Immune checkpoint inhibitors (ICIs) have emerged as a valuable therapeutic option for patients with bladder cancer with the potential to achieve durable responses. However, only 20%-30% of patients will respond to ICIs,^1,2 highlighting the need for biomarkers to guide treatment selection and drug development to combat mechanisms of immune resistance. Epigenetic modulators including the DNA methyltransferase inhibitor 5-azacitidine (AZA) may represent synergistic partners with ICIs to enhance antitumor immunity. AZA has been demonstrated to increase expression of a panel of immunomodulatory pathway genes termed the AZA immune gene panel (AIM) in breast, ovarian, and colorectal cancer cell lines.³ This panel includes gene sets involved in inflammation, interferon, influenza, cytokine/chemokine signaling, antigen presentation, and cancer testis antigens (CTA, a family of immunogenic proteins⁴). We hypothesized that bladder cancer with low AIM expression could be therapeutically converted with epigenetic modulation to “AIM high” for future combination with ICIs.

The Cancer Cell Line Encyclopedia was used to characterize intrinsic AIM gene expression in human bladder cancer cell lines, and 4 lines predicted to be either AIM high (n=1) or AIM low (n=3) were selected for this study. To screen for AZA toxicity, cells were cultured and treated with AZA for 72 hours (Figure 1). A cell viability assay was performed on day 6, and dose curves were generated to identify the dose of AZA with <50% toxicity. For RNA sequencing, cells were seeded in 100 mm² cell culture dishes and treated with AZA or dimethyl sulfoxide for 72 hours. Cells were harvested at days 6, 14, and 21. RNA was extracted from the pellets for whole transcriptomic analysis. For clinical correlation, RNA sequencing data were obtained from The Cancer Genome Atlas (TCGA) and primary (n=6) and metastatic (n=83) bladder cancer specimens for 20 patients through the University of Washington Bladder Cancer Rapid Autopsy Program from 2015-2020 and used to evaluate AIM gene expression.

Figure 1. Experiment design. AZA indicates 5-azacitidine. Figure created with BioRender.com.

Figure 2. The author (right) with Dr Jonathan Wright.

RNA sequencing analysis of these 4 human bladder cancer cell lines following AZA treatment demonstrated increased expression (>2-fold change, P < .05) of a subset of AIM genes involved in inflammation, interferon, cytokine/chemokine signaling, and CTA, whereas the AIM subset categories antigen presentation and influenza remained largely unaltered by AZA. Two intrinsically AIM low cell lines displayed the greatest enrichment in AIM genes after AZA treatment. Unbiased analysis of whole transcriptomic data uncovered additional enrichment in T-cell activation and granulocyte migration pathways, which were not represented in the original AIM gene set. Furthermore, consensus molecular classification subtype⁵ was determined for each sample pre- and post-AZA treatment. Within the 2 basal/squamous and 2 luminal-papillary cell lines, subtype was not changed by AZA treatment and did not correlate with AIM gene expression. Analysis of clinical specimens from both TCGA and the rapid autopsy series demonstrated AIM gene set categories (interferon, cytokine/chemokine, inflammation, and CTA) were upregulated in a subset of patients. AIM gene enrichment displayed a patient-dependent pattern and was consistent across various anatomical metastatic sites within a patient.

We observed that gene demethylation by AZA increased expression of immunomodulatory and T cell—related genes in bladder cancer cell lines with low baseline expression. Further correlation with TCGA and the rapid autopsy series will determine clinical- and treatment-related factors contributing to intrinsic immune-related gene expression. These findings endorse the continued study of sequential therapy to immunologically prime bladder cancer through epigenetic modulation in order to enhance responsiveness to ICIs. We plan to conduct future in vivo and clinical studies to support development of novel therapeutic agents and combination therapies for patients with bladder cancer.

I am immensely grateful to the Urology Care Foundation for selecting me to receive the Outstanding Graduate Scholar Award and for their support of my project, “AIM High: DNA Methyltransferase Inhibition and Immune Stimulation in Bladder Cancer.” My mentors at the University of Washington, Dr Jonathan Wright (Figure 2) and Dr Ming Lam, have been instrumental in creating and carrying out this project, and I am very thankful for their guidance. The Research Scholar Award has provided me an incredible opportunity to develop my translational research skills and foster multidisciplinary collaborations. This experience directly led to my selection to participate in the Society of Urologic Oncology Young Urologic Oncologists Clinical Trials Development Workshop and the Bladder Cancer Advocacy Network Think Tank, which will allow me to continue exploring bench-to-bedside concepts for this project and beyond as I develop my career as a surgeon-scientist in urologic oncology. I sincerely thank the Urology Care Foundation for this formative experience and for supporting me in the pursuit of more effective treatment options for patients with bladder cancer.