Abstract 2988: Patient-survival linked <i>TTLL12</i> isoform switching event sensitizes EAC cells to standard-of-care chemotherapy agents via chaperone-mediated autophagy

Abstract Esophageal adenocarcinoma (EAC) represents a growing health problem characterized by rising incidence and poor prognosis, despite current multimodal therapy. Although EAC has a high mutational burden, it lacks highly prevalent oncogenic drivers that can be therapeutically targeted, as TP53 mutation accounts for over 80% of mutational events in EAC. Recently, our group characterized isoform switching events, defined as a change of dominant gene isoforms between conditions, associated with EAC progression and identified 75 isoform switching events between Barrett’s low-grade dysplasia (BE.LGD) and Barrett’s high-grade dysplasia (BE.HGD) patient samples. Herein, we further investigated whether isoform-switching events may offer prognostic or therapeutic value in EAC. Isoform switching analysis was performed using RNA-seq data derived from 57 patient samples comparing BE.LGD to BE.HGD combined with EAC (BE.HGD+EAC) alone or in combination with TP53 mutations. Patients were further stratified into tertiles based on isoform expression levels, followed by survival analysis. Forty-two isoforms were significantly (P&amp;lt;0.05) linked with all-cause mortality, comparing BE.LGD with BE.HGD+EAC, whereas 39 isoforms were identified when TP53 mutation status was included. Among those identified to be significantly associated with all-cause mortality comparing BE.LGD with BE.HGD+EAC, isoforms of tubulin tyrosine ligase like 12 (TTLL12), a gene linked with aberrant cell proliferation, oxidative stress and cancer progression in other targets, were among the top isoforms switched. Isoform-specific genetic knockdown was performed via siRNA to target the protein-coding isoform of TTLL12 in two EAC cell lines (OE19 and OE33). Knockdown of the TTLL12 protein-coding isoform significantly inhibited cell viability and migration in both cell lines. In addition, synergistic effects on cell viability were observed for TTLL12 knockdown of EAC cells treated with standard-of-care chemotherapeutic agents (Paclitaxel and Carboplatin), suggesting a role for specific isoform-switching events in therapeutic sensitization. Protein-interaction prediction using STRING and immunoblot analysis further supported that TTLL12 is linked with the regulation of chaperone-mediated autophagy (CMA), as evidenced by the induction of LAMP2A and HSC70 in EAC cells. Because CMA is linked with the regulated degradation of CHK1 and mutant TP53 in other cancers, we are currently assessing the role of CMA in regulation of CHK1 and degradation of TP53 in EAC. Future directions include using FDA-approved agents to target CMA in EAC. In conclusion, isoform switching may provide fresh insight for the identification of prognostic markers and inform new potential therapeutic targets for EAC treatment or prevention. Citation Format: Yun Zhang, Rachel Israel, Bryce Vandenburg, Shari Barnett, Jean-Jack Riethoven, Jennifer Clarke, Michelle P. Lee, David A. Ntsiful, Kiran H. Lagisetty, Jules Lin, Rishindra M. Reddy, Chigozrim Ekeke, Andrew C. Chang, David Odell, Laura A. Kresty. Patient-survival linked TTLL12 isoform switching event sensitizes EAC cells to standard-of-care chemotherapy agents via chaperone-mediated autophagy [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 2988.