Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types.1–5 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 analyzed samples from 46 patients with metastatic melanoma obtained before and during anti-PD1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next generation sequencing for T-cell receptors (TCR). In serially sampled tumours, responding patients showed proliferation of intratumoural CD8+ T-cells that directly correlated with radiographic reduction in tumour size. Pre-treatment samples obtained from responding patients showed higher numbers of CD8, PD1, 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 following therapeutic PD-1 blockade requires pre-existing CD8+ T cells that are negatively regulated by PD-1/PD-L1 mediated adaptive immune resistance.

Malignant transformation can result in melanoma cells that resemble different stages of their embryonic development. Our gene expression analysis of human melanoma cell lines and patient tumors revealed that melanoma follows a two-dimensional differentiation trajectory that can be subclassified into four progressive subtypes. This differentiation model is associated with subtype-specific sensitivity to iron-dependent oxidative stress and cell death known as ferroptosis. Receptor tyrosine kinase-mediated resistance to mitogen-activated protein kinase targeted therapies and activation of the inflammatory signaling associated with immune therapy involves transitions along this differentiation trajectory, which lead to increased sensitivity to ferroptosis. Therefore, ferroptosis-inducing drugs present an orthogonal therapeutic approach to target the differentiation plasticity of melanoma cells to increase the efficacy of targeted and immune therapies.

BACKGROUND Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors. METHODS We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed. RESULTS Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L–mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)–pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L–mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor. CONCLUSIONS Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann–La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.)

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.

Combining immunotherapy and BRAF targeted therapy may result in improved antitumor activity with the high response rates of targeted therapy and the durability of responses with immunotherapy. However, the first clinical trial testing the combination of the BRAF inhibitor vemurafenib and the CTLA-4 antibody ipilimumab was terminated early due to substantial liver toxicities. MEK inhibitors can potentiate the MAPK inhibition in BRAF mutant cells, while potentially alleviating the unwanted paradoxical MAPK activation in BRAF wild type cells that lead to side effects when using BRAF inhibitors alone. However, there is the concern of MEK inhibitors being detrimental to T cell functionality. Using a mouse model of syngeneic BRAFV600E driven melanoma, we tested whether addition of the MEK inhibitor trametinib would enhance the antitumor activity of combined immunotherapy with the BRAF inhibitor dabrafenib. Combination of dabrafenib and trametinib with pmel-1 adoptive cell transfer (ACT) showed complete tumor regression, increased T cell infiltration into tumors and improved in vivo cytotoxicity. Single agent dabrafenib increased tumor-associated macrophages and T regulatory cells (Tregs) in tumors, which decreased with the addition of trametinib. The triple combination therapy resulted in increased melanosomal antigen and MHC expression, and global immune-related gene up-regulation. Given the up-regulation of PD-L1 seen with dabrafenib and/or trametinib combined with antigen-specific ACT, we tested combination of dabrafenib, trametinib with anti-PD1 therapy in SM1 tumors, and observed superior anti-tumor effect. Our findings support the testing of triple combination therapy of BRAF and MEK inhibitors with immunotherapy in patients with BRAFV600E mutant metastatic melanoma.