EWS/FLI is a Master Regulator of Metabolic Reprogramming in Ewing Sarcoma

Abstract

Ewing sarcoma is a bone malignancy driven by a translocation event resulting in the fusion protein EWS/FLI1 (EF). EF functions as an aberrant and oncogenic transcription factor that misregulates the expression of thousands of genes. Previous work has focused principally on determining important transcriptional targets of EF, as well as characterizing important regulatory partnerships in EF-dependent transcriptional programs. Less is known, however, about EF-dependent metabolic changes or their role in Ewing sarcoma biology. Therefore, the metabolic effects of silencing EF in Ewing sarcoma cells were determined. Metabolomic analyses revealed distinct separation of metabolic profiles in EF-knockdown vs. control-knockdown cells. Mitochondrial stress tests demonstrated that knockdown of EF increased respiratory as well as glycolytic functions. Enzymes and metabolites in several metabolic pathways were altered, including de novo serine synthesis and elements of one-carbon metabolism. Furthermore, phosphoglycerate dehydrogenase (PHGDH) was found to be highly expressed in Ewing sarcoma and correlated with worse patient survival. PHGDH knockdown or pharmacological inhibition in vitro caused impaired proliferation and cell death. Interestingly, PHGDH modulation also led to elevated histone expression and methylation. These studies demonstrate that the translocation-derived fusion protein EF is a master regulator of metabolic reprogramming in Ewing sarcoma, diverting metabolites toward biosynthesis. As such, these data suggest that the metabolic aberrations induced by EF are important contributors to the oncogenic biology of these tumors.

Menin regulates the serine biosynthetic pathway in Ewing sarcoma

“…We first investigated the baseline expression of the SSP in Ewing sarcoma cell lines and tumors. In the Cancer Cell Line Encyclopedia (http://www.broadinstitute.org/ccle), PHGDH expression was higher in Ewing sarcoma than in any other cancer, an observation recently reported by others . We measured the expression of SSP enzymes in Ewing sarcoma cell lines using qRT‐PCR and western blot, and observed higher protein expression of pathway components in all cell lines compared with mesenchymal stem cells (MSCs), putative cells of origin for Ewing sarcoma (supplementary material, Figure S3A–C).…”

“…Of particular relevance to this study, menin was shown to cooperate with cMYC in the regulation of metabolism‐associated genes, including PSAT1 , in an MLL‐independent fashion . Moreover, the EWS–FLI1 oncoprotein itself contributes to maintenance of SSP gene expression in Ewing sarcoma . The extent to which MYC, EWS–FLI1, or other transcriptional regulators cooperate with menin to hijack and maintain oncogenic activation of the SSP in Ewing sarcoma remains to be determined.…”

EWS‐FLI1 reprograms the metabolism of Ewing sarcoma cells via positive regulation of glutamine import and serine‐glycine biosynthesis

“…Our study complements and extends a recent report by Tanner and colleagues that also described the regulation of de novo serine‐glycine biosynthesis by EWS‐FLI1. Tanner and colleagues presented data using shRNA‐based silencing of EWS‐FLI1 that indicated EWS‐FLI1 regulates PHGDH, PSAT1, and PSPH expression, and that depletion of EWS‐FLI1 decreases the expression of SHMT1/2.…”

“…Depletion of EWS‐FLI1 or the silencing or inhibition of PHGDH reduced the GSH/GSSG and NADPH/NADP ratios in EWS cells resulting in increased production of reactive oxygen species in EWS cells (Figure ). Our findings suggest a mechanistic basis for previous observations that have described mitochondrial stress or DNA damage following depletion of EWS‐FLI1 in EWS cells.…”

“…Our results differ from previously published findings. Tanner and colleagues reported an increased utilization of glucose by glycolysis following depletion of EWS‐FLI1 that they linked to the derepression of the expression of the hexokinase enzyme . A separate study showed glucose deprivation in combination with hypoxia affected the viability of EWS cells .…”