Cucurbitacins, a Family of Natural Compounds That Effectively Disrupt Stat3 Signaling in Antigen-Presenting Cells (APCs) Are Promising Agents To Overcome Tumor-Induced CD4+ T-Cell Tolerance.

Abstract Signal transducer and activator of transcription 3 (Stat3) is a key mediator of several cytokines and growth factors signaling pathways. On myeloid cells, activation of Stat3 to its phosphorylated form (pStat3) has been shown to negatively regulate inflammatory responses. Recently, we have unambiguously demonstrated that Stat3 signaling in APCs also play a central role in the decision leading to immune activation versus immune tolerance of antigen-specific T-cells1. In spite of these advances, there is however a paucity of therapeutic strategies targeting this signaling pathway in immune cells. Using a high throughput cytoblot screening for phospho-Stat3 inhibition, we have recently identified a family of natural compounds known as Cucurbitacins that effectively disrupt Stat3 signaling at different levels2. Three compounds have been identified, Cucurbitacin A (CuA) that inhibits phospho-JAK-2, Cucurbitacin I (CuI) a dual inhibitor of p-JAK2 and p-Stat3 and Cucurbitacin Q (CuQ) a selective inhibitor of p-Stat3. In vitro treatment of peritoneal elicited macrophages (PEM) and bone marrow-derived dendritic cells (DCs) with increasing concentrations of CuA or CuI resulted in inhibition of p-Stat3 and enhanced antigen presentation to naive CD4+ T cells specific for a MHC class II restricted epitope of influenza hemagglutinin (HA). Indeed, these clonotypic T cells displayed increased antigen-specific proliferation and IL-2 production as compared to clonotypic T cells encountering cognate antigen on untreated APCs. Furthermore, unlike untreated PEM or DCs, which are unable to trigger IFN-gamma production by CD4+ T-cells, Cucurbitacin-treated APCs efficiently trigger the production of this cytokine by naïve CD4+ T-cells in response to cognate antigen. Given the above results, we explored next whether inhibition of Stat3 signaling in B-cell lymphomas by Cucurbitacins might increase the intrinsic antigen-presenting capabilities of these malignant B-cells. Reminiscent of our findings with bone marrow derived APCs, Cucurbitacin-treated A20 lymphoma cells also display enhanced antigen-presenting cell function leading to increased proliferation, IL-2 and IFN-gamma by naive antigen-specific CD4+ T-cells. More importantly, tolerant CD4+ T-cells (isolated from lymphoma bearing mice) exposed to Cucurbitacin-treated A20 B-cells regained their ability to proliferate and produce significant amounts of IL-2 and IFN-gamma in response to cognate antigen stimulation. Taken together, the ability of Cucurbitacins to inhibit p-Stat3 in normal APCs as well as in malignant B-cells make these natural compounds a promising agents to overcome the remarkable barrier that tolerance to tumor antigens has imposed to cancer immunotherapeutic strategies.