64 GPR65 is a Novel Therapeutic Target Associated with Immune Exhaustion in Clear Cell Renal Cell Carcinoma

Background Novel therapeutic targets in clear cell renal cell carcinoma (ccRCC) remain a pressing need, particularly for patients who experience progression to immune checkpoint inhibitor (ICI)-based therapies. ccRCC is highly glycolytic, resulting in an acidic tumor microenvironment (TME). GPR65 is a proton-sensing receptor enriched on tumor infiltrating immune cells that has ultimately been connected to immune dysfunction in the TME. Novel GPR65 inhibitors are in clinical development for patients with RCC and other solid tumors. However, the expression patterns, associated TME phenotypes, and clinical implications of GPR65 in RCC tumors remain uncharacterized, as does the biological impact of GPR65 inhibition in the RCC TME. Methods GPR65 expression was interrogated in The Cancer Genome Atlas (TCGA) KIRC and clinical trial cohorts comprising patients with metastatic ccRCC (IMmotion151, Javelin101, and HCRN-GU16-260). Associations of GPR65 with clinicopathological features, co-expressed genes, immune infiltrates, and IMmotion151 NMF TME phenotypes (Motzer et al. Cancer Cell. 2020) were assessed with Pearson correlation coefficients, Mann-Whitney U tests, and Chi-square tests as appropriate. Tumors within each cohort were stratified into GPR65-High and -Low groups by median expression value. Survival analysis employed Kaplan-Meier estimates with comparisons performed using Cox proportional hazard models. Single cell RNA sequencing (scRNAseq) data was analyzed from three cohorts of patients with RCC (Zhang et al. PNAS. 2021; Bi et al. Immunity. 2025; Hugaboom et al. Cancer Discovery. 2025), including in pre- and post-ICI treatment samples. Patient derived xenograft (PDX) models were generated from human RCC tumors and treated with a first-in-class GPR65 inhibitor. Gene expression readouts of tumors were assessed by NanoString nCounter gene expression array. Results Within TCGA KIRC, GPR65 expression was significantly increased in RCC tumor tissues compared to matched normal kidney epithelium (p < 0.0001) and was significantly increased in Stage IV RCC tumors compared to Stage I (p < 0.05). Gene set enrichment analysis revealed significant enrichment of adaptive immune processes in GPR65-High tumors; GPR65 expression also significantly correlated with expression of immune checkpoint molecules and predicted infiltration of numerous pro-inflammatory immune populations by immune deconvolution. scRNAseq of RCC tumors and adjacent normal renal epithelium analysis confirmed GPR65 enrichment in tumor-infiltrating immune populations, with highest expression in proliferating lymphocytes. In clinical trial cohorts, GPR65 expression was significantly increased in PD-L1+ tumors compared to PD-L1- tumors (p = 5.85x10-5 and 7.35x10-7 in IMmotion151 and Javelin101, respectively). The IMmotion151 T-Effector/Proliferative NMF TME cluster was also disproportionately enriched for GPR65-High cases (p < 0.00001). GPR65-High status did not predict improved outcomes with either atezolizumab/bevacizumab or avelumab/axitinib but did portend significantly prolonged progression free survival with nivolumab compared to GPR65-Low in HCRN-GU16-260 (HR = 0.49, p = 0.009). However, scRNAseq analysis of post-progression tumor specimens from four patients (n = 21,521 cells) demonstrated an appreciable increase in GPR65 expression amongst myeloid and natural killer cells in tumors that did not achieve response to nivolumab (n = 2) compared to those that did (n = 2) in HCRN-GU16-260. GPR65 inhibition in RCC PDX models led to heightened expression of pro-inflammatory T cell genes including IL2RG, chemokines (CCL4, CCL5, CXCL10), and antigen processing machinery (PSMB7-10), concurrent with suppression of pro-tumorigenic genes including TGFB and IL10 and achieved tumor control equivalent to or surpassing anti-PD-1 therapy in syngeneic mouse models. Conclusions GPR65 represents a clinically actionable and biologically relevant target in the RCC TME. These data suggest GPR65 is a rational target for patients who experience progression on ICI-based therapy. The ongoing phase I/II RAISIC-1 trial (NCT06634849) is investigating a novel GPR65 inhibitor, PTT-4256, as monotherapy or in combination with anti-PD-1 ICI in patients with solid tumors, including dose expansion cohorts for patients with RCC.