Msi2 Directly Regulates The TGF-β Signaling Pathway and Myeloid Lineage Bias In Hematopoietic Stem Cells

Hematopoiesis is a tightly regulated process in which different cell lineages of the blood and immune system are generated from hematopoietic stem cells (HSC). HSCs can self renew and also give rise to more differentiated progenitor cells through symmetric and asymmetric cell division respectively. Progenitors can further differentiate into more committed cells that can generate the mature lymphoid and myeloid compartments. In order to support a normal hematopoietic system HSCs must maintain normal cell fate decisions between symmetric and asymmetric divisions.

Recent studies from our group and others have implicated Msi2 as a regulator of HSCs. Nevertheless, the exact role for Msi2 in HSCs and critical pathways regulated by Msi2 in these stem cells remains unclear. In addition to its high expression in normal HSC, MSI2 is upregulated in patients with poor clinical prognosis in acute myeloid leukemia and in the blast crisis phase of chronic myelogenous leukemia. To understand the role of Msi2 in normal HSCs, we are characterizing mice with a conditional deletion of Msi2 (Mx1-Cre::Msi2^flox/flox mice).

We previously demonstrated that the conditional ablation of Msi2 results in the failure of HSC maintenance due to a loss of quiescence and increased commitment divisions. Although Msi2 is critical for HSC engraftment, we observed a preferential requirement for Msi2 in the myeloid biased HSCs (My-HSCs). Based on the surface markers LSK CD34- CD150 high we found a ∼3 fold reduction in the frequency of My-HSCs and no differences in the frequency of the unbiased HSCs or in common lymphoid progenitors. Consistent with these results, we observed a dramatic decrease in chimerism in the myeloid versus lymphoid compartment of the Msi2 deleted cells in mice that were transplanted.

In order to understand why hematopoietic stem and progenitors cells require Msi2, we previously performed global transcriptome profiling and RNA target analysis using high throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). This overlap analysis implicated a variety of pathways including RNA translation, HSC self-renewal and TGF-β. The TGF-β pathway has been linked to the maintenance of both normal and leukemia stem cell self-renewal, and the control of myeloid biased HSCs.

We examined the output of the TGF-β pathway in HSCs in the presence and absence of Msi2. We found reduced phosphorylation of Smad2/Smad3 in Msi2^D/D HSCs grown in vitro or directly isolated from bone marrow. Recently, it has been reported that the loss of p57 in HSCs results in reduced quiescence and compensatory up-regulation of p27. Consistent with reduced signaling output of TGF-β, we observed a significant decrease in p57 expression and up-regulation of p27 in Msi2^D/D LSKs. The effect of TGF-β treatment is biphasic in hematopoietic stem cells, with high levels of TGF-β blocking proliferation and low levels of this pathway activation leading to increased proliferation. We therefore examined the functional response of Msi2^D/D HSCs to biphasic TGF-β signaling in vitro. Msi2^D/D HSCs responded normally to high dose of exogenous TGF-β resulting in potent growth suppression. In contrast, when exposed to lower levels of TGF-β that has been shown to expand normal HSCs, Msi2^D/D HSCs failed to respond, consistent with diminished sensitivity to the proliferative effects of TGF-β. These results demonstrate that the proliferative signals downstream of TGF-β signaling are impaired in the Msi2 deleted HSCs.

Most intriguingly, our data suggests that Msi2 is required to maintain the response to TGF-β signaling and this contributes to the maintenance and fate of the My-HSCs. It will be interesting to find out if Msi2 is involved in the prevalence of My-HSCs during aging. Taken together, these results have important implications for understanding how the activity of RNA binding proteins contribute to HSC biology that govern normal tissue homeostasis.