Targeting GABA signaling in cancer associated fibroblasts to reduce immunosuppression in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a devasting disease with few therapeuticoptions. This is due, in large part, to the expansion of the non-tumor cellular compartmentknown as the stroma. Cancer-associated fibroblasts (CAFs) are one of the major celltypes that populate the stroma of PDAC tumors and are extremely immunosuppressivebecause they produce large amounts of cytokines, growth factors and metabolites. Thus,understanding how CAFs impart severe immunosuppression in the tumormicroenvironment is critical to make PDAC amenable to immunotherapies. Using a novel3D culturing method, our data support the notion that PDAC CAFs produce the glutamatemetabolite and inhibitory neurotransmitter gamma aminobutyric acid (GABA), de novo ,and we detected GABA (~27µM) in tumor interstitial fluid (TIF) isolated from patient tissue(10 patients). Surprisingly, CAFs lacked expression of glutamate decarboxylase, thecanonical enzyme responsible for converting glutamate into GABA, which led us toexplore alternative, non-canonical pathways for GABA synthesis. We found that CAFsderived from patient PDAC tumors expressed the enzymes of the non-canonicalsynthesis pathway (ODC1, DAO, ALDH1A1 and ALDH9A1), and to a greater degreewhen compared to normal fibroblasts. In parallel, we demonstrated that, by exploringparacrine signaling, GABA treatment of CAFs resulted in changes in expression of p-p70,p-JNK, p-MTOR and p-AKT proteins, key signaling hubs involved in overcomingmetabolic stress in PDAC. In addition, GABA treatment increased the production of pro-tumor cytokines, such as TGFbeta, IL8, and IL6, by CAFs. In preclinical orthotopic murinetumor models of PDAC, inhibition of GABA synthesis through ALDH1A1, and GABAsignaling through GABAB receptor inhibitors, reduced tumor burden, increased influx ofanti-tumor T and NK cells, and blocked production of pro-tumor cytokines in tumorexplants. Translationally, we have acquired patient matched plasma, TIF, and tissue from10 patients, and will compare their metabolic, immune, and spatial transcriptomic profileswith well annotated clinical parameters, in order to develop improved diagnostic tests.Overall, we have uncovered a novel signaling circuit driven by GABA that plays a role incontrolling the extremely immunosuppressive PDAC microenvironment.