Abstract 3278: Identification of epigenetic therapy induced tumor antigens to promote proliferation and infiltration of antigen specific T cells in non-small cell lung cancer
Authors: An Julia , Anagnostou Valsamo , Marrone Kristen A. , Velculescu Victor E. , Brahmer Julie R. , Baylin Stephen B. , Topper Michael J. 
Published: June 2022

Abstract
Among the hallmarks of non-small cell lung cancer (NSCLC) is cancer immunoediting. Immunoediting results when cancer cells evade immune recognition via positive selection for cells that do not present immunogenic antigens recognized by CD8+ T cells. An epigenetic therapy-based approach has been developed in our lab to enhance expression of tumor antigens using combinatorial DNA methyltransferase inhibition (DNMTi) paired with histone deacetylase inhibition (HDACi). However, durable responses from the application of epigenetic therapy alone or in combination with other therapies have remained elusive in NSCLC. This may be because enhanced proliferation and infiltration of tumor-associated antigen specific CD8+ T cells are necessary to recognize epigenetically induced antigens to halt disease progression. In the current study, we identified epigenetic therapy inducible self-antigens that can potentially be used to activate and guide the differentiation of CD8+ T cells specific to NSCLC tumor antigens. The identification of these candidates was derived from analyses of paired RNAseq data from NSCLC patients treated with combination epigenetic therapy. From these analyses, differentially expressed transcripts selected from total transcriptome data were filtered to remove non-protein coding genes and genes with detectable expression in normal tissues as indicated by GTEx. HLA binding affinity (HLA-A*0201) of selected targets was defined by NetMHC 4.0 artificial neural network-based prediction to identify strong binding peptide candidates. The combined results of these data filtering facilitated the identification of 34 epigenetic therapy induced genes and associated peptides (9mers) with predicted high binding affinity for HLA-A*0201. The predicted peptides of target genes were selected for single peptide pulsing in allogenic dendritic cells to generate antigen specific CD8+ T cells for TCR sequencing (TCRseq). TCRseq libraries derived from peptide stimulation experiments revealed distinct V and J gene frequencies and emergence of antigen specific clonotypes, which suggest clonal expansion. To validate whether epigenetic therapy could induce target genes expression in an independent experimental model, in vitro human NSCLC cell lines were treated with DNMTi and HDACi to mimic the clinical drug paradigm and transcriptional augmentation was assessed by qRT-PCR. In this orthogonal system, we validated eight target genes that were induced by epigenetic therapy in vitro. Altogether, these data suggest that a conserved subset of genes, whose derived proteins are predicted to be presented in class I MHC and act as T cell epitopes, is induced by combination epigenetic treatment. Our studies provide a basis for the development of novel and personalized strategies to improve outcomes and quality of life for more patients with NSCLC.
Citation Format: Julia An, Valsamo Anagnostou, Kristen A. Marrone, Victor E. Velculescu, Julie R. Brahmer, Stephen B. Baylin, Michael J. Topper. Identification of epigenetic therapy induced tumor antigens to promote proliferation and infiltration of antigen specific T cells in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3278.