In melanoma, therapies with inhibitors to oncogenic BRAFV600E are highly effective but responses are often short-lived due to the emergence of drug-resistant tumor subpopulations. We describe here a mechanism of acquired drug resistance through the tumor microenvironment, which is mediated by human tumor-associated B cells. Human melanoma cells constitutively produce the growth factor FGF-2, which activates tumor-infiltrating B cells to produce the growth factor IGF-1. B-cell-derived IGF-1 is critical for resistance of melanomas to BRAF and MEK inhibitors due to emergence of heterogeneous subpopulations and activation of FGFR-3. Consistently, resistance of melanomas to BRAF and/or MEK inhibitors is associated with increased CD20 and IGF-1 transcript levels in tumors and IGF-1 expression in tumor-associated B cells. Furthermore, first clinical data from a pilot trial in therapy-resistant metastatic melanoma patients show anti-tumor activity through B-cell depletion by anti-CD20 antibody. Our findings establish a mechanism of acquired therapy resistance through tumor-associated B cells with important clinical implications.

Anti-PD-1 therapy is used as a front-line treatment for many cancers, but mechanistic insight into this therapy resistance is still lacking. Here we generate a humanized (Hu)-mouse melanoma model by injecting fetal liver-derived CD34+ cells and implanting autologous thymus in immune-deficient NOD-scid IL2Rγnull (NSG) mice. Reconstituted Hu-mice are challenged with HLA-matched melanomas and treated with anti-PD-1, which results in restricted tumor growth but not complete regression. Tumor RNA-seq, multiplexed imaging and immunohistology staining show high expression of chemokines, as well as recruitment of FOXP3+ Treg and mast cells, in selective tumor regions. Reduced HLA-class I expression and CD8+/Granz B+ T cells homeostasis are observed in tumor regions where FOXP3+ Treg and mast cells co-localize, with such features associated with resistance to anti-PD-1 treatment. Combining anti-PD-1 with sunitinib or imatinib results in the depletion of mast cells and complete regression of tumors. Our results thus implicate mast cell depletion for improving the efficacy of anti-PD-1 therapy.

Melanoma is a heterogeneous tumor with different subpopulations showing different proliferation rates. Slow-cycling cells were previously identified in melanoma, but not fully biologically characterized. Using the label-retention method, we identified a subpopulation of slow-cycling cells, defined as label-retaining cells (LRC), with strong invasive properties. We demonstrate through live imaging that LRC are leaving the primary tumor mass at a very early stage and disseminate to peripheral organs. Through global proteome analyses, we identified the secreted protein SerpinE2/protease nexin-1 as causative for the highly invasive potential of LRC in melanomas.

Melanoma cells share many biological properties with neural crest stem cells. Here we show that the homeodomain transcription factor MSX1, which is significantly correlated with melanoma disease progression, reprograms melanocytes and melanoma cells toward a neural crest precursor-like state. MSX1-reprogrammed normal human melanocytes express the neural crest marker p75 and become multipotent. MSX1 induces a phenotypic switch in melanoma, which is characterized by an oncogenic transition from an E-cadherin—high nonmigratory state toward a ZEB1-high invasive state. ZEB1 up-regulation is responsible for the MSX1-induced migratory phenotype in melanoma cells. Depletion of MSX1 significantly inhibits melanoma metastasis in vivo. These results show that neural crest-like reprogramming achieved by a single factor is a critical process for melanoma progression.

Multipotent stem cells with neural crest-like properties have been identified in the dermis of human skin. These neural crest stem cell (NCSC)-like cells display self-renewal capacity and differentiate into neural crest derivatives, including epidermal pigment-producing melanocytes. NCSC-like cells share many properties with aggressive melanoma cells, such as high migratory capabilities and expression of the neural crest markers. However, little is known about which intrinsic or extrinsic signals determine the proliferation or differentiation of these neural crest-like stem cells. Here, we show that in NCSC-like cells, Notch signaling is highly activated, similar to melanoma cells. Inhibition of Notch signaling reduced proliferation of NCSC-like cells, induced cell death, and down-regulated non-canonical Wnt5a, suggesting that the Notch pathway contributes to the maintenance and motility of these stem cells. In three-dimensional skin reconstructs, canonical Wnt signaling promoted the differentiation of NCSC-like cells into melanocytes. This differentiation was triggered by the endogenous Notch inhibitor Numb, which is up-regulated in the stem cells by Wnt7a derived from UV-irradiated keratinocytes. Together, these data reveal a crosstalk between the two conserved developmental pathways in postnatal human skin, and highlight the role of the skin microenvironment in specifying the fate of stem cells.