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 including Id2 and Id3 may regulate cell specification of HSPC. We found that HSCs require Id2 for HSC quiescence and survival using an Id2 conditional (Id2F/F) mouse model to delete Id2 in vivo, by regulating hypoxia inducible factor (HIF) -1a and downstream targets. In more recent studies, we found that ID2-KD in AML cell lines (UT-7, MB-02, and M-07e) significantly reduced their leukemogenic potential in NSG mice, indicating that ID2 is essential for AML cell growth in vivo. Conversely, overexpressing ID2 in the human AML cell line M-07e enhanced their leukemic potential and burden in NSG mice, supporting the notion that ID2 may preserve LSC quiescence. Experiments with primary AML-PDX cells, which are more clinically relevant, also showed reduced leukemic potential in transplanted NSG mice upon ID2 knockdown. Additionally, ID2-KD cells displayed significantly lower levels of reactive oxygen species (ROS) and mitochondrial activity compared to control cells in both AML cell lines and PDX cells. As ROS-low cells are more susceptible to cytarabine (AraC), we investigated whether ID2-KD renders AML cells more susceptible to AraC. Indeed, MB-02-ID2-KD and UT7-ID2-KD cells showed increased sensitivity to AraC, while M-07e cells overexpressing ID2 exhibited resistance to AraC. Furthermore, AraC effectively eliminated ROS-low cells, but ROS-high cells remained resistant, supporting previous findings. To further investigate the effect of ID2-KD in AraC resistance in vivo, UT7-ID-KD and MB-02-ID2-KD transplanted NSG mice were treated with AraC and monitored for their overall survival, and it was observed that ID2-KD transplanted mice have significantly higher overall survival compared to the control cell transplanted mice. Thus, we found that ID2 plays a critical role in maintaining human AML cells. 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. Finally, we have found that reducing reducing Id1 gene expression reduces the mutational load and genomic instability of in Tet2-/- in HSPCs in vivo. We have found that reducing Id1 levels in Tet2-/- mice increases apoptosis and senescence, which could account for the reduced growth of these cells. Mechanistically, we found that Tet2-/- HSPCs show low levels of p16 that are increased by reducing Id1 levels in Tet2-/- mice. KD of p16 expression in Tet2-/-Id1-/- HSPCs reduces senescence and apoptosis and suggest that Id1 reduces p16 levels and apoptosis in Tet2-/- HSPCs.