Combining genetic analyses and pre-clinical studies in melanoma defines multiple sub-populations with differential responses to targeted therapies
Authors: Keiran Smalley, Min Xiao, Maurizia Dalla Palma, Mercedes Lioni, Meenhard Herlyn, Keith Flaherty, Katherine Nathanson
Journal: Clinical Cancer Research (2008)

A30   Identifying genetic changes in subsets of tumors has allowed for the development of therapies targeted specifically to those groups. Genetic analyses of melanomas have stratified them into several groups based on mutations within genes that activate the MAPK signaling pathway, including BRAF, NRAS and KIT. Herein, we describe three groups of melanomas with defined sets of genetic/genomic changes and corresponding pre-clinical studies of therapies targeted to those changes. First, we studied determinants of therapeutic resistance to BRAF-inhibitors in BRAF V600E mutated melanomas. We identified a set of metastatic melanomas and melanoma cell lines, which harbored either concurrent BRAF V600E andCDK4 mutations or concurrent BRAF V600E mutation and cyclin D1 (CCND1) amplification (17% of all BRAF mutated). Increased resistance to a BRAF-inhibitor was not seen in cell lines with a CDK4 mutation alone, but was in a cell line with a CDK mutation and CCND1 amplification. We overexpressed CCND1 alone, and in the presence of CDK4, in a drug sensitive melanoma line. CCND1 overexpression increased resistance, which was enhanced with concurrent overexpression of CDK4. Increased levels of CCND1, resulting from genomic amplification, may contribute to the BRAF-inhibitor resistance of BRAF-V600E mutated melanomas. Secondly, we examined whether non-V600E BRAF mutated melanomas responded differently to therapies than V600E melanomas. We identified a panel of melanoma lines with non-V600E mutations in BRAF (G469E, D549V), which are observed in melanomas. Although the cell lines were highly resistant to MEK inhibition, they were sensitive to the CRAF inhibitor sorafenib, whereas BRAF V600E cell lines were not. Prior studies have shown low-activity mutants of BRAF signal via CRAF. Sorafenib down-regulated CRAF targets, such as Bcl-2, suggesting that drugs targeting CRAF should be used in melanomas with non-V600E BRAF mutations. Lastly, we studied melanomas without activating mutations in BRAF or NRAS, as these constitute a substantial group. We used a genomic strategy to identify a group of melanomas and cell lines with co-overexpression of CDK4 and KIT. Pharmacological studies showed they were resistant to BRAF inhibitors but sensitive to imatinib both in vitro and in vivo. Other melanoma cell lines, which retained some KIT expression but lacked phospho-KIT, were not sensitive to imatinib, suggesting that KIT expression alone is not predictive of response. We suggest that co-overexpression of KIT/CDK4 is a potential mechanism of oncogenic transformation in some BRAF/NRAS wild-type melanomas. Using genetic/genomic studies of melanoma, we have identified three sub-groups of melanoma, two of which are novel. Based on our studies, we can suggest optimal therapies for the two sub-groups of melanomas, sorafenib (or other CRAF inhibitors) in non-V600E BRAF mutated and imatinib (or other KIT inibitors) in KIT/CDK4 co-overexpressed melanomas. In addition, we have demonstrated in patients with BRAF V600E mutations, additional genotyping will need to be done, and in particular concurrent CCND1 amplification appears to confer resistance to BRAF-inhibitors. These studies strongly support the genetic classification of melanomas should be done prior to treatment and demonstrate how they can be used to guide therapeutic selection.