Pharmacological Myristoylation Inhibition by Zelenirstat Disrupts Growth Signalling, Energy Metabolism, and Migration in Triple-Negative Breast Cancer Cells.

Abstract

Background: Metastatic breast cancer cells rely on oxidative phosphorylation (OXPHOS) for energy production, yet selectively targeting mitochondrial metabolism in cancer cells remains a major therapeutic challenge. Myristoylation, the modification of proteins with the 14-carbon fatty acid myristate by N-myristoyltransferases NMT1 and NMT2, dually impacts cancer cell signalling and metabolism. The pan-NMT inhibitor zelenirstat selectively inhibits these enzymes to prevent the myristoylation of target proteins.

Methods: We assessed the effects of zelenirstat on the protein levels and metabolic activity of multiple triple-negative breast cancer (TNBC) cell lines. NDUFAF4 knockout (KO) MDA-MB-231 cells were engineered using CRISPR/Cas9. Functional assays included multiple types of respirometry, cell migration measurements, and spheroid formation.

Results: Zelenirstat treatment depleted myristoylated complex I assembly factor NDUFAF4 levels in four different TNBC cell lines. This loss effectively abrogated mitochondrial OXPHOS through impairment of complex I, resulting in reduced cellular energy production. Zelenirstat treatment resulted in NADH accumulation, loss of AMPK, and decreased glycolysis. Given that zelenirstat impacted cell signalling and OXPHOS, we further demonstrated that it also significantly impaired migration and invasion. Importantly, both zelenirstat treatment and NDUFAF4 KO TNBC MDA-MB-231 cells dramatically reduced mammosphere formation, indicating potent activity against breast cancer stem cells (BCSCs).

Conclusion: In addition to the previously shown inhibitory effect of zelenirstat on TNBC cell growth in vitro and in vivo, our new data demonstrate the widespread effects of zelenirstat on both aggressive TNBC cells and BCSCs. These findings highlight the potential of zelenirstat as an anti-metastatic agent, warranting further clinical trial evaluation in patients with TNBC.