463. ISOFORM SWITCHING EVENTS IN ESOPHAGEAL ADENOCARCINOMA MAY INFORM NEW THERAPEUTIC TARGETS THROUGH IDENTIFYING MOLECULAR MECHANISMS CONTRIBUTING TO POOR PATIENT OUTCOMES
Authors: Zhang Yun , Vandenburg Bryce , Barnett Shari , Sunilkumer Shruthi , Lee Michelle , Riethoven Jean-Jack , Clarke Jennifer , Lagisetty Kiran , Lin Jules , Reddy Rishindra , Chang Andrew , Kresty Associate Laura A 
Published: September 2024

Abstract

Background
Esophageal adenocarcinoma (EAC) represents a growing health problem characterized by rising incidence and poor prognosis due to late-stage diagnosis coupled with therapeutic resistance. Moreover, EAC is a cancer with a high mutational burden but lacks highly prevalent oncogenic drivers that can be successfully targeted. Herein, we investigated whether newly identified isoform switching events, defined as differential usage of gene transcripts between conditions, may reveal new therapeutic targets and inform the molecular mechanisms contributing to EAC resistance.

Methods
We conducted RNA-sequencing followed by isoform switching analysis using IsoformSwitchAnalyzeR on Barrett’s low-grade dysplasia (BE.LGD) compared to Barrett’s with high-grade dysplasia (BE.HGD)+EAC tissues alone or in combination with TP53 mutation. Patients were stratified into tertiles based on isoform fraction levels, followed by overall and cancer-free survival analysis using the Cox proportional-hazards model. To assess whether mortality-linked isoforms influence cancer cell growth, viability, migration, and response to chemotherapy (Paclitaxel and Carboplatin). Isoform-specific siRNA knockdown experiments targeting HM13 were performed in OE19 and OE33 EAC cell lines, with STRING protein prediction interaction analysis and western blots conducted to assess pathway and protein alterations.

Results
Twenty-one isoform switched genes were significantly linked with all-cause mortality in BE.LGD versus BE.HGD+EAC and similarly 20 were significantly associated with cancer-specific patient mortality with 14 shared between the two groups. With inclusion of TP53 mutation status, 13 isoform switched genes were significantly linked with cancer-specific mortality. HM13, DANT2, CFDP1, KRAS, SIMM6 were among the most significantly altered. Knockdown of the pro-cancer isoform of HM13 significantly inhibited EAC cell migration and induced EAC cell death and knockdown of HM13 combined with chemotherapeutic treatment exhibited synergistic effects in EAC cell lines. Protein interaction analysis supported that HM13 is linked to ER-stress associated degradation, cellular response to stress, and ER unfolded protein response which were validated using a panel of relevant proteins (i.e., PERK, IRE-1a, ATF4, BIP, P-JNK).

Conclusions
Our research identified isoform switching events significantly linked to all-cause mortality as well as cancer specific mortality among EAC patients with and without TP53 mutations. Knockdown of the pro-cancer isoform of HM13 isoform significantly inhibited EAC cell migration and induced EAC cell death with synergistic effects observed with chemotherapeutic agent treatment supporting a role for specific isoform switches in therapeutic sensitization and in this case via the ERAD and ER unfolded protein response pathways. Modulation of specific proteins with established roles in ERAD and ER unfolded responses following isoform specific HM13 knockdown lends further support to an important role for this isoform, especially in TP53 mutant EAC. In conclusion, isoform switches may inform molecular mechanisms contributing to poor patient outcomes and reveal new potential therapeutic targets for EAC.