PROJECT SUMMARY Advanced age in the majority of acute myeloid leukemia (AML) patients limits the use of aggressive chemotherapy leading to poor overall survival. Alternative treatment strategies for AML are highly sought after. Our research has revealed vitamin C (ascorbate) to be a potential non-toxic therapeutic adjuvant for the treatment of AML. Ascorbate is an essential micronutrient in humans that, in addition to its role as a cellular antioxidant, participates as a direct cofactor of Ten-eleven (TET) enzymes. Mammalian TET proteins (TET1-3) are tumor suppressors of the hematopoietic lineage that catalyze the oxidation of 5-methylcytosine (5mC) leading to DNA demethylation and the reversal of gene silencing. TET2 loss-of-function mutations are frequently observed in AML patients and are associated with a worse overall prognosis. Importantly, TET2 mutations are almost always heterozygous, suggesting that enhancing residual TET2 activity (encoded by the remaining wild- type TET2 allele) could be a viable therapeutic strategy for the treatment of TET2-mutant AML. We have shown in cellular and animal models that ascorbate, in a TET-dependent manner, reprograms the AML epigenome by increasing DNA hydroxymethylation, leading to DNA demethylation, a block in aberrant self-renewal, increased differentiation, and slowing of leukemia progression. We hypothesize that ascorbate will be an effective therapeutic agent in the treatment of TET2 mutant AML by enhancing residual TET tumor suppressive function. There are currently no other therapeutic agents that target TET proteins for functional restoration. We propose to understand the mechanistic basis of how physiological or pharmacological doses of ascorbate influence TET activity, and whether uptake capacity through sodium-dependent vitamin C transporters or total residual TET activity influences ascorbate treatment efficacy (Aim1). AML progression in the context of TET2 mutation and diverse oncogenic drivers will be tested for sensitivity to ascorbate in combination with standard AML chemotherapy, and data already obtained from our loss of function genetic screens in AML cells will be used to design rational combinatorial treatment strategies that maximize the efficacy of ascorbate as a therapeutic adjuvant (Aim2). Finally, we will explore approaches to enhance the bioavailability of ascorbate as a TET2 cofactor using lipophilic ascorbyl analogs and genetic or pharmacological modulation of vitamin C transporters on AML cells (Aim3). The goal of this proposal is to understand the dose and AML context in which ascorbate treatment will be most efficacious, how to combine ascorbate with existing therapies to improve treatment outcome and identify novel approaches to enhance ascorbate bioavailability for increased TET-activating potential. We believe these studies will provide a strong foundation for clinical translation of ascorbate as a non- toxic adjuvant in combination therapies for the treatment of AML...