Project 1

University Of Virginia, Charlottesville VA

Investigators

Abstract

PROJECT SUMMARY Acute myeloid leukemia (AML) has a 5-year survival rate of ~30%. Despite new treatments, these rates have changed little with virtually all patients developing relapsed/refractory AML. Thus, there is a strong clinical need to improve treatment outcomes for this disease. In this regard, our P01 team has built the foundational research showing that enhancement of the cellular levels of ceramide (Cer), a pro-death sphingolipid (SL), is a therapeutic avenue for AML. Additionally, the investigators of this P01 program developed a novel endogenous Cer generator, Cer nanoLiposomes (CNL), which cooperates with current standard of care (SOC) therapeutics in preclinical AML models. Our goal is to improve the efficacy of both CNL & current SOC AML therapeutics by ablating CNL/Cer “clearance” pathways linked to AML clonogenic survival. One such Cer clearance pathway has been highly overlooked: the conversion of Cer to ceramide-1-phosphate (C1P) by the enzyme ceramide kinase (CERK). In preliminary studies, CERK inhibition (CERKi) dramatically increased Cer levels and significantly reduced the viability & clonogenicity of multiple AML cell models with no effect on normal controls. In collaboration, CERKi was also found to induce a novel form of cell death and cooperate with CNL, venetoclax, and other Cer generators (SACLAC). These data infer that CERK clears a differential cellular “pool” of Cer from those enhanced by SACLAC & CNL, which forms the premise that a combinatorial approach of multiple Cer generators & SOC therapeutics with CERKi will increase the levels of differential Cer “pools” & be highly efficacious in AML. Mechanistic studies showed that CERKi in AML cells also collapsed key aspects of dysregulated AML metabolism required for clonogenic capacity (e.g., pentose phosphate pathway). Intriguingly, these aspects of AML metabolism are regulated by members of the direct C1P interactome. Therefore, we hypothesize that CERK-derived C1P is a “rheostat” for essential aspects of dysregulated AML metabolism via direct and sustained activation of pivotal factors. We further hypothesize that CERK is an advantageous target to induce additional Cer “pools” & novel cell death mechanisms for the enhancement of AML therapeutic efficacy. Our hypotheses will be interrogated by three specific aims: 1) Determine the therapeutic potential of targeting CERK in AML using state-of-the-art preclinical models of AML and a novel CERK inhibitor. Computational models of SL metabolism will be utilized to predict the sensitivity of specific patient AML cells for sensitivity to CERKi and cooperation with other Cer generators; 2) Determine the cellular topology and role of CERK in modulating key aspects of dysregulated AML metabolism & therapy resistance using flux sphingolipidomics and novel molecular constructs to modulate C1P formation in specific cell compartments. Genome-wide metabolic models will identify key vulnerabilities in AML metabolism affected by CERKi; and 3) Determine the C1P interactome modulating AML metabolism and mitochondrial function using Haberkant proteomics coupled to biophysical (e.g., surface plasmon resonance) and molecular methods (e.g., CRISPR/Cas9).