Acute myeloid leukemia (AML) is the most common adult acute leukemia and accounts for 20% of childhood leukemias. Internal tandem duplication (ITD) mutations in FMS-like tyrosine kinase 3 (FLT3) are among the most common mutations in AML and are particularly associated with a poor prognosis. FLT3-ITD causes constitutive activation of FLT3 and the strong evidence that activated FLT3 drives leukemogenesis has led to the development of several FLT3-targeted inhibitors. Among these, the most potent and selective FLT3 inhibitor is AC220 (Quizartinib), which demonstrated a 44 % complete remission rate in AML patients harboring FLT3-ITD. However, remissions achieved by AC220 were short-lived, and AC220 treatment resulted in much more effective clearing of peripheral blasts than bone marrow blasts, implicating the bone marrow microenvironment as an important contributor to drug resistance. Indeed, it has been shown that bone marrow stromal cell factors can mediate resistance to the cytotoxic effects of FLT3 inhibition. Previous studies from our lab have shown that FLT3 inhibition by AC220 impairs glutathione (GSH) metabolism and induces mitochondrial reactive oxygen species (mitoROS) accumulation in FLT3-ITD AML cells, which is causative in apoptotic cell death. In addition, genetic knockdown or pharmacological inhibition of Ataxia Telangiectasia Mutated (ATM) or its downstream target, Glucose-6-Phosphate Dehydrogenase (G6PD), resulted in further impairment of GSH metabolism, more mitoROS accumulation, and enhanced apoptosis upon FLT3 inhibition. However, whether and how these metabolic alterations influence bone marrow stromal cell-mediated protection of FLT3-ITD AML cells from AC220 treatment is not understood. My preliminary data suggest that when FLT3-ITD AML cells are treated with AC220 in conditioned media of bone marrow stromal cells, they fail to induce mitoROS and are protected from the killing effect of AC220, which is associated with maintenance of GSH levels and expression of MYC and its target glutamine transporters, ASCT2 and LAT1. Similar effects are consistently observed in a small subset of cells that are refractory to AC220 treatment in regular media. Interestingly, knockdown of ATM or G6PD in combination with AC220 substantially reverses the protection from cell death mediated by conditioned media. Furthermore, knockdown of ATM or G6PD in combination with AC220 results in significant reduction of MYC expression in cells cultured in conditioned media. Performing both in vitro and in vivo studies, I will determine if maintenance of MYC and its target glutamine transporters, ASCT2 and LAT1 play a key role in maintaining GSH levels and bone marrow stromal cell-mediated protection of FLT3-ITD AML cells from AC220 treatment (Aim 1). To understand how ATM and G6PD are involved in this protection from AC220, I will determine what stromal components are responsible for activation of ATM and G6PD, and what the essential downstream effectors of ATM and G6PD are.
(Aim 2). Findings from this research will provide new insights into the mechanism of bone marrow stromal cell-mediated protection of FLT3-ITD AML from FLT3-targeted therapy, and potentially identify additional targets for combinatorial therapies designed to overcome the protective effects of bone marrow stromal cells and improve patient outcomes.

Public Health Relevance

FLT3-ITD+ AML is associated with a poor prognosis and AC220, a potent FLT3 inhibitor, demonstrates much more effective clearing of peripheral blasts than bone marrow blasts. This proposal attempts to understand the key mechanism of bone marrow stromal cell-mediated protection of FLT3-ITD+ AML from AC220 treatment. Proposed experiments will address molecular and metabolic mechanisms of bone marrow stromal cell- mediated protection from the killing effect of AC220, and will potentially identify novel additional drug targets for an effective combinatorial therapy. !

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
5F30CA236197-03
Application #
10070088
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Damico, Mark W
Project Start
2019-01-01
Project End
2022-12-31
Budget Start
2021-01-01
Budget End
2021-12-31
Support Year
3
Fiscal Year
2021
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045