Abstract 5033: Azacitidine pretreatment sensitizes NSCLC cells to interferon-γ
Authors: He Kai , Zhang Xin , Danilova Ludmila , Pan Xiaoyu , Brahmer Julie , Pardoll Drew , Brock Malcolm , Baylin Stephen , Herman James , Wrangle John 
Published: August 2015

Abstract
Epigenetic treatment and immune checkpoint blockade are novel therapeutic approaches being investigated in NSCLC. Our previous studies showed that DNA methyltransferase inhibitor azacitidine (AZA) treatment in NSCLC cell lines led to up-regulation of genes related to the response to interferon and adaptive immune attack. We hypothesize that Interferon-γ(IFN-γ), an immune effecter made primarily by activated T cells and NK cells, has a direct tumor suppression effect in NSCLC cells, which can be enhanced by the DNA hypomethylation agent.
To test this hypothesis, NSCLC cells (H838 and/or H1299) were first treated with or without 500nM AZA for 3 days, and then received IFN-γ. The samples were subsequently tested for cell viability, apoptosis, cell cycle, and gene expression assays. This preclinical study is part of the collective efforts to investigate the efficacy of combined epigenetic and immune therapy in NSCLC.
IFN-γ causes moderate direct growth inhibition in H838 and H1299 cells, which was enhanced, using cell viability assays as a readout, by pretreatment with AZA. The AZA sensitizing effect lasted at least 3 weeks and was not detectable 8 weeks after stopping treatment. This timing is consistent with initial reprogramming of the cells and eventual waning of this effect. Flow cytometry studies showed that AZA enhances lung cell apoptosis induced by IFN-γ. However, no significant change in cell cycle was observed. To examine the mechanisms behind these effects on cell growth, we further determined how AZA altered IFN-γ induced gene expression in H1299 cells, first using Agilent expression arrays, and then validating specific changes in key IFN and cell death pathway genes with RT-PCR based methods. IFN-γ significantly up-regulated a group of known IFN targeted genes, including IFI27, IFITM1, ISG20, ICAM1, CCL5, CXCL10, MX1 and others, and this upregulation was further enhanced by AZA pretreatment. AZA also enhanced IFN-γ induced expression of CASP1, CASP4, TNFSF 10, and BCL2A. These AZA enhanced IFN-γ induced genes are important in tumor immune response and evasion, cell death, cytokine response and other critical cellular process.
Our studies demonstrated that AZA enhanced lung cancer cell response to the immune effecter IFN-γ. This supports the clinical exploration of epigenetic “priming” and its combination with subsequent immune therapy in NSCLC treatment. It may also provide mechanistically derived biomarkers that can be used to monitor and predict epigenetic and immune response in NSCLC.
Citation Format: Kai He, Xin Zhang, Ludmila Danilova, Xiaoyu Pan, Julie Brahmer, Drew Pardoll, Malcolm Brock, Stephen Baylin, James Herman, John Wrangle. Azacitidine pretreatment sensitizes NSCLC cells to interferon-γ. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5033. doi:10.1158/1538-7445.AM2015-5033