Targeted Therapies in Melanoma

Advances in understanding the biology of melanoma have provided great insights about the mechanisms of chemoresistance and its genetic heterogeneity in parallel with advances in drug design culminating in recent major treatment breakthroughs using small molecules inhibitors in metastatic melanoma (MM). While clinical benefit of targeted therapies has been unquestionable, future advances can only be possible if we better understand the interplay between genetic aberrations and role of other crucial non-genetic changes yet to be identified by such projects as the Cancer Genome Atlas Project (TCGA) in Melanoma. Combination therapies, either among small molecule inhibitors themselves and/or with immunotherapies may be the optimal strategy to prevent development of drug resistance that is inherently linked with such targeted therapies

Use of Susceptibility-Weighted Imaging (SWI) in the Detection of Brain Hemorrhagic Metastases from Breast Cancer and Melanoma

SWI has significantly increased our sensitivity in detecting hemorrhagic brain lesions. We sought to explore the prevalence of intra-tumoral hemorrhage as detected by SWI in brain metastases from melanoma and breast cancer

Mitochondrial respiration - an important therapeutic target in melanoma

The importance of mitochondria as oxygen sensors as well as producers of ATP and reactive oxygen species (ROS) has recently become a focal point of cancer research. However, in the case of melanoma, little information is available to what extent cellular bioenergetics processes contribute to the progression of the disease and related to it, whether oxidative phosphorylation (OXPHOS) has a prominent role in advanced melanoma. In this study we demonstrate that compared to melanocytes, metastatic melanoma cells have elevated levels of OXPHOS. Furthermore, treating metastatic melanoma cells with the drug, Elesclomol, which induces cancer cell apoptosis through oxidative stress, we document by way of stable isotope labeling with amino acids in cell culture (SILAC) that proteins participating in OXPHOS are downregulated. We also provide evidence that melanoma cells with high levels of glycolysis are more resistant to Elesclomol. We further show that Elesclomol upregulates hypoxia inducible factor 1-α (HIF-1α), and that prolonged exposure of melanoma cells to this drug leads to selection of melanoma cells with high levels of glycolysis. Taken together, our findings suggest that molecular targeting of OXPHOS may have efficacy for advanced melanoma. © 2012 Barbi de Moura et al

Importance of glycolysis and oxidative phosphorylation in advanced melanoma

Serum lactate dehydrogenase (LDH) is a prognostic factor for patients with stage IV melanoma. To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma. Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2. Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS), we subsequently determined using tissue microarray (TMA) analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes. To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT) 1 and 4. Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma. Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS. Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy

DNA methylation profiles in primary cutaneous melanomas are associated with clinically significant pathologic features

DNA methylation studies have elucidated a methylation signature distinguishing primary melanomas from benign nevi and provided new insights about genes that may be important in melanoma development. However, it is unclear whether methylation differences among primary melanomas are related to tumor pathologic features with known clinical significance. We utilized the Illumina Golden Gate Cancer Panel array to investigate the methylation profiles of 47 primary cutaneous melanomas. Array-wide methylation patterns revealed a positive association of methylation with Breslow thickness and mutated BRAF, a negative association with mitotic rate, and a weak association with ulceration. Hierarchical clustering on CpG sites exhibiting the most variable methylation (n=235) divided the melanoma samples into three clusters, including a highly-methylated cluster that was positively associated with Breslow thickness and an intermediately-methylated cluster associated with Breslow thickness and mitotic rate. Our findings provide support for the existence of methylation-defined subsets in melanomas, with increased methylation associated with Breslow thickness

HIF1α and HIF2α independently activate SRC to promote melanoma metastases

Malignant melanoma is characterized by a propensity for early lymphatic and hematogenous spread. The hypoxia-inducible factor (HIF) family of transcription factors is upregulated in melanoma by key oncogenic drivers. HIFs promote the activation of genes involved in cancer initiation, progression, and metastases. Hypoxia has been shown to enhance the invasiveness and metastatic potential of tumor cells by regulating the genes involved in the breakdown of the ECM as well as genes that control motility and adhesion of tumor cells. Using a Pten -deficient, Braf -mutant genetically engineered mouse model of melanoma, we demonstrated that inactivation of HIF1α or HIF2α abrogates metastasis without affecting primary tumor formation. HIF1α and HIF2α drive melanoma invasion and invadopodia formation through PDGFRα and focal adhesion kinase–mediated (FAK-mediated) activation of SRC and by coordinating ECM degradation via MT1-MMP and MMP2 expression. These results establish the importance of HIFs in melanoma progression and demonstrate that HIF1α and HIF2α activate independent transcriptional programs that promote metastasis by coordinately regulating cell invasion and ECM remodeling

Targeted next generation sequencing identifies clinically actionable mutations in patients with melanoma

Somatic sequencing of cancers has produced new insight into tumorigenesis, tumor heterogeneity, and disease progression, but the vast majority of genetic events identified are of indeterminate clinical significance. Here we describe a NextGen sequencing approach to fully analyze 248 genes, including all those of known clinical significance in melanoma. This strategy features solution capture of DNA followed by multiplexed, high-throughput sequencing, and was evaluated in 31 melanoma cell lines and 18 tumor tissues from patients with metastatic melanoma. Mutations in melanoma cell lines correlated with their sensitivity to corresponding small molecule inhibitors, confirming, for example, lapatinib sensitivity in ERBB4 mutant lines and identifying a novel activating mutation of BRAF. The latter event would not have been identified by clinical sequencing and was associated with responsiveness to a BRAF kinase inhibitor. This approach identified focal copy number changes of PTEN not found by standard methods, such as comparative genomic hybridization (CGH). Actionable mutations were found in 89% of the tumor tissues analyzed, 56% of which would not be identified by standard-of-care approaches. This work shows that targeted sequencing is an attractive approach for clinical use in melanoma

MERTK receptor tyrosine kinase is a therapeutic target in melanoma

Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. Although recent therapeutic advances have prolonged patient survival, the prognosis remains dismal. C-MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that MERTK expression correlates with disease progression. MERTK expression was highest in metastatic melanomas, followed by primary melanomas, while the lowest expression was observed in nevi. Additionally, over half of melanoma cell lines overexpressed MERTK compared with normal human melanocytes; however, overexpression did not correlate with mutations in BRAF or RAS. Stimulation of melanoma cells with the MERTK ligand GAS6 resulted in the activation of several downstream signaling pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT. MERTK inhibition via shRNA reduced MERTK-mediated downstream signaling, reduced colony formation by up to 59%, and diminished tumor volume by 60% in a human melanoma murine xenograft model. Treatment of melanoma cells with UNC1062, a novel MERTK-selective small-molecule tyrosine kinase inhibitor, reduced activation of MERTK-mediated downstream signaling, induced apoptosis in culture, reduced colony formation in soft agar, and inhibited invasion of melanoma cells. This work establishes MERTK as a therapeutic target in melanoma and provides a rationale for the continued development of MERTK-targeted therapies

InterMEL: An international biorepository and clinical database to uncover predictors of survival in early-stage melanoma

We are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. We also evaluated tissue-derived predictors of extracted nucleic acids’ quality and success in downstream testing. This ongoing study will target 1,000 melanomas within the international InterMEL consortium.Medicin

IL2 Inducible T-cell Kinase, a Novel Therapeutic Target in Melanoma

Interleukin-2 inducible T-cell kinase (ITK) promoter CpG sites are hypomethylated in melanomas compared to nevi. The expression of ITK in melanomas, however, has not been established and requires elucidation