Project Summary/Abstract Advanced gastric adenocarcinoma (GAC) presents significant treatment challenges due to frequent peritoneal metastases, leading to poor survival rates despite existing therapies. Understanding the mechanisms driving advanced GAC progression is crucial, with identified subsets like MSIH, Her2 positive, PDL-1 high, EBV, Claudin18.2 high, and FGFR2b+ tumors leading to patient stratification for customized treatments. We defined two advanced GAC subtypes: Type I, with CDH1/E-cadherin inactivation linked to SOX4 and EZH2 activation, and Type 11, marked by elevated RHOA activity. Unique immune profiles were elucidated for each subtype. We established murine organoid lines, including wild type (WT), KrasG120; Trp53Δ/Δ (KP), and Cdh1 knockout (KO); KrasG120 ; Trp53Δ/Δ (EKP), with EKP resembling Type I advanced GAG with EZH2 activation, inhibited by EZH2 blockade. SOX4 activation was confirmed in genetically engineered mouse models (Tff1-CreERT2; Cdh1MI; KrasG120; Trp5:t1'" [EKPTI), showing notably higher expression post-tamoxifen treatment compared to WT and Tff1-GreERT2; Cdh1MI; Trp5:t1'" (EPT). High SOX4 levels were evident in stage IV advanced GAG patients, correlating with poor prognosis. Functional studies, including SOX4 KO in GA0518 cells (GDH1-inactive human advanced GAG cell line), demonstrated suppressed tumor growth and improved survival rates, while SOX4 overexpression enhanced GDH1-inactive GAC aggressiveness. Further investigations will focus on SOX4 and EZH2-mediated transcriptional reprogramming in GDH1-inactivated advanced GAG. Additionally, we expanded our models to include six murine organoid lines (Trp53Δ/Δ [P], RHOAY420 [R], Cdh1 KO [E], Gdh1 KO; Trp53"" [EP], RHOAY42c; Trp53Δ/Δ [RP], RHOAY42c; KrasG120; Trp53Δ/Δ [RKP]) and three 2D cell lines (KP, EKP, and RKP) to explore subtype-specific responses to immune checkpoint blockade alongside EZH2 blockade for Type I and RHOA blockade for Type II advanced GAG. Herein, we hypothesized GDH1 loss promotes GAG progression via intrinsic (SOX4 and EZH2 mediated transcriptional reprogramming) and extrinsic (immune evasion) dysregulation. Two aims will be pursued: Aim 1. To determine the mechanisms of GDH1-inactivated advanced GAG progression; Aim 2. To determine the molecular subtype-based immune checkpoint blockade response. This study aims to uncover mechanisms of GDH1 loss-associated GAG progression, test molecular signature-based targeted therapy, and establish a new paradigm for GAG patient stratification.

PROJECT NARRATIVE Advanced gastric adenocarcinoma (GAC) presents significant treatment challenges due to peritoneal metastases, necessitating effective patient stratification for targeted therapies. Our study identified two advanced GAC subtypes: Type I and Type II, each with unique molecular characteristics, including CDH1 inactivation in Type I. Understanding the mechanisms, particularly CDH1 loss, is crucial for developing novel therapeutics based on subtypes of GACs.