Michael Kharas - US grants

DESCRIPTION (provided by applicant): Hematopoietic stem cell (HSC) numbers are carefully maintained by switching from symmetric to asymmetric cell divisions. A complex program made of genetic and epigenetic mechanisms that maintain normal HSCs can become dysregulated in hematopoietic disorders and malignancies. Myelodysplastic syndromes (MDS) are characterized as a clonally derived set of heterogeneous diseases demonstrating defective hematopoietic differentiation. Taken together with recent discoveries in the genetic landscape of MDS, the etiology of this disease is considered to be driven by an HSC or an early myeloid progenitor. Thus, dysregulation of cell fate decisions including the balance of asymmetric and symmetric cell division could explain how certain lineages are blocked or why self-renewal is altered. RNA binding proteins in the Msi family have been shown to be important for the switch between symmetric and asymmetric cell division in germ and tissue stem cell function, neural cell differentiation and cell fate determination. Recent studies have implicated MSI2 as a regulator of HSC function and this proposal utilizes Msi2 conditional knockouts and gain of function approaches to dissect the precise role for MSI2 in controlling asymmetric division, self-renewal and cell fate determination. Moreover, this proposal incorporates high throughput sequencing UV-crosslinking immunoprecipitation to identify global RNA targets (HITS-CLIP) that will be used to elucidate the direct mechanism for MSI2 in hematopoietic cells. Furthermore, our global genetic approaches have already uncovered how regulation of translation alters several developmental pathways. MSI2 has also been recently shown to be dysregulated in MDS within low- risk patients, expressing below normal levels and within high-risk patients demonstrating elevated MSI2 expression. This proposal utilizes the NUP98-HOXD13 MDS murine model combined with novel gain and loss of function MSI2 mouse models to study the initial stages of MDS within the HSC. We suggest that MSI2 dysregulation contributes to the biology of hematopoietic disorders and thus a deeper understanding of these mechanisms will provide additional therapeutic approaches.

PROJECT ABSTRACT Molecular and genetic analysis of novel Slicer-dependent miRNA pathways in blood Most conserved microRNAs (miRNAs) are generated by a biogenesis pathway that deposits them into an Argonaute effector, guiding them to broad regulatory target networks. Amongst the cohort of four mammalian Argonautes, only Ago2 has catalytic ability to cleave transcripts, an enzymatic activity known as Slicing that underlies experimental RNA interference. Nevertheless, the endogenous biological usage of mammalian Slicing remains largely mysterious. Our previous and ongoing studies provide the unexpected perspective of multiple Slicing-dependent biogenesis strategies that generate both Dicer-independent and Dicer-dependent erythroid miRNAs. These data strongly support our hypothesis that a dominant usage of Ago2 catalysis is to generate specific conserved miRNAs in the blood system. Our extensive preliminary data are the basis of (1) a series of biochemical and genomic experiments to elucidate a novel Slicing-dependent miRNA biogenesis mechanism, (2) genetic studies of novel knockout animals of erythroid, Slicing-dependent miRNAs in normal development, blood homeostasis and leukemia, and (3) molecular genetic analyses that seek to connect dysregulated processes in Ago2-catalytically defective blood system to specific Slicing- dependent miRNAs. These studies will bring new insights on post-transcriptional control of erythroid development, homeostasis, and blood cancer, as well as pinpoint the functional basis of mammalian RNAi to the generation of erythroid-specific miRNAs.

SUMMARY An important mechanism of gene expression regulation is dynamically regulated, and possibly reversible, nucleotide modifications in mRNA. These modifications can have marked effects on mRNA stability, translation, and other aspects of mRNA metabolism. We had a founding role in this field by developing the technology for transcriptome-wide mapping of N6-methyladenosine (m6A). Our mapping study provided the first evidence that m6A could be dynamically regulated, and potentially impart new functions in mRNA. We recently showed that acute myeloid leukemia (AML) cells exhibit elevated levels of METTL3 and METTL14, the heterodimer that acts as the m6A-forming methyltransferase. We found that m6A promotes self-renewal in AML and in CD34+ stem cells, and depletion of m6A triggers a differentiation program. Thus, m6A has critical roles in hematopoietic differentiation at specific stages of development, and this process is deregulated in AML. Therefore, precise characterization of these stage-specific patterns of m6A at a transcriptome-wide level is critical to understand how m6A affects developmental transitions. Developing new methods to map m6A in the rare cell populations relevant to hematopoiesis and AML would help to reveal how this epitranscriptomic modification is critical for the regulation and deregulation of differentiation seen in AML, and possibly other cancers. Additionally, the effects of m6A are largely thought to reflect the actions of specific ?reader? proteins, which bind m6A in mRNA to affect its fate in cells. The major readers are YTHDC1 in the nucleus, and the YTHDF family in the cytoplasm, which comprise three nearly identical paralogs, and which may have redundant functions. In order to significantly advance our understanding of the role of m6A in AML, the specific aims of this proposal are: (1) To visualize and map m6A in mRNA in a cell-type specific manner. Here we describe the development of methods for detecting and mapping m6A in a cell type-specific manner and their application to understand m6A dynamics in hematopoiesis and AML. (2) To define the functional requirement for the m6A reader YTHDC1 in normal blood cells and in AML. Based on a genome-wide screen and our preliminary data, YTHDC1 is a strong candidate for the reader that may mediate major aspects of the effect of m6A in AML. Here we assess the functional role for YTHDC1 in both normal and malignant hematopoiesis using human cord blood cells, AML cell lines and primary AML patients. (3) To determine the roles and regulation of the YTHDF cytosolic m6A readers on mRNA fate. The YTHDF proteins appear to be the major regulators of m6A mRNAs in the cytosol. We will determine how YTHDF proteins are regulated to mediate their m6A-mRNA destabilizing effects and if YTHDF proteins influence cellular differentiation and proliferation in cancer cell lines and in AML. Overall, our project will develop new enabling technologies for studying m6A in cancer and test mechanisms by which the m6A readers contribute to cancer progression.

PROJECT SUMMARY/ABSTRACT Hematopoietic stem cells (HSCs) must navigate important cellular fate choices that include a symmetric self-renewal, symmetric commitment or undergo an asymmetric cell division where one of the cells is fated to differentiate. Alterations in this homeostatic program can lead to hematopoietic disorders and malignancies. Myelodysplastic syndromes (MDS) are a heterogeneous set of clonal disorders characterized by ineffective blood cell development. A common pathophysiologic mechanism in MDS is the presence of dysregulated hematopoietic stem and progenitor cells that fail to normally develop into the diverse set of blood cells necessary for normal function. Our laboratory and others identified MUSASHI2 (MSI2) as a central regulator of HSC and hematopoietic progenitor cell self-renewal (Kharas et al. Nature Medicine 2010). Additionally, we identified that Msi2 loss results in a defect in controlling symmetric and asymmetric division, failure to engraft and results in defective maintenance of myeloid lineage biased HSCs in part through control of the TGF? pathway (Park et al. 2014 Journal of Experimental Medicine). We found that elevated levels of MSI2 expression predicts poor outcome and using a genetic MDS mouse model that overexpresses MSI2 can drive a more aggressive MDS (Taggart et al. 2016 Nature Communications). To determine if MSI2 is part of a regulatory network, we performed proteomics and in vivo shRNA screen for functional regulators of leukemia self-renewal. Based on this screen, we identified SYNCRIP, an RNA binding protein that shares MSI2 targets and is required in leukemia stem cells (Vu et al., 2017 Nature Genetics). Our preliminary data with a conditional knockout for Syncrip indicates that it is also critical for HSC self-renewal. Our proposal will expand our focus from MSI2 to its associated regulatory network and characterize and identify new molecular determinants for HSC and HSPC symmetric self-renewal and asymmetric fate choice. We have adapted new technologies that include a barcoding, single cell RNA-seq and paired daughter HSC assays (FATE-seq). We have also develop a new way to map direct mRNA targets in HSCs called (HYPERTRIBE), (Nguyen et al. Nature Communications 2020).This proposal will identify new regulators of HSPC fate choice which will lead to novel therapeutic strategies to improve outcomes in MDS patients.