Estrogen-related receptor gamma maintains oxidative metabolism via glutamine-derived anaplerosis

Glutamine (Gln), traditionally considered a nonessential amino acid, becomes essential in cancer cells as the rate of Gln consumption exceeds that of endogenous biosynthesis. This metabolic dependency constitutes a unique “glutamine addiction”. Although many factors, both genetic and environmental, have been shown to contribute to the control of Gln metabolism, the precise regulation of Gln utilization in cancer still remains largely unclear. Here, we identify estrogen-related receptor gamma (ERRgamma) as a critical regulator of glutaminolytic enzymes and cellular Gln utilization. RNA-sequencing of four primary retinoblastoma cells lines with or without shRNA-mediated ERRgamma knockdown showed that, relative to shGFP controls, shERRgamma downregulated genes were enriched for hypoxic adaptation and glycolysis pathways ( FDR < 0.05 ), with no change in oxidative phosphorylation, indicating a shift toward lactate-producing glucose flux. Consistent with this, key genes in lactic acid fermentation (PDK1, LDH, MCT1) were significantly reduced ( p < 0.05 ). shERRgamma also decreased expression of glutaminolysis-related genes ( FDR < 0.05 ), suggesting reliance on Gln-derived carbon to sustain TCA activity in a “Warburg-like” state. Functionally , RB006, a model cancer cell line with high basal ERRgamma expression, requires exogenous Gln for viability, exhibiting marked cell death upon Gln withdrawal and a dose-dependent increase in proliferation, with ≥ 4mM required for maximal growth. Mesenchymal stem cells (MSCs), which express low ERRgamma, were Gln-independent; however, ERRgamma overexpression (MSC[ERRgamma]), induced both Gln dependence and dose-responsive proliferation. To further assess Gln utilization, cells were supplemented with increasing Gln, and TCA intermediates (glutamate, alpha-KG, citrate, pyruvate, ATP) were quantified. All lines converted Gln to glutamate in a dose-dependent manner, but only RB006 and MSC[ERRgamma] showed parallel increases in alpha-KG and citrate, consistent with ERRgamma-mediated control of GLUD1/2 and downstream reductive carboxylation. High- ERRgamma cells demonstrated strong correlations between Gln levels and all metabolites (r > 0.9), produced greater metabolite output per unit Gln than MSCs, and exhibited larger stepwise increases in Gln-derived flux, reflecting enhanced glutamine-handling capacity. Our results indicate ERRgamma may be necessary to support oxidative phosphorylation via glutamine-derived anaplerosis. We provide evidence that ERRgamma is required for glutaminolytic pathways, suggesting that ERRgamma may be a key player in the metabolic adaptation of highly proliferative cell lines.