Abstract 5965: First-in-class chemical induced proximity system achieves dose-dependent control of TP53 activation in gastric cancer

Abstract

Abstract Background: Gastric cancer (GC) remains a common and deadly disease, and inactivating TP53 mutations occur in up to 68% of GC tumors. p53 is a master regulator of multiple cellular pathways, such as the cell cycle, apoptosis, and senescence. Previously, we demonstrated the ability to activate endogenous target gene expression using a novel chemical induced proximity system comprised of deactivated Cas9 (dCas9) and “chemical epigenetic modifiers” or “CEMs” (PMID: 31712774). In this study, we applied our dCas9-CEM system for the first time in oncology to activate TP53 gene expression and selectively kill targeted cells in a preclinical model of GC. Methods: AGS cells were derived from a gastric adenocarcinoma, and harbor wild-type TP53. They were transduced with our dCas9 system and either sgRNAs targeting TP53 or a non-targeting (NT) sgRNA. After 48 h CEM87 incubation (1-25 nM), expression of TP53 and downstream targets (CDKN1A and BBC3) were evaluated by RT-qPCR. After 48 h CEM87 incubation (0.1-25 nM), colony-forming potential was assessed by crystal violet staining after 10 days. After 24 h incubation (5-25 nM), cells were fixed and stained with propidium iodide (PI), and DNA content was assessed by flow cytometry to determine cell cycle fractions. After 48 h incubation (50-200 nM), cells were fixed and stained with Annexin V and PI, and apoptosis was assessed by flow cytometry. Last, cells were treated with nine ascending concentrations (10 pM-100 μM) for 48 h, and cell viability was measured using CellTiter-Glo™. IC50 values were calculated using four-parameter non-linear regression. Results: AGS-TP53 cells show significantly increased expression of TP53 and downstream target genes CDKN1A and BBC3 versus AGS-NT cells after 10 nM CEM87 (105%, 99%, and 43% increase, P=0.004, 0.002, and 0.03, respectively; n=3). Colony formation was significantly reduced in AGS-TP53 versus AGS-NT cells after 10 nM CEM87 (60.2% reduction, P=0.003; n=3). Treatment with 10 nM CEM87 also led to a G2/M stall after significantly reducing cells in both G1 (47.4% vs 38.9%, P<0.0001; n=3) and S-phase (25.1% vs 20.3%%, P<0.0001; n=3) in AGS-TP53 versus AGS-NT cells. Treatment with 200 nM CEM87 significantly increased early apoptosis in AGS-TP53 versus AGS-NT cells (28.7% vs 6.6%, respectively, P<0.0001; n=3). Last, CEM87 was 50 times more potent in AGS-TP53 cells than AGS-NT cells (IC50: 3 vs 146 nM; n=8). Conclusions: These data highlight how our innovative system upregulates TP53 expression and impacts downstream phenotypic effects of TP53 activation in a preclinical model of GC. Citation Format: Ryan M. Kemper, Travis J. Nelson, Nathaniel A. Hathaway, Daniel J. Crona. First-in-class chemical induced proximity system achieves dose-dependent control of TP53 activation in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5965.