Antonella Bacchiocchi

PLX4032, a selective BRAFV600E kinase inhibitor, activates the ERK pathway and enhances cell migration and proliferation of BRAFWT melanoma cells

BRAFV600E/K is a frequent mutationally active tumor‐specific kinase in melanomas that is currently targeted for therapy by the specific inhibitor PLX4032. Our studies with melanoma tumor cells that are BRAFV600E/K and BRAFWT showed that, paradoxically, while PLX4032 inhibited ERK1/2 in the highly sensitive BRAFV600E/K, it activated the pathway in the resistant BRAFWT cells, via RAF1 activation, regardless of the status of mutations in NRAS or PTEN. The persistently active ERK1/2 triggered downstream effectors in BRAFWT melanoma cells and induced changes in the expression of a wide‐spectrum of genes associated with cell cycle control. Furthermore, PLX4032 increased the rate of proliferation of growth factor‐dependent NRAS Q61L mutant primary melanoma cells, reduced cell adherence and increased mobility of cells from advanced lesions. The results suggest that the drug can confer an advantage to BRAFWT primary and metastatic tumor cells in vivo and provide markers for monitoring clinical responses.

Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032

Activating mutations in BRAF kinase are common in melanomas. Clinical trials with PLX4032, the mutant-BRAF inhibitor, show promising preliminary results in patients selected for the presence of V600E mutation. However, activating V600K mutation is the other most common mutation, yet patients with this variant are currently excluded from the PLX4032 trials. Here we present evidence that a patient bearing the BRAF V600K mutation responded remarkably to PLX4032, suggesting that clinical trials should include all patients with activating BRAF V600E/K mutations.

Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas

We report on whole-exome sequencing (WES) of 213 melanomas. Our analysis established NF1, encoding a negative regulator of RAS, as the third most frequently mutated gene in melanoma, after BRAF and NRAS. Inactivating NF1 mutations were present in 46% of melanomas expressing wild-type BRAF and RAS, occurred in older patients and showed a distinct pattern of co-mutation with other RASopathy genes, particularly RASA2. Functional studies showed that NF1 suppression led to increased RAS activation in most, but not all, melanoma cases. In addition, loss of NF1 did not predict sensitivity to MEK or ERK inhibitors. The rebound pathway, as seen by the induction of phosphorylated MEK, occurred in cells both sensitive and resistant to the studied drugs. We conclude that NF1 is a key tumor suppressor lost in melanomas, and that concurrent RASopathy gene mutations may enhance its role in melanomagenesis.

MicroRNA signatures differentiate melanoma subtypes

Melanoma is an aggressive cancer that is highly resistance to therapies once metastasized. We studied microRNA (miRNA) expression in clinical melanoma subtypes and evaluated different miRNA signatures in the background of gain of function somatic and inherited mutations associated with melanoma. Total RNA from 42 patient derived primary melanoma cell lines and three independent normal primary melanocyte cell cultures was evaluated by miRNA array. MiRNA expression was then analyzed comparing subtypes and additional clinicopathologic criteria including somatic mutations. The prevalence and association of an inherited variant in a miRNA binding site in the 3’UTR of the KRAS oncogene, referred to as the KRAS-variant, was also evaluated. We show that seven miRNAs, miR-142-3p, miR-486, miR-214, miR-218, miR-362, miR-650 and miR-31, were significantly correlated with acral as compared to non-acral melanomas (p < 0.04). In addition, we discovered that the KRAS-variant was enriched in non-acral melanoma (25%), and that miR-137 under expression was significantly associated with melanomas with the KRAS-variant. Our findings indicate that miRNAs are differentially expressed in melanoma subtypes and that their misregulation can be impacted by inherited gene variants, supporting the hypothesis that miRNA misregulation reflects biological differences in melanoma.

Genotype-Selective Combination Therapies for Melanoma Identified by High-Throughput Drug Screening

UNLABELLED Resistance and partial responses to targeted monotherapy are major obstacles in cancer treatment. Systematic approaches to identify efficacious drug combinations for cancer are not well established, especially in the context of genotype. To address this, we have tested pairwise combinations of an array of small-molecule inhibitors on early-passage melanoma cultures using combinatorial drug screening. Results reveal several inhibitor combinations effective for melanomas with activating RAS or BRAF mutations, including mutant BRAF melanomas with intrinsic or acquired resistance to vemurafenib. Inhibition of both EGF receptor and AKT sensitized treatment-resistant BRAF mutant melanoma cultures to vemurafenib. Melanomas with RAS mutations were more resistant to combination therapies relative to BRAF mutants, but were sensitive to combinations of statins and cyclin-dependent kinase inhibitors in vitro and in vivo. These results show the use of combinatorial drug screening for discovering unique treatment regimens that overcome resistance phenotypes of mutant BRAF- and RAS-driven melanomas. SIGNIFICANCE We have used drug combinatorial screening to identify effective combinations for mutant BRAF melanomas, including those resistant to vemurafenib, and mutant RAS melanomas that are resistant to many therapies. Mechanisms governing the interactions of the drug combinations are proposed, and in vivo xenografts show the enhanced benefit and tolerability of a mutant RAS -selective combination, which is currently lacking in the clinic.

Preexisting MEK1 Exon 3 Mutations in V600E/KBRAF Melanomas Do Not Confer Resistance to BRAF Inhibitors

UNLABELLED BRAF inhibitors (BRAFi) induce antitumor responses in nearly 60% of patients with advanced V600E/KBRAF melanomas. Somatic activating MEK1 mutations are thought to be rare in melanomas, but their potential concurrence with V600E/KBRAF may be selected for by BRAFi. We sequenced MEK1/2 exon 3 in melanomas at baseline and upon disease progression. Of 31 baseline V600E/KBRAF melanomas, 5 (16%) carried concurrent somatic BRAF/MEK1 activating mutations. Three of 5 patients with BRAF/MEK1 double-mutant baseline melanomas showed objective tumor responses, consistent with the overall 60% frequency. No MEK1 mutation was found in disease progression melanomas, except when it was already identified at baseline. MEK1-mutant expression in V600E/KBRAF melanoma cell lines resulted in no significant alterations in p-ERK1/2 levels or growth-inhibitory sensitivities to BRAFi, MEK1/2 inhibitor (MEKi), or their combination. Thus, activating MEK1 exon 3 mutations identified herein and concurrent with V600E/KBRAF do not cause BRAFi resistance in melanoma. SIGNIFICANCE As BRAF inhibitors gain widespread use for treatment of advanced melanoma, biomarkers for drug sensitivity or resistance are urgently needed. We identify here concurrent activating mutations in BRAF and MEK1 in melanomas and show that the presence of a downstream mutation in MEK1 does not necessarily make BRAF–mutant melanomas resistant to BRAF inhibitors.

Integrative Analysis of Epigenetic Modulation in Melanoma Cell Response to Decitabine: Clinical Implications

Decitabine, an epigenetic modifier that reactivates genes otherwise suppressed by DNA promoter methylation, is effective for some, but not all cancer patients, especially those with solid tumors. It is commonly recognized that to overcome resistance and improve outcome, treatment should be guided by tumor biology, which includes genotype, epigenotype, and gene expression profile. We therefore took an integrative approach to better understand melanoma cell response to clinically relevant dose of decitabine and identify complementary targets for combined therapy. We employed eight different melanoma cell strains, determined their growth, apoptotic and DNA damage responses to increasing doses of decitabine, and chose a low, clinically relevant drug dose to perform whole-genome differential gene expression, bioinformatic analysis, and protein validation studies. The data ruled out the DNA damage response, demonstrated the involvement of p21Cip1 in a p53-independent manner, identified the TGFβ pathway genes CLU and TGFBI as markers of sensitivity to decitabine and revealed an effect on histone modification as part of decitabine-induced gene expression. Mutation analysis and knockdown by siRNA implicated activated β-catenin/MITF, but not BRAF, NRAS or PTEN mutations as a source for resistance. The importance of protein stability predicted from the results was validated by the synergistic effect of Bortezomib, a proteasome inhibitor, in enhancing the growth arrest of decitabine in otherwise resistant melanoma cells. Our integrative analysis show that improved therapy can be achieved by comprehensive analysis of cancer cells, identified biomarkers for patients selection and monitoring response, as well as targets for improved combination therapy.

Phosphoproteomic Screen Identifies Potential Therapeutic Targets in Melanoma

Therapies directed against receptor tyrosine kinases are effective in many cancer subtypes, including lung and breast cancer. We used a phosphoproteomic platform to identify active receptor tyrosine kinases that might represent therapeutic targets in a panel of 25 melanoma cell strains. We detected activated receptors including TYRO3, AXL, MERTK, EPHB2, MET, IGF1R, EGFR, KIT, HER3, and HER4. Statistical analysis of receptor tyrosine kinase activation as well as ligand and receptor expression indicates that some receptors, such as FGFR3, may be activated via autocrine circuits. Short hairpin RNA knockdown targeting three of the active kinases identified in the screen, AXL, HER3, and IGF1R, inhibited the proliferation of melanoma cells and knockdown of active AXL also reduced melanoma cell migration. The changes in cellular phenotype observed on AXL knockdown seem to be modulated via the STAT3 signaling pathway, whereas the IGF1R-dependent alterations seem to be regulated by the AKT signaling pathway. Ultimately, this study identifies several novel targets for therapeutic intervention in melanoma. Mol Cancer Res; 9(6); 801–12. ©2011 AACR.

Plasma markers for identifying patients with metastatic melanoma

Purpose: With the rising incidence of melanoma, more patients are undergoing surveillance for disease recurrence. Our purpose was to study levels of proteins that might be secreted in the blood of patients with metastatic melanoma that can be used for monitoring these individuals. Methods: Genome-wide gene expression data were used to identify abundantly expressed genes in melanoma cells that encode for proteins likely to be present in the blood of cancer patients, based on high expression levels in tumors. ELISA assays were employed to measure proteins in plasma of 216 individuals; 108 metastatic melanoma patients and 108 age- and gender-matched patients with resected stage I/II disease split into equal-sized training and test cohorts. Results: Levels of seven markers, CEACAM (carcinoembryonic antigen–related cell adhesion molecule), ICAM-1 (intercellular adhesion molecule 1), osteopontin, MIA (melanoma inhibitory activity), GDF-15 (growth differentiation factor 15), TIMP-1 (tissue inhibitor of metalloproteinase 1), and S100B, were higher in patients with unresected stage IV disease than in patients with resected stage I/II disease. About 81% of the stage I/II patients in the training set had no marker elevation, whereas 69% of the stage IV patients had elevation of at least one marker (P < 0.0001). Receiver operating characteristic curves for the markers in combination in these two patient populations had an area under curve (AUC) of 0.79 in the training set and 0.8 in the test set. A CART (Classification and Regression Trees) model developed in the training set further improved the AUC in the test set to 0.898. Conclusions: Plasma markers, particularly when assessed in combination, can be used to monitor patients for disease recurrence and can compliment currently used lactate dehydrogenase and imaging studies; prospective validation is warranted. Clin Cancer Res; 17(8); 2417–25. ©2011 AACR.

A Variant in a MicroRNA Complementary Site in the 3'UTR of the KIT Oncogene Increases Risk of Acral Melanoma

MicroRNAs (miRNAs) are small ∼22nt single stranded RNAs that negatively regulate protein expression by binding to partially complementary sequences in the 3′ untranslated region (3′ UTRs) of target gene messenger RNAs (mRNA). Recently, mutations have been identified in both miRNAs and target genes that disrupt regulatory relationships, contribute to oncogenesis and serve as biomarkers for cancer risk. KIT, an established oncogene with a multifaceted role in melanogenesis and melanoma pathogenesis, has recently been shown to be upregulated in some melanomas, and is also a target of the miRNA miR-221. Here, we describe a genetic variant in the 3′ UTR of the KIT oncogene that correlates with a greater than fourfold increased risk of acral melanoma. This KIT variant results in a mismatch in the seed region of a miR-221 complementary site and reporter data suggests that this mismatch can result in increased expression of the KIT oncogene. Consistent with the hypothesis that this is a functional variant, KIT mRNA and protein levels are both increased in the majority of samples harboring the KIT variant. This work identifies a novel genetic marker for increased heritable risk of melanoma.