Abstract 4854: Alleviation of low pH induced immunosuppression through antagonism of the pH sensor GPR65 in highly glycolytic NSCLC tumors
Authors: Prajna Behera, Samantha Buongervino, Kayla Guidry, Kyle Krois, Yanah Hossain, Victor Du, Tamara Ouspenskaia, Hongyu (Annie) Liang, Shu-Yun Zhang, Laura Johnston, Abby Lin, Tolu Aweda, Ramona Plant, Paul Bojczuk, Marica Speranza, Chris Hopson, Charles McHugh, Stacey Kendrew, Bradley Spencer-Dene, Susanne Moore, Deon Hildebrand, Jonathan Lea, Kerena Norris, Antonia Lewis, Jacob Brett, Paru Rao, Carol Harris, Joseph Pero, Laura Ator, Jing Fang, David washburn, Lisa Sloan, Catherine Alder, Richard Lonsdale, Nick Barton, Sue Griffin, Paul Redford, James Smothers
Journal: Cancer Research (2025)

Abstract Out of more than 5683 clinical trials testing anti-PD1/PDL1 mAbs, on average only 20% of patients achieved an objective response, leaving an unmet need for patients who do not respond to immune checkpoint inhibitors (ICIs)(Zhao et al, 2020, Upadhyaya et al, 2022). Independent reports in NSCLC and melanoma suggest that non responders to ICI exhibit elevated levels of tumor “aerobic” glycolysis (Renner et al, 2019, Silva et al, 2022), a phenotype associated with solid and metastatic tumor cells (Hanahan, 2022). Aerobic glycolysis results in accumulation of lactate and acidification of the tumor microenvironment (TME), resulting in immune evasion through inhibition of pro-inflammatory/ anti-tumorigenic responses in surrounding immune cells (Li et al, 2022). GPR65 is a proton (pH) sensing GPCR, expressed on immune cells, signals via cAMP to activate anti-inflammatory pathways in multiple immune cell types (Robert and Mackay, 2018; Wang et al, 2023). In the context of solid tumors, GPR65 activation promotes the increase in anti-inflammatory TAMs, in addition to suppressing anti-tumor responses by NK and cytotoxic-T cell (Raker et al, 2016). GSK analysis of Tempus RW data conducted with TEMPUS LENS software indicated high glycolytic gene signature in NSCLC cohorts is associated with worse prognosis to both chemo and ICI therapies, providing a window of opportunity to target pH sensor GPR65. Ex vivo experiments demonstrated that low pH dampens the proinflammatory phenotype of monocyte-derived macrophages (MDMs), while knockout of GPR65 under these acidic conditions restores secretion of the proinflammatory cytokines CXCL9/10/11 in M1-like polarized MDMs. Compound A, an exemplar compound from our lead series, is a potent GPR65 antagonist with low nanomolar in vitro functional activity (pIC50=8.2) and excellent lipophilic ligand efficiency (6.9). It also features strong correlation between its functional and binding (nanoBRET) activities along with selectivity at GPR65 over GPR4 and GPR68. In vitro translational and cell-based assays demonstrated dose-dependent attenuation of proinflammatory cytokine release by primary CD8+T cells, M1 macrophages at pH 6.8 and in a NSCLC patient-derived tumor fragmentation assay. In vivo, compound A displays attractive pharmacokinetic (PK) properties in rodent with robust oral bioavailability. A preliminary estimate of projected human dose (PHD) is ∼245 mg qd based on liver blood flow scaling of its rat PK properties and a targeted 24 h free drug coverage over functional in vitro IC50. Additional In vivo PD and/or efficacy data in translational models would further refine this calculation. In summary, we hypothesize that restricting immune cells from sensing acidification in the TME via antagonizing GPR65 will prevent the activation of anti-inflammatory programs in immune cells and promote proinflammatory anti-tumor effector mechanisms. Citation Format: Prajna Behera, Samantha Buongervino, Kayla Guidry, Kyle Krois, Yanah Hossain, Victor Du, Tamara Ouspenskaia, Hongyu (Annie) Liang, Shu-Yun Zhang, Laura Johnston, Abby Lin, Tolu Aweda, Ramona Plant, Paul Bojczuk, Marica Speranza, Chris Hopson, Charles McHugh, Stacey Kendrew, Bradley Spencer-Dene, Susanne Moore, Deon Hildebrand, Jonathan Lea, Kerena Norris, Antonia Lewis, Jacob Brett, Paru Rao, Carol Harris, Joseph Pero, Laura Ator, Jing Fang, David washburn, Lisa Sloan, Catherine Alder, Richard Lonsdale, Nick Barton, Sue Griffin, Paul Redford, James Smothers. Alleviation of low pH induced immunosuppression through antagonism of the pH sensor GPR65 in highly glycolytic NSCLC tumors [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 4854.