Defining the role of menin in the Ewing sarcoma stress response

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PROJECT SUMMARY Cancer cells must respond to microenvironmental stress during tumor growth and metastasis in order maintain metabolic homeostasis and support cell survival. ATF4 is a stress-induced master transcription factor that is frequently upregulated in cancer. ATF4 activates gene expression programs that allow cells to respond to stresses, such as nutrient and amino acid limitation, hypoxia, and oxidative stress. Previously published work from the Lawlor lab revealed that the scaffolding protein menin promotes Ewing sarcoma tumorgenicity and identified metabolic and stress response pathways, including the serine biosynthesis pathway, as downstream targets of menin. Ewing sarcoma is a bone and soft tissue tumor that is most commonly driven by the EWS-FLI1 oncogene. Although survival rates for patients with localized tumors have improved, despite maximally intensive therapy, Ewing sarcoma remains lethal for a third of patients, and event free and overall survival rates for patients with metastatic disease are dismal. To this day, efforts to target the pathognomonic EWS-FLI1 fusion protein have been largely unsuccessful, requiring the identification novel therapeutic targets and strategies. The scaffolding protein menin can function as a tumor suppressor or as an oncogene in cancer. Menin can regulate gene expression in both epigenetic-dependent and -independent manners, depending on its interaction with several different protein-binding partners. Our preliminary data show that EWS-FLI1 contributes to regulation of menin and its control of cell metabolism. In particular, we have found that, in Ewing sarcoma, ATF4 is highly expressed and maintains Ewing sarcoma cell proliferation by regulating stress response pathways, including the serine synthesis pathway. In addition, our studies show that ATF4 expression is modulated by menin. This proposal will test the central hypothesis that a novel EWS-FLI1-menin-ATF4 axis functions in Ewing sarcoma to promote cellular adaptation to stress, and that this axis is required for maintenance of metabolic homeostasis and tumorgenicity. This hypothesis will be tested through two specific aims. Aim 1 will investigate the molecular mechanism(s) by which menin regulates ATF4 in Ewing sarcoma, and the biological significance of this axis in response to stress. Aim 2 will elucidate the contribution of the EWS-FLI1 oncogene to maintaining high-level menin expression as part of this stress-response axis. To accomplish these specific aims, menin, ATF4, and EWS-FLI1 gain and loss-of-function in vitro models; metabolic profiling; and in vivo metastasis models will be used to study the impact of EWS-FLI1 and/or menin modulation on ATF4-dependent Ewing sarcoma cell survival and response to stress. The results from these innovative studies will define cooperative mechanisms between EWS-FLI1 and menin in regulation of stress response pathways in Ewing sarcoma, with the overall goal of identifying new therapeutic opportunities. Elucidating the contribution of menin to cellular metabolic homeostasis will be of profound importance to cancer biology more broadly.