Abstract
Epigenomic changes have become an important component of cellular regulation and ultimately, of our understanding of oncogenomics in Multiple Myeloma (MM) as well as in 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 have 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 six MM cells lines studied (MM1S, OPM2, RPMI8226, U266, MOLP8 and NCI-H929) and in primary bone marrow plasma cells (CD138+) from newly diagnosed MM patients (N=3).
To address the functional role of HDAC8 in MM biology and to evaluate its potency as therapeutic target, we used a lentiviral-shRNA delivery system for HDAC8-knockdown in MM1S and OPM2 myeloma cells. The HDAC8 depletion in HMCLs resulted in significant inhibition of proliferation of MM at 1 week 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) and was confirmed to be synergistic using Calcusyn software. Immunoblotting using a panel of 15 antibodies for DNA damage response (DDR) pathway proteins (including γH2Ax, pATM, pATR, pBRCA1, pBRCA2, pCHK2, pCHK1, ku70, RPA70, 53BP1, DNA-PKs, pP53) confirmed increased levels of DNA damage in OPM2 and MM1S cells with HDAC8 depletion. In consistence with this observation HDAC8 knockdown led to decreased homologous recombination (HR) activity as measured by a transient direct repeat DsRED-GFP/I-SceI plasmid-based assay. We performed singe cell electrophoresis under neutral conditions (comet-assay) in OPM2 and MM1S after HDAC8 depletion with or without exposure to gamma irradiation (γ-IR), and in OPM2 and MM1S cells treated and untreated with HDAC8 inhibitor in combination with γ-IR and observed decreased repair of DSBs after γ-IR measured following HDAC8 knockdown as well as following treatment of the cells with HDAC8 inhibitor. Importantly, using laser micro-irradiation in myeloma and U2OS cells, we observed HDAC8 recruitment to DSBs sites. Moreover, the HDAC8 protein was co-localized and co-immunoprecipitated with Rad51 after IR, and with Scm3, member of cohesion complex after mitotic sychronization, suggesting its relation with cytoskeleton. We confirmed the significant alteration in expression of cohesion complex members SMC1 and RAD21 after HDAC8 depletion and re-overexpression in MM cells. In MM1s cells containing a stably integrated Rad51-luciferase reporter construct, the addition of HDAC8 inhibitor resulted in a decrease in Rad51 promoter activity, confirming the immunoblotting findings. An ongoing mass spectromentry-based analysis is expected to identify thoroughly the HDAC8-interacting proteins.
In conclusion, our results demonstrate an impact of aberrant epigenome on DNA integrity through connection between HDAC8 and DDR pathway, and provide insights into the effect of HDAC8 on cellular growth and survival with potent therapeutic implications in MM.
Disclosures
Anderson: Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy.
DNA damage and macrophage infiltration in the ovaries of the long-lived GH deficient Ames Dwarf and the short-lived bGH transgenic mice
