(P 24) THE SIRTUIN SIRT5 SUPPORTS EWING SARCOMA SURVIVAL VIA REGULATION OF EWS-FLI1 STABILITY AND ACTIVITY

Objective: Ewing sarcoma (EWS) is an aggressive tumor of bone or soft tissue that mainly affects children and young adults. With typical treatment modalities, patients with localized disease show favorable outcome; however, those with metastatic/recurrent diseases still have poor prognosis. Moreover, current EWS treatments have many adverse side effects. Therefore, there is an important need to develop improved therapeutic approaches that have fewer detrimental side effects, and that can effectively treat patients with recurrent/metastatic disease. Sirtuins are NAD+-dependent deacylases that regulate diverse cellular processes such as metabolic homeostasis and genomic integrity. Mammals possess seven sirtuin family members, SIRT1-SIRT7, that display diverse subcellular localization patterns, catalytic activities, protein targets, and biological functions. In the current study, we have tested the tumor promoter functions of the sirtuin SIRT5, a desuccinylase, in EWS.

Methods: EWS cell lines were evaluated for their survival dependency on SIRT5 in vitro and in xenograft models via shRNA and CRISPR mediated SIRT5 knockdown (KD). A variety of cutting-edge tools in addition to traditional techniques were employed to decipher the mechanism behind tumor promoting roles of SIRT5 in EWS. These include, immunoblotting, flow cytometry, RNA-Seq analysis, CHIP-Seq, qRT-PCR, site-directed mutagenesis, mass spectrometry-based metabolite analysis, metabolic profiling using the Agilent-Seahorse extracellular flux analyzer, biochemical assays, drug screening via thermostability assay, and crystallography, among others. GraphPad Prism was used to perform all statistical analysis.

Results: Depletion of SIRT5 resulted in reduced cell proliferation and impaired colony formation ability in 6/6 EWS cell lines tested in vitro. These results were further corroborated by the inability of SIRT5 knockdown (KD) EWS cells to form tumors in xenograft models. Loss of SIRT5 resulted in increased apoptosis, as evidenced by increased levels of cleaved PARP and cleaved caspase 3, and increased number of annexin V- and PI-positive cells. It has been reported that silencing the expression or activity of oncogenic driver EWS-FLI1 induces apoptosis and decreases viability as well as tumorigenicity in EWS cells, like the phenotypes that we observe upon SIRT5 loss. Interestingly, we find that EWS-FLI1 is hypersuccinylated, and its levels are reduced, in SIRT5-depleted EWS cells. Consistently, SIRT5 KD EWS cells display reduced chromatin occupancy of EWS-FLI1 and attenuated expression of its target genes. Based on these results, we speculate that SIRT5 regulates succinylation on the oncogenic driver EWS-FLI1 and modulates its stability and activity. Crucially, SIRT5-deficient mice, and normal cells depleted of SIRT5, show normal development and minimal phenotypes. Therefore, a great deal of interest exists in targeting SIRT5 pharmacologically. In this context, we have generated a novel small molecule inhibitor that can suppress SIRT5 activity both in vitro and in vivo. EWS cells treated with this inhibitor exhibited attenuated growth and other signature phenotypes of SIRT5 loss of function, suggesting that pharmacological SIRT5 inhibition may have therapeutic potential in EWS.

Conclusion: In conclusion, we have found that SIRT5 is crucial for the survival of EWS cells and represents an attractive therapeutic target in EWS. Mechanistically, SIRT5 regulates succinylation levels on the oncogenic driver EWS-FLI1 and promotes its stability and activity. SIRT5 depletion/inhibition causes hypersuccinylation on EWS-FLI1, resulting in its reduced levels and decreased chromatin binding, which in turn alters the expression of multiple genes including pro-survival genes that leads to EWS cell death.