Abstract
Background
Deregulation of the DNA damage response (DDR) signaling machinery underlies genomic instability, leading to cancer development and clonal evolution. Multiple Myeloma (MM) remains an incurable disease characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DDR machinery. Mammalian NAD+-dependent deacetylase sirtuin-6 (SIRT6) is emerging as new protein involved in multiple pathways, including maintenance of genome integrity.
Methods
A panel of 18 MM cell lines, both sensitive and resistant to conventional and novel anti-MM therapies, was used in this study. Blood and BM samples from healthy volunteers and MM patients were obtained after informed consent and mononuclear cells (MNCs) separated by Ficoll-Paque density sedimentation. Patient MM cells were isolated from BM MNCs by CD138-positive selection. Lentiviral delivery was used for expression and knock-down of SIRT6 in MM cell lines. The biologic impact of SIRT6 phenotype was evaluated using cell growth, viability and apoptosis assays. DNA Double-Strand Breaks (DSB) repair occurring via homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways was assessed using a transient direct repeat (DR)-GFP/I-SceI system.
Results
A comparative gene expression analysis of 414 newly-diagnosed uniformly-treated MM patients showed high levels of SIRT6 mRNA in MM patients versus MGUS or normal donors; moreover, in active MM elevated SIRT6 expression correlated with adverse clinical outcome. Due to its prognostic significance, we further evaluated its role in MM biology. We found higher SIRT6 nuclear expression in MM cell lines and primary cells compared to PBMCs from healthy donors. Targeting SIRT6 by specific shRNA increased MM cell survival by reducing DNA repair efficiency (HR and NHEJ). Whole genome profiling of three different SIRT6 knockout (Sirt6-/-) MM cell lines identified a restricted effect of SIRT6 silencing on transcription of DNA damage genes, which also represented the most down-regulated genes. Consistent with these data, GSEA algorithm revealed that gene set regulating DNA repair were prominently enriched in SIRT6 depleted cells (p<0.0001 and FDR=0.003), confirming the role of SIRT6 in this pathway. We next examined the therapeutic relevance of SIRT6 inhibition in MM by evaluating the effect of SIRT6 depletion on cytotoxicity induced by genotoxic agents. SIRT6 shRNA impaired DNA DSB repair pathways triggered by DNA damaging agents, thereby enhancing overall anti-MM activity of these agents. Finally, in concert with our in vitro data, studies using our human MM xenograft model confirmed that SIRT6 depletion enhanced anti-MM activity of DNA-damaging agents.
Conclusion
Collectively, our data provide basis for targeting SIRT6 as a novel therapeutic strategy in combination with genotoxic agents to enhance cytotoxicity and improve patient outcome in MM.
Disclosures:
Tai: Onyx: Consultancy. Hideshima:Acetylon Pharmaceuticals: Consultancy. Chauhan:Vivolux: Consultancy. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.
Induction of DNA breakage in X-irradiated nucleoids selectively stripped of nuclear proteins in two mouse lymphoma cell lines differing in radiosensitivity.
