Dissecting innate immune mechanisms of resistance to checkpoint blockade therapy in bladder cancer

PROJECT SUMMARY Bladder cancer is the fifth most common cancer in the United States, accounting for around 47 deaths per day. Promisingly, five PD-1/PD-L1 immune checkpoint blockade (ICB) therapies were approved for bladder cancer in 2016. Although these ICB treatments have achieved durable clinical responses in a subset of patients (15-25%), the majority of patients have still not benefitted from this therapy. This clinical urgency to extend the benefits of ICB to more patients has led to a need to investigate tumor intrinsic mechanisms underlying resistance. Tumor- promoting inflammation, a hallmark of cancer pathogenesis, is known to contribute to cancer growth in multiple ways including restraining antitumor immunity. We discovered a gene signature from pre-treatment tumor associating with myeloid cells that is enriched in inflammation and innate immune genes and predictive of poor ICB outcomes and survival in two ICB clinical trials. I plan to follow up on this work and dissect the innate immune landscape of bladder cancer and investigate mechanisms of myeloid-cell mediated resistance to ICB therapy. Aim 1 seeks to define the landscape of untreated bladder tumors and provide insight into the immune cell subsets underlying ICB resistance. I will construct a transcriptomic and molecular atlas of bladder cancer at a single-cell resolution, a resource that does not currently exist. I will build atlases of patients’ tumor, blood, and urine using single-cell RNA sequencing, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITEseq), spatial transcriptomics, and O-link proteomics and analyze them using Seurat and other R-based tools. I plan to resolve myeloid cells expressing this resistant gene signature and define their cellular interactions. In Aim 2, I will delve into the transcriptional pathways in myeloid cells that are contributing to ICB resistance. We have identified NLRP3 inflammasome activation and IL-1β signaling in tumor monocyte-macrophages (mono- MΦs) as candidate pathways promoting tumor inflammation and progression. I will model these mono-MΦs by differentiating peripheral blood monocytes into MΦ using GM-CSF and M-CSF under hypoxic conditions with IL- 1β and NLRP3 inflammasome activators., I will test effects on adaptive immunity by co-culturing these mono- MΦs with activated autologous CD8+ T cells. I will also use this model to test drug candidates known to modulate IL-1β and NLRP3 inflammasome activity as potential combinatorial treatments with ICB in bladder cancer. This proposal combines direct ex vivo single cell genomics with in vitro functional experiments for a thorough interrogation of the innate immune contribution to ICB resistance in bladder cancer. Combined, these aims will elucidate innate immune pathway driven resistance to PD-1/PD-L1 ICB therapy in bladder cancer, which can be used to identify critical predictive clinical biomarkers and inform new combinatorial treatment strategies.