BRAF targets in melanoma. Biological mechanisms, resistance, and drug discovery. Cancer drug discovery and development. Volume 82. Ed. Ryan J. Sullivan. Springer (2015)
The sentinel event in the development of targeted therapy for melanoma was the discovery of point mutations in the BRAF gene . These mutations were identified as part of a screen for mutations in the genes that encode the RAF kinases, which are part of the RAS-RAF-MEK-ERK signaling cascade (Fig. 6.1). This initial screen of cell lines and tumors identified recurrent point mutations in exon 15 of the BRAF gene, most frequently in the melanomas that were included in the study, but also in colorectal, primary brain, lung, liver, ovarian, and other cancer types. Subsequent studies have demonstrated that more than 90% of the BRAF mutations that are detected in melanoma occur in exon 15 and result in substitutions for the valine at the 600 position (V600) . The most common mutation results in substitution of a glutamic acid (V600E), which in multiple series has been shown to represent = 70% of the detected BRAF mutations [17, 18]. The catalytic activity of the BRAF V600E mutant protein is increased more than 400-fold in comparison to the wild-type BRAF protein and results in constitutive activation of MEK and ERK. Other substitutions at the V600 site, including V600K and V600D, also markedly (more than 100–200-fold) increase the catalytic activity of BRAF. A variety of other rare point mutations in BRAF have also been detected, both in exon 15 (i.e. K601E, L597V) and exon 11 (i.e. G469E, G464E). Interestingly, these mutations are quite variable in their effects on the catalytic activity of BRAF, with some mutations actually resulting in decreased kinase activity (i.e. G466E, D594V, and G596R) . However, expression of essentially any of these mutations results in increased activation of MEK and ERK, as the kinase-inactivating mutations promote the formation of BRAF-CRAF heterodimers that activate the pathway through CRAF’s catalytic activity .
Fig. 6.1 Frequent somatic mutations in signaling pathways in melanoma.
Meta-analyses of large cohorts of melanoma clinical samples have demonstrated that substitutions of the V600 residue of BRAF occur in 40–50% of cutaneous melanomas  (Table 6.1). These mutations are most frequent in cutaneous melanomas arising in areas with intermittent sun exposure, but are less common in tumors that arise in areas of chronic sun exposure and have histologic evidence of chronic sun damage (CSD) [21, 22]. The mutations are less prevalent (10–15%) in acral melanomas, which arise on the relatively sun-protected palms of the hands, soles of the feet, and nailbeds. Mucosal melanomas, which arise from mucosal surfaces throughout the body, have a BRAF mutation rate of < 5%. Finally, BRAF mutations have not been detected in uveal melanomas that arise from melanocytes in the eye.
Activating mutations in NRAS, which also activate the RAS-RAF-MEK-ERK signaling pathway, are the second most common somatic activating mutations detected in melanoma. These mutations occur in 15–20% of cutaneous melanomas, most commonly resulting in substitutions at the Q61 residue of exon 2 (~ 80% of mutations) or the G12/13 residues of exon 1 (~ 20%) . NRAS mutations are also detected in acral and mucosal melanomas, but are not found in uveal melanomas (Table 6.1). In treatment-naпve patients, hotspot NRAS mutations and BRAF V600 mutations are essentially mutually exclusive, with both mutations found in less than 1% of tumors . However, NRAS mutations are frequently detected in melanomas with non-V600 BRAF mutations, particularly those that fail to increase the catalytic activity of BRAF . Similar to NRAS, strong genetic interaction has also been identified for loss of function mutations of the PTEN tumor suppressor . PTEN is a phosphatase that dephosphorylates phospho-lipids in the cell membrane, thereby antagonizing signaling by the oncogenic lipid kinase PI3K. Loss of PTEN results in constitutive signaling through the PI3K-AKT pathway. A number of analyses have demonstrated that loss of function and/or expression of PTEN in melanomas are mutually exclusive with the presence of NRAS mutations [24–26]. In contrast, PTEN can occur in melanomas with activating BRAF mutations, and is detected in 20–30% of BRAF V600-mutant melanomas.
Table 6.1. Prevalence and pattern of common somatic mutations in different melanoma subtypes. “CSD”, chronic sun damaged. “–”, insignificant number reported. “?”, not yet reported.
|Cutaneous (Non-CSD)||45%||15–20%||~ 1%||–||?|
|Uveal||–||–||–||80%||50% (85% of monosomy 3)|
|Melanoma from an unknown primary||50%||20%||–||–||–|
Focused sequencing studies have identified a number of other somatic changes in oncogenes in melanoma in, or downstream of, the canonical RAS-RAF-MEK-ERK and PI3K-AKT pathway, such as rare activating point mutations in AKT1, AKT3, MEK1, and amplifications of cyclin D1 [27–29] (Fig. 6.1). In addition, deletions and inactivating mutations of the CDKN2A gene that cause loss of expression/function of the P16 protein are germline mutations in many cases of familial melanoma, and may also occur somatically . Activating mutations and amplifications of the CDK4 gene are also detected in melanomas as germline or somatic events . In addition to these events in cutaneous tumors, studies have revealed a number of mutations in other melanoma subtypes. Somatic mutations and gene amplifications of the KIT gene on chromosome 4 have been identified as frequent events (10–30%) in acral and mucosal melanomas . Some studies have also suggested that these mutations are also common in cutaneous melanomas with evidence of chronic sun damage (CSD), but this has not been observed in other studies . Molecular characterization of uveal melanomas demonstrated a lack of BRAF, NRAS, or KIT mutations in these tumors, but loss of expression of PTEN has been observed [34, 35]. Uveal melanomas instead have a high prevalence of activating point mutations in the GNaQ (35%) and GNa11 (45) genes, which encode regulatory subunits of G-protein coupled receptors [36–38]. As these mutations are mutually exclusive, altogether they are present in ~ 80% of uveal melanomas, and preclinical studies suggest that they can cause activation of multiple signaling pathways. Approximately 80% of uveal melanomas that have monosomy 3, which correlates with poor prognosis, have inactivating mutations of the BAP1 gene, which is located at 3p21 . Germline mutations in BAP1 have also been identified in families with an increased risk of developing uveal melanoma [40, 41].
Recently, broad sequencing efforts that characterize the entire exome or genome have been initiated melanoma [42–45]. These studies have demonstrated that cutaneous melanomas have an extremely high somatic mutation rate. The majority of the observed somatic mutations were C à T or G à A transitions, which are associated with DNA damage from ultraviolet radiation (UVR) . This data is consistent with multiple functional and epidemiological studies implicating UVR in the development of melanoma . These broad sequencing studies have demonstrated the molecular complexity and heterogeneity of melanomas (Fig. 6.2) . The studies have identified many additional somatic events that occur in melanomas with activating BRAF or NRAS mutations, as well as candidate drivers in melanomas that do not have a hotspot mutation in either of those genes. While these studies have already provided significant insight into the molecular pathogenesis of melanoma, it has also illuminated that a critical challenge to researchers will be to determine which mutations are clinically significant. In addition to being therapeutic targets, mutations may have clinical utility if they add to risk prediction models that are used to guide the selection of treatments for patients, or to inform the appropriate design of clinical trials [17, 18, 48]. While the large number of alterations observed in melanoma makes this overall appear to be a daunting challenge, the clinical experience with BRAF V600-mutant melanomas has already demonstrated the tremendous clinical impact such findings can have.
Fig 6.2. Pattern of novel and known somatic alterations in a cohort of 121 melanomas.
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