Project Summary This proposal outlines a 5-year research and career development plan designed to support the candidate’s trajectory towards an independent academic career. The proposed research project, which focuses on acute myeloid leukemia (AML)-associated MYC mutations and their key target genes in AML cells, will capitalize on critical expertise and resources available at Washington University School of Medicine. The career development plan and didactic work will provide the candidate with a variety of skills that will enable a successful transition to independence. This proposal is founded on our recent observation that a unique set of recurrent MYC missense mutations are significantly enriched in the dominant clones of normal karyotype AML patients who have very long first remissions (>5 years) with chemotherapy only; in our series, 6/6 MYC mutations co- occurred with NPM1 mutations (NPMc), suggesting a unique form of cooperativity. Based on our preliminary findings, we hypothesize that: 1) AMLs overexpressing WT or mutant MYC proteins are biologically different; 2) each MYC mutation may have unique effects on its transcriptional targets; and 3) MYC mutations may cooperate with NPMc mutations. To address these hypotheses, we propose the following specific aims: Specific Aim 1: We will examine the function of AML-associated MYC mutations alone, or in cooperation with NPMc. We have developed a doxycycline-inducible system that allows us to regulate Myc expression in hematopoietic stem and progenitor cells (HSPCs) that do or do not contain the Npmc mutation. Using this system, we will evaluate how WT or mutant Myc, with or without Npmc, impacts AML development, progression, and AraC responsiveness both in vitro and in vivo. We are also developing a more physiologic, conditional knock-in model of MYCT58N (a recurrent mutation both in AML and B cell malignancies) that will allow us to explore the influence of physiologic doses of mutant Myc and Npmc on AML pathogenesis and AraC sensitivity. Specific Aim 2: We will define the transcriptional outputs and genomic binding sites of WT vs. mutant Myc proteins in AMLs arising in WT vs. Npmc mice. To identify Myc dysregulated genes, we will use scRNA- seq to determine the transcriptional profiles of AMLs (obtained in Aim 1) initiated by WT vs. mutant Myc over- expression, either alone or in combination with Npmc. We will also perform ChIP-seq studies to characterize the genomic binding sites of WT vs. mutant Myc, and perform an integrated analysis of these datasets to define the relative contributions of direct vs. indirect effects of Myc on transcriptional outputs in AML cells. By combining the results of the functional studies (Aim 1) with the expression and DNA binding studies (Aim 2), we hope to better understand how Myc mutations alter transcription in hematopoietic cells, and how Npmc affects this output. If these studies are successful, they may provide important insights on MYC-mediated transfo...