RXRA regulates transcription as part of a heterodimer with 14 other nuclear receptors, including the peroxisome proliferator-activated receptors (PPARs). Analysis from TCGA raised the possibility that hyperactive PPAR signaling, either due to PPAR gamma gene amplification or RXRA hot-spot mutation (S427F/Y) drives 20–25% of human bladder cancers. Here, we characterize mutant RXRA, demonstrating it induces enhancer/promoter activity in the context of RXRA/PPAR heterodimers in human bladder cancer cells. Structure-function studies indicate that the RXRA substitution allosterically regulates the PPAR AF2 domain via an aromatic interaction with the terminal tyrosine found in PPARs. In mouse urothelial organoids, PPAR agonism is sufficient to drive growth-factor-independent growth in the context of concurrent tumor suppressor loss. Similarly, mutant RXRA stimulates growth-factor-independent growth of Trp53/Kdm6a null bladder organoids. Mutant RXRA-driven growth of urothelium is reversible by PPAR inhibition, supporting PPARs as targetable drivers of bladder cancer.

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Disruption of KDM6A, a histone lysine demethylase, is one of the most common somatic alternations in bladder cancer. Insights into how KDM6A mutations impact the epigenetic landscape to promote carcinogenesis could help reveal potential new treatment approaches. Here, we demonstrated that KDM6A loss triggers an epigenetic switch that disrupts urothelial differentiation and induces a neoplastic state characterized by increased cell proliferation. In bladder cancer cells with intact KDM6A, FOXA1 interacted with KDM6A to activate genes instructing urothelial differentiation. KDM6A-deficient cells displayed simultaneous loss of FOXA1 target binding and genome-wide redistribution of the bZIP transcription factor ATF3, which in turn repressed FOXA1-target genes and activated cell cycle progression genes. Importantly, ATF3 depletion reversed the cell proliferation phenotype induced by KDM6A deficiency. These data establish that KDM6A loss engenders an epigenetic state that drives tumor growth in an ATF3-dependent manner, creating a potentially targetable molecular vulnerability.

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To explore mechanisms of response to combined PD-1/CTLA-4 immune checkpoint blockade (ICB) treatment in individual cell types, we generated scRNA-seq using a mouse model of invasive urothelial carcinoma with three conditions: untreated tumor, treated tumor, and tumor treated after CD4+ T cell depletion. After classifying tumor cells based on detection of somatic variants and assigning non-tumor cell types using SingleR, we performed differential expression analysis, overrepresentation analysis, and gene set enrichment analysis (GSEA) within each cell type. GSEA revealed that endothelial cells were enriched for upregulated IFN-g response genes when comparing treated cells to both untreated cells and cells treated after CD4+ T cell depletion. Functional analysis showed that knocking out IFNgR1 in endothelial cells inhibited treatment response. Together, these results indicated that IFN-g signaling in endothelial cells is a key mediator of ICB induced anti-tumor activity.

Bladder cancer is the tenth most common cancer worldwide, accounting for over 500,000 new cancer cases and 200,000 cancer-related deaths per year. Depending on the degree of muscle invasiveness and disease spread at the time of diagnosis, 5-year survival rates can range from as high as 96% to as low as 6%. In recent years, use of checkpoint inhibitor treatments for bladder cancer has become more common. Currently, there are five PD-1/PD-L1 checkpoint inhibitors approved by the FDA for treatment of bladder cancer and these treatments have been used across the spectrum of disease, from non-muscle invasive to metastatic disease. However, many patients still do not receive benefit from checkpoint inhibitor treatments. For example, clinical trials have shown response rates of only 20-30% in advanced disease. To improve treatment responses and identify patients who will benefit most from checkpoint inhibitor treatment, a deeper understanding of mechanisms of treatment response is needed. In an effort to explore mechanisms of response to combined PD-1/CTLA-4 checkpoint inhibitor treatment, we generated single cell RNA expression sequencing from a carcinogen-induced mouse model of muscle invasive urothelial carcinoma that included three conditions: untreated tumor, tumor treated with combination treatment, and tumor treated with combination treatment after Cd4+ T-cell depletion. After classifying tumor cells based on per cell detection of somatic variants identified using exome sequencing and assigning non-tumor cell types using SingleR, we performed differential expression, overrepresentation, and gene set enrichment analysis (GSEA) within each cell type, comparing each pairwise set of conditions. This analysis revealed that MSigDB’s hallmark interferon gamma response gene set was overrepresented across multiple cell types. Furthermore, GSEA indicated that with combined checkpoint blockade treatment, endothelial cells showed significant enrichment of upregulated interferon gamma response genes. Functional analysis demonstrated that knocking out Ifngr1 in endothelial cells inhibited the treatment response seen in wild type treated mice. Together, these results indicate that interferon gamma response in endothelial cells is a key mediator of combined PD-1/CTLA-4 checkpoint inhibitor treatment response. Citation Format: Sharon L. Freshour, Bryan Fisk, Timothy Hung-Po Chen, Haolin Shen, Matthew Mosior, Catrina Fronick, Jennifer Bolzenius, Obi L. Griffith, Vivek Arora, Malachi Griffith. Comprehensive single-cell transcriptomic analysis of a mouse model of bladder cancer identifies endothelial cells as a principal target of interferon gamma during response to combined PD-1, CTLA-4 immune checkpoint inhibitor treatment [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 6.

<div>Abstract<p>Disruption of KDM6A, a histone lysine demethylase, is one of the most common somatic alternations in bladder cancer. Insights into how KDM6A mutations affect the epigenetic landscape to promote carcinogenesis could help reveal potential new treatment approaches. Here, we demonstrated that KDM6A loss triggers an epigenetic switch that disrupts urothelial differentiation and induces a neoplastic state characterized by increased cell proliferation. In bladder cancer cells with intact KDM6A, FOXA1 interacted with KDM6A to activate genes instructing urothelial differentiation. KDM6A-deficient cells displayed simultaneous loss of FOXA1 target binding and genome-wide redistribution of the bZIP transcription factor ATF3, which in turn repressed FOXA1-target genes and activated cell-cycle progression genes. Importantly, ATF3 depletion reversed the cell proliferation phenotype induced by KDM6A deficiency. These data establish that KDM6A loss engenders an epigenetic state that drives tumor growth in an ATF3-dependent manner, creating a potentially targetable molecular vulnerability.</p>Significance:<p>A gain-of-function epigenetic switch that disrupts differentiation is triggered by inactivating KDM6A mutations in bladder cancer and can serve as a potential target for novel therapies.</p></div>