Immune-checkpoint inhibitors (ICI) have emerged as valuable treatment options for patients with bladder cancer, particularly those who are ineligible for other therapies or have experienced disease progression on treatment. Response rates are only 20-30%, highlighting the need for biomarkers to predict treatment response and strategies to overcome resistance. Epigenetic modulators including the DNA methyltransferase (DNMT) inhibitor 5-azacitidine (AZA) and histone deacetylase inhibitors (HDACi) may represent co-targeting therapies in combination with ICI to enhance antitumor immunity. AZA has been demonstrated to increase the expression of a panel of immunomodulatory genes termed the “AZA Immune gene panel (AIM)” in breast, colorectal, and ovarian cancer cell lines. We hypothesized that bladder cancer with low AIM expression can be therapeutically converted by epigenetic modulation to “AIM-high” for future combination with ICI.
Methods
Five human bladder cancer cell lines were selected: SCaBER, SW780, T24, RT4 and one developed at our institution (CoCaB1). The Cancer Cell Line Encyclopedia (CCLE) was used to screen the four included lines for intrinsic AIM expression, which were predicted to be AIM-high (n=1) and AIM-low (n=3). A cell viability assay was performed to screen for AZA and HDACi toxicity. Cells were treated with drug or vehicle for 72 hours and harvested at day 6, 14, and 21. RNA was extracted for RNA sequencing (RNA-seq) analyses. For analysis of AIM gene expression in clinical specimens, RNA-seq data was obtained from primary (n=6) and metastatic (n=83) bladder cancer specimens for 20 patients participating in the University of Washington Bladder Cancer Rapid Autopsy Program.
Results
RNA-seq analyses of 5 cell lines demonstrated increased expression (>log 2-fold change, p<0.05) of a subset of AIM genes involved in inflammation, interferon, cytokine/chemokine signaling, and cancer testis antigens (CTA, a family of immunogenic proteins) with AZA treatment. Co-treatment with HDACi showed additional upregulation of AIM genes. Gene set enrichment analyses uncovered enrichment in T-cell pathway activation. In metastatic bladder cancer tissues, interferon, cytokine/chemokine, inflammation, and CTA AIM gene set categories were upregulated in a subset of patients. AIM gene enrichment displayed a patient-dependent pattern and was consistent across metastatic sites within a patient.
Conclusions
Epigenetic priming therapy increases the expression of immunomodulatory and T-cell related genes in bladder cancer cell lines with low baseline AIM expression. Additional correlative studies in the rapid autopsy series will further determine clinical- and treatment-related factors contributing to intrinsic immune-related gene expression. Building on this paradigm, future in vivo and clinical studies could lead to novel combination therapies for patients with bladder cancer.
- Elizabeth Leone Koehne, University of Washington, Seattle, WA
- Sonali Arora, Fred Hutchinson Cancer Research Center, Seattle, WA
- Kevin Wang, Fred Hutchinson Cancer Center, Seattle, WA
- Yan Wang, University of Washington, Seattle, WA
- Martine Roudier, Fred Hutchinson Cancer Center, Seattle, WA
- Khursheed Ali, University of Washington, Seattle, WA
- Andrew Caleb Hsieh, Fred Hutchinson Cancer Research Center, Seattle, WA
- James Dai, Fred Hutchinson Cancer Research Center, Seattle, WA
- Michael C. Haffner, Fred Hutchinson Cancer Center, Seattle, WA
- Hung-Ming Lam, University of Washington, Seattle, WA
- Jonathan L. Wright, University of Washington, Seattle, WA; Fred Hutchinson Cancer Research Center, Seattle, WA; Fred Hutchinson Cancer Center, Seattle, WA