Abstract 4876: DZNep enhances apoptosis and augments cancer-testis antigen expression mediated by DNA demethylating agents and histone deacetylase inhibitors in lung cancer cells

Abstract

Abstract Cancer/testis (C-T) genes encode a heterogeneous group of immunogenic proteins expressed almost exclusively in normal testis, which are aberrantly expressed in various human malignancies. On the basis of their tissue specificity and immunogenicity, C-T antigens are considered to be promising targets for cancer immunotherapy. Previous studies from our laboratory have demonstrated that the DNA demethylating agent, 5-aza-2′-deoxycytidine (DAC) and the histone deacetylase inhibitor, depsipeptide (DP), synergistically induce CT-X genes such as NY-ESO-1 and MAGE-A3 expression in lung cancer cells, but not cultured normal human respiratory epithelia. In the present study, we have extended our analysis of epigenetic mechanisms mediating expression of NY-ESO-1 and MAGE-A3 in a panel of cultured lung cancer lines and normal human bronchial epithelial (NHBE) cells. Pyrosequencing experiments revealed that NY-ESO-1 and MAGE-A3 promoters were hypermethylated in H841 lung cancer cells and NHBE cells that do not express these C-T genes, but were hypomethylated in H1299 cells exhibiting high-level NY-ESO-1 and MAGE-A3 expression. Repression of NY-ESO-1 and MAGE-A3 in NHBE cells, as well as A549, Calu-6, and H841 lung cancer cells coincided with expression of the polycomb repression mark, H3K27Me3 within the NY-ESO-1 and MAGE-A3 promoters. In contrast, euchromatin marks such as H3K4Me2, H3K4Me3, and H3K9Ac were increased within the NY-ESO-1 and MAGE-A3 promoters in H1299 lung cancer cells expressing these C-T genes. Knock-down of EZH2 was insufficient to induce NY-ESO-1 and MAGE-A3 expression in H841 cells; however, knock-down of EZH2 markedly enhanced DAC-mediated activation of these C-T genes in lung cancer cells. DZNep, a novel inhibitor of EZH2 expression enhanced NY-ESO-1 and MAGE-A3 activation by DAC in cultured lung cancer cells, but not NHBE cells. Expression of NY-ESO-1 and MAGE-A3 in untreated H1299 cells or H841 cells following sequential DAC/DZNep exposure coincided with marked DNA demethylation, and reduced H3K27Me3 levels within the promoter of these C-T genes. In addition, DZNep mediated dose-dependent apoptosis, and enhanced cytotoxicity mediated by DAC and DP as well as trichostatin A (TSA) in lung cancer cells. Cytokine and chromium release experiments revealed that following sequential DAC/DZNep/TSA exposure, HLA-A0201+ lung cancer cells were recognized and lysed by allogeneic peripheral blood lymphocytes transduced with retroviral vectors encoding HLA-A0201-specific T cell receptors recognizing peptide epitopes of NY-ESO-1 and MAGE-A3, thus confirming the relevance of C-T gene activation in tumor targets. Collectively, these data suggest that combining DZNep with DNA demethylating agents and HDAC inhibitors may be a novel epigenetic strategy to augment antitumor immunity in lung cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4876.