Role of Activin-A signaling in melanoma

Cutaneous melanomas arise from transformed skin melanocytes. While accounting for only 1% of skin malignancies, melanoma is responsible for the majority of skin cancer-related deaths. Owing to lifestyle changes and increased exposure to sunlight, melanoma occurrence is on the rise in developed countries. Identification of the molecular mechanisms driving melanoma formation led to the development of new classes of therapeutics which revolutionized the standard of care. Among these, small molecule inhibitors targeting the MAPK pathway and immune checkpoint inhibitors resulted in unprecedented clinical success. Nevertheless, many patients still do not benefit from these therapies. Activin-A, a secreted ligand of the TGF-Î² superfamily, was recently found to be overexpressed in melanoma. However, whether Activin-A signaling promotes melanoma progression or therapy resistance is unknown. In this thesis, the contributions of autocrine and paracrine Activin-A signaling to melanoma growth are analyzed using lentiviral expression in melanoma cells of a constitutively active mutant type I Activin receptor (caALK4), of secreted Activin-A (gain-of-function), or of a ligand trap (loss-of-function). Paradoxically, while forced ALK4 signaling in melanoma grafts restored tumor suppression by inhibiting tumor cell survival and proliferation, transgenic Activin-A accelerated tumor growth. Conversely, expression of a ligand trap comprising the extracellular domain of Activin receptor IIB fused to human Fc (AIIB-Fc) slowed melanoma growth. Importantly, in immunodeficient Rag1-/- mice, effects of paracrine Activin-A gain and loss-of-function were abrogated, suggesting that Activin-A promotes melanoma progression by inhibiting the adaptive anti-tumor response. Flow cytometry analysis of tumor immune infiltrates combined with antibody-mediated cell depletion experiments confirmed that Activin-A promotes melanoma progression by inhibiting CD8+ tumor-infiltrating lymphocytes (TILs). Furthermore, inhibition of endogenous Activin-A sensitized a treatment-resistant melanoma model to immune checkpoint inhibition by anti-PD1 and anti-CTLA4 antibodies. Activin-A therefore emerges as a promising target to improve immunotherapy outcome in melanoma.

Immunosuppressive roles of Activin-A in melanoma and characterization of its precursor cleavage

Katarina Pinjusic

Activin-A, a transforming growth factor êµ family member, is a pleiotropic cytokine with diverse functions in development, fertility, adult tissue homeostasis, and aging. Accordingly, deregulation of Activin-A signaling has been associated with many pathological conditions, including cancer and cancer-associated muscle wasting (cachexia). Activin-A was shown to signal in endocrine, but also local autocrine and paracrine manner. Its secretion in many cancer types is associated with a poor prognosis. Previous studies suggest that Activin-A expression in melanoma promotes tumor growth and metastasis indirectly by inhibiting adaptive anti-tumor immunity, leading to a decrease in the intratumoral frequency of cytotoxic CD8+ T cells (CTLs). However, the underlying mechanisms and the relevant direct target cells of Activin-A and their significance for cancer therapies remained unknown. Similar to other TGFêµ-related ligands, proActivin-A is cleaved by proprotein convertases (PCs) to release mature ligands. However, unlike other family members whose signaling is tightly regulated, Activin-A apparently does not have latency.In this thesis, I characterize proActivin-A cleavage and show that one prodomain can be cleaved independently of known PCs. The resulting hemi-cleaved Activin-A can bind activin type II receptors and induce SMAD3 signaling in reporter cells. A protein interactome screen and analysis of cleavage intermediates in plasma revealed that the hemi-cleaved Activin-A likely interacts with extracellular matrix proteins via the remaining prodomain where it promotes local signaling and regulates trafficking. Luciferase reporter assay showed that hemi-cleaved Activin-A can avoid, at least partially, inhibition by endogenous inhibitors FSTL3 and FST, and enable parallel activation of BMP and Activin signaling pathways. Finally, analysis of B16-F1 melanoma models revealed that only fully cleaved Activin-A in source cells increased tumor growth by acting locally in the tumor microenvironment suggesting that the context of Activin-A precursor processing regulates its tumor growth-promoting effect.The second part of this thesis addresses the mechanisms of Activin-induced immunosuppression in melanoma. We found that Activin-A impairs CTL function indirectly and independently of CD4+ T cell, CSF1R+ macrophages, and IL4 signaling. At least in part, anti-tumor CTL responses are impaired by the inhibitory effects of Activin-A on the secretion of critical CTL-recruiting chemokines CXCL9 and CXCL10 by macrophages and dendritic cells (DCs). Furthermore, Activin-A conferred resistance to immune checkpoint blockade therapy. Characterization of Activin-induced changes in the gene expression of tumor-infiltrating cells by Single cell RNA sequencing revealed that INHBA expression induced the most changes in DCs and least in T cells. Interestingly, the most prominently induced hallmark in Activin-secreting tumors across cell types was the IFN signature. In vitro analyses showed that Activin-A increases IFNÎ³-induced activation of STAT1 and confers resistance to cytostatic IFN signaling in cancer cells.Altogether, the findings of this thesis expand our knowledge on the regulation of Activin-A signaling by precursor cleavage and offer insights into mechanisms of Activin-induced immunosuppression in melanoma. Inhibition of Activin-A maturation thus emerges as a potential new strategy to improve responses to immunotherapies in melanoma and likely other cancers

EPFL2022

Tumor lymphangiogenesis promotes T cell infiltration and potentiates immunotherapy in melanoma

Natacha Anne-Sophie Bordry, Maria Anna Sofia Broggi, Elodie Da Costa, Manuel Andreas Fankhauser, Douglas Hanahan, Sylvie Hauert, Krisztian Homicsko, Laura Jeanbart, Amanda Waite Lund, Olivier Michielin, Lambert Charles François Potin, Marcela Rincón-Restrepo, Melody Swartz, Christopher Claude Giuseppe Tremblay

In melanoma, vascular endothelial growth factor-C (VEGF-C) expression and consequent lymphangiogenesis correlate with metastasis and poor prognosis. VEGF-C also promotes tumor immunosuppression, suggesting that lymphangiogenesis inhibitors may be clinically useful in combination with immunotherapy. We addressed this concept in mouse melanoma models with VEGF receptor-3 (VEGFR-3)-blocking antibodies and unexpectedly found that VEGF-C signaling enhanced rather than suppressed the response to immunotherapy. We further found that this effect was mediated by VEGF-C-induced CCL21 and tumor infiltration of naive T cells before immunotherapy because CCR7 blockade reversed the potentiating effects of VEGF-C. In human metastatic melanoma, gene expression of VEGF-C strongly correlated with CCL21 and T cell inflammation, and serum VEGF-C concentrations associated with both T cell activation and expansion after peptide vaccination and clinical response to checkpoint blockade. We propose that VEGF-C potentiates immunotherapy by attracting naive T cells, which are locally activated upon immunotherapy-induced tumor cell killing, and that serum VEGF-C may serve as a predictive biomarker for immunotherapy response.

Amer Assoc Advancement Science2017

Engineering Lymphangiogenesis

Manuel Andreas Fankhauser

Melanoma causes the vast majority of skin cancer deaths, and there is currently no conventional treatment available that is able to cure metastatic melanoma patients. However, a novel treatment modality has recently emerged: cancer immunotherapy. Cancer immunotherapy aims at raising potent, tumor-specific T cell responses that are able to recognize and eliminate cancerous cells. Despite the fact that for the first time in history some metastatic melanoma patients can be cured, it is currently unclear why a large fraction of patients does not respond to immunotherapy. Thus, the overarching goal of this thesis was to gain a better understanding of the molecular mechanisms dictating the outcome to immunotherapy. Pre-existing inflammation within the tumor microenvironment is associated with good clinical prognosis following immunotherapy. Because our lab has previously shown that tumor associated lymphatic vessels (LVs) can modulate tumor inflammation, we hypothesized that LVs might be involved in modulating the outcome of cancer immunotherapy. To explore this hypothesis, we characterized two mouse models that recapitulate primary human lymphangiogenic melanoma. Using these models, we found that tumor-associated lymphatics secrete CCL21 to actively attract naïve T cells into the tumor microenvironment. Interestingly, we found that antigen-specific immunotherapy induced activation of these naïve T cell infiltrates, which not only resulted in eradication of the primary melanoma tumors, but also conferred the mice with long-term protection against tumor re-challenge. We thus demonstrate that LVs increase the quantity and quality of tumor inflammation, thereby establishing a microenvironment that is able to potentiate antigen-specific immunotherapy. We then show that controlled release of VEGFC from injectable hydrogels leads to lymphangiogenesis in the mouse dermis. Similarly to what we found in lymphangiogenic tumors, engineered lymphangiogenic skin sites displayed increased expression of CCL21, and increased infiltrations of CD4+ and CD8+ T cells. Delivery of the model antigen ovalbumin into lymphangiogenic sites led to enhanced release of the effector cytokine interferon-¿ upon antigen-restimulation, suggesting that engineered lymphangiogenic sites could be exploited for therapeutic immunomodulation. Finally, to better understand mechanisms underlying successful cancer immunotherapy, we established a tool for the observation of anti-tumor immune responses in the context of the native tumor microenvironment. By combining existing methods in a novel way, we developed an intravital microscopy method based on immunofluorescence that allows simultaneous, high resolution and dynamic visualization of the tumor microenvironment including immune cells and extracellular matrix proteins. In conclusion, this thesis demonstrates that lymphatic endothelial cells (LECs) orchestrate immune cell recruitment into peripheral tissues by secretion of CCL21 chemokine, both in steady-state and disease. Our findings add more evidence to the hypothesis that LECs might play an underappreciated role in driving the formation of peripheral sites of immunomodulation. Our findings have translational potential, as immune cell infiltrates attracted by local lymphangiogenesis can be exploited for therapeutic immune regulation, such as to improving the efficacy of cancer immunotherapy.