Abstract 5332: ST316, a clinical peptide antagonist of beta-catenin, induces anti-tumor immune responses by multiple mechanisms of action
Authors: Scuoppo Claudio , Mendelson Karen , Ramirez Ricardo , Koester Mark , Diehl Julia , Gallagher Erin , Leong Siok , Gonzales Jerel , Mattes Zach , Ghamsari Lila , Merutka Gene , Kappel Barry J. , Vainstein-Haras Abi , Rotolo Jim A. 
Published: March 2024

Abstract
Wnt/β-catenin plays several important roles in cancer, including driving oncogenesis via cell proliferation, survival and metabolic reprogramming of cancer cells, and enhancing immune-desertification of the tumor immune microenvironment (TIME). ST316 is a clinical stage cell-penetrating peptide antagonist of the interaction of β-catenin with its co-activator BCL9, selectively impairing a subset of Wnt target genes and demonstrating potent anti-tumor activity against Wnt-driven tumors in vivo. ST316 is currently being evaluated in a Phase 1-2 study (NCT05848739) enrolling patients with selected advanced solid tumors likely to harbor abnormalities of the Wnt/β-catenin signaling pathway. Here we explore the potential of ST316 to activate the TIME and provide data to support combination treatment with anti-PD-1 and anti-TIGIT therapies. Macrophages derived from human peripheral blood mononuclear cells (hPBMCs) were activated by LPS and IFNγ (M1) or IL-4 (M2) in the presence of ST316 or control and were immunophenotyped by expression of CD80 and CD163 to assess M1 and M2 macrophages, respectively. ST316 induced marked repolarization of hPBMC-derived M2 macrophages to the M1 identity in vitro, as shown by increased CD80 and decreased CD163 staining. Increasing concentration of ST316 in co-cultures of M2 cells with CD8+ T cells induced up to a three-fold increase in IFN-γ expressing cells relative to controls. Additionally, ST316 exposure increased CD155/PVR expression on 4T1 Triple Negative Breast Cancer (TNBC) cells, but not MV411 TNBC that are not Wnt-dependent. When combined with anti-TIGIT treatment, ST316 led to increased T cell activation in an ex vivo assay where 4T1 cells were co-incubated with syngeneic splenic CD8+ cells and frequency of IFN-γ producing cells was assessed. In in vivo experiments, Balb/C mice bearing syngeneic 4T1 TNBC orthotopic tumors were treated with vehicle, ST316, anti-PD-1 or combination. Sub-pharmacologic ST316 enhanced anti-PD-1 suppression of 4T1 tumor growth and induced a substantial decrease of the M2 marker CD209 (DC-SIGN) in the Tumor Associated Macrophage (TAMs). These data support two novel mechanisms of action for the β-catenin antagonist peptide ST316. First, ST316 exposure results in macrophage repolarization towards an immune-active M1 program, both in vitro and in vivo, and increases T-cell activation in co-culture assays. Second, ST316 induced CD155/PVR upregulation and T-cell activation in the presence of anti-TIGIT antibody. Collectively these results suggest a novel immune-modulatory role for ST316 in the TIME and provide rational for combination therapy with checkpoint inhibitors.
Citation Format: Claudio Scuoppo, Karen Mendelson, Ricardo Ramirez, Mark Koester, Julia Diehl, Erin Gallagher, Siok Leong, Jerel Gonzales, Zach Mattes, Lila Ghamsari, Gene Merutka, Barry J. Kappel, Abi Vainstein-Haras, Jim A. Rotolo. ST316, a clinical peptide antagonist of beta-catenin, induces anti-tumor immune responses by multiple mechanisms of action [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 5332.