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Ewing sarcomas are driven by EWS–ETS fusions, most commonly EWS-FLI1, which promotes widespread metabolic reprogramming, including activation of serine biosynthesis. We previously reported that serine biosynthesis is also activated in Ewing sarcoma by the scaffolding protein menin through as yet undefined mechanisms. Here, we investigated whether EWS-FLI1 and/or menin orchestrate serine biosynthesis via modulation of ATF4, a stress-response gene that acts as a master transcriptional regulator of serine biosynthesis in other tumors. Our results show that in Ewing sarcoma, ATF4 levels are high and that ATF4 modulates transcription of core serine synthesis pathway (SSP) genes. Inhibition of either EWS-FLI1 or menin leads to loss of ATF4, and this is associated with diminished expression of SSP transcripts and proteins. We identified and validated an EWS–FLI1 binding site at the ATF4 promoter, indicating that the fusion can directly activate ATF4 transcription. In contrast, our results suggest that menin-dependent regulation of ATF4 is mediated by transcriptional and post-transcriptional mechanisms. Importantly, our data also reveal that the downregulation of SSP genes that occurs in the context of EWS-FLI1 or menin loss is indicative of broader inhibition of ATF4-dependent transcription. Moreover, we find that menin inhibition similarly leads to loss of ATF4 and the ATF4-dependent transcriptional signature in MLL-rearranged B-cell acute lymphoblastic leukemia, extending our findings to another cancer in which menin serves an oncogenic role. Implications: These studies provide new insights into metabolic reprogramming in Ewing sarcoma and also uncover a previously undescribed role for menin in the regulation of ATF4.

s: AACR Special Conference on the Advances in Pediatric Cancer Research; September 17-20, 2019; Montreal, QC, Canada Ewing sarcoma (ES) is an aggressive bone and soft-tissue tumor, most commonly driven by the EWS/FLI1 fusion oncogene. Previous work has revealed that the scaffolding protein menin functions to promote ES tumorgenicity. Additionally, our recent studies revealed a previously undescribed role for menin in the activation of the serine biosynthetic pathway (SSP), a critical metabolic pathway that is aberrantly activated in many human cancers. The transcription factor ATF4 has been identified in other tumor types as a master transcriptional regulator of the SSP. Furthermore, regulation of the SSP by EWS/FLI1 has been described, although the mechanism of this regulation remains unknown. The biologic functions of menin are largely determined by its protein-binding partners, the best characterized of which is MLL in epigenetic trithorax complexes. In the current study, we are investigating the mechanistic link between menin, EWS/FLI1, and SSP hyperactivation to test the hypothesis that, in the context of ES, these transcriptional regulators cooperate to promote metabolic reprogramming via ATF4. Through gain- and loss-of-function studies, our results show that ATF4 regulates the SSP in ES cells, and that loss of ATF4 impairs ES cell proliferation. Additionally, both pharmacologic inhibition with the menin:MLL interaction inhibitor, MI-503, and doxycycline-inducible shRNA knockdown of menin lead to loss of ATF4, coincident with loss of SSP expression. Knockdown of EWS/FLI1 similarly results in loss of both ATF4 and SSP expression. Interrogation of publicly available ChIP-seq data further shows enrichment for EWS/FLI1 binding at the ATF4 gene promoter that is lost with EWS/FLI1 knockdown, and ChIP-qPCR shows menin binding at the ATF4 gene promoter in association with H3K4me3 enrichment. ChIP-qPCR also shows enrichment of ATF4 binding at SSP gene promoters, which is diminished upon menin inhibition with MI-503. Preliminary studies suggest that ATF4 overexpression may rescue the effects of menin loss of function on the SSP. Together these findings support the hypothesis that menin hijacks the SSP via aberrant activation of ATF4, and that this depends on cooperation with EWS/FLI1. Ongoing studies are assessing whether this is mediated by trithorax-dependent or -independent functions of menin. Citation Format: Jennifer A. Jimenez, Ramon Ocadiz Ruiz, April Apfelbaum, Abhijay Kumar, Samuel Kerk, Jolanta Grembecka, Costas A. Lyssiotis, Elizabeth R. Lawlor. Menin and EWS/FLI1 activate serine biosynthesis via ATF4 in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B37.