Abstract
2 min readAbstract Epigenomic changes have emerged as an important player of cellular regulation and understanding of pathogenesis of Multiple Myeloma (MM) as well as of other cancers. In recent years, both clinical and preclinical studies have confirmed that MM is vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets. Although Pan-HDAC inhibitors are effective as therapeutic agents, there is increasing emphasis on understanding the biological and molecular roles of individual HDACs. Here we evaluated the role of HDAC8, a member of Class I HDAC isoenzymes in MM. First, we evaluated the expression of HDAC8 in 172 newly-diagnosed MM patients from the IFM myeloma dataset and observed HDAC8 overexpression as well as its significant correlation with poor survival outcome (P<0.0015). We further evaluated the expression of HDAC8 in HMCLs (probe ID_223909-s_at, 223345_at) and confirmed the high expression and its cytoplasmic and nuclear localization in all MM cells lines studied. The HDAC8 depletion (lentiviral-shRNA)in HMCLs resulted in significant inhibition of proliferation of MM cells as measured by 3[H]-thymidine assay, and as decrease in colony formation evaluated after 3 weeks post transfection (P<.001). We observed similar cell growth inhibition using PCI-34051, a small molecule HDAC8 inhibitor. Interestingly, the combination of HDAC8 inhibitor with melphalan or bendamustine enhanced the anti-MM effects of the DNA damaging agents (all p<0.01). Immunoblotting analysis using a panel of 15 antibodies for DNA damage response (DDR) pathway confirmed increased levels of DNA damage in OPM2 and MM1S cells lacking HDAC8. Consistent with this observation HDAC8 depletion led to decreased homologous recombination (HR) activity as measured by plasmid-based assay. We performed singe cell electrophoresis (Comet-assay) and observed decreased repair of DSBs after IR in OPM2-HDAC8 depleted cells as well as after pharmacologic inhibition of HDAC8. Importantly, using laser micro-irradiation in myeloma and U2OS cells, we observed HDAC8 recruitment to DSBs sites. The HDAC8 was co-localized and co-immunoprecipitated with Rad51 after IR, and with Scm3, member of cohesion complex suggesting its relation with cytoskeleton. In MM1s cells containing a stably integrated Rad51-luciferase reporter construct, the addition of HDAC8 inhibitor suppressed Rad51, confirming the immunoblotting findings. A mass spectromentry-based analysis identified the HDAC8-interacting complexes with cohesion- (cohesin subunit SA-2, Condensin-2) and DDR-key components (Mre11a, XRCC1, Rad50). In conclusion, our results demonstrate impact of epigenomic change on DNA integrity through connection between HDAC8 and DNA damage response pathway, and provide insights into the effect of HDAC8 on DNA stability and cell growth and survival that may have therapeutic implications in MM. Citation Format: Maria Gkotzamanidou, Masood Shammas, Jesús Martín Sánchez, Mehmet Kemal Samur, Stephane Minvielle, Florence Magrangeas, Herve Avet-Loiseau, Athanasios-Meletios Dimopoulos, Kenneth C. Anderson, Nikhil C. Munshi. HDAC8 is recruited to DNA double strand breaks sites and affects the homologous recombination efficiency in multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3035. doi:10.1158/1538-7445.AM2015-3035
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