DEFINING FATTY ACID DESATURASE 2 (FADS2) AS A NOVEL THERAPEUTIC TARGET IN EWING SARCOMA - PROJECT SUMMARY: EWING SARCOMA IS A DEVASTATING PEDIATRIC MALIGNANCY CHARACTERIZED BY PATHOLOGICAL EWSR1-ETS FUSION TRANSCRIPTION FACTORS AND REMAINS DEVOID OF MOLECULARLY TARGETED THERAPIES. THROUGH UNBIASED SMALL MOLECULE SCREENING, TRANSCRIPT-LEVEL CORRELATIONAL ANALYSES, AND MECHANISTIC VALIDATION, I HAVE IDENTIFIED A CLASS OF COMPOUNDS-FADS2-DEPENDENT CYTOTOXINS (FDCS)-THAT SELECTIVELY KILL EWING SARCOMA CELLS BY CO-OPTING THE OXIDATIVE CATALYTIC ACTIVITY OF FATTY ACID DESATURASE 2 (FADS2). MECHANISTIC STUDIES SUGGEST THESE AGENTS ACT AS NON-NATIVE SUBSTRATES THAT INDUCE A GAIN-OF-TOXIC-FUNCTION EFFECT ON FADS2, LEVERAGING ITS CATALYTIC ACTIVITY TO GENERATE SUPEROXIDE AND DRIVE REDOX COLLAPSE. THERE IS A DIRECT POSITIVE CORRELATION BETWEEN FADS2 EXPRESSION LEVEL AND INCREASED TOXICITY TO FDCS. NOTABLY, EWING SARCOMA EXHIBITS UNIQUELY AND CONSISTENTLY HIGH FADS2 EXPRESSION ACROSS ESTABLISHED CANCER CELL LINES AND PATIENT-DERIVED TUMORS, WITH COMPARABLY LOW EXPRESSION IN NON-DISEASED TISSUE-SUGGESTING AN EXPLOITABLE THERAPEUTIC WINDOW. YET, THE MECHANISTIC UNDERPINNINGS OF FDC- INDUCED CYTOTOXICITY AND THEIR TRANSLATIONAL POTENTIAL IN EWING SARCOMA REMAIN UNCHARACTERIZED. THIS PROPOSAL TESTS THE CENTRAL HYPOTHESIS THAT ABERRANTLY HIGH FADS2 EXPRESSION CREATES A LINEAGE-SPECIFIC VULNERABILITY THAT CAN BE PHARMACOLOGICALLY EXPLOITED WITH FDCS. IN AIM 1, I WILL DEFINE THE REQUIREMENT OF FADS2 EXPRESSION IN MEDIATING FDC TOXICITY USING GENETIC LOSS-OF-FUNCTION. IN AIM 2, I WILL DEFINE THE OXIDATIVE INTERMEDIATES FORMED IN RESPONSE TO FDC TREATMENT IN A FADS2-DEPENDENT MANNER. IN AIM 3, I WILL EVALUATE THE IN VIVO EFFICACY OF FDCS IN EWING SARCOMA ORTHOTOPIC XENOGRAFT MODELS. TOGETHER, THESE STUDIES WILL ESTABLISH A MECHANISTIC FRAMEWORK FOR FADS2-DEPENDENT CYTOTOXICITY, PROVIDE PRECLINICAL RATIONALE FOR TARGETING THIS METABOLIC LIABILITY IN EWING SARCOMA, AND ADVANCE A NOVEL THERAPEUTIC STRATEGY FOR A DISEASE WITH URGENT UNMET CLINICAL NEED.