(P 253) ELUCIDATING THE ROLE OF SIRT7 IN EWING SARCOMA

Objective: SIRT7 is a member of the Sirtuin family, a group of NAD+-dependent deacylases involved in the regulation of various cellular processes. Predominantly localized to the nucleolus, SIRT7 regulates RNA transcription, DNA repair, and chromatin remodeling. SIRT7 deacetylates H3K18 and H3K36, two epigenetic marks associated with tumorigenesis. Ewing Sarcoma (EWS) is a rare, aggressive tumor of the bone or soft tissue that predominantly affects children and young adults. Localized EWS has a 70–80% 5-year survival rate with standard treatment, but metastatic cases carry a poor prognosis, and current therapies often cause significant short- and long-term side effects that impact survivors' quality of life. In a preliminary analysis, we found that high SIRT7 expression in metastatic EWS patients correlates with poor survival. This work aims to assess the response of EWS cells to SIRT7 depletion and elucidate the mechanisms by which SIRT7 promotes tumor growth in EWS.

Methods: We used short hairpin RNA (shRNA) and a SIRT7 enzymatic inhibitor to deplete or inhibit SIRT7 in Ewing Sarcoma (EWS) cell lines (A673, A4573, TC32). Cell proliferation and colony formation assays were performed to evaluate growth. Transcriptomic changes were assessed via RNA sequencing, while metabolic alterations were analyzed using LC-MS/MS-based metabolomics and Seahorse XF extracellular flux analysis. ATF4 expression was measured at the mRNA and protein levels by qRT-PCR and Western blotting, respectively.

Results: SIRT7 depletion or inhibition impaired the growth of EWS cell lines (A673, A4573, TC32) in vitro, as shown by proliferation and colony formation assays. RNA-seq analysis of SIRT7-depleted or SIRT7-inhibited EWS cells revealed that the most significantly affected pathways were related to metabolism. Consistently, we observed that the loss of SIRT7 altered metabolite levels and reduced both glycolytic rates and mitochondrial respiration in SIRT7-depleted EWS cells. Furthermore, loss of SIRT7 in EWS cells leads to a significant reduction in activating transcription factor 4 (ATF4) in both gene expression and protein levels.

Conclusion: We hypothesize that loss of SIRT7 may be regulating EWS cell survival by modulating ATF4 expression, either through direct regulation at the chromatin level or indirectly by disrupting stress response signaling. This dysregulation may, in turn, alter metabolic homeostasis, ultimately compromising EWS cell viability and leading to cell death.