HDAC8 Regulates Long-Term Hematopoietic Stem Cell Maintenance Under Stress By Modulating p53 Activity

The functional integrity and maintenance of long-term hematopoietic stem cells (LT-HSC) is dependent on a critical balance of quiescence, apoptosis and proliferation states. As a member of the class I histone deacetylase (HDAC), HDAC8 modulates acetylation of lysine residues, a protein modification important for regulation of numerous biological processes. In this study, we show that Hdac8 is highly expressed in the phenotypic LT-HSC (Lin-cKit+Sca1+CD150+CD48-) population in the bone marrow (BM) of 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 (Hdac8 ^△/△ ) after induction with polyinosinic polycytidylic acid (14 mg/kg, 7 doses). We observed largely normal hematopoietic differentiation capacity of Hdac8 ^△/△ cells. However, the frequency of phenotypic LT-HSC population (0.04968% vs. 0.03575%; n=10-13; p=0.0038) was significantly higher 6 weeks after Hdac8 deletion and the expansion shifted to the phenotypic multipotent progenitor population by one year (0.1571% vs. 0.06683%; n=5-8; p=0.0025). In vitro colony-forming cell (CFC) assay demonstrated that while Hdac8 ^△/△ and control progenitorsgenerated similar numbers of total CFC in the first plating, significantly reduced Hdac8 ^△/△ CFC were seen in secondary and tertiary platings. Using Mx1-Cre/Hdac8^f/f(y) mice with a dual fluorescence Rosa26^mT/mG (mT/mG) Cre reporter allele (GFP+ after deletion) in hematopoietic repopulation assays, we found similar frequencies and numbers of GFP⁺ donor-derived lineage populations and phenotypic stem and progenitor cells. However, Hdac8 ^△/△ GFP⁺ donor-derived multi-lineage repopulation was significantly compromised upon secondary (p=0.0227, n=3) and tertiary (p=0.0109, n=5-8) transplantation. Cell cycle analysis by in vivo BrdU labeling revealed that Hdac8 deficient LT-HSCs display reduced quiescence (35.36% vs. 56.15% G₀; n=5-6; p=0.01918) and increased cycling (45.36% vs. 28.03% S phase; n=5-6; p=0.0077), consistent with significantly reduced expression of Cdkn1a (p=0.0025) and Necdin (p=0.019). These results indicate that Hdac8 deletion perturbs homeostasis of LT-HSC and hematopoietic output over time.

We previously showed that Hdac8 functions to deacetylate p53 protein in inv(16) acute myeloid leukemia. Thus, we hypothesize that Hdac8 could perhaps modulate p53 activity in LT-HSC. Co-immunoprecipitation assay revealed that Hdac8 binds to the p53 protein, which is strongly enhanced upon irradiation (IR; 3Gy). Enhanced p53 acetylation and increased p53 target gene expression including Bax, Noxa, Puma and Cdkn1a were seen after IR in Hdac8 ^△/△ BM cells. Specifically, Hdac8 ^△/△ LT-HSCs are significantly more sensitive to apoptosis after IR. We crossed Mx1-Cre/Hdac8^f/f(y) mice with the p53ER^TAM knock-in (KI) mice carrying a p53 fusion protein completely dependent on ectopic 4-hydroytamoxifen. Inactivation of p53 in Hdac8 ^△/△ /p53ER^KI/KI LT-HSCs reversed the increased IR-induced apoptosis and elevated expression of Noxa and Puma seen in Hdac8 ^△/△ LT-HSCs. To test whether Hdac8 modulates p53 activation via its deacetylase activity, we treated control BM cells with an HDAC8-selective inhibitor (HDAC8i) targeting HDAC8 catalytic site before exposure to IR (3Gy). Treatment with HDAC8i significantly enhanced the responsiveness of p53 targets to IR, supporting that these effects are dependent on HDAC8 deacetylase activity. Loss of quiescence and hyperactivation of p53 could render LT-HSC higher sensitivity to hematopoietic stressors such as ablation with 5-fluorouracil (5-FU). We tested this by treating control, Hdac8 ^△/△, or Hdac8 ^△/△ /p53ER^KI/KI mice with serial 5-FU (100 mg/kg) every 7 days. Dramatically impaired hematopoietic recovery (white blood cell, red blood cell and platelet counts) and increased lethality was seen in Hdac8 ^△/△ mice challenged with serial 5-FU treatment (75% Hdac8 ^△/△ , n=30 vs. 5.9% control n=34). This hypersensitivity to hematopoietic ablation is completely rescued in Hdac8^Δ/Δ/p53ER^KI/KI mice (no lethality seen,n=7), indicating that this effect is indeed mediated by p53. Altogether, this study highlights HDAC8 as a novel regulator of LT-HSC in that Hdac8 functions to modulate p53 activity in LT-HSCs to ensure cell survival and maintenance under stress.