Nature Genetics | Free Association

Melanoma sequencing identifies new druggable targets

Next-generation sequencing technologies are enabling unbiased searches for new cancer genes at an unprecedented scale. In 2011, a flurry of cancer exome and whole-genome papers have been published in high-impact journals, with more in the pipeline. The first genes to be targeted for personalized treatment will be ones harboring recurrent mutations at a high frequency and those with already known inhibitors/modulators. The delivery of personalized therapies in cancer will no longer be bottlenecked by a lack of targets; the development of effective therapies will require new insights into how cancers become resistant to drugs and hopefully, therapeutic interventions that bypass acquired resistance.

Melanomas are tumors of melanocytes, the pigment-producing cells in skin, and occasionally in the iris of the eye (uveal melanoma). Every year, melanoma is diagnosed in approximately 160,000 new cases and leads to death in 48,000 people worldwide. Exposure to UV radiation is a major risk factor for melanoma, and the incidence of melanoma is rising. Patients with stage IV disease have a poor prognosis, with a median survival of 8-18 months post-diagnosis. Currently, dacarbazine is the only chemotherapeutic drug approved by the FDA for metastatic melanoma and has a median overall survival of 5.6-7.8 months after treatment begins.

Phase III clinical trial results for PLX4032 show remarkable clinical activity in melanomas with BRAF mutations.

In 2002, mutations in the protein kinase BRAF were identified in 66% of malignant melanoma tumors analyzed; subsequent studies estimate that 40-60% of melanomas have mutations in BRAF. Nearly 90% of mutations in BRAF lead to a valine to glutamate change (p.Val600Glu). In order to develop small molecule inhibitors of p.Val600Glu, Gideon Bollag of Plexxikon, Inc. in Berkeley, CA and colleagues screened a library of 20,000 compounds using a structure-guided approach. In 2008, the scientists reported that the compound PLX4720 specifically inhibits BRAF p.Val600Glu with potent anti-melanoma effects in in vitro and in vivo models.

Recently, the long-awaited results of phase III clinical trials of the BRAF kinase inhibitor (PLX4032, an analogue of PLX4720) were published ), showing increased overall survival in patients on PLX4032 compared to the chemotherapeutic agent dacarbazine. In the study, 675 patients with metastatic melanoma carrying the p.Val600Glu mutation were randomly assigned to treatment with PLX4032 or dacarbazine. After six months, overall survival in the PLX4032 group was 84%, but only 64% in the group receiving dacarbazine. Of all patients treated with PLX4032, 48% had a confirmed response, although the authors report that most patients showed tumor shrinkage. Despite the obvious benefit of PLX4032 treatment, the effectiveness of the treatment is short-lived, as many responsive tumors become resistant to treatment within 8-12 months. Determining the mechanisms responsible for acquired resistance to PLX4032 is a top priority for further investigation, and some insights have already been made. Surprisingly, in three recent studies

resistance has not been found to be caused by second-site mutations in BRAF, but activation of alternative compensatory pathways.

The promise of personalized treatment in cancer is another step closer with the identification of mutations in ERBB4 in ~20% of melanoma tumors by Yardena Samuels and colleagues.

In a study published in this journal, Yardena Samuels of the National Human Genome Research Institute and colleagues sequenced 86 members of the protein tyrosine kinase gene family in 29 metastatic melanomas. They identified 19 genes with 30 somatic mutations and sequenced these 19 genes in a larger number of melanomas. The most highly mutated gene in this screen was ERBB4, with mutations in 19% of melanomas. Crystal structure analysis showed that positioning of mutations found in ERBB4 were reminiscent of mutations found in the ERBB family members EGFR and ERBB2 in lung cancer, glioblastoma and gastric cancer.

Lapatinib is a dual receptor tyrosine kinase inhibitor that targets EGFR and HER2; this drug was approved by the FDA in 2010 for treatment of HER2-overexpressing metastatic breast cancer. Functional analysis of a subset of the ERBB4 mutations led Samuels and colleagues to hypothesize that mutant ERBB4 might be inhibited by the pan-ERBB inhibitor lapatinib. The scientists found that this small molecule can reduce melanoma cell proliferation in vitro. Armed with this pre-clinical validation data, a multi-center phase II clinical trial at the National Institutes of Health Clinic Center (Principal Investigator Dr. Udo Rudloff) and Memorial Sloan-Kettering Cancer Center (Principal Investigator Dr. Mark Dickson) is currently underway. The trial is testing lapatinib for stage IV melanomas harboring ERBB4 mutations (Clinical Trials.gov Identifier NCT01264081 and Protocol 10-222, respectively).

In the first exome analysis of melanoma also published in this journal and available free for download, Samuels and colleagues identified 68 genes with somatic mutations. They found that GRIN2A is mutated in 25% (34 mutations in 135 samples) of melanomas analyzed. GRIN2A is a glutamate (NMDA) receptor subunit that also is mutated in neurodevelopmental phenotypes and reported in this journal last year. The germline mutations in neurodevelopmental disorders do not overlap with the somatic mutations in melanoma, but mutations in both diseases lead to early stop codons that presumably are null alleles. In melanoma, the spectrum of mutations in GRIN2A cluster to two regions of the protein. The authors also observed three recurrent alterations in residues that are evolutionarily conserved.

The glutamate signaling pathway is likely to be functionally significant in melanoma, since another highly mutated gene was PLCB4, a phosopholipase isozyme known to signal through metabotropic glutamate receptors. Adding another connection between phospholipase activity and melanocytic-derived tumors, Boris Bastian and colleagues recently reported recurrent somatic mutations in the G-proteins GNA11 and GNAQ in uveal melanoma. Somatic mutations in GNAQ were also found in blue naevi; mutations in GNAQ were limited to codon 209 and lead to overactivation of the MAP kinase pathway. Both GNA11 and GNAQ are G-proteins (alpha subunits) that stimulate phospholipase C-beta.

In another link between melanoma and glutamate signaling, Samuels and colleagues note that GRM3, a metabotropic glutamate receptor activated by glutamate, is mutated in 16% of melanoma tumors (unpublished data). A role for glutamate signaling has been seen in neuronal tumors, with an excess of glutamate associated with more aggressive growth of tumor cells. There are published reports of glutamate inhibitors suppressing tumor growth; further investigation of the possible therapeutic benefit of modulating glutamate signaling in GRIN2A, PLCB4 and GRM3 melanomas seems like a natural next step.

Finally, Samuels and colleagues identified a recurrent mutation in TRRAP in 4% (6/167) of melanomas (p.Ser722Phe). The likelihood of this occurrence is ~ 5 X 10-20, suggesting this mutation is functionally significant. TRRAP is part of a protein complex that has histone acetyltransferase activity and is an essential cofactor for the oncogenic transcription factors c-Myc and E1A/E2F. Functional experiments show that the p.Ser722Phe mutation is transforming and that mutant TRRAP is required for survival of melanoma cells; insights into how mutant TRRAP contributes to carcinogenesis is an important area for future research. The identification of TRRAP mutations adds melanoma to the long list of cancers harboring mutations in proteins with histone modifying activity.

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