Managing leptomeningeal melanoma metastases in the era of immune and targeted therapy.

Melanoma frequently metastasizes to the brain, with CNS involvement being clinically evident in ∼30% of patients (as high as 75% at autopsy). In ∼5% cases melanoma cells also metastasize to the leptomeninges, the sub-arachnoid space and cerebrospinal fluid (CSF). Patients with leptomeningeal melanoma metastases (LMM) have the worst prognosis and are characterized by rapid disease progression (mean survival 8-10 weeks) and a death from neurological causes. The recent years have seen tremendous progress in the development of targeted and immune therapies for melanoma that has translated into an increased survival benefit. Despite these gains, the majority of patients fail therapy and there is a suspicion that the brain and the leptomeninges are a "sanctuary" sites for melanoma cells that escape both targeted therapy and immunologic therapies. Emerging evidence suggests that (1) Cancer cells migrating to the CNS may have unique molecular properties and (2) the CNS/leptomeningeal microenvironment represents a pro-survival niche that influences therapeutic response. In this Mini-Review, we will outline the clinical course of LMM development and will describe how the intracranial immune and cellular microenvironments offer both opportunities and challenges for the successful management of this disease. We will further discuss the latest data demonstrating the potential use of BRAF inhibitors and immune therapy in the management of LMM, and will review future potential therapeutic strategies for the management of this most devastating complication of advanced melanoma.

Evaluating melanoma drug response and therapeutic escape with quantitative proteomics.

The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 µg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor ß signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic.

Phosphoproteomic analysis of basal and therapy-induced adaptive signaling networks in BRAF and NRAS mutant melanoma.

Basal and kinase inhibitor driven adaptive signaling has been examined in a panel of melanoma cell lines using phosphoproteomics in conjunction with pathway analysis. A considerable divergence in the spectrum of tyrosine-phosphorylated peptides was noted at the cell line level. The unification of genotype-specific cell line data revealed the enrichment for the tyrosine-phosphorylated cytoskeletal proteins to be associated with the presence of a BRAF mutation and oncogenic NRAS to be associated with increased receptor tyrosine kinase phosphorylation. A number of proteins including cell cycle regulators (cyclin dependent kinase 1, cyclin dependent kinase 2, and cyclin dependent kinase 3), MAPK pathway components (Extracellular signal regulated kinase 1 and Extracellular signal regulated kinase 2), interferon regulators (tyrosine kinase-2), GTPase regulators (Ras-Rasb interactor 1), and controllers of protein tyrosine phosphorylation (dual specificity tyrosine (Y) phosphorylation regulated kinase 1A and protein tyrosine phosphatase receptor type A) were common to all genotypes. Treatment of a BRAF-mutant/phosphatase and tensin homologue (PTEN) null melanoma cell line with vemurafenib led to decreased phosphorylation of ERK, phospholipase C1, and ß-catenin with increases in receptor tyrosine kinase phosphorylation, signal transduction and activator of signaling 3, and glycogen synthase kinase 3α noted. In NRAS-mutant melanoma, MEK inhibition led to increased phosphorylation of epidermal growth factor receptor signaling pathway components, Src family kinases, and protein kinase Cδ with decreased phosphorylation seen in STAT3 and ERK1/2. Together these data present the first systems level view of adaptive and basal phosphotyrosine signaling in BRAF- and NRAS-mutant melanoma.

Melanoma genotypes and phenotypes get personal.

Traditionally, the diagnosis of metastatic melanoma was terminal to most patients. However, the advancements towards understanding the fundamental etiology, pathophysiology, and treatment have raised melanoma to the forefront of contemporary medicine. Indeed, the evidence of durable remissions are being heard ever more frequently in clinics around the globe. Despite having more gene mutations per cell than any other type of cancer, investigators are overcoming complex genomic landscapes, signaling pathways, and immune checkpoints by generating novel technological methods and clinical protocols with breath-taking speed. Significant progress in deciphering molecular genetics, epigenetics, kinase-driven networks, metabolomics, and immune-enhancing pathways to achieve personalized and positive outcomes has truly provided new hope for melanoma patients. However, obstacles requiring breakthroughs include understanding the influence of sunlight exposure on melanoma etiology, and overcoming all too frequently acquired drug resistance, complicating targeted therapy. Pathologists continue to have critically important roles in advancing the field, particularly in the area of transitioning from microscope-based diagnostic reports to pharmacogenomics through molecularly informed tumor boards. Although melanoma is no longer considered just 'one disease', pathologists will continue this rapidly progressing and exciting journey to identify tumor subtypes, to utilize tumorgraft or so-called patient-derived xenograft (PDX) models, and to develop companion diagnostics to keep pace with the bewildering breakthroughs occurring on a regular basis. Exactly which combination of drugs will ultimately be required to eradicate melanoma cells remains to be determined. However, it is clear that pathologists who are as dedicated to melanoma as the pioneering pathologist Dr Sidney Farber was committed to childhood cancers, will be required as the battle against melanoma continues. In this review, we describe what sets melanoma apart from other tumors, and demonstrate how lessons learned in the melanoma clinic are being transferred to many other types of aggressive neoplasms.

A rare case of leptomeningeal carcinomatosis in a patient with uveal melanoma: case report and review of literature.

Uveal melanoma is a rare subtype of melanoma, accounting for only 3-5% of all melanoma cases in the USA. Although fewer than 4% of uveal melanoma patients present with metastasis at diagnosis, approximately half will develop metastasis, more than 90% of which disseminate to the liver. Infrequently, a number of malignancies can lead to leptomeningeal metastases, a devastating and terminal complication. In this case report, we present an exceedingly rare case of a patient with uveal melanoma who developed leptomeningeal carcinomatosis as the sole site of metastasis. After conventional methods to diagnose leptomeningeal carcinomatosis fell short, a diagnosis was confirmed on the basis of identification and genomic analysis of melanoma circulating tumor cells in the cerebrospinal fluid.

Inhibition of BRAF and BRAF+MEK drives a metastatic switch in melanoma.

Recent analyses by our group and others showed that the majority of melanoma patients who fail BRAF inhibitor therapy do so at new disease sites. Using phosphoproteomics we showed that BRAF inhibition mediates a switch to an aggressive/metastatic melanoma phenotype that is driven by ligand-independent erythropoietin-producing hepatocellular receptor A2 (EphA2) signaling.

BRAF Inhibition Generates a Host-Tumor Niche that Mediates Therapeutic Escape.

The current study defines a fibroblast-derived niche that facilitates the therapeutic escape of melanoma cells from BRAF inhibition. Vemurafenib treatment led to the release of transforming growth factor-ß (TGF-ß) from the melanoma cells that increased the differentiation state of the fibroblasts, an affect associated with fibronectin deposition, increase in α-smooth muscle actin expression, and the release of neuregulin (NRG). At the same time, vemurafenib directly activated the fibroblasts through paradoxical stimulation of the mitogen-activated protein kinase pathway, causing them to secrete hepatocyte growth factor (HGF). Treatment with the BRAF/MEK inhibitor combination reversed the release of HGF. Adhesion of melanoma cells to fibronectin was critical in amplifying the fibroblast-derived NRG- and HGF-mediated PI3K/AKT (phosphatidylinositol 3'-kinase/AKT) survival signaling in the melanoma cells following BRAF inhibition. In coculture studies, combination treatment with inhibitors of BRAF/MET/HER kinase was ineffective at reversing the fibroblast-mediated therapeutic escape from BRAF inhibition. Instead, it was noted that combined BRAF/PI3K inhibition overcame fibroblast-mediated drug resistance in vitro and was associated with enhanced antitumor effects in an in vivo xenograft model. Thus, we show that melanoma cells and fibroblasts remodel their microenvironment in response to BRAF inhibition and that these adaptations allow tumor cells to evade therapy through increased PI3K/AKT survival signaling.

Ligand-independent EPHA2 signaling drives the adoption of a targeted therapy-mediated metastatic melanoma phenotype.

UNLABELLED: Many patients with BRAF inhibitor resistance can develop disease at new sites, suggesting that drug-induced selection pressure drives metastasis. Here, we used mass spectrometry-based phosphoproteomic screening to uncover ligand-independent EPHA2 signaling as an adaptation to BRAF inhibitor therapy that led to the adoption of a metastatic phenotype. The EPHA2-mediated invasion was AKT-dependent and readily reversible upon removal of the drug as well as through PI3K and AKT inhibition. In xenograft models, BRAF inhibition led to the development of EPHA2-positive metastases. A retrospective analysis of patients with melanoma on BRAF inhibitor therapy showed that 68% of those failing therapy develop metastases at new disease sites, compared with 35% of patients on dacarbazine. Further IHC staining of melanoma specimens taken from patients on BRAF inhibitor therapy as well as metastatic samples taken from patients failing therapy showed increased EPHA2 staining. We suggest that inhibition of ligand-independent EPHA2 signaling may limit metastases associated with BRAF inhibitor therapy. SIGNIFICANCE: This study provides evidence that BRAF inhibition promotes the adoption of a reversible, therapy-driven metastatic phenotype in melanoma. The cotargeting of ligand-independent EPHA2 signaling and BRAF may be one strategy to prevent the development of therapy-mediated disease at new sites.

Long-term effects of BRAF inhibitors in melanoma treatment: friend or foe?

The clinical development of selective BRAF inhibitors for metastatic BRAF V600 mutant melanoma patients has been a major breakthrough in targeted therapeutics. Objective response rates of approximately 50% have been observed in the Phase III studies of the BRAF inhibitors vemurafenib and dabrafenib. The side effects can be relatively common, including proliferative skin toxicities. The latter range from hyperkeratosis and keratoacanthomas (KAs) to squamous cell carcinomas (SCCs) and new primary melanomas. In addition, case reports on the emergence of gastric/colonic polyps and RAS mutant malignancies have been described during BRAF inhibitor therapy. These events have been attributed to paradoxical activation of the MAPK pathway in BRAF wild-type cells exposed to selective BRAF inhibitors in addition to increased RAS activity. Combined BRAF and MEK inhibition appears to improve clinical outcomes and reduce cutaneous proliferation events as fewer KAs and SCCs have been observed with combination therapy. Next-generation pan-RAF inhibitors ('paradox breakers') and ERK inhibitors may further enhance clinical activity in metastatic BRAF-mutant melanoma patients and mitigate this paradoxical oncogenesis. Further investigation into the potential long-term effects of selective BRAF inhibitors is warranted as expanded use of these agents is expected in patients with BRAF-mutant melanoma and other malignancies.

Amuvatinib has cytotoxic effects against NRAS-mutant melanoma but not BRAF-mutant melanoma.

Effective targeted therapy strategies are still lacking for the 15-20% of melanoma patients whose melanomas are driven by oncogenic NRAS. Here, we report on the NRAS-specific behavior of amuvatinib, a kinase inhibitor with activity against c-KIT, Axl, PDGFRα, and Rad51. An analysis of BRAF-mutant and NRAS-mutant melanoma cell lines showed the NRAS-mutant cohort to be enriched for targets of amuvatinib, including Axl, c-KIT, and the Axl ligand Gas6. Increasing concentrations of amuvatinib selectively inhibited the growth of NRAS-mutant, but not BRAF-mutant melanoma cell lines, an effect associated with induction of S-phase and G2/M-phase cell cycle arrest and induction of apoptosis. Mechanistically, amuvatinib was noted to either inhibit Axl, AKT, and MAPK signaling or Axl and AKT signaling and to induce a DNA damage response. In three-dimensional cell culture experiments, amuvatinib was cytotoxic against NRAS-mutant melanoma cell lines. Thus, we show for the first time that amuvatinib has proapoptotic activity against melanoma cell lines, with selectivity observed for those harboring oncogenic NRAS.

Inhibition of autophagy enhances the effects of the AKT inhibitor MK-2206 when combined with paclitaxel and carboplatin in BRAF wild-type melanoma.

This study investigates the mechanism of action behind the long-term responses (12-16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK-2206 in combination with paclitaxel and carboplatin. Although single agent MK-2206 inhibited phospho-AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK-2206 with paclitaxel and carboplatin was cytotoxic in long-term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase-dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti-oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs.

Paradoxical oncogenesis--the long-term effects of BRAF inhibition in melanoma.

The clinical benefits of BRAF inhibition in patients with advanced-stage BRAF-mutant melanoma are now well established. Although the emergence of cutaneous squamous-cell carcinomas (SCCs) and secondary melanomas in patients on BRAF-inhibitor therapy have been well described, reports are emerging of additional secondary premalignant and malignant events, including RAS-mutant leukaemia, the metastatic recurrence of RAS-mutant colorectal cancer and the development of gastric and colonic polyps. In most cases, paradoxical MAPK activation--resulting from the BRAF-inhibitor-mediated homodimerization and heterodimerization of nonmutant RAF isoforms--seems to underlie the development of these secondary tumours. Although evidence supports that therapy with the simultaneous administration of BRAF and MEK inhibitors abrogates the onset of treatment-induced SCCs, whether combination treatment will limit the emergence of all BRAF-inhibitor-driven pathologies is unclear. In this Review, we describe the clinical and mechanistic manifestations of secondary cancers that have thus far been observed to arise as a consequence of BRAF inhibition. We discuss the concept of pre-existing populations of partly transformed cells with malignant potential that might be present in various organ systems, and the rationale for novel therapeutic strategies for the management of BRAF-inhibitor-induced neoplasia.

Senescent fibroblasts in melanoma initiation and progression: an integrated theoretical, experimental, and clinical approach.

We present an integrated study to understand the key role of senescent fibroblasts in driving melanoma progression. Based on the hybrid cellular automata paradigm, we developed an in silico model of normal skin. The model focuses on key cellular and microenvironmental variables that regulate interactions among keratinocytes, melanocytes, and fibroblasts, key components of the skin. The model recapitulates normal skin structure and is robust enough to withstand physical as well as biochemical perturbations. Furthermore, the model predicted the important role of the skin microenvironment in melanoma initiation and progression. Our in vitro experiments showed that dermal fibroblasts, which are an important source of growth factors in the skin, adopt a secretory phenotype that facilitates cancer cell growth and invasion when they become senescent. Our coculture experiments showed that the senescent fibroblasts promoted the growth of nontumorigenic melanoma cells and enhanced the invasion of advanced melanoma cells. Motivated by these experimental results, we incorporated senescent fibroblasts into our model and showed that senescent fibroblasts transform the skin microenvironment and subsequently change the skin architecture by enhancing the growth and invasion of normal melanocytes. The interaction between senescent fibroblasts and the early-stage melanoma cells leads to melanoma initiation and progression. Of microenvironmental factors that senescent fibroblasts produce, proteases are shown to be one of the key contributing factors that promoted melanoma development from our simulations. Although not a direct validation, we also observed increased proteolytic activity in stromal fields adjacent to melanoma lesions in human histology. This leads us to the conclusion that senescent fibroblasts may create a prooncogenic skin microenvironment that cooperates with mutant melanocytes to drive melanoma initiation and progression and should therefore be considered as a potential future therapeutic target. Interestingly, our simulations to test the effects of a stroma-targeting therapy that negates the influence of proteolytic activity showed that the treatment could be effective in delaying melanoma initiation and progression.

Acquired and intrinsic BRAF inhibitor resistance in BRAF V600E mutant melanoma.

The discovery of activating BRAF V600E mutations in 50% of all cutaneous melanomas has revolutionized the understanding of melanoma biology and provided new strategies for the therapeutic management of this deadly disease. Highly potent small molecule inhibitors of BRAF are now showing great promise as a novel therapeutic strategy for melanomas harboring activating BRAF V600E mutations and are associated with high levels of response. This commentary article discusses the latest data on the role of mutated BRAF in the development and progression of melanoma as the basis for understanding the mechanism of action of BRAF inhibitors in the preclinical and clinical settings. We further address the issue of BRAF inhibitor resistance and outline the latest insights into the mechanisms of therapeutic escape as well as describing approaches to prevent and abrogate the onset of both intrinsic and acquired drug resistance. It is likely that our evolving understanding of melanoma genetics and signaling will allow for the further personalization of melanoma therapy with the goal of improving clinical responses.

PTEN loss confers BRAF inhibitor resistance to melanoma cells through the suppression of BIM expression.

This study addresses the role of PTEN loss in intrinsic resistance to the BRAF inhibitor PLX4720. Immunohistochemical staining of a tissue array covering all stages of melanocytic neoplasia (n = 192) revealed PTEN expression to be lost in >10% of all melanoma cases. Although PTEN expression status did not predict for sensitivity to the growth inhibitory effects of PLX4720, it was predictive for apoptosis, with only limited cell death observed in melanomas lacking PTEN expression (PTEN-). Mechanistically, PLX4720 was found to stimulate AKT signaling in the PTEN- but not the PTEN+ cell lines. Liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM) was performed to identify differences in apoptosis signaling between the two cell line groups. PLX4720 treatment significantly increased BIM expression in the PTEN+ (>14-fold) compared with the PTEN- cell lines (four-fold). A role for PTEN in the regulation of PLX4720-mediated BIM expression was confirmed by siRNA knockdown of PTEN and through reintroduction of PTEN into cells that were PTEN-. Further studies showed that siRNA knockdown of BIM significantly blunted the apoptotic response in PTEN+ melanoma cells. Dual treatment of PTEN- cells with PLX4720 and a PI3K inhibitor enhanced BIM expression at both the mRNA and protein level and increased the level of apoptosis through a mechanism involving AKT3 and the activation of FOXO3a. In conclusion, we have shown for the first time that loss of PTEN contributes to intrinsic BRAF inhibitor resistance via the suppression of BIM-mediated apoptosis.

Enhanced imaging and accelerated photothermalysis of A549 human lung cancer cells by gold nanospheres.

BACKGROUND & AIMS: Gold nanoparticles are excellent photon-thermal energy converters. The purpose of this work was to investigate the influence of gold nanoparticles on the photothermalysis of A549 lung tumor cells. MATERIALS & METHODS: A549 lung tumor cells were exposed to goat antihuman immunoglobulin (Ig)G-conjugated gold nanospheres (40 nm) and were then imaged under a dark-field microscope. The live cells were then subjected to photoirradiation using a 633-nm laser at different power levels. The viability of tumor cells under laser irradiation was monitored by confocal microscopy using a viability-assay kit. RESULTS & DISCUSSION: The death rates of A549 lung tumor cells after gold nanoparticle exposure increased significantly under laser irradiation. The maximum initial cell death rate was observed at a laser power level of 3.75 mW, with the initial deactivation rate accelerated by a factor of 6.6 and a total loss of 92% of cell viability. CONCLUSION: This work demonstrated potential applications of gold nanospheres as both imaging probes and enhancing agents for photothermal therapy of cancer.