Targeting the IL-2 inducible kinase in melanoma; a phase 2 study of ibrutinib in systemic treatment-refractory distant metastatic cutaneous melanoma: preclinical rationale, biology, and clinical activity (NCI9922).

BACKGROUND: IL-2 inducible kinase (ITK) is highly expressed in metastatic melanomas and its inhibition suppresses melanoma cell proliferation. We hypothesize that ibrutinib has a direct antitumor effect in melanoma cell lines and that treatment of metastatic melanomas with ibrutinib induces antitumor responses. METHODS: We assessed the ibrutinib effect on melanoma cell proliferation, apoptosis, and motility. Patients with metastatic melanoma refractory to PD-1 and MAPK inhibitors (if BRAFV600-mutant) were treated with ibrutinib, 840 mg PO QD, as part of a phase II clinical trial (clinicaltrials.gov NCT02581930). RESULTS: Melanoma cell lines frequently express ITK, YES1, and EGFR. Ibrutinib suppressed cell motility and proliferation in most cell lines. Eighteen patients (13 male; median age 63.5 years, range 37-82; 12 with ipilimumab resistance) were enrolled. The most frequent side effects were fatigue (61%), anorexia (50%), hyponatremia (28%), nausea, and vomiting (22% each). No antitumor responses were seen. At a median follow-up of 6 months (0.3-35.8 months), the median progression-free survival was 1.3 months (range 0.2-5.5 months). Fifteen patients were discontinued from the study due to progression, and 14 patients had died from metastatic melanoma. All archived tumors expressed ITK, 41% had no expression of p16 and PTEN, and 61% had absent tumor-infiltrating lymphocytes (TILs). Ibrutinib significantly suppressed proliferating (Ki67+) CD19+ peripheral blood mononuclear cells and had no significant effect on other lymphocyte subsets. CONCLUSION: Ibrutinib did not induce any meaningful clinical benefit. ITK expression may not be clinically relevant. Treatment-refractory metastatic melanomas have other fundamental defects (i.e. absent PTEN and p16 expression, absent TILs) that may contribute to an adverse prognosis.

Identification of a Robust Methylation Classifier for Cutaneous Melanoma Diagnosis.

Early diagnosis improves melanoma survival, yet the histopathological diagnosis of cutaneous primary melanoma can be challenging, even for expert dermatopathologists. Analysis of epigenetic alterations, such as DNA methylation, that occur in melanoma can aid in its early diagnosis. Using a genome-wide methylation screening, we assessed CpG methylation in a diverse set of 89 primary invasive melanomas, 73 nevi, and 41 melanocytic proliferations of uncertain malignant potential, classified based on interobserver review by dermatopathologists. Melanomas and nevi were split into training and validation sets. Predictive modeling in the training set using ElasticNet identified a 40-CpG classifier distinguishing 60 melanomas from 48 nevi. High diagnostic accuracy (area under the receiver operator characteristic curve = 0.996, sensitivity = 96.6%, and specificity = 100.0%) was independently confirmed in the validation set (29 melanomas, 25 nevi) and other published sample sets. The 40-CpG melanoma classifier included homeobox transcription factors and genes with roles in stem cell pluripotency or the nervous system. Application of the 40-CpG melanoma classifier to the diagnostically uncertain samples assigned melanoma or nevus status, potentially offering a diagnostic tool to assist dermatopathologists. In summary, the robust, accurate 40-CpG melanoma classifier offers a promising assay for improving primary melanoma diagnosis.

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

PURPOSE: IL2 inducible T-cell kinase (ITK) promoter CpG sites are hypomethylated in melanomas compared with nevi. The expression of ITK in melanomas, however, has not been established and requires elucidation. EXPERIMENTAL DESIGN: An ITK-specific monoclonal antibody was used to probe sections from deidentified, formalin-fixed paraffin-embedded tumor blocks or cell line arrays and ITK was visualized by IHC. Levels of ITK protein differed among melanoma cell lines and representative lines were transduced with four different lentiviral constructs that each contained an shRNA designed to knockdown ITK mRNA levels. The effects of the selective ITK inhibitor BI 10N on cell lines and mouse models were also determined. RESULTS: ITK protein expression increased with nevus to metastatic melanoma progression. In melanoma cell lines, genetic or pharmacologic inhibition of ITK decreased proliferation and migration and increased the percentage of cells in the G0-G1 phase. Treatment of melanoma-bearing mice with BI 10N reduced growth of ITK-expressing xenografts or established autochthonous (Tyr-Cre/Pten(null)/Braf(V600E)) melanomas. CONCLUSIONS: We conclude that ITK, formerly considered an immune cell-specific protein, is aberrantly expressed in melanoma and promotes tumor development and progression. Our finding that ITK is aberrantly expressed in most metastatic melanomas suggests that inhibitors of ITK may be efficacious for melanoma treatment. The efficacy of a small-molecule ITK inhibitor in the Tyr-Cre/Pten(null)/Braf(V600E) mouse melanoma model supports this possibility.

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 GoldenGate Cancer Panel array to investigate the methylation profiles of 47 primary cutaneous melanomas. Arraywide 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.

Mechanisms of chromosomal instability in melanoma.

A systems biology approach was applied to investigate the mechanisms of chromosomal instability in melanoma cell lines. Chromosomal instability was quantified using array comparative genomic hybridization to identify somatic copy number alterations (deletions and duplications). Primary human melanocytes displayed an average of 8.5 alterations per cell primarily representing known polymorphisms. Melanoma cell lines displayed 25 to 131 alterations per cell, with an average of 68, indicative of chromosomal instability. Copy number alterations included approximately equal numbers of deletions and duplications with greater numbers of hemizygous (-1,+1) alterations than homozygous (-2,+2). Melanoma oncogenes, such as BRAF and MITF, and tumor suppressor genes, such as CDKN2A/B and PTEN, were included in these alterations. Duplications and deletions were functional as there were significant correlations between DNA copy number and mRNA expression for these genes. Spectral karyotype analysis of three lines confirmed extensive chromosomal instability with polyploidy, aneuploidy, deletions, duplications, and chromosome rearrangements. Bioinformatic analysis identified a signature of gene expression that was correlated with chromosomal instability but this signature provided no clues to the mechanisms of instability. The signature failed to generate a significant (P = 0.105) prediction of melanoma progression in a separate dataset. Chromosomal instability was not correlated with elements of DNA damage response (DDR) such as radiosensitivity, nucleotide excision repair, expression of the DDR biomarkers γH2AX and P-CHEK2, nor G1 or G2 checkpoint function. Chromosomal instability in melanoma cell lines appears to influence gene function but it is not simply explained by alterations in the system of DDR.

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 analyzing 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.

Genome-wide regulatory analysis using en masse nuclear run-ons and ribonomic profiling with autoimmune sera.

Coordinated gene expression is influenced by transcriptional and posttranscriptional events and is necessary for efficient cell growth and differentiation. Genomic array technologies have afforded great advances in identifying global changes of gene expression in response to a variety of environmental stimuli. However, it has been a challenge to assess whether a concomitant effect on protein expression reflects the coordinated regulation of distinct subsets of mRNAs detected by cDNA arrays [Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 7018]. We have expanded the utility of cDNA arrays by using them to assist in elucidating combinatorial posttranscriptional eukaryotic operons [Mol. Cell 9 (2002) 1161]. In this study, we have used two mRNA partitioning methods in which: (1) subsets of mRNAs are isolated as endogenous mRNP complexes using autoimmune patient sera, and (2) transcriptional contributions to gene expression are assessed using cDNA array analysis of an en masse nuclear run-on assay (emRUN). The combination of these methods can provide an additional 'systems biology' discovery approach to gene expression analysis based upon the physical partitioning of mRNA subsets, as well as a functional partitioning of transcriptional and posttranscriptional processes. We demonstrate how these approaches can reduce transcriptomic complexity by partitioning mRNAs into biologically relevant subsets in order to derive information about the expression of multiple, but functionally linked, genes.

Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays.

Although in vitro methods have been used to identify putative targets of mRNA-binding proteins, direct in vivo methods are needed to identify endogenously associated mRNAs and their cognate proteins. Therefore, we have developed high-throughput methods to identify structurally and/or functionally related mRNA transcripts through their endogenous association with RNA-binding proteins. We have termed the identification and analysis of mRNA subsets using RNA-associated proteins ribonomics, and have established four primary steps for the method: (1) isolation of endogenous mRNA-protein complexes (mRNPs) under optimized conditions, (2) the en masse characterization of the protein and mRNA components associated with the targeted mRNP complexes, (3) identification of sequences or structural similarities among members of the mRNA subset, and (4) determination of functional relationships among the protein products coded for by members of the mRNA subset. We have hypothesized that mRNAs are organized into structurally and functionally linked groups to better affect information transfer through coordinate gene expression. The functional consequences of such organization would be to facilitate the production of proteins that regulate processes necessary for growth and differentiation. This article describes a series of biochemical techniques that deal with the first two steps of ribonomic profiling: purifying endogenous mRNP complexes and identifying multiple mRNA targets using microarray analysis.