Abstract B081: A novel GPR68 negative allosteric modulator shows efficacy in syngeneic mouse models of colorectal cancer and modulates the immunosuppressive phenotype of colorectal fibroblasts

Abstract GPR68 negative allosteric modulators (NAMs) are predicted to be of therapeutic relevance in colorectal cancer through modulation of tumor microenvironment signalling between cancer associated fibroblasts (CAFs), infiltrating immune cells and tumor cells. GPR68 is an acid-sensing GPCR and contributes to tumor growth and immune suppression under acidic conditions. Its expression is upregulated in acidic tumor microenvironments in both CAFs and tumor cells, and in immune cells exposed to low pH. Additionally, elevated GPR68 expression is observed in various human cancers (e.g., colorectal, breast, pancreatic, prostate) and is associated with poor prognosis. Genetic deletion or knockdown of Gpr68 in mouse models reduces tumour progression and inflammatory signalling. In this work we have identified a novel, potent and highly selective NAM of GPR68. 1321N1 cells expressing recombinant human or mouse GPR68 receptor were stimulated with the historical EC80 pH6.8 shown to activate the cAMP response, or EC80 pH 7.4 shown to activate the IP response in the cells, in the presence of test compound. Compound 1 was shown to inhibit signalling via both Gs and Gq/11. The primary assay was also run in a NAM activity mode to establish alpha cooperativity factor and the compound KB (nM). Using the NAM assay mode, cells were stimulated with a dose response of pH6.2 - 8.0 in the presence of 3 concentrations of compound from 31nM to 3.1μM for activity against both mouse and human GPR68 variants. We also demonstrate that Compound 1 produces similar data in colorectal fibroblasts to data that has been reported for GPR68 knockdown experiments in pancreatic cancer CAFs; where inhibition of GPR68 signalling reduces pro-inflammatory cytokine release and production of fibrotic markers. Moreover, Compound 1 is orally bioavailable in mice and inhibits tumor growth in a mouse MC38 syngeneic model at concentrations that were shown to have no effect on MC38 proliferation in vitro. In summary, we have demonstrated that a GPR68 NAM replicates the effects of GPR68 knockdown on pro-inflammatory and immunosuppressive signalling in CAFs, and in a mouse in vivo tumor model. Additionally, we have shown that GPR68 is a viable target of therapeutic relevance in metastatic colorectal cancer through modulation of CAF and immune cell signalling in the tumor microenvironment. Citation Format: Matilda Bingham, Christoph P. Sager, Luboš Remen, Werner Neidhart, H Nikki March, Laura Gallagher, Daragh Campbell, Michael Hennig, Clare Doris, Robert Cheng. A novel GPR68 negative allosteric modulator shows efficacy in syngeneic mouse models of colorectal cancer and modulates the immunosuppressive phenotype of colorectal fibroblasts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B081.