Abstract 5627: PPARG-high circulating monocytes exhibit an immunosuppressive phenotype in urothelial cancer patients treated with anti-PD1

Abstract

Abstract Background: PPARG is expressed in a variety of immune cells such as monocytes, macrophages, and lymphocytes, where it plays a role in their maturation and function. PPARG has been implicated in immune mediated resistance but to date, a comprehensive analysis of PPARG expression in peripheral blood mononuclear cells (PBMCs) in urothelial cancer (UC) patients is lacking. In this study, we identified the immunophenotypes associated with PPARG expression in UC patients treated with anti-PD1 therapy. Methods: We performed single-cell RNA sequencing of PBMCs from 5 UC patients and 3 normal healthy volunteers (NHV). Real-world samples (Discovery Life Sciences) were collected from patients who received prior Pembrolizumab. Time post-treatment to collection ranged from 46 to 307 days. An average of 9,500 cells were recovered per donor reflecting an ~80% QC pass rate. Count matrices were generated using 10x Genomics Cell Ranger pipeline (v7.0.0; GRCh38 reference). Downstream data processing, analysis and visualization were done on Cellenics software. Results: We evaluated 75,725 cells and identified 9 distinct cell populations. The proportion of T-cells was significantly decreased in UC samples compared to NHVs (40% vs 60%, p=0.03). Highest PPARG expression was observed on Classical Monocytes (CMs) cell population. PPARG expression was higher on CMs in UC compared to NHV samples (1.74 vs 0.11 logcounts; padj<0.001). Unsupervised clustering of all monocytes revealed two distinct CM clusters. In cluster A, 95% of the cells originated from UC samples, while most cells in cluster B (81%) originated from NHV samples. PPARG was the top differentially expressed gene upregulated in cluster A (PPARG-high) vs cluster B (PPARG-low) (1.89 vs 0.23 logcounts; padj<0.001). PPARG-high CMs are characterized by a gene signature inclusive of GPRIN3, RGCC, MAFB and IRF2BP, which have been shown to be upregulated in M2 polarized macrophages [1]. Moreover, pathway analysis using GSEA highlighted that PPARG-high CMs are enriched for TREM1 signaling genes (padj<0.001), whose high expression has been associated with immunosuppressive phenotypes in myeloid cells [2] and shorter survival of patients across other cancers [3]. Conclusion: PPARG-high circulating CMs in UC patients exhibit a transcriptomic profile associated with immunosuppression and M2 macrophage polarization. FX-909, a first-in-class PPARG inverse agonist, is being evaluated in the clinic and the study includes an exploratory biomarker approach to assess the potential effect of PPARG inhibition on the CM immune-profile of advanced UC patients enrolled in the Ph1 study [4].