Molecular-Cellular Regulation of Hematopoietic Stem Cells

Division Of Basic Sciences - Nci

Investigators

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Abstract

During the last year, we continued to define the molecular events that regulate hematopoietic stem and progenitor cell (HSPC) quiescence, survival, self-renewal and, myeloid cell lineage commitment and differentiation. We have found that the helix loop helix (HLH) transcription factor, inhibitor of differentiation 1 (Id1), is induced during the early stages of myeloid development, and can instruct hematopoietic stem cells toward a myeloid versus lymphoid/erythroid cell fates suggesting that this gene and other family members Id2 and Id3 may regulate cell specification of HSPC. In this regard, we found that Id2 regulates the early stages of myeloid and lymphoid development by inhibiting the transcriptional activities of E2A and PU.1 proteins respectively. We found that HSCs express ID2 using an Id2 reporter mouse model (Id2-EYFP/+ mice) developed in our laboratory, which directly correlates with Id2 RNA expression by Q-RT-PCR analysis. To test if ID2 functions in HSC we developed an Id2 conditional (Id2F/F) mouse model, and bred these to transgenic Mx1-Cre+ mice to delete Id2 in vivo. The number of HSCs and ST-HSCs were significantly reduced in Id2-/- mice compared to controls, and mice that lack Id2 show reduced survival over time demonstrating that Id2 is essential to maintain HSCs numbers during steady state hematopoiesis. To identify the molecular mechanism(s) that mediate HSC exhaustion in Id2-/- mice we examined the transcriptome of purified Id2-/- and Id2+/+ HSCs by single cell RNA-seq and RNA-seq. Bioinformatic analysis of differentially expressed genes revealed that Id2-/- HSCs show increased gene expression in pathways that promote growth/proliferation and oxidative phosphorylation, and decreased expression of hypoxia response pathway genes compared to Id2+/+ HSCs. These results suggest that loss of Id2 may lead to HSC activation/proliferation and decreased quiescence via down regulation of hypoxia inducible factor (HIF) and downstream targets. We confirmed that Id2-/- HSCs show significantly reduced levels of HIF-1a protein expression by flow cytometry and immunohistochemistry (IHC), while Hif-1a RNA levels were not changed. HIF-1a promotes HSC quiescence and is required to maintain steady state numbers of HSCs. We found that Id2-/- HSCs show increased cycling 2 weeks after Id2 ablation in vivo by Ki-67 staining and flow cytometry. We determined that ID2 binds to the VHL protein, which interferes with the ubiquitination and degradation of HIF-1a. Furthermore, we demonstrated that stabilization of HIF-1a rescues the exhaustion of Id2-/- HSCs in vitro and in vivo. In other studies, we discovered that mice that lack Id1 show increased quiescence during BMT and are protected from exhaustion during serial BMT and in other models of chronic stress including genotoxic and inflammatory stress and aging. These studies laid the foundation for our current and future goals to determine if Id1 promotes clonal hematopoiesis, a preleukemic phase that proceeds leukemia, and if reducing Id1 expression can reduce hematopoietic stem and progenitor (HSPC) cell cycling and clonal expansion and reduce the incidence of hematopoietic malignancies. We found that Id gene expression is increased in HSPC progenitor cells from Tet2-/- mice, a murine model of clonal hematopoiesis. Furthermore, we have determined that reducing Id1 levels in Tet2-/- mice rescues enhanced donor repopulation during BMT, reduces HSPC expansion and myeloid skewing of Tet2-/- HSPCs. These results suggest we are reducing clonal expansion in Tet2-/- mice. We have shown that reducing Id1 levels in Tet2-/- mice delays the onset of leukemia. Future studies are aimed at demonstrating that reducing Id1 gene expression reduces the mutational load and genomic instability that is observed in Tet2-/- in HSPCs in vivo. We are also pursuing studies to determine if Id1 promotes relapse of acute myeloid leukemia.