The therapeutically actionable long non-coding RNA 'T-RECS' is essential to cancer cells' survival in NRAS/MAPK-driven melanoma.

Finding effective therapeutic targets to treat NRAS-mutated melanoma remains a challenge. Long non-coding RNAs (lncRNAs) recently emerged as essential regulators of tumorigenesis. Using a discovery approach combining experimental models and unbiased computational analysis complemented by validation in patient biospecimens, we identified a nuclear-enriched lncRNA (AC004540.4) that is upregulated in NRAS/MAPK-dependent melanoma, and that we named T-RECS. Considering potential innovative treatment strategies, we designed antisense oligonucleotides (ASOs) to target T-RECS. T-RECS ASOs reduced the growth of melanoma cells and induced apoptotic cell death, while having minimal impact on normal primary melanocytes. Mechanistically, treatment with T-RECS ASOs downregulated the activity of pro-survival kinases and reduced the protein stability of hnRNPA2/B1, a pro-oncogenic regulator of MAPK signaling. Using patient- and cell line- derived tumor xenograft mouse models, we demonstrated that systemic treatment with T-RECS ASOs significantly suppressed the growth of melanoma tumors, with no noticeable toxicity. ASO-mediated T-RECS inhibition represents a promising RNA-targeting approach to improve the outcome of MAPK pathway-activated melanoma.

The therapeutically actionable long non-coding RNA 'T-RECS' is essential to cancer cells' survival in NRAS/MAPK-driven melanoma.

Finding effective therapeutic targets to treat NRAS-mutated melanoma remains a challenge. Long non-coding RNAs (lncRNAs) recently emerged as essential regulators of tumorigenesis. Using a discovery approach combining experimental models and unbiased computational analysis complemented by validation in patient biospecimens, we identified a nuclear-enriched lncRNA (AC004540.4) that is upregulated in NRAS/MAPK-dependent melanoma, and that we named T-RECS. Considering potential innovative treatment strategies, we designed antisense oligonucleotides (ASOs) to target T-RECS. T-RECS ASOs reduced the growth of melanoma cells and induced apoptotic cell death, while having minimal impacton normal primary melanocytes. Mechanistically, treatment with T-RECS ASOs downregulated the activity of pro-survival kinases and reduced the protein stability of hnRNPA2/B1, a pro-oncogenic regulator of MAPK signaling. Using patient- and cell line- derived tumor xenograft mouse models, we demonstrated that systemic treatment with T-RECS ASOs significantly suppressed the growth of melanoma tumors, with no noticeable toxicity. ASO-mediated T-RECS inhibition represents a promising RNA-targeting approach to improve the outcome of MAPK pathway-activated melanoma.

Deconstructing the role of MALAT1 in MAPK-signaling in melanoma: insights from antisense oligonucleotide treatment.

The long non-coding RNA (lncRNA) MALAT1 is a regulator of oncogenesis and cancer progression. MAPK-pathway upregulation is the main event in the development and progression of human cancer, including melanoma and recent studies have shown that MALAT1 has a significant impact on the regulation of gene and protein expression in the MAPK pathway. However, the role of MALAT1 in regulation of gene and protein expression of the MAPK-pathway kinases RAS, RAF, MEK and ERK in melanoma is largely unknown. We demonstrate the impacts of antisense oligonucleotide (ASO)-based MALAT1-inhibition on MAPK-pathway gene regulation in melanoma. Our results showed that MALAT1-ASO treatment decreased BRAF RNA expression and protein levels, and MALAT1 had increased correlation with MAPK-pathway associated genes in melanoma patient samples compared to healthy skin. Additionally, drug-induced MAPK inhibition upregulated MALAT1-expression, a finding that resonates with a paradigm of MALAT1-expression presented in this work: MALAT1 is downregulated in melanoma and other cancer types in which MALAT1 seems to be associated with MAPK-signaling, while MALAT1-ASO treatment strongly reduced the growth of melanoma cell lines, even in cases of resistance to MEK inhibition. MALAT1-ASO treatment significantly inhibited colony formation in vitro and reduced tumor growth in an NRAS-mutant melanoma xenograft mouse model in vivo, while showing no aberrant toxic side effects. Our findings demonstrate new insights into MALAT1-mediated MAPK-pathway gene regulation and a paradigm of MALAT1 expression in MAPK-signaling-dependent cancer types. MALAT1 maintains essential oncogenic functions, despite being downregulated.

Melanoma risk during immunomodulating treatment.

Immunosuppressive therapy is standard for the treatment of inflammatory diseases and for minimizing rejection in transplant patients. However, immunosuppressant drugs are associated with an increased risk of certain cancers. In particular, melanoma is an immunogenic tumor and as such, is strongly influenced by the immune system. We performed this literature review to summarize the effects of commonly used immunomodulating agents on melanoma development, recurrence and progression. We outline the mechanism of action of each drug and discuss the available evidence on its influence on melanoma. Based on existing literature, we recommend avoiding the following agents in patients with a history of invasive melanoma: cyclosporine, sirolimus, natalizumab, IL-6 inhibitors, cyclophosphamide, methotrexate and the tumor necrosis factor-alpha inhibitors infliximab and etanercept. If there are no viable alternative agents, we recommend for these patients to see a dermatologist every 6 months for a thorough skin examination.

MEK/CDK4,6 co-targeting is effective in a subset of NRAS, BRAF and 'wild type' melanomas.

Targeted therapy has become a cornerstone for the treatment of melanoma patients. Targeting NRAS function is particularly challenging. To date, only single MEK inhibitor treatment was able to show minimal clinical efficacy. The discovery that co-targeting of MEK and CDK4,6 has antitumor activity created excitement for patients and clinicians; however, it is largely unknown if only NRAS mutant patients might benefit from MEK/CDK4,6 blockade. In this study we investigate response patterns of NRAS, BRAF mutant and 'wild type' melanoma cells in vitro and in vivo when challenged with inhibitors of MEK, CDK4,6 and the combination of both. Data revealed, that in vitro growth response patterns of cells treated with the MEK/CDK4,6 combination correspond to in vivo efficacy of MEK/CDK4,6 co-targeting in melanoma xenograft models. Strikingly, this was consistently observed in NRAS and BRAF mutant, as well as in 'wild type' melanoma cells. Additionally, cells displaying elevated p-Rb levels after single MEK inhibition, showed more effective growth reduction with MEK/CDK4,6 co-targeting compared to single MEK inhibitor treatment in vivo. Findings indicate that combined MEK/CDK4,6 inhibition could offer an effectively therapeutic modality in a subset of BRAF and NRAS mutant, as well as 'wild type' melanoma patients.

The lincRNA MIRAT binds to IQGAP1 and modulates the MAPK pathway in NRAS mutant melanoma.

Despite major advances in targeted melanoma therapies, drug resistance limits their efficacy. Long noncoding RNAs (lncRNAs) are transcriptome elements that do not encode proteins but are important regulatory molecules. LncRNAs have been implicated in cancer development and response to different therapeutics and are thus potential treatment targets; however, the majority of their functions and molecular interactions remain unexplored. In this study, we identify a novel cytoplasmic intergenic lincRNA (MIRAT), which is upregulated following prolonged MAPK inhibition in NRAS mutant melanoma and modulates MAPK signaling by binding to the MEK scaffold protein IQGAP1. Collectively, our results present MIRAT's direct modulatory effect on the MAPK pathway and highlight the relevance of cytoplasmic lncRNAs as potential targets in drug resistant cancer.

Dual MEK/AKT inhibition with trametinib and GSK2141795 does not yield clinical benefit in metastatic NRAS-mutant and wild-type melanoma.

Aberrant MAPK and PI3K pathway signaling may drive the malignant phenotype in NRAS-mutant and BRAFWT NRASWT metastatic melanoma. To target these pathways, NRAS-mutant and BRAFWT NRASWT patients received oral trametinib at 1.5 mg daily and GSK2141795 at 50 mg daily in a two-cohort Simon two-stage design. Participants had adequate end-organ function and no more than two prior treatment regimens. Imaging assessments were performed at 8-week intervals. A total of 10 NRAS-mutant and 10 BRAFWT NRASWT patients were enrolled. No objective responses were noted in either cohort. The median PFS and OS were 2.3 and 4.0 months in the NRAS-mutant cohort and 2.8 and 3.5 months in the wild-type cohort. Grade 3 and grade 4 adverse events, primarily rash, were observed in 25% of patients. We conclude that the combination of trametinib and GSK2141795 does not have significant clinical activity in NRAS-mutant or BRAFWT NRASWT melanoma.

Phosphoproteomic Analyses of NRAS(G12) and NRAS(Q61) Mutant Melanocytes Reveal Increased CK2α Kinase Levels in NRAS(Q61) Mutant Cells.

In melanoma, mutant and thereby constantly active neuroblastoma rat sarcoma (NRAS) affects 15-20% of tumors, contributing to tumor initiation, growth, invasion, and metastasis. Recent therapeutic approaches aim to mimic RAS extinction by interfering with critical signaling pathways downstream of the mutant protein. This study investigates the phosphoproteome of primary human melanocytes bearing mutations in the two hot spots of NRAS, NRAS(G12) and NRAS(Q61). Stable isotope labeling by amino acids in cell culture followed by mass spectrometry identified 14,155 spectra of 3,371 unique phosphopeptides mapping to 1,159 proteins (false discovery rate < 2%). Data revealed pronounced PI3K/AKT signaling in NRAS(G12V) mutant cells and pronounced mitogen-activated protein kinase (MAPK) signaling in NRAS(Q61L) variants. Computer-based prediction models for kinases involved, revealed that CK2α is significantly overrepresented in primary human melanocytes bearing NRAS(Q61L) mutations. Similar differences were found in human NRAS(Q61) mutant melanoma cell lines that were also more sensitive to pharmacologic CK2α inhibition compared with NRAS(G12) mutant cells. Furthermore, CK2α levels were pronounced in patient samples of NRAS(Q61) mutant melanoma at the mRNA and protein level. The preclinical findings of this study reveal that codon 12 and 61 mutant NRAS cells have distinct signaling characteristics that could allow for the development of more effective, mutation-specific treatment modalities.

Pharmacological inhibitors of c-KIT block mutant c-KIT mediated migration of melanocytes and melanoma cells in vitro and in vivo.

Mutations in the receptor tyrosine kinase c-KIT (KIT) are frequent oncogenic alterations in melanoma and are predominantly detected in tumors of acral, mucosal, and chronically sun-damaged skin. Research indicates that melanocytes with aberrant KIT signaling can be found in the distant periphery of the primary tumor; However, it is hitherto unknown whether KIT might confer a migratory advantage, thereby enabling genetically abnormal cells to populate a distal area. In this study, we investigated the role of mutant KIT in melanocyte- and melanoma cell migration using KIT mutant lines as well as genetically manipulated murine and primary human melanocytes. Our results revealed that melanocytes, stably transduced with mutant KIT closed a gap inflicted on cell monolayers faster than wild-type controls. Similarly, KIT mutant human melanoma lines were able to populate a larger area in a 3D in vitro skin model compared to KIT wild type and BRAF mutant lines. Genomic profiling revealed that genes associated with increased cell-dispersal of KIT mutant variants were linked to a statistically significant up-regulation of 60 migratory genes (z-score 1.334; p=0.0001). In addition, in vivo experiments harnessing a mouse xenograft model of early melanoma development demonstrated rapid lateral migration of KIT mutant cells compared to respective controls. The specific kinase inhibitors imatinib and nilotinib, could abrogate this migratory advantage in vitro and in vivo. Our work suggests that KIT inhibition might help to target migratory active, KIT mutant melanoma cells, thus representing a potential strategy to reduce spread and local recurrence.

Searching for the Chokehold of NRAS Mutant Melanoma.

Up to 18% of melanomas harbor mutations in the neuroblastoma rat-sarcoma homolog (NRAS). Yet, decades of research aimed to interfere with oncogenic RAS signaling have been largely disappointing and have not resulted in meaningful clinical outputs. Recent advances in disease modeling, structural biology, and an improved understanding of RAS cycling as well as RAS signaling networks have renewed hope for developing strategies to selectively block hyperactive RAS function. This review discusses direct and indirect blocking of activated RAS with a focus on current and potential future therapeutic approaches for NRAS mutant melanoma.

Acyl protein thioesterase 1 and 2 (APT-1, APT-2) inhibitors palmostatin B, ML348 and ML349 have different effects on NRAS mutant melanoma cells.

Oncogenic NRAS mutations are frequent in melanoma and lead to increased downstream signaling and uncontrolled cell proliferation. Since the direct inhibition of NRAS is not possible yet, modulators of NRAS posttranslational modifications have become an area of interest. Specifically, interfering with NRAS posttranslational palmitoylation/depalmitoylation cycle could disturb proper NRAS localization, and therefore decrease cell proliferation and downstream signaling. Here, we investigate the expression and function of NRAS depalmitoylating acyl protein thioesterases 1 and 2 (APT-1, APT-2) in a panel of NRAS mutant melanoma cells. First, we show that all melanoma cell lines examined express APT-1 and APT-2. Next, we show that siRNA mediated APT-1 and APT-2 knock down and that the specific APT-1 and -2 inhibitors ML348 and ML349 have no biologically significant effects in NRAS mutant melanoma cells. Finally, we test the dual APT-1 and APT-2 inhibitor palmostatin B and conclude that palmostatin B has effects on NRAS downstream signaling and cell viability in NRAS mutant melanoma cells, offering an interesting starting point for future studies.

The burden of malignant melanoma--lessons to be learned from Austria.

AIM OF STUDY: Incidence rates of melanoma, generated by cancer registries (CRs), are susceptible to reporting inconsistencies due to increasing decentralisation of diagnosis. We therefore independently assessed the burden of melanoma in Austria. METHODS: We collected histopathological reports on melanoma of all patients diagnosed in Austria in 2011. Demographic and clinical characteristics, histopathological tumour stages were assessed. Their regional distributions and incidence rates were analysed and compared with data of national and international CRs. RESULTS: A total of 5246 patients were diagnosed with 1951 in-situ and 3295 invasive melanomas in Austria in 2011 (population 8.4 million). Age, sex and anatomic distribution corresponded to findings in other European countries, however, the incidence of 25/100,000 (world age-standardised rate) for invasive melanomas was two-fold higher than published by the Austrian CR (12/100,000). Varying frequencies in diagnosing thin melanomas (≤1 mm; n = 4415) accounted exclusively for significant regional disparities, while advanced tumours (>1 mm; n = 761) were evenly distributed. Western Austria showed the highest rates (36/100,000). Patients from eastern Austria whose melanomas were diagnosed in laboratories in western Austria (n = 76) showed significantly higher proportions of in-situ lesions (n = 43; 57%) compared to those whose tumours were diagnosed in eastern Austria (n = 4014; in-situ = 1369; 34%) (p < 0.0001). CONCLUSIONS: In Austria, the melanoma burden and its potential socio-economic implications are significantly underestimated. Similarities of incidences indicate this could affect other European countries with well-established CRs and compromise international comparability of data. Austrian regional disparities suggest overdiagnosis of thin melanomas due to the variability of pathologists' thresholds for the diagnosis of early stage tumours.

New insights in melanoma biomarkers: long-noncoding RNAs.

Melanoma is one of the leading cancers worldwide, distinguished for its malignancy and low survival rates. Although the poor outcome could improve with an early diagnosis and a good monitoring of the disease, current melanoma biomarkers display several limitations which make them useless. Interestingly, long-noncoding RNAs are secreted into the bloodstream inside exosomes by a wide range of malignant cells, and several of them have been validated as promising circulating molecular signatures of other tumors, but not melanoma. In this review we propose to explore the booming field of long-noncoding RNAs in order to find potential candidates to be tested as novel melanoma biomarkers, with the ultimate goal of improving melanoma detection, diagnosis and prognosis.

Melanoma risk perception and prevention behavior among African-Americans: the minority melanoma paradox.

INTRODUCTION: Melanoma is the most deadly type of skin cancer with 75% of all skin cancer deaths within the US attributed to it. Risk factors for melanoma include ultraviolet exposure, genetic predisposition, and phenotypic characteristics (eg, fair skin and blond hair). Whites have a 27-fold higher incidence of melanoma than African-Americans (AA), but the 5-year survival is 17.8% lower for AA than Whites. It is reported continuously that AA have more advanced melanomas at diagnosis, and overall lower survival rates. This minority melanoma paradox is not well understood or studied. OBJECTIVE: To explore further, the possible explanations for the difference in melanoma severity and survival in AA within the US. METHODS: Qualitative review of the literature. RESULTS: Lack of minority-targeted public education campaigns, low self-risk perception, low self-skin examinations, intrinsic virulence, vitamin D differences, and physician mistrust may play a role in the melanoma survival disparity among AA. CONCLUSION: Increases in public awareness of melanoma risk among AA through physician and media-guided education, higher index of suspicion among individuals and physicians, and policy changes can help to improve early detection and close the melanoma disparity gap in the future.

Pembrolizumab Cutaneous Adverse Events and Their Association With Disease Progression.

IMPORTANCE: Immunomodulatory anticancer drugs, such as the anti-programmed death-1 drug pembrolizumab, have shown promising results in trials, and more patients will receive such treatments. Little is known about cutaneous adverse events (AEs) caused by these drugs and their possible correlation with treatment response. OBJECTIVE: To describe the frequency and spectrum of cutaneous AEs linked with pembrolizumab and their possible correlation with treatment response. DESIGN, SETTING, AND PARTICIPANTS: A single-institution, retrospective medical record review was conducted of patients with cancer who were treated with pembrolizumab from March 1, 2011, to May 28, 2014. The review comprised 83 consecutive patients who were enrolled in 2 clinical trials, received at least 1 dose of pembrolizumab, and had at least 1 follow-up visit. Patients were grouped according to the following therapeutic regimen for pembrolizumab: 43 received 10 mg/kg every 3 weeks, 24 received 10 mg/kg every 2 weeks, and 16 received 2 mg/kg every 3 weeks. Sixty-six patients were treated for melanoma, 15 patients for lung cancer, 1 patient for prostate cancer, and 1 patient for Merkel cell carcinoma. Median follow-up was 15 weeks (range, 2-105 weeks). The analysis was conducted from March 1 to September 30, 2014. MAIN OUTCOMES AND MEASURES: Occurrence, severity, and type of cutaneous AEs, as well as disease progression and response to pembrolizumab treatment. RESULTS: Thirty-five patients (42%) developed cutaneous AEs attributed to pembrolizumab. The most common cutaneous AEs were macular papular eruption (24 [29%]), pruritus (10 [12%]), and hypopigmentation (7 [8%]). All 7 patients who developed hypopigmentation were treated for melanoma. Survival analyses showed that patients who developed cutaneous AEs had significantly longer progression-free intervals in all 3 groups (pembrolizumab, 10 mg/kg, every 3 weeks, P = .001; pembrolizumab, 10 mg/kg, every 2 weeks, P = .003; pembrolizumab, 2 mg/kg, every 3 weeks, P = .009) compared with patients who did not develop cutaneous AEs. CONCLUSIONS AND RELEVANCE: Pembrolizumab therapy was associated with cutaneous AEs in 42% of patients. The development of cutaneous AEs, especially of hypopigmentation in patients with melanoma, could point toward better treatment response.