Abstract 5437: N-myristoylation inhibitor zelenirstat reduces mitochondrial complex I activity, OXPHOS, glycolysis, and cancer stem cell growth in triple-negative breast cancer (TNBC) cells

Two mammalian N-myristoyltransferases (NMT1 and NMT2) catalyze the addition of the fatty acid myristate to >200 different proteins and regulate cellular processes such as signal transduction, organelle trafficking, and metabolism. The pan-NMT inhibitor zelenirstat preferentially killed hematologic cancer cells while sparing normal cells, was safely administered, and showed early signs of efficacy in cancer patients during phase I clinical trials. Normally-myristoylated proteins exposing their un-myristoylated N-terminal glycine resulting from zelenirstat treatment are susceptible for degradation by a glycine-specific N-degron. Using differential mass spectrometry proteomics of NMT1/NMT2 CRISPR/Cas9 knockout (KO) HAP1 cells or HAP1 cells treated with zelenirstat compared to untreated WT HAP1 cells, we identified multiple NMT-specific substrates. Surprisingly, NMT1 KO statistically reduced the protein levels of only a few signaling proteins, while preferentially reducing the levels of 36 mitochondrial respiratory proteins including the myristoylated complex I assembly factor NDUFAF4. MISS-54, a gene signature that predicts the sensitivity of cancer or normal cells to zelenirstat, is significantly higher in TNBC than in other breast cancer subtypes or normal breast cells. Zelenirstat abrogated mitochondrial complex I assembly and activity as well as oxidative phosphorylation (OXPHOS) in MDA-MB-231 TNBC cells. The KO of NDUFAF4 in these cells recapitulated all these results but also increased glucose utilization measured through extracellular acidification rates (ECAR). Zelenirstat significantly decreased respiration through complex I, overall oxygen consumption, ECAR, and the use of glucose as an alternate energy source. Zelenirstat treatment also resulted in pyruvate dehydrogenase inhibition, decreased AMPKβ levels, and decreased the NAD/NADH ratio. Thus, zelenirstat caused significant metabolic disruption that drastically impaired energy production in TNBC cells. We hypothesize that the inability of TNBC cells to switch to glucose utilization upon zelenirstat treatment is due to the loss of myristoylated AMPKβ. Cancer stem cells (CSCs) have metabolic plasticity and can use either OXPHOS or glycolysis. Since zelenirstat reduced both pathways, we hypothesized that zelenirstat could also reduce the growth of CSCs in vitro. Both zelenirstat and NDUFAF4 KO individually reduced mammosphere formation and produced dose-dependant additive effects on CSC growth when combined. Overall, our results indicate that N-myristoylation is required for optimal energy production, cancer cell respiration, and CSC growth. In addition to inhibiting cell growth signaling, our data indicate that zelenirstat reduces OXPHOS and glycolysis, which may reduce CSC survival and prevent metastasis. Citation Format: Rony Pain, Erwan Beauchamp, Morris A. Kostiuk, Claudia Y. Acevedo-Morantes, Luc G. Berthiaume. N-myristoylation inhibitor zelenirstat reduces mitochondrial complex I activity, OXPHOS, glycolysis, and cancer stem cell growth in triple-negative breast cancer (TNBC) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5437.