Abstract Ewing sarcoma (EWS) is an aggressive tumor arising in soft tissue and bone of children and young adults. EWS is treated with a combination of cytotoxic chemotherapy, local radiation, and/or surgery. Patients with localized disease show a favorable overall survival rate. However, there is still a pressing need for new therapeutic approaches for EWS. Patients with metastatic or recurrent EWS have a very poor prognosis. Moreover, current EWS treatments are associated with many short- and long-term sequelae, e.g. accelerated cardiovascular disease and secondary cancers. EWS-FLI1 is the fusion oncoprotein present in most cases of EWS. It functions as a pioneer transcription factor to affect expression of many target genes. The aberrant EWS transcriptome represents a potential therapeutic target in EWS. This proposal focuses on the sirtuin SIRT5 as a novel therapeutic target in EWS. SIRT5 is found throughout the cell, and regulates protein targets in diverse pathways by removing negatively charged modifications on lysine residues, including succinylation. Although normal cell types and whole mice tolerate loss of SIRT5 with minimal phenotypes, we have found that specific cancers, notably including EWS, are exquisitely dependent on SIRT5, and rapidly undergo apoptosis following SIRT5 depletion. We have linked this effect to a role for SIRT5 in desuccinylating nuclear histones, thereby modulating gene expression in EWS. We and others have shown that SIRT5 is in principle amenable to selective inhibition or degradation with small molecules. Our long-term goal is to evaluate SIRT5 as a potential therapeutic target for EWS. The objective of this proposal is to generate new biological insights into SIRT5 function in EWS, and characterize SIRT5 inhibitors and SIRT5 PROTAC-based degraders. The central hypotheses of this application are that: 1) SIRT5 is required for EWS cell survival via histone desuccinylation and regulation of gene expression; and 2) SIRT5 inhibitors and degraders will represent useful tool compounds to interrogate SIRT5 biology, and a starting point for potential future EWS therapeutics. The rationale for this application is that EWS cells show exquisite vulnerability to SIRT5 loss-of-function, while other cell types and whole mice show no major ill effects. Hence, SIRT5 inhibition would likely be well tolerated clinically. The work will take place in the context of two Specific Aims. First, we will elucidate the impact of SIRT5 and Ksucc on histones and gene expression, using mass spectrometry along with transcriptomic and epigenomic approaches. Second, we will optimize and validate SIRT5 inhibitors and degraders, using medicinal chemistry approaches and based in part on SIRT5-inhibitor co-crystal structures. The application is innovative, in that no published data currently link SIRT5 to EWS, and no potent and selective SIRT5 inhibitors or PROTACs have as yet been described. The work is significant, since there is an unmet clinic...