Last update:
15-Jan-2020

OBJECTIVE To investigate the function of histone deacetylase 8 (HDAC8) in the biology of multiple myeloma (MM) and to evaluate its potential as a therapeutic target.

METHOD The lentiviral-shRNA delivery system was used for knockdown of HDAC8 in OPM2 and U266 cells. The HDAC8 inhibitor PCI-34051 was used as a chemical inhibitor. A panel of 15 antibodies was used in immunoblot analysis, immunofluorescence staining was performed and the images were analyzed with confocal microscopy. Single cell electrophoresis under neutral conditions (comet-assay) was performed in OPM2-HDAC8 knockdown cells with or without exposure to gamma irradiation (IR), and in treated and untreated cells with HDAC8 inhibitor in combination with IR. Co-immunoprecipitation assay was performed for investigation of interactions of HDAC8 after induction of DNA damage. DNA double-strand break (DSB) repair occurring via homologous recombination (HR) pathway was assessed using a transient direct repeat DsRED-GFP/I-SceI plasmid-based system. Expression of DNA damage and repair pathway (DDR) genes was evaluated using a high-throughput polymerase chain reaction (PCR) assay. Cellular senescence was assessed with SA-β-galactosidase staining.

RESULTS In 172 newly-diagnosed patients with MM from the IFM myeloma dataset HDAC8 overexpression was observed, with significant correlation with poor survival outcome (p<0.0015). The high expression of HDAC8 in human myeloma cell lines (HMCLs) was confirmed in its cytoplasm and nuclear localization in all five MM cell lines studied. HDAC8 depletion in two MM cells lines resulted in significant inhibition of proliferation of MM cells at one week, and decrease in colony formation (p<0.001). The combination of HDAC8 inhibitor with melphalan or bendamustine enhanced the anti-MM effects of the genotoxic agents (all p<0.01). U266 cells with HDAC8 depletion exhibited raised levels of markers of DNA damage. Consistent with this observation, HDAC8 knockdown led to decreased HR activity and decreased repair of DSBs after IR. Similar results were obtained with HDAC8 inhibitor. The HDAC8 protein co-localized and co-immunoprecipitated with p53 after IR, and with SCM3, a member of cohesin. Finally, depletion of HDAC8 resulted in a higher prevalence of senescence associated with β-Gal-positive cells 3 weeks post transduction.

CONCLUSIONS These results demonstrate a mechanistic connection between HDAC8 and the DNA damage repair pathway, and provide insight into the effect of HDAC8 on the cytoskeleton, which may have therapeutic implications in MM.

Key words: Deacetylation, Histone, Multiple myeloma.