Mechanisms and In Vivo Efficacy of Synergistic Acid Ceramidase and Bcl-2 Inhibition in Acute Myeloid Leukemia

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PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) is an aggressive, heterogeneous myeloid malignancy that results in bone marrow failure. The Bcl-2 antagonist/BH3-mimetic, venetoclax (VEN), is used in combination with chemotherapy or hypomethylating agents to treat newly diagnosed AML patients unable to tolerate induction chemotherapy. However, relapse rates remain high due to de novo and acquired resistance attributed to mitochondrial reprogramming and upregulation of compensatory survival proteins like Mcl-1 and Bcl-xL. Strategies that antagonize mitochondrial respiration, induce the integrated stress response (ISR), and/or reduce Mcl-1/Bcl-xL protein expression are known to improve VEN sensitivity. Importantly, sphingolipid (SL) metabolism, mitochondrial respiration, the ISR, and Mcl-1 protein levels are closely linked, making SL metabolism an attractive therapeutic target in combination with VEN. The balance of pro-apoptotic ceramides and pro-survival sphingosine-1-phosphate form the bioactive core of sphingolipid signaling. Acid ceramidase (AC) is a lipid hydrolase that catabolizes pro-apoptotic ceramides. We previously demonstrated that AML blasts rely on AC for survival. Because we and others showed that AC inhibition severely impairs mitochondrial respiration, we sought to determine whether combining AC inhibitors with VEN could be effective for treating AML. Our preliminary data show that AC inhibition improves VEN sensitivity in human AML cell lines and patient samples. However, the mechanisms underlying the synergy from this novel combination are unknown, and the in vivo efficacy of combinatorial AC and Bcl-2 inhibition has not been explored. We hypothesize that synergy between AC and Bcl- 2 inhibition results from SL-mediated impairment of mitochondrial form, respiration, and ISR overactivation. Aim 1 (F99 phase) is designed to complete my dissertation work by characterizing the mechanism, biomarkers, and in vivo efficacy of combined AC and Bcl-2 inhibition in AML. To accomplish these goals, we will analyze mitochondrial form and function, interrogate the role of the ISR for synergy, and utilize computational approaches to define biomarkers of drug responses. My overarching career goal is to develop into a highly productive, independent cancer researcher focused on developing novel SL-based therapeutics and precision medicine. To best prepare myself to start an independent cancer research program, I will utilize the K00 period to i) learn new skills in dynamic BH3 profiling (a precision medicine assay), CRISPR-Cas9 screening, and genomic data analysis; and ii) expand my cancer research portfolio into solid tumors by studying triple-negative breast cancer. Thus, Aim 2 (K00 phase) will evaluate the relationship between SLs and mitochondrial apoptotic priming using dynamic BH3 profiling and will utilize CRISPR screening to identify novel SL-based combination therapies to treat triple-negative breast cancer. The training plans in the F99/K00 phases will help me successfully achieve my career goals and thrive as an independent cancer researcher.