Abstract A37: Epigenetic modification of ovarian cancer immunogenicity
Authors: Spiliopoulou Pavlina , Walton Josephine , Dowson Suzanne , Binks Alex , Maddocks Oliver , Adams Peter , McNeish Iain 
Published: August 2018

Background: Ovarian high-grade serous carcinoma (HGSC) remains a poor prognosis disease due to its late presentation and lack of “actionable” mutations to target pharmacologically. The host immune system, however, plays a pivotal role with the presence of cytotoxic T lymphocytes and chemokines that regulate T-cell trafficking both having positive effect on survival in preclinical and clinical studies. HGSC cells can achieve immune escape via epigenetic silencing of key immune-related genes, including via DNA methylation; when DNA methylation is pharmacologically reversed, genes involved in antigen presentation, receptor-dependent immune cell stimulation, and chemokine release are reactivated. One of these genes encodes the angiostatic chemokine Cxcl10, which is a T-lymphocyte attractant. In order to elucidate other potential epigenetic mechanisms directly or indirectly involved in Cxcl10 gene silencing, we will screen a library of novel epigenetic probes that target bromodomains, methyltransferases, demethylases, and others epigenetic pathways (Structural Genomic Consortium library).

Aims: We will use Trp53-/- ID8 ovarian cancer cells to perform a medium-throughput screening of 40 novel epigenetic probes, measuring cxcl10 production, with the demethylating drug decitabine as positive control.

Methods: Trp53-/- ID8 cells were treated with decitabine for up to 72 hours. Cell-cycle analysis was assessed via BrdU incorporation assay. Cytokine/chemokine transcription was assessed using RT2 profiler 84 gene chemo/cytokine PCR array (Qiagen), and verified using qRT-PCR.  Acid-hydrolysed DNA was subjected to mass spectrometry/liquid chromatography to quantify the mean ratio of methyl-cytosine/cytosine. Western immunoblotting was performed to test DNMT1 protein levels in protein lysates after decitabine treatment. Cxcl10 levels in supernatant were quantified by ELISA.

Results: Treatment of Trp53-/- ID8 cells with decitabine (DAC), at non-cytostatic/cytotoxic doses causes upregulation of multiple chemokines, specifically Cxcl10. 200nM DAC for 48 hours increased Cxcl10 transcription 22-fold (95% CI 20.6-21.1-fold, p=0.0001, values normalized to Gapdh control), and protein levels in supernatant three-fold (t test p&lt;0.0001). At this dose, DNTM1, the target enzyme of DAC, is downregulated on Western-blot analysis, and the methyl-cytosine/cytosine ratio decreases significantly (means compared with t test, p =0.002). Other statistically significant upregulated chemokine genes are Cxcl1 (p=0.002), Cxcl3 (p&lt;0.001), Cxcl4 (p&lt;0.001), Cxcl5 (p=0.03), and Ccl4 (p=0.01).

Conclusions: Abrogation of DNA methylation with DAC in Trp53-/- ID8 cells induces an increase in the chemokine cxcl10, at both transcriptional and protein level. This provides a robust control for a medium-throughput screening of other epigenetic modifiers that are still at an early drug development stage, such as probes against bromodomains, methyltransferases, and demethylases. Such screening can unmask novel epigenetic mechanisms involved in immune-related gene transcription and offer new ways to potentiate immunotherapy in ovarian cancer.

Citation Format: Pavlina Spiliopoulou, Josephine Walton, Suzanne Dowson, Alex Binks, Oliver Maddocks, Peter Adams, Iain McNeish. Epigenetic modification of ovarian cancer immunogenicity. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A37.