The loss-of-function mutation of the tumor suppressing Krebs cycle enzyme fumarate hydratase (FH) is a driver of Hereditary Leiomyomatosis and Renal Cell Carcinoma (HLRCC). Accumulation of fumarate results in impaired energy metabolism and activation of stress related mechanisms leading to activation of the mTORC1 pathway and stabilization of HIF1a following by upregulation of angiogenesis and cell survival related genes. To better understand how cells compensate for the loss of FH in HLRCC, we studied the nutrient requirements of the FH-deficient UOK262 cell line (UOK262) and its FH-repleted control (UOK262WT). We found that asparagine, in addition to glutamine, increased the growth rate of both cell lines in vitro. Glutamine, but not asparagine, increased oxygen consumption rates and glycolytic reserve of both cell lines. To better understand the mechanism of asparagine-stimulated growth, we performed an mRNA-Seq analysis of both cell lines in the presence and absence of glutamine and asparagine. Surprisingly, asparagine in combination with glutamine, but not asparagine or glutamine alone, stimulated expression of genes associated with the endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in UOK262 to a greater extent than in FH-restored cells. The changes in expression of these genes were confirmed by RT-PCR, and the stimulation of the UPR was confirmed orthogonally by demonstration of an increase in spliced XBP1 (sXBP1) in UOK262 cells under these conditions. To further understand the role of asparagine in UOK262 cells, we conducted stable isotope based metabolic studies using 13C,15N labeled glutamine or asparagine in these cell lines. Although Asn was taken up by the cells, there was little evidence of asparagine-derived label in asparagine pathways cellular catabolites, indicating that asparagine was not catabolized, consistent with the absence of asparaginase in mammalian cells. However, asparagine strongly stimulated glutamine labeling of uracil and precursors, uridine phosphates and hexosamine metabolites in the UOK262 cells and to a much lesser extent in the UOK262WT cells, indicating an activation of the hexosamine biosynthetic pathway by asparagine. To determine the mechanism of uridine and hexosamine labeling we examined the expression of key uracil synthetic pathway enzymes reported to be regulated by sXBP1, and found that asparagine exposure increased the RNA and protein expression of GFPT2, the Gln-fructose - 6- phosphate amidotransferase which is a transcriptional target of sXBP1. In summary, asparagine in the presence of glutamine induces an ER stress response in FH-deficient UOK262 cells and stimulates increased synthesis of UDP-acetyl glycans indicative of hexosamine biosynthetic pathway activity. These data demonstrate a novel effect of asparagine on cellular metabolism in FH-deficient cells that could be exploited therapeutically.
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