Abstract PR12: Immunomodulatory effects of 5-Azacyditine in ovarian cancer cell lines
Authors: Chiappinelli Katherine B. , Yen Ray-Whay Chiu , Li Huili , Topper Michael , Zahnow Cynthia A. , Baylin Stephen B. 
Published: August 2014

Abstract
Epithelial ovarian cancer is a deadly disease due to late detection and lack of targeted therapies. Novel therapies for ovarian cancer are clearly needed and epigenetic agents have emerged as promising new therapies for ovarian cancer and other solid tumors. Clinical success observed for patients with lung cancer suggests that the actions of the DNA methyltransferase inhibitor 5-azacytidine (AZA) may stimulate the immune response and target immune cells to clear tumors. Ovarian cancer is a good candidate for immune therapy, as infiltrating lymphocytes predict longer time to recurrence. Therapies that upregulate the immune system have been shown to be effective in mouse and human ovarian cancers.
To determine how epigenetic therapy affects ovarian cancer cells, we utilized genome-wide methylation and expression profiling on 17 ovarian cancer cell lines treated with low-dose AZA. We discovered upregulation of immunomodulatory pathways including viral defense, type I interferon signaling, antigen processing and presentation, and immune evasion via Gene Set Enrichment Analysis. In addition, we observed strong upregulation of cancer testis antigens and molecules that attract and activate natural killer cells. Endogenous retroviruses were also increased; increased transcription of these elements caused by reversal of DNA methylation may trigger the viral defense/ interferon response.
This was a specific, not an off-target, effect of AZA, as a colon cancer cell line (DKO) genetically haploinsufficient for DNMT1 and lacking DNMT3b showed a similar upregulation of immune genes. Treatment with the chemotherapeutic agent carboplatin did not upregulate the same immune response genes. The cell lines that had the highest upregulation of immune genes after AZA treatment were often the best responders to AZA when grown as xenografts in NOD/SCID mice. We hypothesize that AZA activates the immune response in cancer cells, resulting in tumor cell killing.
Validation of immune target genes showed that AZA treatment activates the interferon response, including transcription of interferon beta. Media transferred from AZA-treated cells to naïve cells was sufficient to cause an interferon response in the target cells, as evidenced by increased levels of interferon-stimulated genes. FACS staining confirmed the upregulation of antigen-presenting MHC Class I molecules on the cell surface of AZA-treated ovarian cancer cells. Adding the histone deacetylase inhibitor MS275 (entinostat) to AZA treatment increased AZA-induced expression of interferon-stimulated genes IRF7 and IFI27. This combination treatment also caused an increase in the activating chromatin mark H3Ac at the IRF7 promoter.
AZA increased transcript levels and cell surface expression of the PD-L1 molecule, ligand for PD-1 on lymphocytes and responsible for evasion of the host immune system by tumors, in ovarian cancer cell lines. We predict that AZA plus anti-PD-L1 (or PD-1) treatment, which has shown success in non-small cell lung cancer, might be effective for ovarian cancers. Future work will involve combination treatment of AZA and anti-PD-1 in immune competent ovarian cancer mouse models.
The immune pathways upregulated by AZA were used to query hundreds of primary ovarian cancer samples from the Cancer Genome Atlas project (TCGA). Ovarian tumors classified clearly into “high” and “low” interferon gene expression subsets. The “high” interferon group was associated with higher levels of antigen presentation as well as an expression signature associated with better prognosis. The “low” interferon group indicates a group of solid tumors that could be targeted by AZA to upregulate levels of immunomodulatory pathways, and thus apoptosis or targeting by host immune cells. These preliminary results point to an “immune priming” role for the DNA methyltransferase inhibitor 5-azacytidine and could lead to clinical trials with combined AZA and immune therapies in epithelial ovarian cancer.
This abstract is also presented as Poster B79.
Citation Format: Katherine B. Chiappinelli, Ray-Whay Chiu Yen, Huili Li, Michael Topper, Cynthia A. Zahnow, Stephen B. Baylin. Immunomodulatory effects of 5-Azacyditine in ovarian cancer cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR12.