SUMMARY N6-methyladenosine (m6A) is a modified nucleotide in mRNA whose transcriptome-wide expression and distribution are altered in various cancers, including acute myeloid leukemia (AML) cells. m6A has a pivotal role in controlling cell fate decisions especially in the hematopoietic lineage. Our recent work has revealed that this effect of m6A is mediated in part by its ability to regulate “symmetric commitment,” in which a stem cell differentiates into two daughter cells that both adopt the same new cell identity. We recently identified SON, one of the most highly methylated transcripts in hematopoietic stem cells, as a major mediator of m6A-dependent control of symmetric commitment. However, the mechanism by which SON regulates these phenomena is unclear, and moreover it is unclear if deregulated m6A control over SON contributes to the undifferentiated phenotype of AML cells. Inhibitors of METTL3, the m6A biosynthetic enzyme, are a promising approach to target AML, but appear to exhibit diverse effects in cells not seen with METTL3 knockout. Also, unlike METTL3 knockout, METTL3 inhibitors also appear to preferentially target cancer cells. A major goal is to understand the basis for the actions of METTL3 inhibitors compared to METTL3 knockout and to determine if specific cellular pathways can influence whether cells, and ultimately patients, would be more or less sensitive to METTL3 inhibitor treatment. To significantly advance our understanding of epitranscriptomic regulation in cancer, the specific aims of this proposal are: (1) To define the functional requirement for SON and nuclear RNA methylation on cell fate in leukemia. In this aim we will use a new conditional knockout mouse model to dissect SON's stage-specific role, clarifying its impact on METTL3-related blood phenotypes. We will determine SON's importance in leukemia and unravel its downstream targets, mRNA regulatory mechanisms, and links to the RNA methylation. (2) To identify pathways and mechanisms that make cancer cells sensitive to METTL3 inhibition. Our preliminary data show that METTL3 inhibitors cause marked reorganization of nuclear architecture, and their effects can be overcome by forced expression of YTHDC1. We have also identified additional suppressors of METTL3 inhibitors by performing a base editor screen to generate >600 oncogenic mutations and probing how the mutations affect sensitivity to METTL3 inhibitors. We will broadly identify how YTHDC1 and these newly discovered oncogenic pathways influence the m6A pathway and METTL3 inhibitors. (3) To determine the function of m66A in MYC mRNA. Our studies revealed the presence of a previously unidentified epitranscriptomic mark, N6,N6-dimethyladenosine (m6,6A) as a highly prevalent evolutionarily conserved modification in MYC mRNA. We will determine the prevalence and functional significance of this new modification, and determine how it impacts the oncogenic function of MYC. Overall, this project develops new techno...