• ¹School of Basic Medicine, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China.
• ²Department of Pharmacology, School of Pharmacy, Hubei University of Science & Technology, Xianning 437100, China.
• ³School of Pharmacy, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China.
• ⁴Medical Research Institute, Wuhan University, Wuhan 430071, China.

Deamidation of glutamine to glutamate byglutaminase1 (GLS1, also called GLS) and GLS2 is an essential step in bothglutaminolysisand glutathione (GSH) biosynthesis. However, mechanisms whereby cancer cells regulate glutamine catabolism remains largely unknown. We report here that N-Myc, an essentialMycfamily member,promotesconversion of glutamine to glutamate in MYCN-amplifiedneuroblastomacells by directly activating GLS2, but not GLS1, transcription. Abrogation of GLS2 function profoundly inhibitedglutaminolysis, which resulted in feedback inhibition of aerobic glycolysis likely due to thioredoxin-interacting protein (TXNIP)activation, dramatically decreasing cell proliferation and survival in vitro and in vivo. Moreover, elevated GLS2 expression is significantly elevated in MYCN-amplified neuroblastomas in comparison with non-amplified ones, correlating with unfavorable patient survival. In aggregate, these results reveal a novel mechanism deciphering context-dependent regulation of metabolic heterogeneities, uncovering a previously unsuspected link betweenMyc, GLS2 and tumor metabolism.