Abstract 4322: NSC59984 is a radiosensitizer and cytotoxic agent in <i>in vitro</i> and <i>in vivo</i> models of <i>TP53mut</i> endometrial cancer

Abstract Background: Endometrial cancer (EC) mortality is associated with high grade histologies harboring TP53 mutations. NSC59984 is a small molecule drug hypothesized to restore p53 signaling in TP53mut cancers, but has not been explored in EC. We aimed to evaluate the cytotoxic and radiosensitizing potential of NSC59984 in TP53mut EC cell lines in vitro and in vivo and elucidate its mechanism of action. Methods: In vitro experiments used 2 EC cell lines with differing TP53 status: JHUEM2 (WT TP53) and Hec1B (homozygous R248Q). Dose response curves were generated by treating cells with NSC59984 for 24hrs and assessing viability with CellTiter-Glo after 9 days. To assess for mutation specific effects, we used CRISPR/Cas9 modified variants of JHUEM2 cells lacking WT p53 and harboring 1 of 5 TP53 mutations (R248Q, R248W, R273C, R273H, Y220C). Western blot analysis assessed p53, p21, PUMA, GADD45, and NOXA signaling using whole cell lysates after treatment with 25μM of NSC59984 for 24hrs. Radiosensitizing effect was assessed using JHUEM2 TP53 variants and Hec1B treated with increasing doses of NSC59984 and a single fraction of RT (0, 0.5, 1, 2, 4 and 8Gy.) In vivo experiments used HEC1B xenograft tumors generated in female NSG mice. 2 x106 cells were injected SQ in the flanks of 6 week old mice. 2 mice (4 tumors) were included in each arm: vehicle, RT, NSC59984, and NSC59984 + RT. Once tumors were 150mm3, mice were treated with a 2Gy fraction of RT and NSC59984 (45mcg/kg) every 5 days for 23 days via IP injection. Synergy was tested using HSA and BLISS modeling. Results: NSC59984 had a negative, dose dependent effect on cell viability. IC50s for JHUEM2 and Hec1B were 4.2μM and 5.7μM. IC50s for NTC, TP53 KO and variants were similar (4.2-6.1μM). In Western blot analysis, NSC59984 restored p53 signaling via p21, PUMA, and NOXA with decreased mutant p53 signal across all variant cell lines. Radiosensitization assays demonstrated synergy of NSC59984 + RT in all cell lines with maximum synergy at 5μM of NSC59984 and 2Gy (p&amp;lt;0.05) based on HSA modeling. In vivo pilot experiments confirmed in vitro findings, showing tumor growth suppression with NSC59984 + RT compared to vehicle or single agent treatment. On day 20, mean tumor size in the NSC59984 + RT group was 103.4mm, significantly smaller than vehicle (384.5mm, p=0.004), RT (506.2mm, p=0.03), and NSC59984 (394.8mm, p=0.02) groups. Tumor growth inhibition synergy analysis using the Bliss model demonstrated synergy of NSC59984 + RT on days 20-23 with a mean synergy score of 101.6. Conclusions: NSC59984 has cytotoxic and radiosensitizing effects in in vitro and in vivo models of TP53mut EC. These effects are independent of specific TP53 mutations, suggesting it bypasses inactive p53 to drive p21, PUMA, and NOXA signaling. The synergistic effect is within biologically relevant doses and supports the need for further preclinical investigation in a disease with significant unmet need and poor outcomes. Citation Format: Camilla Yu, Aaron Petty, Theresa D'Silva, Roberto Vargas. NSC59984 is a radiosensitizer and cytotoxic agent in in vitro and in vivo models of TP53mut endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4322.