Acute myeloid leukemia (AML) arises through multistep clonal evolution characterized by stepwise accumulation of successive alterations affecting the homeostasis of differentiation, proliferation, self-renewal, and survival programs. The persistence and dynamic clonal evolution of leukemia-initiating cells and preleukemic stem cells during disease progression and treatment are thought to contribute to disease relapse and poor outcome. Inv(16)(p13q22) or t(16;16)(p13.1;q22), one of the most common cytogenetic abnormalities in AML, leads to expression of a fusion protein CBFβ-SMMHC (CM) known to disrupt myeloid and lymphoid differentiation. Anemia is often observed in AML but is presumed to be a secondary consequence of leukemic clonal expansion. Here, we show that CM expression induces marked deficiencies in erythroid lineage differentiation and early preleukemic expansion of a phenotypic pre-megakaryocyte/erythrocyte (Pre-Meg/E) progenitor population. Using dual-fluorescence reporter mice in lineage tracking and repopulation assays, we show that CM expression cell autonomously causes expansion of abnormal Pre-Meg/E progenitors with compromised erythroid specification and differentiation capacity. The preleukemic Pre-Meg/Es display dysregulated erythroid and megakaryocytic fate-determining factors including increased Spi-1, Gata2, and Gfi1b and reduced Zfpm1, Pf4, Vwf, and Mpl expression. Furthermore, these abnormal preleukemic Pre-Meg/Es have enhanced stress resistance and are prone to leukemia initiation upon acquiring cooperative signals. This study reveals that the leukemogenic CM fusion protein disrupts adult erythropoiesis and creates stress-resistant preleukemic Pre-Meg/E progenitors predisposed to malignant transformation. Abnormality in Meg/E or erythroid progenitors could potentially be considered an early predictive risk factor for leukemia evolution.
The maintenance and functional integrity of long-term hematopoietic stem cells (LT-HSCs) is critical for lifelong hematopoietic regeneration. Histone deacetylases (HDACs) modulate acetylation of lysine residues, a protein modification important for regulation of numerous biological processes. Here, we show that is most highly expressed in the phenotypic LT-HSC population within the adult hematopoietic hierarchy. Using an-floxed allele and a dual-fluorescence Cre reporter allele, largely normal hematopoietic differentiation capacity of deficient cells was observed. However, the frequency of phenotypic LT-HSC population was significantly higher shortly after deletion, and the expansion had shifted to the phenotypic multipotent progenitor population by 1 year. We show that -deficient hematopoietic progenitors are compromised in colony-forming cell serial replating in vitro and long-term serial repopulating activity in vivo. Mechanistically, we demonstrate that the HDAC8 protein interacts with the p53 protein and modulates p53 activity via deacetylation.-deficient LT-HSCs displayed hyperactivation of p53 and increased apoptosis under genotoxic and hematopoietic stress. Genetic inactivation of p53 reversed the increased apoptosis and elevated expression of proapoptotic targets and seen in -deleted LT-HSCs. Dramatically compromised hematopoietic recovery and increased lethality were seen in-deficient mice challenged with serial 5-fluorouracil treatment. This hypersensitivity to hematopoietic ablation was completely rescued by inactivation of p53. Altogether, these results indicate that HDAC8 functions to modulate p53 activity to ensure LT-HSC maintenance and cell survival under stress.
Acute myeloid leukemia (AML) harboring inv(16)(p13q22) expresses high levels of miR-126. Here we show that the CBFB-MYH11 (CM) fusion gene upregulates miR-126 expression through aberrant miR-126 transcription and perturbed miR-126 biogenesis via the HDAC8/RAN-XPO5-RCC1 axis. Aberrant miR-126 upregulation promotes survival of leukemia-initiating progenitors and is critical for initiating and maintaining CM-driven AML. We show that miR-126 enhances MYC activity through the SPRED1/PLK2-ERK-MYC axis. Notably, genetic deletion of miR-126 significantly reduces AML rate and extends survival in CM knock-in mice. Therapeutic depletion of miR-126 with an anti-miR-126 (miRisten) inhibits AML cell survival, reduces leukemia burden and leukemia stem cell (LSC) activity in inv(16) AML murine and xenograft models. The combination of miRisten with chemotherapy further enhances the anti-leukemia and anti-LSC activity. Overall, this study provides molecular insights for the mechanism and impact of miR-126 dysregulation in leukemogenesis and highlights the potential of miR-126 depletion as a therapeutic approach for inv(16) AML.
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/Hdac8f/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/Hdac8f/f(y) mice with a dual fluorescence Rosa26mT/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/Hdac8f/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. Disclosures No relevant conflicts of interest to declare.
<p>Table S1. Differentially expressed genes for c_1 vs c_1^* Table S2. Differentially expressed genes for c_2 vs c_1^* Table S3. Differentially expressed genes for c_3 vs c_1^* Table S4. Differentially expressed genes for c_2 vs c_1 Table S5. Differentially expressed genes for c_3 vs c_1 Table S6. Differentially expressed genes for c_3 vs c_2 Tables S7-S10. Differentially expressed genes for early, transition, persistent, and leukemia events. Tables S11-S14. GO terms enriched for early, transition, persistent, and leukemia events. Table S15. Top 1% of eigengenes.</p>
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