HDAC8 Regulates Long-Term Hematopoietic Stem Cell Quiescence and Maintenance

Long-term (LT) hematopoietic stem cells (HSC) are responsible for life-long production of mature blood cells of all lineages through tightly concerted cell fate decisions including quiescence, self-renewal, differentiation and apoptosis. Histone deacetylase 8 (HDAC8) is a member of class I HDAC enzymes that remove acetyl moieties from lysine residues on histones and a variety of non-histone proteins. Specifically, HDAC8 has been shown to modulate the acetylation cycle of cohesin complex protein SMC3. Loss-of-function mutations in HDAC8, located on the X chromosome q13, have been found in patients with Cornelia de Lange Syndrome (CdLS) and those with CdLS-like features. These HDAC8 mutations are associated with severely skewed X-inactivation (100% wild type allele) in the peripheral blood of female patients, possibly due to selection against the mutant alleles. However, the expression and function of HDAC8 in normal HSCs and hematopoiesis remain unknown. In this study, we show that Hdac8 is highly expressed in the phenotypic LT-HSC (Lin^-cKit⁺Sca1⁺CD150⁺CD48^-) population in adult mice. To determine the function of HDAC8 in adult hematopoiesis, we generated conditional Hdac8 deficient mice using the Mx1-Cre and a floxed Hdac8 allele (Mx1-Cre/Hdac8^f/f(y)) andconfirmed that Hdac8 is successfully deleted by polyinosinic-polycytidylic acid [poly (I:C)] treatment. Phenotypic analysis of Hdac8 deficient mice showed increased LT-HSC population compared to similarly treated control mice. However, largely normal steady state hematopoietic profile was found in Hdac8 deficient mice at 6 weeks and 1 year after induction. To further track Hdac8-deleted cells, we generated Cre/Hdac8^f/f(y) mice with a dual fluorescence Rosa26^mT/mG (mT/mG) Cre reporter allele, which expresses dTomato prior to Cre induction and becomes GFP⁺ after Cre-mediated recombination. We assessed hematopoietic repopulation by transplanting bone marrow cells from Cre/Hdac8^f/f(y)/mTmG⁺mice (2 x 10⁵) together with wild type support cells (2 x 10⁵) into lethally irradiated CD45.1⁺ congenic recipients. Hdac8 deletion was induced by treating the recipients with 7 does (14 m▢g/kg/dose) of poly (I:C). We found that Hdac8 deletion did not affect CD45.2⁺ or GFP⁺ donor-derived overall engraftment or lineage repopulation up to 16 weeks. There was also no change in the frequency or number of GFP⁺ donor-derived phenotypic LT-HSCs in the bone marrow. Serial transplantation was performed to further assess long-term repopulating activity of HSCs. Hdac8 deficient cells were significantly (p=0.019; n=3) compromised in multi-lineage repopulation in secondary transplant recipients. Except a modest reduction in Pre-GM, there was no change in the overall composition of Hdac8 deficient CD45.2⁺-derived populations. Upon tertiary transplantation, no donor engraftment was observed for Hdac8 deficient cells (0 out of 4) compared to 50% positive engraftment in control group (4 out of 8). These results indicate that HDAC8 is crucial for maintaining long-term serial-repopulating activity over time. Cell cycle analysis revealed that Hdac8 deficient LT-HSCs display reduced quiescence and increased cycling, consistent with the increased number of phenotypic LT-HSC seen in Hdac8 deleted mice. Therefore, we further tested the sensitivity of Hdac8 deficient mice to serial ablation with 5-fluorouracil (5FU), an S phase-specific cytotoxic chemotherapeutic agent. Impaired hematopoietic recovery and increased lethality (p<0.001; n=23) was seen in Hdac8 deficient mice treated with 5-FU (100 mg/kg) every 7 days, indicating that Hdac8 deletion renders hypersensitivity to serial ablation. There were significnatly less phenotypic LT-HSCs in Hdac8 deficient mice 6 days after 5-FU treatment (p<0.01; n=4). In parallel, we observed increased DNA strand beaks as indicated by γ-H2AX staining and comet assays (p<0.001; n>100 cells). Analysis of p53 activation, cell cycle regulators and DNA dmage response are ongoing. Collectively, our study indicates that HDAC8 plays a pivotal role in LT-HSC quiescence and maintenance.