Immunotherapies such as adoptive cell therapy (ACT) are promising treatments for solid cancers. However, many patients develop resistance to immunotherapy. Here we utilise two preclinical models of ACT to study the evolution of acquired resistance in relapse tumours. In these models, ACT consistently induces initial robust tumour regression, however resistant tumour cells invariably emerge, mimicking the disease progression in relapsing patients. Analysis of tumour cells that escape ACT revealed a loss of immunogenic antigen expression but retention of the antigen-encoding genomic sequence in resistant cells suggesting epigenetic silencing following immune selection in both models. Accordingly, treatment of escaped tumour cells with DNA hypomethylating agents Azacytidine (AZA) and Decitabine (DEC) restored antigen expression in a proportion of the tumour population. Antigen-negative cells were recovered after treatment with DNA hypomethylating agents and clones screened against a panel of nine small molecule epigenetic modifying agents (EMAs) targeting a wide range chromatin regulatory complexes including heterochromatin proteins (e.g. GLP1/EHMT1 and G9a/EHMT2), euchromatic repressors (e.g. LSD1, EZH2, HDAC and Jarid1a/b/c), euchromatic activators (e.g. BRD4, CREBBP/ EP300 and UTX/JMJD3), elongation complexes (e.g. DOT1L). None of these individual EMAs restored antigen expression. Our data show that relapsing tumour cells descend from immune escapees with reduced antigen expression, however the resistant clones display epigenetic heterogeneity imposing a potent barrier to the efficacy of combined epigenetic and immuno-therapy.

We further examined if resistance to ACT could be overcome by re-wiring T cells with nine EMAs. Single agent EMAs reduced T cell proliferation in-vitro, but did not alter differentiation into memory phenotype (CD44, CD62L, Bcl-2, Sca1 expression). Subsequently, ACT with epigenetically modified T cells did not improve efficacy of treatment, and bromodomain inhibitor treated T cells reduced treatment efficacy. Together, our data suggests the complexity of combining epigenetic and immuno-therapy in cancer, and the need to understand these mechanisms in-depth.