Project Summary/Abstract Translation of messenger RNAs (mRNA) is commonly dysregulated in cancer cells, highlighting regulatory mechanisms in translation as potential targets for cancer treatment. Chemical modifications of mRNA, known as the epitranscriptome, have emerged as a new layer of translation regulation. Yet, the types and prevalence of mRNA modifications as well as their roles in cancer remain poorly understood. N4-acetylcytidine (ac4C) is a novel mRNA modification that directly enhances translation efficiency of target mRNAs, representing a new and significant layer of translational regulation. Of substantial relevance to cancer, the enzyme that catalyzes the deposition of ac4C, N-acetyltransferase 10 (NAT10), is commonly dysregulated in cancers, whereas NAT10 depletion specifically decreases growth of cancer cells, including acute myeloid leukemia (AML) cells. In light of these observations, this proposal stipulates that NAT10-catalyzed ac4C promotes proliferation of AML cells by enhancing translation efficiency. During the K99 phase, this study aims to investigate the molecular mechanisms of mRNA acetylation (Aim 1) and the mechanisms by which ac4C promotes translation efficiency (Aim 2). In the R00 phase, this proposal aims to evaluate whether NAT10-catalyzed ac4C promotes AML growth by enhancing translation of proliferation-promoting factors (Aim 3). The overall long-term goals of this study are to expand our understanding of the epitranscriptome and the mechanisms that dynamically regulate translation efficiency in cancer cells, using this knowledge to implement novel therapeutic strategies for human conditions where translation is altered, including AML and other cancers. My career goal is to develop an independent high-quality research program for the benefit of human health. To achieve these goals, I built a career development plan which includes this research proposal and a plan for training, mentoring and networking to expand my research, bioinformatical, leadership and mentoring skills. The K99 phase will be developed in the Intramural Research Program of the National Cancer Institute (NCI) and will be mentored by Dr. Shalini Oberdoerffer (NCI), and co- mentored by Dr. Jeffery Coller (Case Western Reserve University). The NCI Intramural Research Program offers strong well-established research programs and multiple resources for career development and transition of postdoctoral fellows.