Ewing sarcoma (EWS) is a soft tissue and bone tumor found primarily in adolescents and young adults. In most cases of EWS, the chimeric transcription factor, EWS-FLI1 is the primary oncogenic driver. The expression profile of EWS cells reflects EWS-FLI1 binding and activation or repression of transcription. Recent studies have shown EWS-FLI1 deregulates proteins that function in tryptophan metabolism (Mutz et al., 2016, FEBS Lett. 590, 2063) or serine-glycine biosynthesis (Tanner et al., 2017, Mol. Cancer Res., 11, 1517). In our study, we extend these findings by establishing that EWS-FLI1 transcriptionally regulates the expression of enzymes required for serine-glycine biosynthesis and the mitochondrial one-carbon cycle, and the uptake of the alternative nutrient source glutamine, directly. To identify metabolic pathways regulated by EWS-FLI1, we interrogated the expression profiles of EWS-FLI1-silenced TC32 EWS cells. We observed that silencing of EWS-FLI1 results in the decreased expression of genes required for serine-glycine biosynthesis specifically, PHGDH, PSAT1, PSPH, and SHMT2. Using ChIP analysis, we demonstrated that EWS-FLI1 binds specific regulatory regions within these four serine-glycine biosynthesis genes. Also, ectopic expression in 293T cells results in increased expression of PHGDH, PSAT1, PSPH, and SHMT2. Consistent with the ability of EWS cells to synthesize serine and glycine de novo, EWS cells grown in medium depleted of these amino acids exhibit no change in viability. However, EWS cells were sensitive to the depletion of glutamine. Analysis of the expression profiles of EWS-FLI1-silenced TC32 EWS cells showed EWS-FLI1 positively regulates expression of the glutamine transporter, SLC1A5 and that EWS cells require glutamine for glycine synthesis. Next, we examined metabolic processes downstream of serine-glycine biosynthesis and showed that enzymes associated with the mitochondrial one-carbon cycle, MTHFD2 and MTHD1L, are transcriptional targets of EWS-FLI1, but not their cytosolic counterpart MTHFD1. Our data suggests EWS-FLI1 favors activation of enzymes associated with the mitochondrial one-carbon cycle, which provides the reducing equivalents GSH and NADPH. Depletion of EWS-FLI1 or the inhibition of PHGDH reduced the GSH/GSSG and NADPH/NADP ratios in EWS cells resulting in increased production of reactive oxygen species, inducing DNA damage and apoptosis. Importantly, analysis of EWS primary tumor transcriptome data confirmed that the aforementioned metabolism genes we identified as regulated by EWS-FLI1 exhibit increased expression compared with normal tissues. In summary, our study demonstrates that EWS-FLI1 reprograms the metabolism of EWS cells and that serine-glycine metabolism and glutamine uptake are potential vulnerabilities in this tumor type.
Citation Format: Nirmalya Sen, Allison M. Cross, Philip L. Lorenzi, Javed Khan, Berkley E. Gryder, Suntae Kim, Natasha J. Caplen. EWS-FLI1 reprograms the metabolism of Ewing sarcoma cells via positive regulation of glutamine import and serine-glycine biosynthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5471.