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Increased expression of the Microphthalmia-associated transcription factor (MITF) contributes to melanoma progression and resistance to BRAF pathway inhibition. Here we show that the lack of MITF is associated with more severe resistance to a range of inhibitors, while its presence is required for robust drug responses. Both in primary and acquired resistance, MITF levels inversely correlate with the expression of several activated receptor tyrosine kinases, most frequently AXL. The MITF-low/AXL-high/drug-resistance phenotype is common among mutant BRAF and NRAS melanoma cell lines. The dichotomous behaviour of MITF in drug response is corroborated in vemurafenib-resistant biopsies, including MITF-high and -low clones in a relapsed patient. Furthermore, drug cocktails containing AXL inhibitor enhance melanoma cell elimination by BRAF or ERK inhibition. Our results demonstrate that a low MITF/AXL ratio predicts early resistance to multiple targeted drugs, and warrant clinical validation of AXL inhibitors to combat resistance of BRAF and NRAS mutant MITF-low melanomas. Increased expression of MITF transcription factor is thought to promote melanoma progression and kinase inhibitor resistance. Here Muller et al . show that MITF loss is also common in melanomas and confers kinase inhibitor resistance due to upregulation of AXL and other receptor tyrosine kinases.

Significance MITF, a master regulator of melanocytes and a major melanoma oncogene amplified in 30-40% of melanomas, determines proliferative or invasive phenotypes. Previously unrecognized as a driver of lysosomal biogenesis, we found that MITF expression correlates with many lysosomal genes and generates late endosomes that are not functional in proteolysis. This accumulation of incomplete organelles expands the late endosomal compartment, enhancing Wnt signaling by entrapping the Wnt machinery in multivesicular bodies. Wnt signaling can stabilize many proteins besides β-catenin. Our study identifies MITF as an oncogenic protein stabilized by Wnt, and describes three novel glycogen synthase kinase 3-regulated phosphorylation sites in this oncogene. This study deepens our knowledge on proliferative stages of melanoma: MITF, multivesicular bodies, and Wnt may form a feedback loop that drives proliferation. Canonical Wnt signaling plays an important role in development and disease, regulating transcription of target genes and stabilizing many proteins phosphorylated by glycogen synthase kinase 3 (GSK3). We observed that the MiT family of transcription factors, which includes the melanoma oncogene MITF (micropthalmia-associated transcription factor) and the lysosomal master regulator TFEB, had the highest phylogenetic conservation of three consecutive putative GSK3 phosphorylation sites in animal proteomes. This finding prompted us to examine the relationship between MITF, endolysosomal biogenesis, and Wnt signaling. Here we report that MITF expression levels correlated with the expression of a large subset of lysosomal genes in melanoma cell lines. MITF expression in the tetracycline-inducible C32 melanoma model caused a marked increase in vesicular structures, and increased expression of late endosomal proteins, such as Rab7, LAMP1, and CD63. These late endosomes were not functional lysosomes as they were less active in proteolysis, yet were able to concentrate Axin1, phospho-LRP6, phospho-β-catenin, and GSK3 in the presence of Wnt ligands. This relocalization significantly enhanced Wnt signaling by increasing the number of multivesicular bodies into which the Wnt signalosome/destruction complex becomes localized upon Wnt signaling. We also show that the MITF protein was stabilized by Wnt signaling, through the novel C-terminal GSK3 phosphorylations identified here. MITF stabilization caused an increase in multivesicular body biosynthesis, which in turn increased Wnt signaling, generating a positive-feedback loop that may function during the proliferative stages of melanoma. The results underscore the importance of misregulated endolysosomal biogenesis in Wnt signaling and cancer.

The determination of individual cell trajectories through a high-dimensional cell-state space is an outstanding challenge for understanding biological changes ranging from cellular differentiation to epigenetic responses of diseased cells upon drugging. We integrate experiments and theory to determine the trajectories that single BRAF V600E mutant melanoma cancer cells take between drug-naive and drug-tolerant states. Although single-cell omics tools can yield snapshots of the cell-state landscape, the determination of individual cell trajectories through that space can be confounded by stochastic cell-state switching. We assayed for a panel of signaling, phenotypic, and metabolic regulators at points across 5 days of drug treatment to uncover a cell-state landscape with two paths connecting drug-naive and drug-tolerant states. The trajectory a given cell takes depends upon the drug-naive level of a lineage-restricted transcription factor. Each trajectory exhibits unique druggable susceptibilities, thus updating the paradigm of adaptive resistance development in an isogenic cell population. Detailed understanding of how cancer cells transition from a drug sensitive to a tolerant state is lacking. Here, using single cell proteomic and metabolic data the authors uncover that isogenic BRAF mutant melanoma cells can take two distinct paths to become tolerant to BRAF inhibition.

Phenotypic plasticity is associated with non-genetic drug tolerance in several cancers. Such plasticity can arise from chromatin remodeling, transcriptomic reprogramming, and/or protein signaling rewiring, and is characterized as a cell state transition in response to molecular or physical perturbations. This, in turn, can confound interpretations of drug responses and resistance development. Using BRAF-mutant melanoma cell lines as the prototype, we report on a joint theoretical and experimental investigation of the cell-state transition dynamics associated with BRAF inhibitor drug tolerance. Thermodynamically motivated surprisal analysis of transcriptome data was used to treat the cell population as an entropy maximizing system under the influence of time-dependent constraints. This permits the extraction of an epigenetic potential landscape for drug-induced phenotypic evolution. Single-cell flow cytometry data of the same system were modeled with a modified Fokker-Planck-type kinetic model. The two approaches yield a consistent picture that accounts for the phenotypic heterogeneity observed over the course of drug tolerance development. The results reveal that, in certain plastic cancers, the population heterogeneity and evolution of cell phenotypes may be understood by accounting for the competing interactions of the epigenetic potential landscape and state-dependent cell proliferation. Accounting for such competition permits accurate, experimentally verifiable predictions that can potentially guide the design of effective treatment strategies.

Background In melanoma, dysregulation of the MAPK pathway, usually via BRAF V600 or NRAS Q61 somatic mutations, leads to constitutive ERK signaling. While BRAF inhibitors are initially effective for BRAF -mutant melanoma, no FDA-approved targeted therapies exist for BRAF-inhibitor-resistant BRAF V600 , NRAS mutant, or wild-type melanoma. Methods The 50% inhibitory concentration (IC50) of SCH772984, a novel inhibitor of ERK1/2, was determined in a panel of 50 melanoma cell lines. Effects on MAPK and AKT signaling by western blotting and cell cycle by flow cytometry were determined. Results Sensitivity fell into three groups: sensitive, 50% inhibitory concentration (IC 50 ) < 1 μM; intermediately sensitive, IC 50 1-2 μM; and resistant, >2 μM. Fifteen of 21 (71%) BRAF mutants, including 4 with innate vemurafenib resistance, were sensitive to SCH772984. All three (100%) BRAF/NRAS double mutants, 11 of 14 (78%) NRAS mutants and 5 of 7 (71%) wild-type melanomas were sensitive. Among BRAF V600 mutants with in vitro acquired resistance to vemurafenib, those with MAPK pathway reactivation as the mechanism of resistance were sensitive to SCH772984. SCH772984 caused G1 arrest and induced apoptosis. Conclusions Combining vemurafenib and SCH722984 in BRAF mutant melanoma was synergistic in a majority of cell lines and significantly delayed the onset of acquired resistance in long term in vitro assays. Therefore, SCH772984 may be clinically applicable as a treatment for non- BRAF mutant melanoma or in BRAF -mutant melanoma with innate or acquired resistance, alone or in combination with BRAF inhibitors.

Background The clinical use of BRAF inhibitors for treatment of metastatic melanoma is limited by the development of drug resistance. In this study we investigated whether co-targeting the MAPK and the PI3K-AKT pathway can prevent emergence of resistance or provide additional growth inhibitory effects in vitro . Methods Anti-tumor effects of the combination of the BRAF inhibitor (BRAFi) dabrafenib and GSK2141795B (AKTi) in a panel of 23 BRAF mutated melanoma cell lines were evaluated on growth inhibition by an ATP-based luminescent assay, on cell cycle and apoptosis by flow cytometry and on cell signaling by western blot. Moreover, we investigated the possibilities of delaying or reversing resistance or achieving further growth inhibition by combining AKTi with dabrafenib and/or the MEK inhibitor (MEKi) trametinib by using long term cultures. Results More than 40% of the cell lines, including PTEN-/- and AKT mutants showed sensitivity to AKTi (IC 50  < 1.5 μM). The combination of dabrafenib and AKTi synergistically potentiated growth inhibition in the majority of cell lines with IC 50  > 5 nM dabrafenib. Combinatorial treatment induced apoptosis only in cell lines sensitive to AKTi. In long term cultures of a PTEN-/- cell line, combinatorial treatment with the MAPK inhibitors, dabrafenib and trametinib, and AKTi markedly delayed the emergence of drug resistance. Moreover, combining AKTi with the MAPK inhibitors from the beginning provided superior growth inhibitory effects compared to addition of AKTi upon development of resistance to MAPK inhibitors in this particular cell line. Conclusions AKTi combined with BRAFi-based therapy may benefit patients with tumors harboring BRAF mutations and particularly PTEN deletions or AKT mutations.

Patients with melanoma who are treated with a BRAF inhibitor have a high incidence of keratoacanthomas. Most of the tumors have oncogenic mutations in HRAS that probably preceded the use of the BRAF inhibitor. In animal models, second tumors are blocked when a MEK inhibitor is added to the BRAF inhibitor. The t→a transversion at position 1799 of BRAF ( BRAF V600E ) is present in approximately 50% of patients with metastatic melanoma. 1 , 2 BRAF V600E induces constitutive signaling through the mitogen-activated protein kinase (MAPK) pathway, stimulating cancer-cell proliferation and survival. 2 The clinical development of inhibitors of oncogenic BRAF, termed type I BRAF inhibitors, which block the active conformation of the BRAF kinase, has led to a high rate of objective tumor responses and improvement in overall survival, as compared with standard chemotherapy. 3 – 5 However, nonmelanoma skin cancers — well-differentiated cutaneous squamous-cell carcinomas and keratoacanthomas — have developed in approximately 15 . . .

The dynamics of T-cell responses are investigated in tumour tissue from patients with advanced melanoma who were treated with a PD-1-blocking monoclonal antibody, revealing that clinical efficacy of the treatment correlates with increased frequencies of pre-existing CD8 + T cells and PD-1 and PD-L1 expression. PD-1 blockade inhibits adaptive immune resistance Therapies that target the human cell-surface programmed death-1 (PD-1) receptor have shown unprecedented clinical responses in a variety of cancer types. Here Paul Tumeh et al . investigate the dynamics of T-cell responses in tumour tissues of patients with advanced melanoma treated with pembrolizumab, a humanized monoclonal antibody directed against human PD-1. Clinical efficacy is shown to correlate with increased frequencies of pre-existing CD8 + T cells and PD-1 and PD-L1 expression at the invasive tumour margin and within tumours. Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types 1 , 2 , 3 , 4 , 5 . One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8 + T cells (termed adaptive immune resistance) 6 , 7 . Here we show that pre-existing CD8 + T cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy. We analysed samples from 46 patients with metastatic melanoma obtained before and during anti-PD-1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next-generation sequencing for T-cell antigen receptors (TCRs). In serially sampled tumours, patients responding to treatment showed proliferation of intratumoral CD8 + T cells that directly correlated with radiographic reduction in tumour size. Pre-treatment samples obtained from responding patients showed higher numbers of CD8-, PD-1- and PD-L1-expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire. Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients. Our findings indicate that tumour regression after therapeutic PD-1 blockade requires pre-existing CD8 + T cells that are negatively regulated by PD-1/PD-L1-mediated adaptive immune resistance.

Continuous BRAF inhibition of BRAF mutant melanomas triggers a series of cell state changes that lead to therapy resistance and escape from immune control before establishing acquired resistance genetically. We used genome-wide transcriptomics and single-cell phenotyping to explore the response kinetics to BRAF inhibition for a panel of patient-derived BRAFV600 -mutant melanoma cell lines. A subset of plastic cell lines, which followed a trajectory covering multiple known cell state transitions, provided models for more detailed biophysical investigations. Markov modeling revealed that the cell state transitions were reversible and mediated by both Lamarckian induction and nongenetic Darwinian selection of drug-tolerant states. Single-cell functional proteomics revealed activation of certain signaling networks shortly after BRAF inhibition, and before the appearance of drug-resistant phenotypes. Drug targeting those networks, in combination with BRAF inhibition, halted the adaptive transition and led to prolonged growth inhibition in multiple patient-derived cell lines.