Melanoma-derived Wnt5a conditions dendritic cells to promote regulatory T cell differentiation via the upregulation of indoleamine 2,3-dioxygenase: novel pharmacological strategies for augmenting immunotherapy efficacy

Previous studies have shown the β-catenin signaling pathway to promote the development of tolerogenic dendritic cells (DCs) that are capable of driving regulatory T cell (Treg) differentiation. Interestingly, tolerogenic DCs have recently been described to play a role in carcinogenesis. However, the molecular mechanisms underlying the establishment of immune tolerance by this DC population are poorly understood and the methods by which developing cancers can co-opt this pathway to subvert immune surveillance are unknown. Using a genetically engineered model, we demonstrate that melanoma-derived Wnt5a ligand is a novel regulator of indoleamine 2,3-dioxygenase-1 (IDO) expression in local myeloid DCs and that Wnt5a induces the durable expression and enzymatic activity of IDO via β-catenin (Figures ​(Figures11,​,2).2). Further, we show that Wnt5a-conditioned DCs promote Treg differentiation in an IDO-dependent manner and that melanoma secretion of Wnt5a both suppresses the generation of anti-tumor immunity and promotes melanoma progression in vivo (Figure ​(Figure3).3). By genetically silencing the PORCN acyl transferase which is necessary for Wnt ligand secretion, we confirm the role of the soluble Wnt ligands in directing DC tolerization both in vitro and in vivo while also establishing a potential pharmacologic target for manipulating this novel pathway (Figure ​(Figure4).4). Indeed, utilizing a small molecule inhibitor of PORCN, we are able to reverse Wnt5a-mediated IDO upregulation by DCs both in vitro and in vivo. These findings were expanded in further melanoma studies where we demonstrated small molecule PORCN inhibition to synergistically suppress melanoma progression while also enhancing anti-melanoma immunity in the setting of combination anti-CTLA-4 therapy (Figure ​(Figure5).5). Additional work in human melanoma confirms the existence of this Wnt5a-mediated paracrine signaling pathway in DCs and reveals by microarray dataset analysis that human melanoma co-expression of Wnt5a and FoxP3 is highly significant. These data prompted us to hypothesize that a Wnt5a-induced gene signature in the melanoma microenvironment may be indicative of immune tolerance. Indeed, we have found that a Wnt5a gene signature identified in purified sentinel lymph node-derived DCs is associated with an inferior clinical prognosis in melanoma patients. This work emphasizes the importance of DC populations in directing tumor immune surveillance and illustrates that the molecular mechanisms involved in DC physiology represent potential targets for pharmacologically enhancing anti-tumor immunity. Figure 1 Wnt3a and Wnt5a upregulate IDO expression and activity in DCs. A. Wnt5a induces durable IDO expression by BMDCs based on Western blot analysis at 24 and 48 hrs. Representative of 3 independent experiments. UT, untreated. XAV939, β-catenin inhibitor. ... Figure 2 Murine melanomas induce paracrine β-catenin signaling activation in tumor and TDLN DCs in vivo. A.Tumor-infiltrating DCs (TIDCs) isolated from Tyr::CreER;BrafCA;Ptenlox/lox primary melanomas exhibit increased expression levels of β-catenin ... Figure 3 Wnt5a conditions DCs to drive regulatory T-cell cifferentiation. A. Schematic of in vitro Treg assay. B. Wnt5a-conditioned DCs stimulate CD4+FoxP3+ Treg differentiation in vitro following co-incubation with total splenic CD4+ T cells at a 1:1, 5:1 or ... Figure 4 Silencing PORCN expression in the B16 melanoma model enhances anti-tumor immunity and suppresses tumor progression in vivo. A. Silencing PORCN expression by B16/F10 cells suppresses paracrine β-catenin activation in a 293T-TCF/LEF1-luciferase ... Figure 5 C59 Inhibition of the Wnt-β-catenin signaling pathway synergistically enhances the efficacy of anti-CTLA-4 antibody immunotherapy in the B16 melanoma model. A. Combination inhibition of Wnt-mediated signaling and anti-CTLA-4 blockade synergistically ...