Germline BAP1 Inactivation Is Preferentially Associated with Metastatic Ocular Melanoma and Cutaneous-Ocular Melanoma Families

Background: BAP1 has been shown to be a target of both somatic alteration in high-risk ocular melanomas (OM) and germline inactivation in a few individuals from cancer-prone families. These findings suggest that constitutional BAP1 changes may predispose individuals to metastatic OM and that familial permeation of deleterious alleles could delineate a new cancer syndrome. Design: To characterize BAP1’s contribution to melanoma risk, we sequenced BAP1 in a set of 100 patients with OM, including 50 metastatic OM cases and 50 matched non-metastatic OM controls, and 200 individuals with cutaneous melanoma (CM) including 7 CM patients from CM-OM families and 193 CM patients from CM-non-OM kindreds. Results: Germline BAP1 mutations were detected in 4/50 patients with metastatic OM and 0/50 cases of non-metastatic OM (8 % vs. 0%, p = 0.059). Since 2/4 of the BAP1 carriers reported a family history of CM, we analyzed 200 additional hereditary CM patients and found mutations in 2/7 CM probands from CM-OM families and 1/193 probands from CM-non-OM kindreds (29 % vs. 0.52%, p =.003). Germline mutations co-segregated with both CM and OM phenotypes and were associated with the presence of unique nevoid melanomas and highly atypical nevoid melanoma-like melanocytic proliferations (NEMMPs). Interestingly, 7/14 germline variants identified to date reside in C-terminus suggesting that the BRCA1 binding domain i

Vemurafenib Synergizes with Nutlin-3 to Deplete Survivin and Suppresses Melanoma Viability and Tumor Growth

Purpose: For patients with advanced melanoma, primary and secondary resistance to selective BRAF inhibition remains one of the most critically compelling challenges. One rationale argues that novel biologically informed strategies are needed to maximally cripple melanoma cells up front before compensatory mechanisms emerge. As p53 is uncommonly mutated in melanoma, restoration of its function represents an attractive adjunct to selective BRAF inhibition. Experimental Design: Thirty-seven BRAF(V600E)-mutated melanoma lines were subjected to synergy studies in vitro using a combination of vemurafenib and nutlin-3 (Nt-3). In addition, cellular responses and in vivo efficacy were also determined. We also analyzed changes in the levels of canonical apoptotic/survival factors in response to vemurafenib. Results: Dual targeting of BRAF(V600E) and Hdm2 with vemurafenib and Nt-3, respectively, synergistically induced apoptosis and suppressed melanoma viability in vitro and tumor growth in vivo. Suppression of p53 in melanoma cells abrogated Nt-3's effects fully and vemurafenib's effects partially. A survey of canonical survival factors revealed that both vemurafenib and Nt-3 independently attenuated levels of the antiapoptotic protein, survivin. Genetic depletion of survivin reproduces the cytotoxic effects of the combination strategy. Conclusion: These results show preclinical feasibility for overcoming primary vemurafenib resistance by restoring p53 function. Moreover, it identifies survivin as one downstream mediator of the observed synergism and a potential secondary target. (C)2013 AACR

MITF Modulates Therapeutic Resistance through EGFR Signaling.

Response to targeted therapies varies significantly despite shared oncogenic mutations. Nowhere is this more apparent than in BRAF(V600E)-mutated melanomas where initial drug response can be striking and yet relapse is commonplace. Resistance to BRAF inhibitors have been attributed to the activation of various receptor tyrosine kinases (RTKs) though the underlying mechanisms have been largely uncharacterized. Here, we found that EGFR induced vemurafenib resistance is ligand dependent. We then employed whole-genome expression analysis and discovererd that vemurafenib resistance correlated with the loss of MITF, along with its melanocyte lineage program, and with the activation of EGFR signaling. An inverse relationship between MITF, vemurafenib resistance and EGFR was then observed in patient samples of recurrent melanoma and was conserved across melanoma cell lines and patients' tumor specimens. Functional studies revealed that MITF depletion activated EGFR signaling and consequently recapitulated the resistance phenotype. In contrast, forced expression of MITF in melanoma and colon cancer cells inhibited EGFR and conferred sensitivity to BRAF/MEK inhibitors. These findings indicate that an "autocrine drug resistance loop" is suppressed by melanocyte lineage signal(s), such as MITF. This resistance loop modulates drug response and could explain the unique sensitivity of melanomas to BRAF inhibition.Journal of Investigative Dermatology accepted article preview online, 19 March 2015. doi:10.1038/jid.2015.105

BAP1 Mutations Identified In Study.

<p>Oc mel = ocular melanoma, Cut mel = cutaneous melanoma, NEMMP = nevoid melanoma-like melanocytic proliferation, SSM = superficial spreading melanoma; DCIS = breast ductal carcinoma-in-situ; DOD = died of disease; mat = maternal; pat = paternal; GM = grandmother.</p>*<p>found to be part of the same kindred.</p>#<p>see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035295#pone-0035295-g001" target="_blank">Figure 1</a>.</p

Distribution of <i>BAP1</i> mutations.

<p>Inactivating germline mutations identified in this study are indicated by the red arrows. Reported somatic missense mutations (blue bars) and indels (blue arrows) from ocular melanoma specimens (COSMIC database; <a href="http://www.sanger.ac.uk/genetics/CGP/cosmic/" target="_blank">http://www.sanger.ac.uk/genetics/CGP/cosmic/</a>) and germline variants (blue lines) from other families are also shown. Half of the germline variants occur in the terminal 150 amino acids while the somatic changes are more scattered.</p

Histologic and molecular analyses of tumors from Fam-562.

<p>(<b>A</b>)–(<b>F</b>): Histology of 2 distinct NEMMPs. (<b>A</b>) Scanning view of the first lesion showing two expansile dermal nodules (H&E, 2×,) with a (<b>B</b>) benign nevoid appearance (H&E, 10×). (<b>C</b>) Atypical cytological features including nuclear pleomorphism and prominent nucleoli and a dermal mitotic figure (arrow) (H&E, 40×) along with focal increases in Ki67 staining (inset). (<b>D</b>) In the second lesion, there is an expansile dermal proliferation (H&E, 4×). (<b>E</b>) Detail of a field populated by dermal nevic cells with bland nuclear features (H&E,20×). (<b>F</b>) A proliferative area showing marked nuclear atypia and hyperchromasia along with elevated Ki 67 staining (inset). Biallelic inactivation of <i>BAP1</i> in two tumors through (<b>G</b>) loss of the wildtype allele in a nevoid melanoma (ie. LOH; arrow) or (<b>H</b>) a secondary mutation (p.Ser123Lysfs*3) in a NEMMP that did not exhibit LOH.</p

Cohorts and pedigrees.

<p>(<b>A</b>). Ocular melanoma and cutaneous melanoma cohorts used in this study. (<b>B</b>). Fam-562 pedigree and clinical images of two nevoid melanoma-like melanocytic proliferations (NEMMPs) diagnosed in Fam729. (<b>C</b>). Fam-729 pedigree. One carrier had a Spitz nevus, which has been reported to harbor somatic <i>BAP1</i> mutations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035295#pone.0035295-Wiesner1" target="_blank">[4]</a>. Abbreviations: M, mutation carrier; wt, wildtype germline sequence; Ob, obligate carrier; OCMEL, ocular melanoma; MEL, cutaneous melanoma; NEMMPs, nevoid melanoma-like melanocytic proliferations; BR, breast cancer; CHL, cholangiocarcinoma; LG, lung cancer; KID, kidney cancer; UNP, melanoma of unknown primary site; CNS, central nervous system tumor; LK, leukemia. Crosses indicate CM with a nevoid pattern. The numbers next to the “MEL” indicate ages of diagnosis. <i>For the sake of confidentiality, the pedigrees have been masked for some non-affected individuals and siblings. Nonessential gender information has also been disguised by a diamond; the number of individuals collapsed into the diamond is indicated.</i></p

DNA promoter hypermethylation of melanocyte lineage genes determines melanoma phenotype

Cellular stress contributes to the capacity of melanoma cells to undergo phenotype switching into highly migratory and drug tolerant dedifferentiated states. Such dedifferentiated melanoma cell states are marked by loss of melanocyte specific gene expression and increase of mesenchymal markers. Two crucial transcription factors, MITF and SOX10, important in melanoma development and progression have been implicated in this process. In this study we describe that loss of MITF is associated with a distinct transcriptional program, MITF promoter hypermethylation and poor patient survival in metastatic melanoma. From a comprehensive collection of melanoma cell lines, we observed that MITF methylated cultures were subdivided in two distinct subtypes. Examining mRNA levels of neural crest associated genes we found that one subtype had lost the expression of several lineage genes including SOX10. Intriguingly, SOX10 loss was associated with SOX10 gene promoter hypermethylation and distinct phenotypic and metastatic properties. Depletion of SOX10 in MITF methylated melanoma cells using CRISPR/Cas9 confirmed these findings. In conclusion, this study describes the significance of melanoma state and the underlying functional properties explaining the aggressiveness of such states