Immunomodulatory Effect of SGI-110, a Novel Hypomethylating Agent in Acute Myeloid Leukemia (AML)

Abstract

Introduction: We have developed a promising leukemia vaccine in which patient derived AML cells are fused with autologous dendritic cells (DCs) effectively presenting a broad array of antigens that capture the heterogeneity of the leukemia cell population. A major challenge of developing effective immunotherapy is overcoming the immunosuppressive milieu that characterizes patients with AML. AML cells evade immune targeting, in part, through the limited presentation of antigen by primitive leukemia progenitors. In contrast, induction of differentiation through upregulation of reactive oxygen species may enhance antigen presentation and the capacity to target AML cell by a tumor vaccine. Hypomethylating agents have been shown to be effective therapeutic agents for patients with AML and MDS, in part due to immunomodulatory effects. In the current study, we have examined the immunomodulatory properties of the novel DNA hypomethylating agent SGI-110, a dinucleotide of decitabine (DAC) and deoxyguanosine, which is resistant to in-vivo inactivation by cytidine deaminase. We have explored the in-vitro effect of treatment with SGI-110 on ROS, AML immunogenicity and efficacy of the AML/DC fusion vaccine.Methods and results: AML blasts were isolated by ficoll density centrifugation of bone marrow mononuclear cells (BMMCs) from patients with AML at presentation. AML blasts were cultured for 4 days in the presence or absence of 1 uM SGI-110, added twice daily on days 1 and 2 of culture. ROS as detected by levels of H2O2 expression demonstrated a 40% increase after treatment with SG1-110 (n=2). We subsequently evaluated whether the increase in ROS levels correlated with enhanced targeting by immune effector cells. SGI-110 or control treated AML cells were assessed for their susceptibility to T cell mediated targeting, using a standardized flourochrome CTL assay. Remarkably, a 72% increase in autologous cytotoxic T lymphocyte-mediated lysis as measured by Granzyme B activity, was demonstrated following SGI-110 treatment of AML blasts (n=3). The expression of co-stimulatory molecules CD80 and CD86, were unchanged following treatment with SGI-110 (n=3). In contrast, exposure to SGI-110 resulted in increased expression of the antigen processing TAP proteins by immunocytochemical analysis.We have previously demonstrated that DC/AML fusion cells potently stimulate the expansion of leukemia specific T cells. The effect of SGI-110 on T cell response to DC/AML fusion vaccine stimulation in vitro was assessed. Autologous Dendritic cells (DCs) were generated by culture of adherent peripheral mononuclear cells obtained from AML patients following remission in the presence of GM-CSF, IL-4 and TNF-α. DCs were fused with AML blasts by co-culture at a 1:1 ratio in the presence of polyethylene glycol (PEG). DC/AML fusions were cultured at a 1:10 ratio with autologous T cells for 5-6 days in the presence and absence of 1uM of SGI-110. Exposure of fusion stimulated autologous T cells to SGI-110 resulted in an increase in T cell expression of IFN-γ, with mean fold increase of 2.2 and 2.3 for CD4 and CD8 T cells respectively (n=4). The percentage of CD4+CD25+FOXP3+ regulatory T cells (TRegs) and T cell expression of PD1 was not significantly changed in the presence of SGI-110. The effect of SGI-110 on CTL mediated killing by vaccine stimulated T cells was assessed. Following stimulation with the DC/AML fusion vaccine, T cells mediated killing of autologous AML blasts increased to 11% from 4% following co-culture with unstimulated T cells. Interestingly, exposure of the AML cells to SGI-110 led to a further increase in T cells mediated killing with mean levels of 20% tumor lysis (n=2).Conclusions: The results demonstrate that treatment with SGI-110 results in increased ROS levels and an associated enhanced susceptibility of AML blasts to immune mediated targeting. The addition of SGI-110 to T cell stimulation by an autologous DC/AML fusion vaccine results in an increase in interferon gamma and increased susceptibility of AML cells to T cell mediated killing. The immunomodulatory properties of SGI-110, combined with its favorable pharmacologic and pharmacokinetic features, identify SGI-110 as a useful agent to implement novel combined epigenetic–immunotherapeutic strategies in AML. A clinical trial evaluating SGI-110 in combination with DC/AML fusion cell vaccination is planned. DisclosuresTaverna:Astex Pharmaceuticals, Inc.: Employment. Avigan:Astex Pharmaceuticals: Research Funding.