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Myeloid cells orchestrate the antitumor immune response and influence the efficacy of immune checkpoint blockade (ICB) therapies. We and others have previously shown that IL-32 mediates DC differentiation and macrophage activation. Here, we demonstrate that IL-32 expression in human melanoma positively correlates with overall survival, response to ICB, and an immune-inflamed tumor microenvironment (TME) enriched in mature DC, M1 macrophages, and CD8+ T cells. Treatment of B16F10 murine melanomas with IL-32 increased the frequencies of activated, tumor-specific CD8+ T cells, leading to the induction of systemic tumor immunity. Our mechanistic in vivo studies revealed a potentially novel role of IL-32 in activating intratumoral DC and macrophages to act in concert to prime CD8+ T cells and recruit them into the TME through CCL5. Thereby, IL-32 treatment reduced tumor growth and rendered ICB-resistant B16F10 tumors responsive to anti-PD-1 therapy without toxicity. Furthermore, increased baseline IL-32 gene expression was associated with response to nivolumab and pembrolizumab in 2 independent cohorts of patients with melanoma, implying that IL-32 is a predictive biomarker for anti-PD-1 therapy. Collectively, this study suggests IL-32 as a potent adjuvant in immunotherapy to enhance the efficacy of ICB in patients with non-T cell-inflamed TME.

Interleukin-2 (IL-2) is a component of most protocols of adoptive cell transfer (ACT) therapy for cancer, but is limited by short exposure and high toxicities. NKTR-214 is a kinetically-engineered IL-2 receptor βγ (IL-2Rβγ)-biased agonist consisting of IL-2 conjugated to multiple releasable polyethylene glycol chains resulting in sustained signaling through IL-2Rβγ. We report that ACT supported by NKTR-214 increases the proliferation, homing and persistence of anti-tumor T cells compared to ACT with IL-2, resulting in superior antitumor activity in a B16-F10 murine melanoma model. The use of NKTR-214 increases the number of polyfunctional T cells in murine spleens and tumors compared to IL-2, and enhances the polyfunctionality of T and NK cells in the peripheral blood of patients receiving NKTR-214 in a phase 1 trial. In conclusion, NKTR-214 may have the potential to improve the antitumor activity of ACT in humans through increased in vivo expansion and polyfunctionality of the adoptively transferred T cells.

PD-L1 and PD-L2 are ligands for the PD-1 immune inhibiting checkpoint that can be induced in tumors by interferon exposure, leading to immune evasion. This process is important for immunotherapy based on PD-1 blockade. We examined the specific molecules involved in interferon-induced signaling that regulates PD-L1 and PD-L2 expression in melanoma cells. These studies revealed that the interferon-gamma-JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis primarily regulates PD-L1 expression, with IRF1 binding to its promoter. PD-L2 responded equally to interferon beta and gamma and is regulated through both IRF1 and STAT3, which bind to the PD-L2 promoter. Analysis of biopsy specimens from patients with melanoma confirmed interferon signature enrichment and upregulation of gene targets for STAT1/STAT2/STAT3 and IRF1 in anti-PD-1-responding tumors. Therefore, these studies map the signaling pathway of interferon-gamma-inducible PD-1 ligand expression., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Therapeutic resistance is a major obstacle to achieving durable clinical responses with targeted therapies, highlighting a need to elucidate the underlying mechanisms responsible for resistance and identify strategies to overcome this challenge. An emerging body of data implicates the tyrosine kinase MET in mediating resistance to BRAF inhibitors in BRAFV600E mutant melanoma. In this study we observed a dominant role for the HGF/MET axis in mediating resistance to BRAF and MEK inhibitors in models of BRAFV600E and NRAS mutant melanoma. In addition, we showed that MAPK pathway inhibition induced rapid increases in MET and GAB1 levels, providing novel mechanistic insight into how BRAFV600E mutant melanoma is primed for HGF-mediated rescue. We also determined that tumor-derived HGF, not systemic HGF, may be required to convey resistance to BRAF inhibition in vivo and that resistance could be reversed following treatment with AMG 337, a selective MET inhibitor. In summary, these findings support the clinical evaluation of MET-directed targeted therapy to circumvent resistance to BRAF and MEK inhibitors in BRAFV600E mutant melanoma. In addition, the induction of MET following treatment with BRAF and MEK inhibitors has the potential to serve as a predictive biomarker for identifying patients best suited for MET inhibitor combination therapy.

The programmed cell death protein 1 (PD-1) limits effector T-cell functions in peripheral tissues, and its inhibition leads to clinical benefit in different cancers. To better understand how PD-1 blockade therapy modulates the tumor-host interactions, we evaluated three syngeneic murine tumor models, the BRAF V600E -driven YUMM1.1 and YUMM2.1 melanomas, and the carcinogen-induced murine colon adenocarcinoma MC38. The YUMM cell lines were established from mice with melanocyte-specific BRAF V600E mutation and PTEN loss (BRAF V600E /PTEN -/- ). Anti-PD-1 or anti-PD-L1 therapy engendered strong antitumor activity against MC38 and YUMM2.1, but not YUMM1.1. PD-L1 expression did not differ between the three models at baseline or upon interferon stimulation. Whereas mutational load was high in MC38, it was lower in both YUMM models. In YUMM2.1, the antitumor activity of PD-1 blockade had a critical requirement for both CD4 and CD8 T cells, as well as CD28 and CD80/86 costimulation, with an increase in CD11c + CD11b + MHC-II high dendritic cells and tumor-associated macrophages in the tumors after PD-1 blockade. Compared with YUMM1.1, YUMM2.1 exhibited a more inflammatory profile by RNA sequencing analysis, with an increase in expression of chemokine-trafficking genes that are related to immune cell recruitment and T-cell priming. In conclusion, response to PD-1 blockade therapy in tumor models requires CD4 and CD8 T cells and costimulation that is mediated by dendritic cells and macrophages. Cancer Immunol Res; 4(10); 845-57. ©2016 AACR., Competing Interests: The authors declare no conflicts of interest., (©2016 American Association for Cancer Research.)

Immune-regulatory mechanisms are used by cancer to hide from the immune system. Advances and in-depth understanding of the biology of melanoma and its interaction with the immune system have led to the development of some of antagonistic antibodies to the programmed death 1 pathway (PD-1) and one of its ligands, programmed death ligand 1 (PD-L1), which are demonstrating high clinical benefit rates and tolerability. Blocking the immune-regulatory checkpoints that limit T-cell responses to melanoma upon PD-1/PD-L1 modulation has provided clinically validated targets for cancer immunotherapy. Combinations with other anti-melanoma agents may result in additional benefits. Nivolumab, pembrolizumab (formerly known as MK-3475 and lambrolizumab), and pidilizumab are anti-PD-1 antibodies in clinical development for melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck cancers, lymphoma, and several other cancers. Long-term survivors already have been reported with these therapies. In this review, we discuss the current state of anti-PD-1 agents, the evidence in the literature to support the combination of anti-PD-1 antibodies with other anti-cancer agents and discuss the future directions for rational design of clinical trials that keep on increasing the number of long-term survivors., (Copyright © 2015. Published by Elsevier Inc.)

Recurrent metastatic melanoma provides a unique opportunity to analyze disease evolution in metastatic cancer. Here, we followed up eight patients with an unusually prolonged history of metastatic melanoma, who developed a total of 26 recurrences over several years. Cell lines derived from each metastasis were analyzed by comparative genomic hybridization and global transcript analysis. We observed that conserved, patient-specific characteristics remain stable in recurrent metastatic melanoma even after years and several recurrences. Differences among individual patients exceeded within-patient lesion variability, both at the DNA copy number (P<0.001) and RNA gene expression level (P<0.001). Conserved patient-specific traits included expression of several cancer/testis antigens and the c-kit proto-oncogene throughout multiple recurrences. Interestingly, subsequent recurrences of different patients did not display consistent or convergent changes toward a more aggressive disease phenotype. Finally, sequential recurrences of the same patient did not descend progressively from each other, as irreversible mutations such as homozygous deletions were frequently not inherited from previous metastases. This study suggests that the late evolution of metastatic melanoma, which markedly turns an indolent disease into a lethal phase, is prone to preserve case-specific traits over multiple recurrences and occurs through a series of random events that do not follow a consistent stepwise process.

Prognostic molecular markers are urgently needed for allowing to discriminate the clinical course of disease of melanoma patients, which is highly heterogeneous and unpredictable also within a specific clinicopathological stage and substage of disease. Alterations in DNA methylation have been reported to be widely present in cutaneous melanoma, profoundly impacting its biology. In line with this notion, we have identified methylation markers as independent prognostic factors in stage IIIC melanoma patients. In this chapter we describe the measurement of the methylation of the Long Interspersed Nucleotide Element-1 sequences in laser capture microdissected tumor tissues as a prognostic tool in stage III melanoma patients, which could help in achieving a more appropriate and patient-tailored clinical management of cutaneous melanoma.

Malignant melanoma is a complex disease that arises and evolves due to a myriad of genetic and epigenetic events. Among these, the interaction between epigenetic alterations (i.e., histone modifications, DNA methylation, mRNA silencing by miRNAs and nucleosome repositioning) has been recently identified as playing an important role in melanoma development and progression by affecting key cellular pathways such as cell cycle regulation, DNA repair, apoptosis, invasion and immune recognition. Differently to genetic lesions, epigenetic changes are potentially pharmacologically reversible by using epigenetic drugs. Along this line, preclinical and clinical findings indicate that these drugs, given alone or in combination therapies, can efficiently modulate the immunophenotype of melanoma cells. The aim of this review is to provide a comprehensive summary of melanoma epigenetics and the current use of epigenetic drugs in the clinical setting.

Background: The clinical course of cutaneous melanoma (CM) can differ significantly for patients with identical stages of disease, defined clinico-pathologically, and no molecular markers differentiate patients with such a diverse prognosis. This study aimed to define the prognostic value of whole genome DNA methylation profiles in stage III CM., Methods: Genome-wide methylation profiles were evaluated by the Illumina Human Methylation 27 BeadChip assay in short-term neoplastic cell cultures from 45 stage IIIC CM patients. Unsupervised K-means partitioning clustering was exploited to sort patients into 2 groups based on their methylation profiles. Methylation patterns related to the discovered groups were determined using the nearest shrunken centroid classification algorithm. The impact of genome-wide methylation patterns on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analyses., Results: Unsupervised K-means partitioning by whole genome methylation profiles identified classes with significantly different OS in stage IIIC CM patients. Patients with a "favorable" methylation profile had increased OS (P = 0.001, log-rank = 10.2) by Kaplan-Meier analysis. Median OS of stage IIIC patients with a "favorable" vs. "unfavorable" methylation profile were 31.5 and 10.4 months, respectively. The 5 year OS for stage IIIC patients with a "favorable" methylation profile was 41.2% as compared to 0% for patients with an "unfavorable" methylation profile. Among the variables examined by multivariate Cox regression analysis, classification defined by methylation profile was the only predictor of OS (Hazard Ratio = 2.41, for "unfavorable" methylation profile; 95% Confidence Interval: 1.02-5.70; P = 0.045). A 17 gene methylation signature able to correctly assign prognosis (overall error rate = 0) in stage IIIC patients on the basis of distinct methylation-defined groups was also identified., Conclusions: A discrete whole-genome methylation signature has been identified as molecular marker of prognosis for stage IIIC CM patients. Its use in daily practice is foreseeable, and promises to refine the comprehensive clinical management of stage III CM patients.

Background: The prognosis of cutaneous melanoma (CM) differs for patients with identical clinico-pathological stage, and no molecular markers discriminating the prognosis of stage III individuals have been established. Genome-wide alterations in DNA methylation are a common event in cancer. This study aimed to define the prognostic value of genomic DNA methylation levels in stage III CM patients., Methods: Overall level of genomic DNA methylation was measured using bisulfite pyrosequencing at three CpG sites (CpG1, CpG2, CpG3) of the Long Interspersed Nucleotide Element-1 (LINE-1) sequences in short-term CM cultures from 42 stage IIIC patients. The impact of LINE-1 methylation on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analysis., Results: Hypomethylation (i.e., methylation below median) at CpG2 and CpG3 sites significantly associated with improved prognosis of CM, CpG3 showing the strongest association. Patients with hypomethylated CpG3 had increased OS (P = 0.01, log-rank = 6.39) by Kaplan-Meyer analysis. Median OS of patients with hypomethylated or hypermethylated CpG3 were 31.9 and 11.5 months, respectively. The 5 year OS for patients with hypomethylated CpG3 was 48% compared to 7% for patients with hypermethylated sequences. Among the variables examined by Cox regression analysis, LINE-1 methylation at CpG2 and CpG3 was the only predictor of OS (Hazard Ratio = 2.63, for hypermethylated CpG3; 95% Confidence Interval: 1.21-5.69; P = 0.01)., Conclusion: LINE-1 methylation is identified as a molecular marker of prognosis for CM patients in stage IIIC. Evaluation of LINE-1 promises to represent a key tool for driving the most appropriate clinical management of stage III CM patients.

Cutaneous melanoma is a very aggressive neoplasia of melanocytic origin with constantly growing incidence and mortality rates world-wide. Epigenetic modifications (i.e., alterations of genomic DNA methylation patterns, of post-translational modifications of histones, and of microRNA profiles) have been recently identified as playing an important role in melanoma development and progression by affecting key cellular pathways such as cell cycle regulation, cell signalling, differentiation, DNA repair, apoptosis, invasion and immune recognition. In this scenario, pharmacologic inhibition of DNA methyltransferases and/or of histone deacetylases were demonstrated to efficiently restore the expression of aberrantly-silenced genes, thus re-establishing pathway functions. In light of the pleiotropic activities of epigenetic drugs, their use alone or in combination therapies is being strongly suggested, and a particular clinical benefit might be expected from their synergistic activities with chemo-, radio-, and immuno-therapeutic approaches in melanoma patients. On this path, an important improvement would possibly derive from the development of new generation epigenetic drugs characterized by much reduced systemic toxicities, higher bioavailability, and more specific epigenetic effects.

Interleukin-2 (IL-2) is a cytokine required for effector T cell expansion, survival, and function, especially for engineered T cells in adoptive cell immunotherapy, but its pleiotropy leads to simultaneous stimulation and suppression of immune responses as well as systemic toxicity, limiting its therapeutic use. We engineered IL-2 cytokine-receptor orthogonal (ortho) pairs that interact with one another, transmitting native IL-2 signals, but do not interact with their natural cytokine and receptor counterparts. Introduction of orthoIL-2Rβ into T cells enabled the selective cellular targeting of orthoIL-2 to engineered CD4+ and CD8+ T cells in vitro and in vivo, with limited off-target effects and negligible toxicity. OrthoIL-2 pairs were efficacious in a preclinical mouse cancer model of adoptive cell therapy and may therefore represent a synthetic approach to achieving selective potentiation of engineered cells.

Loss-of-function mutations in JAK1/2 can lead to acquired resistance to anti-programmed death protein 1 (PD-1) therapy. We reasoned that they may also be involved in primary resistance to anti-PD-1 therapy. JAK1/2-inactivating mutations were noted in tumor biopsies of 1 of 23 patients with melanoma and in 1 of 16 patients with mismatch repair-deficient colon cancer treated with PD-1 blockade. Both cases had a high mutational load but did not respond to anti-PD-1 therapy. Two out of 48 human melanoma cell lines had JAK1/2 mutations, which led to a lack of PD-L1 expression upon interferon gamma exposure mediated by an inability to signal through the interferon gamma receptor pathway. JAK1/2 loss-of-function alterations in The Cancer Genome Atlas confer adverse outcomes in patients. We propose that JAK1/2 loss-of-function mutations are a genetic mechanism of lack of reactive PD-L1 expression and response to interferon gamma, leading to primary resistance to PD-1 blockade therapy.SignificanceA key functional result from somatic JAK1/2 mutations in a cancer cell is the inability to respond to interferon gamma by expressing PD-L1 and many other interferon-stimulated genes. These mutations result in a genetic mechanism for the absence of reactive PD-L1 expression, and patients harboring such tumors would be unlikely to respond to PD-1 blockade therapy. Cancer Discov; 7(2); 188-201. ©2016 AACR.See related commentary by Marabelle et al., p. 128This article is highlighted in the In This Issue feature, p. 115.

The programmed cell death protein 1 (PD-1) limits effector T-cell functions in peripheral tissues, and its inhibition leads to clinical benefit in different cancers. To better understand how PD-1 blockade therapy modulates the tumor-host interactions, we evaluated three syngeneic murine tumor models, the BRAFV600E-driven YUMM1.1 and YUMM2.1 melanomas, and the carcinogen-induced murine colon adenocarcinoma MC38. The YUMM cell lines were established from mice with melanocyte-specific BRAFV600E mutation and PTEN loss (BRAFV600E/PTEN-/-). Anti-PD-1 or anti-PD-L1 therapy engendered strong antitumor activity against MC38 and YUMM2.1, but not YUMM1.1. PD-L1 expression did not differ between the three models at baseline or upon interferon stimulation. Whereas mutational load was high in MC38, it was lower in both YUMM models. In YUMM2.1, the antitumor activity of PD-1 blockade had a critical requirement for both CD4 and CD8 T cells, as well as CD28 and CD80/86 costimulation, with an increase in CD11c+CD11b+MHC-IIhigh dendritic cells and tumor-associated macrophages in the tumors after PD-1 blockade. Compared with YUMM1.1, YUMM2.1 exhibited a more inflammatory profile by RNA sequencing analysis, with an increase in expression of chemokine-trafficking genes that are related to immune cell recruitment and T-cell priming. In conclusion, response to PD-1 blockade therapy in tumor models requires CD4 and CD8 T cells and costimulation that is mediated by dendritic cells and macrophages. Cancer Immunol Res; 4(10); 845-57. ©2016 AACR.

Surgical resection represents the treatment of choice for primary melanoma, providing the best results, in terms of long-term survival, for thin lesions. However, surgery has limited efficacy in the management of locoregional and distant metastases, as do chemotherapy and radiotherapy. In this context, immunotherapy is a promising and additional therapeutic option to cure melanoma patients and to prevent relapse of the disease. The identification and characterization of immunogenic tumor-associated antigens (TAAs) has prompted the design and development of a plethora of therapeutic vaccines to drive the host immune system to specifically recognize and eradicate neoplastic cells. Although encouraging, clinical results obtained with TAA-based vaccination are not yet satisfactory. In this regard, a great number of experimental data indicate that neoplastic cells are capable of escaping the host's immune surveillance and impairing the efficacy of immunotherapeutic approaches by different mechanisms. Among these, the epigenetic editing of gene-expression patterns by aberrant DNA hypermethylation and/or histone deacetylation is emerging as a frequent and important tool employed by melanoma cells to trigger the downregulation of the components of the tumor presentation/recognition complex. This review will summarize the available data derived from preclinical and clinical research using epigenetic drugs, such as histone deacetylase and DNA methyltransferase inhibitors, to revert epigenetic alterations, and the current knowledge of the potential immunotherapeutic implications of epigenetic treatment in the melanoma clinic. [ABSTRACT FROM AUTHOR]