Funded by the National Cancer Institute

ABSTRACT:

Synergistic with the P01's overall goal to weaponize Cer against cancer, Project 3 aims to improve the efficacy of Cer-elevating therapies by targeting resistance mechanisms that develop in the mitochondria when Cer is elevated – specifically, the pro-survival pathways that sustain mitochondrial membrane potential (MMP). Prior research shows that mitochondria are a primary target of Cer-induced cell death. Cer affects mitochondrial function, especially respiration, through both indirect and direct mechanisms. Collaborative studies with Projects 1 & 2 reveal that increasing cellular Cer levels, via ceramide nanoliposome (CNL) or ceramide-kinase/acid ceramidase (CERK/AC) inhibitors, consistently impairs mitochondrial respiration. However, we recently discovered that AML cells can survive Cer-induced respiratory damage by activating non-canonical mechanisms to sustain MMP. Instead of relying on the proton-pumping respiratory complexes of the electron transport chain, these cells sustain MMP through alternative processes like hydrolyzing ATP via ATP synthase. Notably, inhibiting ATP hydrolysis or collapsing MMP with protonophores significantly enhances Cer's cytotoxicity by up to 14-fold. These findings suggest that AML cells counteract Cer-induced apoptosis by adapting their mitochondria to preserve MMP independently of respiration. Our overarching hypothesis is that reliance on non-canonical mechanisms to sustain MMP represents actionable resistance mechanisms that can be exploited to improve the efficacy of Cer-elevating therapies. This hypothesis will be tested in two specific aims. Related to Aim 1, preliminary data suggest that different Cer therapeutics induce divergent effects on the mitochondrial proteome, suggesting that mitochondrial pro-survival mechanisms are influenced by Cer's source, sub-cellular location, and metabolites. Aim 1A-B will explore this concept by manipulating Cer metabolism using our Cer therapies (CNL, CERK/AC inhibitors), as well as lentiviral manipulation of Project 1 & 2 Cer clearance pathways. In collaboration with Cores A & C, we'll integrate comprehensive mitochondrial phenotyping with quantitative sphingolipidomics to define how variations in Cer's source, content, sub-cellular location, and metabolites interface with AML cell mitochondrial bioenergetics to support/suppress AML cell survival. In Aim 1C an alternative strategy will be tested, targeting Cer specifically to mitochondria by conjugating Cer to the triphenylphosphonium cation (TPP+). In Aim 2, we will determine if targeting MMP generated by ATP synthase can sensitize AML cells to Cer therapy. Specifically, in Aim 2A we will block MMP generation by ATP synthase by driving the expression of ATP inhibitory factor 1 (ATPIF1) and determine the impact on AML cell sensitivity to Cer-elevating therapies. Aim 2B will determine if matrix ATP hydrolysis can be targeted by manipulating the matrix ATP generating enzyme succinyl- CoA lyase. Although the field is replete with studies on Cer's impact on mitochondria, the mechanism underlying mitochondrial resistance to Cer therapies is unknown. Successful completion of this project will fill this knowledge gap and identify the combinatorial approaches needed to improve the efficacy of Cer treatment.