Msi2 Maintains Normal Hematopoietic Stem Cell Self-Renewal

Abstract 222 Genes that regulate normal hematopoietic stem cells are commonly dysregulated in hematopoietic malignancies. Recently we published that the Msi2 RNA binding protein is an important modulator in both normal hematopoietic stem cells and leukemia (Kharas et al, Nat. Medicine 2010). The closely related Msi1 protein has been shown to regulate mRNA translation through binding to the 39UTR. Based on the high homology in the RNA recognition motifs, Msi2 has been considered to have similar functions. Moreover, increased MSI2 expression in chronic myelogenous leukemia blast crisis and acute myeloid leukemia predicts a worse clinical prognosis. Previous studies have mainly utilized shRNAs to functionally assess the role of Msi2 in the hematopoietic compartment. However, it remains unclear how Msi2 affects hematopoietic stem cells (HSC) and what are its critical mRNA targets. To develop a model focusing on the HSC compartment and to avoid potential compensatory mechanisms during development, we created Msi2 conditional knockout mice and crossed them with Mx1-Cre mice. We induced excision with poly(I):poly(C), (pIpC), and tested the peripheral blood, bone marrow cells and splenocytes by Southern blotting and QPCR analysis to verify Msi2 deletion. Loss of Msi2 mRNA was confirmed in the Lineage lo , Sca1 + and c-Kit + (LSK) population. Msi2 deleted bone marrow contained reduced myeloid colony forming capacity and replating efficiency. Mice conditionally deleted for Msi2 had normal white blood cell counts but smaller spleens. In addition, we observed normal percentages of the mature hematopoietic populations, including the myeloid and lymphoid compartments. Nevertheless, absolute numbers of long-term HSCs in the bone marrow were reduced by 3-fold. Bone marrow cells non-competitively transplanted into primary and secondary recipient mice showed a dramatic reduction in HSC chimerism. This defect was also observed when bone marrow was transplanted first to allow engraftment followed by Msi2 deletion. Furthermore, we were able to recapitulate this defect in vitro using the cobblestone-forming activity assay. These results indicate that Msi2 is both an important regulator of normal HSC maintenance and required for efficient engraftment. Most interestingly, Msi2 HSCs failed to maintain a normal quiescent HSC population. We performed microarrays to identify the pathways altered in the LSK population. The Msi2 deficient LSKs showed a reduced self-renewal and increased differentiation gene signature. Gene expression analysis indicates a defective self-renewal program in Msi2-deficient HSCs that is identical to the program gained in leukemic stem cells. These data suggest that MSI2 is a critical modulator of HSCs and may help explain its requirement in the most aggressive myeloid leukemias. Disclosures: Daley: iPierian, Inc: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity9s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity9s Board of Directors or advisory committees.