Mentor Name: Poul Sorensen

Aggressive cancer cells must overcome diverse forms of stress in the tumor microenvironment and blood circulation, such as oxidative stress, to survive and metastasize. We previously showed that EWSR1-ETS oncofusions found in Ewing Sarcoma (EwS) directly induce surface expression of IL1 receptor accessory protein (IL1RAP). IL1RAP is highly expressed in EwS and shows limited expression in healthy tissues except placenta, nominating IL1RAP as a promising EwS immunotherapy target. We therefore engineered antibody-drug conjugates (ADCs) with different cytotoxic payloads targeting IL1RAP. ADCs potently blocked tumor growth and induced durable regression of EwS xenografts in mice, and diminished metastatic dissemination in vivo. We found that IL1RAP is a critical regulator of cysteine and glutathione (GSH) metabolism in EwS. Indeed, IL1RAP acts by directly associating with the CD98/xCT cystine/glutamate antiporter to enhance cystine uptake, an essential precursor for the synthesis of GSH, a major cellular antioxidant and key metabolic co-factor, including as a source of sulphur to generate iron-sulphur (Fe-S) clusters that are essential for many mitochondrial enzymes including electron transport complexes. We evaluated if EwS cells could tolerate reduced levels of GSH, using either xCT genetic or pharmacologic inhibition, or by blocking downstream enzymes in the GSH synthesis pathway, including GCLC, using buthionine sulfoximine (BSO), or glutaminase using CB-839. We demonstrated the presence of BSO and CB-839 resistant cells surviving under low levels of GSH. These cells somehow maintained not only redox homeostasis, but also showed functional mitochondrial respiration (oxidative phosphorylation). Using data from CRISPR dropout screens that the Sorensen team recently performed, we plan to identify how these cells are surviving in the absence of GSH, identifying specific dependencies of these BSO and CB-839 resistant cells as part of this project. Genes identified as being essential for the survival of low GSH-resistant cells will be individually depleted in the BSOR/CB-839-resistant cells. We will evaluate if these depletions are mimicking the phenotypes observed in the BSOR/CB-839-resistant cells, performing assays such as Incucyte live-cell analysis that monitors cell growth and death, Seahorse metabolic analysis that measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells, flow cytometry analysis that evaluates markers indicative of cell survival, mitochondrial fitness, and redox homeostasis. These studies will provide opportunities for synthetic lethal interventions for potential therapeutic applications.