Protein glutathionylation is essential for leukemia initiating cell survival.

The goal of this project is to develop a strategy to effectively eradicate leukemia-initiating cells. Leukemia- initiating cells are responsible for tumor initiation and recurrence in acute myeloid leukemia (AML), making it critically important to understand and target the biology required for leukemia-initiating cell survival. LICs are characterized by their self-renewal capacity, block in differentiation, and quiescent nature making them therapy resistant. A well characterized vulnerability of leukemia-initiating cells is oxidative phosphorylation (OxPhos) a pathway responsible for energy production. Direct OxPhos inhibition has been toxic in cancer patients. Thus, the development of approaches to target processes that regulate OxPhos in leukemia-initiating cells that are dispensable in normal cells is required. Our preliminary data shows that OxPhos is regulated by a post- translational modification called protein glutathionylation in AML cells and leukemia-initiating cells but not in normal hematopoietic stem and progenitor cells (HSPCs). These data indicate that protein glutathionylation regulation may represent a mechanism for decreasing OxPhos that could be LIC/AML specific and therefore targeting protein glutathionylation may be an approach to kill LICs with a more favorable therapeutic window than other approaches. Importantly, our data suggests that depletion of mitochondrial proteins that regulate protein glutathionylation results in reduced LIC function, induction of myeloid cell differentiation and sensitizes primary human AML cells to commonly used AML therapies but does not impact HSPCs. These data further support the potential for a therapeutic window may exist to target protein glutathionylation in AML. Based on these findings, we hypothesize that the regulation of mitochondrial protein glutathionylation is essential for LIC function by regulating OxPhos. We will examine this hypothesis by determining the molecular and biological role of protein glutathionylation in regulating leukemia-initiating cells and HSPC function using primary AML specimens, patient derived xenograft (PDX) models, and normal bone marrow specimens from healthy donors. Specifically, we will quantify leukemia-initiating cell phenotypes and function upon genetic depletion of proteins that regulate glutathionylation. Further, we will interrogate the mechanism(s) by which protein glutathionylation regulates mitochondrial energy production in leukemia-initiating cells. Taken together, our studies will be the first to establish protein glutathionylation as a novel regulator of 1) leukemia-initiating cells function and 2) OxPhos in cancer. Project Number: 1R01CA311131-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Courtney Jones (+1 co-PI) | Institution: CINCINNATI CHILDRENS HOSP MED CTR, CINCINNATI, OH | Award Amount: $533,245 | Activity Code: R01 | Study Section: Cancer Cell Biology Study Section[CCB] View on NIH RePORTER: https://reporter.nih.gov/project-details/11336711