Uncovering Roles For Myristoylation in Cancer, and NMTs as a New Therapeutic Target in Acute Myeloid Leukemia

Abstract

Protein myristoylation is a form of modification by the attachment of the fatty acid myristate to the N-terminus, helping to regulate protein localization, stability, and function. Myristoylation has been proposed as a therapeutic target in cancer for decades, but only recently have high-quality compounds targeting N-myristoyltransferase 1 and 2 (NMTs, the enzymes catalyzing protein myristoylation) been developed to enable the targeting of this process. Hematopoietic cancers such as Acute Myeloid Leukemia (AML) have been proposed as vulnerable to NMT inhibitors (NMTi) such as zelenirstat. AML is an aggressive cancer with poor clinical outcomes tied to disease relapse and resistance to therapy. Relapses are believed to be driven by leukemic stem cells (LSCs) which are highly resistant to conventional therapies and expand to re-establish the disease. Patient survival rates upon relapse are extremely low, highlighting a need for new therapies capable of both targeting LSCs, and overcoming common mechanisms of resistance. The primary goal of this study was to conduct a pre-clinical evaluation of zelenirstat as a novel therapeutic for AML, providing proof of concept and the necessary investigation to initiate clinical trials. Zelenirstat inhibits signaling through Src-family kinases necessary for oncogenic signaling through clinically relevant FLT3 and KIT receptors, in addition to inhibiting oxidative phosphorylation and AMPK activity necessary for LSC function. AML cell stress and apoptosis was induced by zelenirstat, and cell killing observed in vitro and in vivo, with an apparently selectivity for LSC-enriched populations of the OCI-AML-22 cell model. Zelenirstat also inhibited glycolysis in AML cells, presenting a potent multi-pathway inhibitor capable of targeting LSCs with high potential for synergy with venetoclax. Analysis of AML patient data revealed NMT2 expression functions as a prognostic marker in AML patients experiencing poor outcomes under current standards of care were associated with both low NMT2 expression and high MISS-54 score, predictive of positive response to zelenirstat. Following the broad metabolic impacts of zelenirstat in AML, we further explored the impacts upon mitochondria in HeLa cells. We confirmed inhibition of oxidative phosphorylation and glycolysis in this model, and demonstrated induction of cellular calcium leakage and suppression of glutathione metabolism as a consequence of zelenirstat treatment. Additionally, we provide the first evidence of NMTi disrupting mitochondrial structure, with loss of mitochondrial cristae organization and density upon zelenirstat treatment. Additional validation of the impacts of zelenirstat on AMPK show near-complete loss of activation, compromising the ability of the cell to respond to energetic stress. Next we attempted to contextualize the sensitivity of cancer cells to zelenirstat through pan-cancer analysis of NMT levels, response to zelenirstat, and differential analysis of multi-omics in the context of zelenirstat treatment or genetic NMT ablation. We find that genes related to oxidative phosphorylation are among the most responsive to disruption of protein myristoylation, and present myristoylation inhibition sensitivity signature MISS-54. Developed from differential genomic analysis of more than 1200 cell lines screened for sensitivity against NMTis, MISS-54 allows for prediction of tumor sensitivity to NMTi. MISS-54 is also demonstrated to be lower in cognate healthy tissues than in cancer, supporting the notion of increased sensitivity of cancer to NMTi. Collectively, this work identifies a critical role for myristoylation in oxidative phosphorylation and mitochondria as a significant, pan-cancer target of NMTi. This represents a key mechanism by which NMTi could target both LSCs and cancer stem cells at large, helping to improve and lengthen patient remissions. The large number of pathways in which myristoylated proteins participate give zelenirstat a pleiotropic function to tackle genetically and functionally diverse cancers, smothering resistance. In AML, zelenirstat is an exciting new therapeutic option starting clinical trials with predicted benefit to patients in desperate need of improved outcomes. This study identifies key mechanisms and provides rationale necessary for clinical trials and beyond