The role of GATA2 in myeloid malignancies and MonoMAC syndrome - PROJECT SUMMARY/ABSTRACT The long-term goal of this project is to better define the role of GATA2 in the pathogenesis of acute myeloid leukemia (AML) and the MonoMAC syndrome, and to use this information for therapeutic benefit. Somatic GATA2 mutations occur in -3-4% of AML cases, and inherited mutations are the cause of the MonoMAC syndrome, which is associated with the development of AML in most patients. We recently showed that Gata2 can act as a tumor suppressor in AML. However, the mechanisms by which GATA2wr and GATA2 mutations influence AML pathogenesis are currently unclear. In preliminary studies, we found that AMLassociated GA TA2 mutations dysregulate gene expression, and alter GAT A2 protein interactions. Based on these findings, we hypothesize that GATA2wr normally acts as a tumor suppressor as part of an interacting set of proteins that regulate a specific set of target genes; GA TA2 mutations may promote AML and MonoMAC by altering these functions, We will test these hypotheses via the following aims: Specific Aim 1: We will define the genes that are regulated by Gata2wr, and how their expression is altered by AML- and MonoMAC-associated Gata2 mutations, We generated a novel Gata27354M knock-in mouse- the first to model a MonoMAC- and AML-associated GATA2 mutation. In preliminary studies, we used single-cell RNA sequencing (scRNA-seq) and flow cytometry to find that these mice have decreases in key hematopoietic populations similar to that of the MonoMAC syndrome. ScRNA-seq also allowed us to identify 346 genes that are differentially expressed in myeloid progenitor cells from Gata2'354M mice vs. littermates. The Gata27354M mice will be fully characterized for changes in hematopoiesis and AML development We will also use an overexpression model to define alterations in gene regulation caused by other AML-associated Gata2 mutations including A318V, L321 F, T354M, and R362G in human and mouse hematopoietic stem/progenitor cells (HSPCs). If successful, these studies will allow us to identify the genes that are regulated by Gata2wr in primary HSPCs, and to determine pathways that are disrupted by Gata2 mutations. Specific Aim 2: We will identify the GATA2 protein interactome , and how it is altered by AML- and MonoMAC-associated GATA2 mutations, We developed a proximity labeling system in which the Gata2 cDNA is fused to an enhanced biotin ligase, called TurbolD . GATA2-TurboID biotinylates proximal proteins, allowing for the identification of interacting proteins by mass spectrometry. In preliminary studies, we identified the protein interactome of GA TA2wr in primary mouse and human HSPCs, and determined that GATA2T354 M alters many of these interactions. We will apply this system to study how other GATA2 mutations (A318V, G320D, L321 F, R362G, and R398W) affect protein interactions. Co-immunoprecipitation assays in HSPCs from Gata27354M mice will be used to orthogonally validate our results. If successful, these studies may reveal mutant GAT A2 protein interactions that can be targeted with novel therapeutic approaches to treat AML.
