The impact of DNA methyltransferase inhibitor treatment on tumor cell recognition by CD8+ T cells and their effector function
Paluch, Benjamin Edward
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The azanucleosides, azacitidine (AZ) and decitabine (DA), are FDA approved and have become the standard of care for the treatment of myelodysplastic syndrome (MDS) and low blast count acute myeloid leukemia (AML). About 40% of patients respond to therapy, associated with reduced transfusion dependence, improved quality of life, decreased AML transformation, and prolonged survival. Azanucleosides were originally developed as cytotoxic agents for the treatment of cancer. During DNA replication, they are incorporated in place of deoxycytidine inhibiting DNA methyltransferases ((DNMTs), referred to as DNMT inhibitors (DNMTis)) resulting in both DNA damage and depletion of DNMTs. Although they are also referred as hypomethylating agents (HMAs), reactivation of silent tumor suppressor genes does not predict clinical response to therapy, nor DNA damage. Therefore, it is important to determine the mechanism of action of these compounds in order to identify patients who will respond to therapy. Interestingly, DA treatment has also been shown to induce the expression of immunogenic antigens, such as genes within the cancer testis antigen (CTA) family like NY-ESO-1 (New York Esophageal Squamous Cell Carcinoma 1). Moreover, it has been shown to upregulate the expression of human leukocyte antigen (HLA) major histocompatibility (MHC) class I and intercellular adhesion molecule 1 (ICAM-1) enhancing tumor cell recognition by NY-ESO-1-specific CD8+ T cells in a variety of tumor models. Furthermore, the frequency of CTA-specific CD8+ T cells correlates with the clinical response to combination DNMTi and histone deacetylase inhibitor treatment; however the mechanism underlying the expansion of CTA-specific CD8+ T cells has not been investigated. One of the major challenges treating patients with solid tumors using DA is postulated to be its short half-life. SGI-110 is a dinucleotide, consisting of DA coupled to deoxyguanosine, rationally designed to prolong DA exposure. Importantly, the impact of SGI-110 treatment relative to AZ and DA has not been investigated in leukemia or ovarian cancer with respect to tumor cell recognition. We hypothesized DNMTi treatment enhances the immunogenicity of tumor cells and modulates the effector function of CD8+ T cells. Furthermore, we hypothesized DNMTi treatment using DA increases the number and improves the function of CD8+ T cells. To test the hypothesis, we proposed the following aims: Aim 1: evaluate the impact of DNMTi treatment on tumor cell recognition focusing on antigen presentation and adhesion, which contribute to an improved antitumor response. Tumor cell recognition was evaluated by measuring CD8+ T cell effector function following DNMTi-pretreatment of leukemic (KG1a) and epithelial ovarian cancer (EOC, OVCAR3) cells. Antigen presentation and adhesion were evaluated by measuring NY-ESO-1, HLA-ABC, and ICAM-1 expression in leukemic (HL60, U937, KG1a) and EOC (A2780, OVCAR3) cell lines. Antitumor response was evaluated by measuring tumor growth and survival using an OVCAR3 xenograft model. Aim 2: investigate the mechanism underlying the impact of DNMTi treatment on CD8+ T cell number and function. CD8+ T cell number was investigated by measuring CD8+ T cell lymph node homing and proliferation in healthy mice. Fold change of the number of CD8+ and regulatory T cells following DA treatment of MLL-AF9+ leukemic mice was also investigated. CD8+ T cell function was investigated by measuring tumor cell killing and IFN-γ secretion using the ovalbumin-specific, class I-restricted TCR transgenic (OT-I) model system. Our results indicate SGI-110 treatment, like DA, enhances NY-ESO-1 expression via promoter hypomethylation as well as HLA-ABC expression in both leukemic and EOC models, contributing to enhanced tumor cell recognition by HLA-restricted NY-ESO-1-specific CD8+ T cells in vitro. Furthermore, SGI-110 treatment promoted an antitumor response, significantly prolonging survival of mice bearing an OVCAR3 xenograft. DA treatment enhanced naive CD8+ T cell lymph node homing and proliferation occurring in lymph nodes. Importantly, DA treatment did not impair antigen-specific tumor cell killing or IFN-γ secretion, in vitro. Thus, DA can be used in combination with vaccine strategies harnessing CD8+ T cells for the treatment of human malignancy.