✍🏼 Jin Heon Jeon, Mark D. Slayton, Ben Krinkel, Olamide Animasahun, Ajay Shankaran, Fulei Wuchu, Minal Nenwani, Zackariah Farah, Julia Burke, Abhinav Achreja, Brisilda Nilaj, Kerslee Kohagen, Yi-Hsien Eu, Alyssa Rosenfeld, Mason Collard, Liwei Bao, Xu Cheng, Celina Kleer, Christopher Squire, Kerry Loomes, Deepak Nagrath & Sofia D. Merajver

🏠 Department of Internal Medicine, Michigan Medicine, University of Michigan, Ann Arbor, MI, USA

📑  Cancer Metabolism (2025)

Abstract
Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous subtype of breast cancer with poor clinical outcomes. Malic enzyme 2 (ME2) is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and has been proposed as a therapeutic target. ME2 is highly expressed in many cell types including TNBC cells. We sought to define the molecular and cellular consequences of ME2 inhibition to facilitate its clinical translation. Here, we systematically evaluated the cellular and molecular effects of ME2 knockdown (ME2kd) in multiple TNBC models. ME2kd had heterogeneous effects on proliferation, migration, and metabolic flexibility in TNBC cell lines. ME2kd MDA-MB-468 xenografts in nude mice grew significantly slower and conferred prolonged host survival. ME2kd caused distinct shifts in mitochondrial respiration and glycolysis, whereas metabolomic and transcriptomic analyses revealed altered tricarboxylic acid (TCA) cycle flux, glutamine consumption, and serine/glycine metabolism, partly through changes in malate-aspartate shuttle (MAS) activity. The interplay between ME2, the serine synthesis pathway and the MAS was investigated with metabolite deprivation and co-knockdown assays. Importantly, we determined the crystal structure of ME2 bound to the small-molecule inhibitor NPD-389 and identified the binding interactions that drive the inhibitory response. These findings help to clarify the role of ME2 in TNBC phenotypes and highlight the therapeutic potential of ME2 inhibition in precision oncology.

How the WOLF was used in this study
In this study, the NanoCellect WOLF G2 Cell Sorter was used to enrich for cells successfully transduced with lentiviral constructs targeting ME2. Cells were transduced with lentiviral vectors carrying either scrambled shRNA or ME2-targeting shRNA, both co-expressing TurboRFP as a fluorescent reporter. After 72 hours of transduction and induction of shRNA expression with doxycycline, the WOLF G2 was employed to fluorescence-activate sort TurboRFP-positive cells, effectively removing unmodified cells from the population. This sorting step ensured that downstream experiments were performed on a pure population of successfully transduced cells, improving the accuracy and reproducibility of functional analyses.