SummaryOnce melanomas have progressed with acquired resistance to mitogen-activated protein kinase (MAPK)-targeted therapy, mutational heterogeneity presents a major challenge. We therefore examined the therapy phase before acquired resistance had developed and discovered the melanoma survival oncogene MITF as a driver of an early non-mutational and reversible drug-tolerance state, which is induced by PAX3-mediated upregulation of MITF. A drug-repositioning screen identified the HIV1-protease inhibitor nelfinavir as potent suppressor of PAX3 and MITF expression. Nelfinavir profoundly sensitizes BRAF and NRAS mutant melanoma cells to MAPK-pathway inhibitors. Moreover, nelfinavir is effective in BRAF and NRAS mutant melanoma cells isolated from patients progressed on MAPK inhibitor (MAPKi) therapy and in BRAF/NRAS/PTEN mutant tumors. We demonstrate that inhibiting a driver of MAPKi-induced drug tolerance could improve current approaches of targeted melanoma therapy.

Recently the rationale for combining targeted therapy with immunotherapy has come to light, but our understanding of the immune response during MAPK pathway inhibitor treatment is limited. We discovered that the immune-microenvironment can act as source of resistance to MAPK pathway-targeted therapy, and moreover during treatment this source becomes reinforced. In particular, we identified macrophage-derived TNFα as a crucial melanoma-growth factor that provides resistance to MAPK pathway inhibitors through the lineage-transcription factor MITF. Most strikingly, in BRAF mutant melanomas of patients and BRafV600E-melanoma allografts MAPK pathway inhibitors increased the number of tumor-associated macrophages, and TNFα and MITF expression. Inhibiting TNFα-signaling with IκB-kinase inhibitors profoundly enhanced the efficacy of MAPK pathway inhibitors by targeting not only the melanoma cells, but also the microenvironment. In summary, we identify the immune-microenvironment as a novel source of resistance and reveal a new strategy to improve the efficacy of targeted therapy in melanoma.

SummaryClonal selection and transcriptional reprogramming (e.g., epithelial-mesenchymal transition or phenotype switching) are the predominant theories thought to underlie tumor progression. However, a “division of labor” leading to cooperation among tumor-cell subpopulations could be an additional catalyst of progression. Using a zebrafish-melanoma xenograft model, we found that in a heterogeneous setting, inherently invasive cells, which possess protease activity and deposit extracellular matrix (ECM), co-invade with subpopulations of poorly invasive cells, a phenomenon we term “cooperative invasion”. Whereas the poorly invasive cells benefit from heterogeneity, the invasive cells switch from protease-independent to an MT1-MMP-dependent mode of invasion. We did not observe changes in expression of the melanoma phenotype determinant MITF during cooperative invasion, thus ruling out the necessity for phenotype switching for invasion. Altogether, our data suggest that cooperation can drive melanoma progression without the need for clonal selection or phenotype switching and can account for the preservation of heterogeneity seen throughout tumor progression.

Human exposure to polycyclic aromatic hydrocarbons (PAH) occurs through complex mixtures such as coal tar. The effect of complex PAH mixtures on the activation of carcinogenic PAH to DNA-binding derivatives and carcinogenesis were investigated in mice treated topically with NIST (National Institute of Standards and Technology) Standard Reference Material 1597 (SRM), a complex mixture of PAH extracted from coal tar, and either additional benzo[a]pyrene (B[a]P) or dibenzo[a,l]pyrene (DB[a,l]P). In an initiation-promotion study using 12-O-tetradecanoylphorbol-13-acetate as the promoter for 25 weeks, the SRM and B[a]P co-treated mice had a similar incidence of papillomas per mouse compared with the group exposed to B[a]P alone as the initiator. PAH-DNA adduct analysis of epidermal DNA by 33P-post-labeling and reversed-phase high-performance liquid chromatography found the SRM co-treatment led to a significant decrease in the total level of DNA adducts and B[a]P-DNA adducts to less than that observed in mice treated with B[a]P alone at 6, 12 and 72 h exposure. After 24 and 48 h exposure, there was no significant difference in the levels of adducts between these groups. In the DB[a,l]P initiation-promotion study, the co-treated group had significantly fewer papillomas per mouse than mice treated with DB[a,l]P alone as initiator. Averaging over the times of exposure gave strong evidence that mice co-treated with SRM and DB[a,l]P had a significantly lower level of PAH-DNA adducts than mice treated with DB[a,l]P alone. Western immunoblots showed that both cytochrome P450 (CYP) 1A1 and 1B1 were induced by the SRM. These results are consistent with the hypothesis that two major factors determining the carcinogenic activity of PAH within a complex mixture are (i) the persistence of certain PAH-DNA adducts as well as total adduct levels, and (ii) the ability of the components present in the mixture to inhibit the activation of carcinogenic PAH by the induced CYP enzymes.

The target of rapamycin (TOR) kinase regulates cell growth and division. Rapamycin only inhibits a subset of TOR activities. Here we show that in contrast to the mild impact of rapamycin on cell division, blocking the catalytic site of TOR with the Torin1 inhibitor completely arrests growth without cell death in Schizosaccharomyces pombe. A mutation of the Tor2 glycine residue (G2040D) that lies adjacent to the key Torin-interacting tryptophan provides Torin1 resistance, confirming the specificity of Torin1 for TOR. Using this mutation, we show that Torin1 advanced mitotic onset before inducing growth arrest. In contrast to TOR inhibition with rapamycin, regulation by either Wee1 or Cdc25 was sufficient for this Torin1-induced advanced mitosis. Torin1 promoted a Polo and Cdr2 kinase-controlled drop in Wee1 levels. Experiments in human cell lines recapitulated these yeast observations: mammalian TOR (mTOR) was inhibited by Torin1, Wee1 levels declined and mitotic commitment was advanced in HeLa cells. Thus, the regulation of the mitotic inhibitor Wee1 by TOR signalling is a conserved mechanism that helps to couple cell cycle and growth controls.

Despite the general focus on an invasive and de-differentiated phenotype as main driver of cancer metastasis, in melanoma patients many metastatic lesions display a high degree of pigmentation, indicative for a differentiated phenotype. Indeed, studies in mice and fish show that melanoma cells switch to a differentiated phenotype at secondary sites, possibly because in melanoma differentiation is closely linked to proliferation through the lineage-specific transcriptional master regulator MITF. Importantly, while a lot of effort has gone into identifying factors that induce the de-differentiated/invasive phenotype, it is not well understood how the switch to the differentiated/proliferative phenotype is controlled. We identify collagen as a contributor to this switch. We demonstrate that collagen stiffness induces melanoma differentiation through a YAP/PAX3/MITF axis and show that in melanoma patients increased collagen abundance correlates with nuclear YAP localization. However, the interrogation of large patient datasets revealed that in the context of the tumour microenvironment, YAP function is more complex. In the absence of fibroblasts, YAP/PAX3-mediated transcription prevails, but in the presence of fibroblasts tumour growth factor-β suppresses YAP/PAX3-mediated MITF expression and induces YAP/TEAD/SMAD-driven transcription and a de-differentiated phenotype. Intriguingly, while high collagen expression is correlated with poorer patient survival, the worst prognosis is seen in patients with high collagen expression, who also express MITF target genes such as the differentiation markers TRPM1, TYR and TYRP1, as well as CDK4. In summary, we reveal a distinct lineage-specific route of YAP signalling that contributes to the regulation of melanoma pigmentation and uncovers a set of potential biomarkers predictive for poor survival.

Approaches to prolong responses to BRAF targeting drugs in melanoma patients are challenged by phenotype heterogeneity. Melanomas of a “MITF‐high” phenotype usually respond well to BRAF inhibitor therapy, but these melanomas also contain subpopulations of the de novo resistance “AXL‐high” phenotype. > 50% of melanomas progress with enriched “AXL‐high” populations, and because AXL is linked to de‐differentiation and invasiveness avoiding an “AXL‐high relapse” is desirable. We discovered that phenotype heterogeneity is supported during the response phase of BRAF inhibitor therapy due to MITF‐induced expression of endothelin 1 (EDN1). EDN1 expression is enhanced in tumours of patients on treatment and confers drug resistance through ERK re‐activation in a paracrine manner. Most importantly, EDN1 not only supports MITF‐high populations through the endothelin receptor B (EDNRB), but also AXL‐high populations through EDNRA, making it a master regulator of phenotype heterogeneity. Endothelin receptor antagonists suppress AXL‐high‐expressing cells and sensitize to BRAF inhibition, suggesting that targeting EDN1 signalling could improve BRAF inhibitor responses without selecting for AXL‐high cells.

BackgroundThe mitogen-activated protein–kinase pathway consisting of the kinases RAF, MEK, and ERK is central to cell proliferation and survival and is deregulated in more than 90% of melanomas. MEK inhibitors are currently trialled in the clinic, but despite efficient target inhibition, cytostatic rather than cytotoxic activity limits their efficacy.MethodsWe assessed the cytotoxicity to MEK inhibitors (PD184352 and selumetinib) in melanoma cells by toluidine-blue staining, caspase 3 cleavage, and melanoma-sphere growth. Western blotting and quantitative real-time polymerase chain reaction were applied to determine SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2), PAX3, and MITF expression. Human melanoma samples (n = 77) from various stages were analyzed for SMURF2 and PAX3 expression. RNA interference was performed to target SMURF2 during MEK inhibition in vivo in melanoma xenografts in mice and zebrafish. All statistical tests were two-sided.ResultsActivation of transforming growth factor β (TGF-β) signalling sensitized melanoma cells to the cytotoxic effects of MEK inhibition. Melanoma cells resistant to the cytotoxic effects of MEK inhibitors counteracted TGF-β signalling through overexpression of the E3 ubiquitin ligase SMURF2, which resulted in increased expression of the transcription factors PAX3 and MITF. High MITF expression protected melanoma cells against MEK inhibitor cytotoxicity. Depleting SMURF2 reduced MITF expression and substantially lowered the threshold for MEK inhibitor–induced apoptosis. Moreover, SMURF2 depletion sensitized melanoma cells to the cytotoxic effects of selumetinib, leading to cell death at concentrations approximately 100-fold lower than the concentration required to induce cell death in SMURF2-expressing cells. Mice treated with selumetinib alone at a dosage of 10mg/kg body weight once daily produced no response, but in combination with SMURF2 depletion, selumetinib suppressed tumor growth by 97.9% (95% confidence interval = 38.65% to 155.50%, P = .005).ConclusionsTargeting SMURF2 may be a novel therapeutic approach for increasing the antitumor efficacy of MEK inhibitors.