Abstract 454: Validation of GPR35 as a novel cancer target in digestive tract cancers and discovery of potent, selective GPR35 inverse agonists
Authors: Westcott James , Luckhurst Christopher A. , McKenzie Grahame , Memon Danish , Lin Li-Chiung , Knight Sinead , Blaikley Elizabeth J. , Pearce Martin , Warren Hannah R. , Parker Eleanor , Milligan Graeme , Hughes Stuart W. , McCarthy Tom 
Published: April 2023

Abstract
Cancers of the digestive tract are a major area of unmet clinical need with incidence rates of some cancers increasing and early onset forms becoming more prevalent. Environmental factors, such as poor diet and microbiota, as well as inflammatory conditions, are strongly associated with occurrence of digestive system cancers. Obesogenic diet combined with microbiotic metabolism can lead to high levels of secondary bile acids in the human gut. Secondary bile acids deoxy- and litho-cholic acid have been shown to have oncogenic potential and their levels are increased in the intestines of cancer patients relative to healthy controls. GPR35 is an orphan class A G protein-coupled receptor primarily expressed in digestive system epithelial tissues and cells of myeloid lineage. GPR35 polymorphisms are strongly associated with inflammatory bowel diseases. In particular, rs37947171, a missense variant that codes for a threonine to methionine substitution at position 108 (T108M) and has been shown to have hypermorphic function, is strongly associated with Crohn’s disease and ulcerative colitis. GPR35 is also overexpressed in adenocarcinomas of the digestive tract, including those of esophageal, hepatic/bile ductal, pancreatic, gastric and colorectal origin, and high expression has been shown to confer poor prognosis in various digestive system cancers. Consistent with a proposed role in digestive tract cancers we now show that GPR35 is activated by lithocholic acid. Furthermore, we demonstrate through CRISPR-gene editing of cancer cells and RNAseq analysis that GPR35 can regulate transcriptional and cytoskeletal modules associated with hallmarks of cancer, including chemokine and growth factor expression and F-actin formation. GPR35 activates a transcriptional program that is enriched for genes containing pro-oncogenic serum response factor response element in their upstream promoters. High throughput screening and medicinal chemistry optimization has led to the discovery of potent antagonists of GPR35 signalling. Pharmacological characterisation of lead series has shown that they are able to prevent Gα and β-arrestin protein binding, while also inhibiting phospho-ERK, calcium flux, receptor internalisation and serum response factor-induced gene transcription. Furthermore, the series are competitive with predicted orthosteric agonist, can block activation of receptor signalling by lithocholic acid and act as inverse agonists of constitutive receptor tone. Profiling these inhibitors in cancer models is currently ongoing. This work provides the basis for the pre-clinical development of GPR35 inverse agonists as anti-cancer drugs.
Citation Format: James Westcott, Christopher A. Luckhurst, Grahame McKenzie, Danish Memon, Li-Chiung Lin, Sinead Knight, Elizabeth J. Blaikley, Martin Pearce, Hannah R. Warren, Eleanor Parker, Graeme Milligan, Stuart W. Hughes, Tom McCarthy. Validation of GPR35 as a novel cancer target in digestive tract cancers and discovery of potent, selective GPR35 inverse agonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 454.