Alisha Holtzhausen

Type III TGF-β receptor downregulation generates an immunotolerant tumor microenvironment

Cancers subvert the host immune system to facilitate disease progression. These evolved immunosuppressive mechanisms are also implicated in circumventing immunotherapeutic strategies. Emerging data indicate that local tumor-associated DC populations exhibit tolerogenic features by promoting Treg development; however, the mechanisms by which tumors manipulate DC and Treg function in the tumor microenvironment remain unclear. Type III TGF-β receptor (TGFBR3) and its shed extracellular domain (sTGFBR3) regulate TGF-β signaling and maintain epithelial homeostasis, with loss of TGFBR3 expression promoting progression early in breast cancer development. Using murine models of breast cancer and melanoma, we elucidated a tumor immunoevasion mechanism whereby loss of tumor-expressed TGFBR3/sTGFBR3 enhanced TGF-β signaling within locoregional DC populations and upregulated both the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in plasmacytoid DCs and the CCL22 chemokine in myeloid DCs. Alterations in these DC populations mediated Treg infiltration and the suppression of antitumor immunity. Our findings provide mechanistic support for using TGF-β inhibitors to enhance the efficacy of tumor immunotherapy, indicate that sTGFBR3 levels could serve as a predictive immunotherapy biomarker, and expand the mechanisms by which TGFBR3 suppresses cancer progression to include effects on the tumor immune microenvironment.

Novel bone morphogenetic protein signaling through Smad2 and Smad3 to regulate cancer progression and development

The bone morphogenetic protein (BMP) signaling pathways have important roles in embryonic development and cellular homeostasis, with aberrant BMP signaling resulting in a broad spectrum of human disease. We report that BMPs unexpectedly signal through the canonical transforming growth factor β (TGF‐β)‐responsive Smad2 and Smad3. BMP‐induced Smad2/3 signaling occurs preferentially in embryonic cells and transformed cells. BMPs signal to Smad2/3 by stimulating complex formation between the BMP‐binding TGF‐β superfamily receptors, activin receptor‐like kinase (ALK)3/6, and the Smad2/3 phosphorylating receptors ALK5/7. BMP signaling through Smad2 mediates, in part, dorsoventral axis patterning in zebrafish embryos, whereas BMP signaling through Smad3 facilitates cancer cell invasion. Consistent with increased BMP‐mediated Smad2/3 signaling during cancer progression, Smad1/5 and Smad 2/3 signaling converge in human cancer specimens. Thus, the signaling mechanisms used by BMPs and TGF‐β superfamily receptors are broader than previously appreciated.—Holtzhausen, A., Golzio, C., How, T., Lee, Y.‐H., Schiemann, W. P., Katsanis, N., Blobe, G. C. Novel bone morphogenetic protein signaling through Smad2 and Smad3 to regulate cancer progression and development. FASEB J. 28, 1248–1267 (2014). www.fasebj.org

Melanoma-Derived Wnt5a Promotes Local Dendritic-Cell Expression of IDO and Immunotolerance: Opportunities for Pharmacologic Enhancement of Immunotherapy

Melanomas produce Wnt5a, which promotes the ability of local DCs to drive regulatory T-cell expansion and to generate an immunosuppressive tumor microenvironment. Targeting this system could lead to the development of novel immunotherapeutic strategies. The β-catenin signaling pathway has been demonstrated to promote the development of a tolerogenic dendritic cell (DC) population capable of driving regulatory T-cell (Treg) differentiation. Further studies have implicated tolerogenic DCs in promoting carcinogenesis in preclinical models. 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 currently unknown. This work demonstrates that melanoma-derived Wnt5a ligand upregulates the durable expression and activity of the indoleamine 2,3-dioxygenase-1 (IDO) enzyme by local DCs in a manner that depends upon the β-catenin signaling pathway. These data indicate that Wnt5a-conditioned DCs promote the differentiation of Tregs in an IDO-dependent manner, and that this process serves to suppress melanoma immune surveillance. We further show that the genetic silencing of the PORCN membrane–bound O-acyl transferase, which is necessary for melanoma Wnt ligand secretion, enhances antitumor T-cell immunity, and that the pharmacologic inhibition of this enzyme synergistically suppresses melanoma progression when combined with anti–CTLA-4 antibody therapy. Finally, our data suggest that β-catenin signaling activity, based on a target gene expression profile that includes IDO in human sentinel lymph node–derived DCs, is associated with melanoma disease burden and diminished progression-free survival. This work implicates the Wnt–β-catenin signaling pathway as a novel therapeutic target in the melanoma immune microenvironment and demonstrates the potential impact of manipulating DC function as a strategy for optimizing tumor immunotherapy. Cancer Immunol Res; 3(9); 1082–95. ©2015 AACR.

Early Carcinogenesis Involves the Establishment of Immune Privilege via Intrinsic and Extrinsic Regulation of Indoleamine 2,3-dioxygenase-1: Translational Implications in Cancer Immunotherapy.

Although prolonged genetic pressure has been conjectured to be necessary for the eventual development of tumor immune evasion mechanisms, recent work is demonstrating that early genetic mutations are capable of moonlighting as both intrinsic and extrinsic modulators of the tumor immune microenvironment. The indoleamine 2,3-dioxygenase-1 (IDO) immunoregulatory enzyme is emerging as a key player in tumor-mediated immune tolerance. While loss of the tumor suppressor, BIN-1, and the over-expression of cyclooxygenase-2 have been implicated in intrinsic regulation of IDO, recent findings have demonstrated the loss of TβRIII and the upregulation of Wnt5a by developing cancers to play a role in the extrinsic control of IDO activity by local dendritic cell populations residing within tumor and tumor-draining lymph node tissues. Together, these genetic changes are capable of modulating paracrine signaling pathways in the early stages of carcinogenesis to establish a site of immune privilege by promoting the differentiation and activation of local regulatory T cells. Additional investigation of these immune evasion pathways promises to provide opportunities for the development of novel strategies to synergistically enhance the efficacy of the evolving class of T cell-targeted “checkpoint” inhibitors.

Tumor-secreted Pros1 inhibits macrophage M1 polarization to reduce antitumor immune response

Tyro3, Axl, Mer (TAM) receptor tyrosine kinases reduce inflammatory, innate immune responses. We demonstrate that tumor-secreted protein S (Pros1), a Mer/Tyro3 ligand, decreased macrophage M1 cytokine expression in vitro and in vivo. In contrast, tumor cells with CRISPR-based deletion of Pros1 failed to inhibit M1 polarization. Tumor cell–associated Pros1 action was abrogated in macrophages from Mer- and Tyro3- but not Axl-KO mice. In addition, several other murine and human tumor cell lines suppressed macrophage M1 cytokine expression induced by IFN-&ggr; and LPS. Investigation of the suppressive pathway demonstrated a role for PTP1b complexing with Mer. Substantiating the role of PTP1b, M1 cytokine suppression was also lost in macrophages from PTP1b-KO mice. Mice bearing Pros1-deficient tumors showed increased innate and adaptive immune infiltration, as well as increased median survival. TAM activation can also inhibit TLR-mediated M1 polarization. Treatment with resiquimod, a TLR7/8 agonist, did not improve survival in mice bearing Pros1-secreting tumors but doubled survival for Pros1-deleted tumors. The tumor-derived Pros1 immune suppressive system, like PD-L1, was cytokine responsive, with IFN-&ggr; inducing Pros1 transcription and secretion. Inhibition of Pros1/TAM interaction represents a potential novel strategy to block tumor-derived immune suppression.

Role of the Wnt-β-catenin signaling pathway in melanoma-mediated dendritic cell tolerization

Recent studies have shown that tumor immune evasion mechanisms significantly contribute to immunotherapy failure. Emerging data is indicating that tumor-associated dendritic cells (DCs) undergo phenotypic tolerization and promote the differentiation and activation of regulatory T cell (Treg) populations to generate local immune subversion. The critical role of DCs in orchestrating anti-tumor immunity suggests that the signaling pathways that regulate DC tolerogenesis may be promising targets for pharmacologically augmenting immunotherapy efficacy. Previous work has shown that the β-catenin signaling pathway plays a potential role in the DC tolerization process. Others have demonstrated that the DC-expressed enzyme, indoleamine 2,3-dioxygenase (IDO), promotes tumor-induced immune tolerance by catalyzing the conversion of the amino acid tryptophan into kynurenine. Other investigators have demonstrated melanomas to upregulate the expression of several Wnt ligands during their malignant transformation. Using a transgenic murine model of melanoma, we have determined that melanoma-associated DCs exhibit elevated levels of expression of IDO and other β-catenin target genes and demonstrate diminished T cell stimulatory capacity in vitro. Further studies have shown that Wnt3a and Wnt5a induce the expression and enzymatic activity of IDO in myeloid DCs (mDCs) in a β-catenin-dependent fashion while IDO reporter assays suggest this process to be mediated by direct promoter activation. This data suggest the Wnt-β-catenin signaling pathway to be a potential target for suppressing melanoma-induced DC tolerization and enhancing anti-tumor immunity. Indeed, both small molecule inhibition and genetic silencing of the PORCN acyl transferase enzyme which is necessary for Wnt secretion, abrogated B16 melanoma paracrine induction of β-catenin signaling and IDO expression by local mDCs. Further studies have shown PORCN-silenced B16 melanomas to exhibit reduced tumorigenecity in vivo and that this effect correlated with decreased mDC IDO expression and reduced levels of local Tregs. We therefore hypothesize that melanoma-expressed Wnt ligands induce DC tolerogenesis and stimulate β-catenin-dependent IDO expression and subsequent Treg generation in the tumor microenvironment, resulting in the development of an immunotolerant state and ultimately allowing for disease progression. Further, we propose that the pharmacological inhibition of this pathway can reverse this mechanism of immune evasion and enhance other immunotherapeutic approaches. Future work will include testing the ability of the small molecule PORCN inhibitor to stimulate the generation of tumor antigen-specific T cell responses in an autochthonous transgenic melanoma model.

Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

Lung cancer is the leading cause of cancer-related deaths worldwide, and lung squamous carcinomas (LUSC) represent about 30% of cases. Molecular aberrations in lung adenocarcinomas have allowed for effective targeted treatments, but corresponding therapeutic advances in LUSC have not materialized. However, immune checkpoint inhibitors in sub-populations of LUSC patients have led to exciting responses. Using computational analyses of The Cancer Genome Atlas, we identified a subset of LUSC tumors characterized by dense infiltration of inflammatory monocytes (IMs) and poor survival. With novel, immunocompetent metastasis models, we demonstrated that tumor cell derived CCL2-mediated recruitment of IMs is necessary and sufficient for LUSC metastasis. Pharmacologic inhibition of IM recruitment had substantial anti-metastatic effects. Notably, we show that IMs highly express Factor XIIIA, which promotes fibrin cross-linking to create a scaffold for LUSC cell invasion and metastases. Consistently, human LUSC samples containing extensive cross-linked fibrin in the microenvironment correlated with poor survival.Lung squamous carcinomas (LUSC) are poorly molecularly characterized, but sub-populations show promising response to immune checkpoint inhibitors. Here, the authors identify a subset of LUSC characterized by infiltration of inflammatory monocytes, where metastasis is linked to Factor XIIIA promoting fibrin cross-linking.

Paracrine Wnt5a-β-Catenin Signaling Triggers a Metabolic Program that Drives Dendritic Cell Tolerization

Summary Despite recent advances, many cancers remain refractory to available immunotherapeutic strategies. Emerging evidence indicates that the tolerization of local dendritic cells (DCs) within the tumor microenvironment promotes immune evasion. Here, we have described a mechanism by which melanomas establish a site of immune privilege via a paracrine Wnt5a‐&bgr;‐catenin‐peroxisome proliferator‐activated receptor‐&ggr; (PPAR‐&ggr;) signaling pathway that drives fatty acid oxidation (FAO) in DCs by upregulating the expression of the carnitine palmitoyltransferase‐1A (CPT1A) fatty acid transporter. This FAO shift increased the protoporphyrin IX prosthetic group of indoleamine 2,3‐dioxgenase‐1 (IDO) while suppressing interleukin(IL)‐6 and IL‐12 cytokine expression, culminating in enhanced IDO activity and the generation of regulatory T cells. We demonstrated that blockade of this pathway augmented anti‐melanoma immunity, enhanced the activity of anti‐PD‐1 antibody immunotherapy, and suppressed disease progression in a transgenic melanoma model. This work implicates a role for tumor‐mediated metabolic reprogramming of local DCs in immune evasion and immunotherapy resistance. Graphical Abstract Figure. No Caption available. HighlightsMelanomas metabolically reprogram local DCs to induce immune toleranceMelanoma‐derived Wnt5a triggers DC FAO via a &bgr;‐catenin‐PPAR‐&ggr; pathwayDC FAO drives IDO enzymatic activity by promoting protoporphyrin IX synthesisInhibiting the Wnt5a‐&bgr;‐catenin‐CPT1A pathway enhances anti‐PD‐1 antibody activity &NA; Previous studies suggest that DC tolerization plays a role in tumor‐mediated immune evasion. The mechanism by which cancers promote this process remains poorly understood. Zhao et al. demonstrate that melanomas generate a site of immune privilege by driving DC fatty acid oxidation via a Wnt5a‐&bgr;‐catenin‐PPAR‐&ggr; signaling pathway that culminates in the induction of IDO enzyme activity. Inhibiting this pathway reverses DC tolerization and enhances anti‐PD‐1 antibody efficacy in a transgenic model of melanoma.

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 ...

Abstract 3035: Bone morphogenetic proteins signal through Smad2 and Smad3 to regulate cell migration and proliferation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The TGF-β superfamily consists of a large number of growth factors, including transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) that regulate cellular proliferation, differentiation, invasion, migration and apoptosis. TGF-β activates the transcription factors Smad2/3, as well as Smad1/5/8, while BMP stimulation results in the activation of Smad1/5/8. Unexpectedly, we have demonstrated that BMP family members, including BMP2, 3, 4, 7, 9 and GDF5 can also induce Smad2/3 phosphorylation and downstream gene transcription in ovarian, pancreatic and breast cancer cell lines. BMP2-induced Smad2 phosphorylation can be attenuated by expression of dominant negative TβRII or shRNA-mediated knockdown of TβRII, while BMP2-induced Smad3 phosphorylation can be attenuated by expression of dominant negative BMPRII or shRNA-mediated knockdown of BMPRII, suggesting different mechanisms of BMP-mediated activation of Smad2 and Smad3. BMP2-induced Smad1, Smad2 and Smad3 phosphorylation increase cell invasion, migration and proliferation, respectively. BMP stimulated Smad2/3 phosphorylation suggests that BMP-mediated effects on cancer progression may be regulated, in part, through these novel signaling pathways. Current studies are focused on further elucidating the role of BMP-induced Smad2 and Smad3 phosphorylation on human cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3035. doi:1538-7445.AM2012-3035