PROJECT SUMMARY One hallmark of cancer is a tumor’s ability to evade immune detection and elimination. Immune checkpoint inhibitors (ICIs) are an attractive therapeutic strategy for many cancers. After many clinical trials targeting various solid cancer types, however, only 15-40% of patients benefited from ICI therapy. Shockingly, 4-29% of non- responsive patients developed hyperprogressive disease (HPD), a condition characterized by accelerated tumor growth and rapid clinical deterioration. Very little is known about what causes HPD or how to prevent it, but recent work suggests that tumor hyperproliferation is an immune-related adverse event (irAE). As part of the VAI-SU2C Epigenetic Dream Team, we discovered that activating the aryl hydrocarbon receptor (AHR) pathway and its downstream target gene CYP1A1 are prognostic indicators for the hyperproliferative-irAE (HP-irAE), and that HP-irAE bladder tumors show a metabolic switch towards fatty acid and xenobiotic metabolism. AHR is known to have an oncogenic role in tumorigenesis and immune dysfunction, which leads to the working hypothesis that AHR activation before ICI therapy preconditions the cancer cells or tumor microenvironment for rapid proliferation, where HP-irAE is then triggered by intracellular PD-L1 signaling in immune cells and/or cancer cells after ICI therapy. To test this hypothesis, we will first use the NanoString GeoMX platform to spatially profile RNA and protein expression in CYP1A1-positive bladder tumor microenvironments to resolve the spatial heterogeneity of immune and cancer cell dependencies (Aim 1). In Aim 2, we will determine how ICI treatment and AHR activation transcriptionally and metabolically reprograms bladder cancer and immune cells. These experiments will separate the cancer-intrinsic, immune-related, and metabolic consequence of AHR and PD-L1 signaling, and provide powerful models for future genetic- or drug-screening studies. Finally (Aim 3), we will use a bladder cancer mouse models to define how AHR and ICI treatment impacts the evolution of the tumor microenvironment in vivo. These data will provide valuable insight into how AHR activation might influence ICI efficacy and adverse events. The K99 phase of this project will provide vital training in spatial transcript/proteomics, metabolomics, and multimodal single-cell technologies, and how to process and integrate high-dimensional data to understand (bladder) cancer evolution and irAEs. This effort will be guided by an outstanding, multidisciplinary advisory committee, led by Dr. Peter Jones at Van Andel Institute, that amalgamate basic and translational science across cancer biology, immunology, epigenetic, and metabolism fields. These training and new skill sets will be leveraged to ultimately transition into an independent research career (R00 phase) and validate in vitro findings with in vivo mouse models. Together, this project will provide the first mechanistic insights into how the AHR pathway exacerbates bladder cancer response to ICI treatment, which will provide new opportunities for future drug discovery and precision medicine efforts to minimize HP-irAE in ICI-treated patients.

PROJECT NARRATIVE A subgroup of bladder cancer patients who are treated with immune checkpoint inhibitors (ICI) develop and succumb to hyperproliferative immune-related adverse events (HP-irAEs), for reasons that are unknown. I discovered a genetic and metabolic signature for HP-irAE that seems to pre-condition the tumor environment for HP-irAE before ICI treatment. By understanding how the genetic and metabolic pathway is differentially activated in tumor and immune cells within the tumor microenvironment, we will be able to better stratify bladder cancer patients before ICI treatment, and identify new opportunities for future drug discovery and precision medicine efforts to minimize HP-irAE in ICI-treated patients.