Abstract 1395: HDAC6 and DNMT inhibition affect immunogenicity of ovarian cancer cells: A rationale for combining epigenetic and immune therapy in ovarian cancer
Authors: Srivastava Aneil P. , Moufarrij Sara M. , Hadley Melissa , Chisholm Sarah , Lopez-Acevedo Micael , Villagra Alejandro , Chiappinelli Katherine B. 
Published: August 2018

Abstract
Background: Therapies that activate the immune system to fight cancer have shown robust responses in solid tumors. However, most patients, including those with ovarian cancer, do not respond to these therapies alone. Drugs that inhibit epigenetic modifiers increase immune signaling from cancer cells. Epigenetic modifiers DNA methyltransferase inhibitors (DNMTi) and selective histone deacetylase inhibitors (HDACi),in particular selective HDAC6i, modulate immune-related pathways involved in anti-tumor immune responses. HDAC6i downregulate immunosuppressive ligands PD-L1 and PD-L2 via dephosphorylating pSTAT3 and upregulate tumor associated antigens (TAA) and antigen presentation machinery. Similarly, DNMTi activate anti-viral signaling via expression of Endogenous Retroviruses (ERVs) to trigger the type I interferon response, upregulate tumor antigen processing and presentation, and stimulate pro-inflammatory cytokines. The aim of our study is to test if the combination of epigenetic modulators Nexturastat A (Next A), a selective HDAC6i, and 5-azacytidine (AZA), a DNMTi, can be safely used to increase an immune response in ovarian cancer. We hypothesize that these drugs will enhance tumor immunity alone and when combined with immune checkpoint blockades targeting PD-1. Results: HDAC enzymes are differentially expressed in A2780, HEY, OVCAR3, SKOV3, and TYKNu human ovarian cancer cell lines. HDAC1 and HDAC2 proteins were similarly expressed in HEY and SKOV3 whereas HDAC6 was expressed at lower levels in HEY and TYKNu but at higher levels in SKOV3, OVCAR3, and A2780. As previously reported we believe this may be due to the presence of the chromatin modifier ARID1A mutation in the SKOV3 and A2780 cell lines. The upregulation of HDAC6 also correlated with a higher IC50 for NextA treatment in those particular cell lines. Further immunoblots showed that PD-L1, a marker of poor prognosis in ovarian cancer, decreased after treatment with NextA and even more in combination with AZA. Additionally, DNMT1, the known target of AZA, was decreased after treatment with AZA and NextA, both independently and in combination, a finding that has not been previously reported. Conclusions: As shown previously, HDAC6 enzyme levels are higher in cell lines with ARID1A mutations. DNMT1 was decreased after treatment with AZA, as expected, but surprisingly also after treatment with NextA. PD-L1 decreased after treatment with NextA and even more so when combined with AZA. We thus believe that combining these two epigenetic modifiers will lead to an additive effect on immune signaling through stimulation of antiviral signaling (DNMTi), which can upregulate the immunosuppressive ligand PD-L1, which is then reduced by HDAC6 inhibition. We are currently testing the combination of both epigenetic modifiers with anti-PD-1 in an immunocompetent mouse model of ovarian cancer.
Citation Format: Aneil P. Srivastava, Sara M. Moufarrij, Melissa Hadley, Sarah Chisholm, Micael Lopez-Acevedo, Alejandro Villagra, Katherine B. Chiappinelli. HDAC6 and DNMT inhibition affect immunogenicity of ovarian cancer cells: A rationale for combining epigenetic and immune therapy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1395.