Once 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 a 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 (microphthalmia transcription factor). Most strikingly, in BRAF-mutant melanomas of patients and BRAF^V600E 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. Significance: This study identifies the immune microenvironment as a source of resistance to MAPK pathway inhibitors through macrophage-derived TNFα, and reveals that in patients on treatment this source becomes reinforced. Inhibiting IκB kinase enhances the efficacy of MAPK pathway inhibitors, which identifies this approach as a potential novel strategy to improve targeted therapy in melanoma. Cancer Discov; 4(10); 1214–29. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 1103

Increasing evidence indicates that success of targeted therapies in the treatment of cancer is context-dependent and is influenced by a complex crosstalk between signaling pathways and between cell types in the tumor. The Fibroblast Growth Factor (FGF)/FGF receptor (FGFR) signaling axis highlights the importance of such context-dependent signaling in cancer. Aberrant FGFR signaling has been characterized in almost all cancer types, most commonly non-small cell lung cancer (NSCLC), breast cancer, glioblastoma, prostate cancer and gastrointestinal cancer. This occurs primarily through amplification and over-expression of FGFR1 and FGFR2 resulting in ligand-independent activation. Mutations and translocations of FGFR1-4 are also identified in cancer. Canonical FGF-FGFR signaling is tightly regulated by ligand-receptor combinations as well as direct interactions with the FGFR coreceptors heparan sulfate proteoglycans (HSPGs) and Klotho. Noncanonical FGFR signaling partners have been implicated in differential regulation of FGFR signaling. FGFR directly interacts with cell adhesion molecules (CAMs) and extracellular matrix (ECM) proteins, contributing to invasive and migratory properties of cancer cells, whereas interactions with other receptor tyrosine kinases (RTKs) regulate angiogenic, resistance to therapy, and metastatic potential of cancer cells. The diversity in FGFR signaling partners supports a role for FGFR signaling in cancer, independent of genetic aberration.

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.

The 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. We 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. Activation 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). Targeting SMURF2 may be a novel therapeutic approach for increasing the antitumor efficacy of MEK inhibitors.

Mitogen-activated protein kinase (MAPK) pathway antagonists induce profound clinical responses in advanced cutaneous melanoma, but complete remissions are frustrated by the development of acquired resistance. Before resistance emerges, adaptive responses establish a mutation-independent drug tolerance. Antagonizing these adaptive responses could improve drug effects, thereby thwarting the emergence of acquired resistance. In this study, we reveal that inflammatory niches consisting of tumor-associated macrophages and fibroblasts contribute to treatment tolerance through a cytokine-signaling network that involves macrophage-derived IL-1β and fibroblast-derived CXCR2 ligands. Fibroblasts require IL-1β to produce CXCR2 ligands, and loss of host IL-1R signaling in vivo reduces melanoma growth. In tumors from patients on treatment, signaling from inflammatory niches is amplified in the presence of MAPK inhibitors. Signaling from inflammatory niches counteracts combined BRAF/MEK (MAPK/extracellular signal–regulated kinase kinase) inhibitor treatment, and consequently, inhibiting IL-1R or CXCR2 signaling in vivo enhanced the efficacy of MAPK inhibitors. We conclude that melanoma inflammatory niches adapt to and confer drug tolerance toward BRAF and MEK inhibitors early during treatment.