Abstract 4169: Coordination of malic enzyme 2 and the malate-aspartate shuttle supports growth and survival in triple-negative breast cancer

Abstract Background & Hypothesis: Triple-negative breast cancer (TNBC) presents therapeutic challenges due to the lack of hormone-targeted treatments. Malic enzyme 2 (ME2), crucial for pyruvate and NADPH production, regulates cancer metabolism and redox balance. Elevated ME2 expression correlates with poor outcomes in multiple cancers. We hypothesized that ME2 knockdown (ME2kd) would impair TNBC growth by disrupting metabolic homeostasis, with the malate-aspartate shuttle (MAS) playing a complementary role. Methods: TNBC cell lines (MDA-MB-468, HCC70, HCC1806, Hs578T, BT20) and a control line (MCF10a) were analyzed under ME2kd using siRNA and shRNA. Growth, migration, and metabolic flux were assessed in vitro. Metabolite profiling was performed using GC-MS and Metabolite-Glo assays. Xenograft models of MDA-MB-468 and HCC70 evaluated tumor growth in vivo. ME2kd effects on mitochondrial function were measured via Seahorse XF96 assays. Growth under serine, glycine, and glucose deprivation was also assessed. The role of MAS in supporting ME2kd cells was evaluated with double knockdown experiments. Results: ME2kd reduced proliferation by over 50% in MDA-MB-468 and HCC1806 cells with minimal effects on MCF10a. Tumors derived from MDA-MB-468 ME2kd cells grew significantly slower, with extended survival in vivo. In contrast, ME2kd had no impact on HCC70 growth or migration. Metabolite profiling revealed elevated malate and reduced NADH/NADPH levels under ME2kd, with compensatory upregulation of serine biosynthesis genes. Deprivation of serine, glycine, or glucose with or without ME2kd impaired growth in 4 of 5 TNBC lines, with minimal effects on MCF10a. Combined ME2 and MAS knockdown further suppressed growth across TNBC lines, emphasizing MAS’s role in maintaining cytosolic NAD+/NADH balance under ME2kd. Conclusions: ME2 is essential for supporting the metabolic and proliferative demands in TNBC. Loss of ME2 disrupts key metabolic pathways, leading to significant reductions in cellular proliferation and migratory capacity. The observed increase in malate levels and altered NADH/NADPH ratios under ME2kd highlight its central role in maintaining metabolic homeostasis. Our data shows that ME2, de novo serine synthesis, and glycolysis are closely co-regulated in TNBC cell lines. The ability of the cell lines to compensate for ME2kd correlated with their inherent de novo serine synthesis capacity. Furthermore, the interdependence of MAS and ME2 suggests that MAS may act as a bridge between ME2 and de novo serine synthesis via regulation of cytosolic NAD+/NADH ratio. These findings confirm that ME2 is involved in TNBC metabolism and establish it as a compelling target for therapeutic intervention. Future studies could explore synergistic strategies combining ME2 inhibition with interventions targeting complementary metabolic pathways. Citation Format: Mark Daniel Slayton, Jin Heon Jeon, Zackariah Farah, Abhinav Achreja, Ben Krinkel, Yi-Hsien Eu, Alyssa Rosenfeld, Kerslee Kohagen, Liwei Bao, Xu Cheng, Kerry Loomes, Deepak Nagrath, Sofia Merajver. Coordination of malic enzyme 2 and the malate-aspartate shuttle supports growth and survival in triple-negative breast cancer [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 4169.