizing effect was also observed in MM cells resistant to doxorubicin (RPMI-Doxo40) or melphalan (LR5), indicating that Nam increases chemosensitivity in both drug-sensitive and –resistant MM cells. Similarly, lentivirus-mediated shRNA knockdown of SIRT-6 and −7 sensitized MM cells to melphalan and doxorubicin. Finally, both chemical and genetic approaches improved the efficiency of DNA DSB repair mechanisms (Homologous and non-Homologous end-joining Recombination) in MM cell lines containing chromosomally integrated green fluorescent protein-based reporter constructs. Ongoing in vivo experiments are assessing how the chemical susceptibility of SIRT6 and/or 7-deficient cells can be exploited therapeutically.

Our study demonstrates a link between nuclear sirtuins and DNA instability in MM cells, providing the basis for incorporation of inhibitors of these SIRTs into innovative anti-MM therapeutic approaches.

Munshi:Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.