Abstract 2361: An <i>ex vivo</i> organoid study to identify biomarkers of sensitivity to ST316, a clinical-stage β-catenin antagonist peptide

Abstract Background. The WNT/β-catenin pathway, a key oncogenic driver in various cancers, was long deemed undruggable. Historically, attempts at targeting its components resulted in toxicity due to β-catenin's role in homeostatic functions such as intestinal stem cell renewal and bone osteogenesis. ST316 is a β-catenin antagonist peptide that disrupts its interaction with co-activator BCL9, targeting oncogenic signaling and immune exclusion while preserving homeostatic functions. ST316 reduces β-catenin transcriptional activity and tumor growth in vitro and in vivo, yet GLP-toxicology studies and Phase 1 clinical trial data confirm no WNT-inhibition-associated toxicity. ST316 has now progressed to a Phase 2 trial in patients with colorectal cancer (CRC), an indication with a high prevalence of WNT-related mutations. Here, we evaluated the anti-tumor activity of ST316 in 60 tumor-derived organoids with diverse genetic backgrounds to identify pathways associated with treatment responses. RNA sequencing and qRT-PCR identified transcriptional signatures of cell lines treated with ST316 or vehicle. Results. The biomarker study of 60 organoids identified genetic backgrounds linked to ST316 responses, with TP53 WT models showing the highest sensitivity. Further analysis revealed higher ST316 sensitivity in models with BRAF, NRAS, and NF1 mutations, suggesting co-dependency between MAPK and WNT/β-catenin pathways. Given the importance of MAPK signaling in cancer, we evaluated dual inhibition of β-catenin and BRAF by combining ST316 with the BRAF inhibitor encorafenib in BRAF-mutant organoids. This combination showed additive/synergistic anti-tumor effects, highlighting new therapeutic opportunities for BRAFV600E-mutant CRC, a subgroup with limited treatment options. Conclusions. ST316, a novel β-catenin antagonist, preserves WNT/β-catenin homeostasis while targeting oncogenic and immune-suppressive β-catenin/BCL9 signaling. Analysis on 60 patient-derived tumor organoids revealed that tumors with TP53 WT and mutations in the MAPK pathway significantly correlate with sensitivity to ST316. Additionally, combining ST316 with encorafenib showed additive/synergistic effects in BRAFV600E-mutant CRC organoids. β-catenin has been linked to resistance to BRAF inhibitors, and combination therapies targeting both β-catenin and BRAF are proposed to overcome resistance. Given the poor prognosis and limited treatment options for BRAFV600E-mutant CRC, ST316 offers a promising new therapeutic approach for this difficult-to-treat cancer. Our findings support rational combinations of ST316 with clinically relevant MAPK inhibitors. Citation Format: Franco Abbate, Lila Ghamsari, Claudio Scuoppo, Zachary Mattes, Rick Ramirez, Mark Koester, Siok Leong, Barry Kappel, Abi Vainstein-Haras, Jim Rotolo. An ex vivo organoid study to identify biomarkers of sensitivity to ST316, a clinical-stage β-catenin antagonist peptide [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 2361.