Mesenchymal-epithelial transition factor (MET) is a proto-oncogene located on chromosome 7 that encodes a receptor tyrosine kinase expressed on the surface of epithelial cells (1, 2). Activation by extracellular hepatocyte growth factor (HGF) binding induces dimerization and autophosphorylation of MET’s cytoplasmic kinase domain and subsequent activation of downstream pathways that regulate cell survival, motility and proliferation (3-6)(reviewed in (7)) (Figure 1). MET aberrations resulting in constitutive or prolonged activation confer an aggressive phenotype in cancer, promoting tumor proliferation, invasive growth and angiogenesis (reviewed in (7, 8)). Originally identified in papillary renal cell carcinoma (PRC), MET aberrations have now been implicated in a variety of neoplasms, including non-small cell lung cancer (NSCLC), gastric adenocarcinoma and pancreatic, esophageal, thyroid, colorectal, ovarian and breast cancers (9-11). Furthermore, aberrant MET activation often confers acquired resistance to EGFR inhibitors in EGFR-driven cancers, primarily due to cross-talk between these two tyrosine kinase signaling pathways (12-15). MET activation can be inhibited in multiple ways: 1) inhibition of HGF maturation which prevents HGF ligand binding and subsequent MET activation; 2) small molecule or antibody-mediated neutralization of HGF-MET binding; and 3) intracellular inhibition of MET kinase activity by both selective and nonselective tyrosine kinase inhibitors (reviewed in (11, 16)).
Constitutive or prolonged MET activation and subsequent tumorigenesis can result from genetic MET alterations, MET overexpression or aberrant HGF-dependent autocrine/paracrine signaling (reviewed in (17)). Genetic alterations of MET include gene fusions, kinase-activating point mutations, mutations that drive the production of an isoform lacking exon 14 (aka “exon 14 skipping”) and gene amplifications. Detecting these genetic alterations as well as the expression level of MET is necessary to identify the underlying cause of MET deregulation, which dictates which inhibitors effectively block MET activation. As detailed below, the Archer™ VariantPlex™ and FusionPlex™ assays can detect all known types of MET aberrations and can therefore be used to determine the cause of MET deregulation.
Since the original in vitro characterization of the oncogenic TPR-MET fusion in human osteogenic sarcoma cells, several MET fusions have been identified in an array of human cancers, including low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, thyroid carcinoma and PRC (18, 19). As a shared mechanism, MET fusion proteins lack a transmembrane domain as well as the portion of the protein that is normally targeted for ubiquitin-mediated degradation (Figure 2). Furthermore, the fusion partner contains a dimerization motif that induces ligand-independent dimerization and thus autophosphorylation and constitutive activation of the MET kinase domain. Therefore, MET fusion proteins are constitutively active, intracellular proteins with prolonged stability. These intracellular fusion proteins are inaccessible to inhibitors of HGF ligand binding, thereby limiting MET inhibition to intracellular kinase inhibitors. MET fusions can be detected using the following Archer FusionPlex kits:
Activating point mutations in the tyrosine kinase domain of MET were originally discovered in hereditary papillary renal carcinoma (PRC) and somatic activating mutations have since been identified in a subset of sporadic PRC, NSCLC, gastric, head and neck, liver, ovarian, thyroid carcinomas (reviewed in (20)). Data obtained from hereditary PRC tumors indicate that multiple copies of the mutated MET gene are necessary to confer an oncogenic phenotype, as trisomy of chromosome 7 accompanies heterozygous MET mutations in this disease (9). Importantly, mutations occurring in the kinase domain can alter the sensitivity of MET to kinase inhibitors (21). The Archer VariantPlex CTL and Solid Tumor assays cover hot spots in the kinase domain of MET, thus detecting activating point mutations in MET.
METex14, an isoform of MET which lacks exon 14, is an important driver of tumorigenesis in lung adenocarcinoma, other lung neoplasms and brain glioma (22-25). Similar to MET fusion proteins described above, METex14 proteins exhibit prolonged stability due to the loss of their ubiquitin-binding domain, which is encoded by exon 14 (Figure 3). This increased stability of METex14 membrane-bound proteins results in increased HGF/MET-dependent signaling, thereby promoting tumorigenesis. Importantly, a recent study by G.M. Frampton and colleagues demonstrated that MET sequence variants driving exon 14 skipping are highly diverse, with 126 distinct sequence variants detected in 221 cases of METex14 expression (22). More than half of these variants were insertions and deletions (indels) of varying sizes and the rest were base substitutions most frequently occurring in the vicinity of splice acceptor and splice donor sites. As MET mRNA sequence information can be used to detect exon skipping regardless of underlying genomic alterations, RNA-seq (mRNA sequencing) is invaluable in detecting METex14. The aforementioned Archer FusionPlex assays can detect exon 14 skipping leading to expression of the METex14 isoform. MET DNA sequence variants can also be detected by Archer VariantPlex Solid Tumor and Archer VariantPlex CTL assays. MET mRNA and DNA sequence information obtained using these kits provides a means to detect METex14 as well as identify underlying driver mutations.
MET gene amplifications have been detected in a variety of cancers, yet perhaps the most interesting finding is that these amplifications have been observed in liver metastases from colon carcinoma and in cancers with acquired EGFR drug-resistance (13, 15, 26). This indicates that MET amplifications can arise throughout the course of tumor progression, conferring a more aggressive phenotype. MET amplifications result in increased protein expression levels and subsequent increased MET-dependent signaling. MET amplifications can be detected by the VariantPlex Solid Tumor and VariantPlex CTL assays and the resulting increased expression levels of MET mRNA can be detected by the FusionPlex CTL assay.
Transcriptional upregulation of MET is the most frequent cause of constitutive MET activation in human tumors and has been implicated in thyroid, colorectal, ovarian, pancreatic, lung and breast carcinomas (27). Genetic alterations frequently found in cancers, such as RAS mutations and TP53 loss-of-function, result in activation of MET transcription (28). However, one of the most common causes of transcriptional upregulation is hypoxia, which often occurs at the center of a rapidly growing tumor (29). Hypoxia-inducible factor 1α (HIF1α), a transcription factor of MET, induces MET transcription in response to decreases in intracellular oxygen levels (30). As with MET gene amplifications, transcriptional upregulation of MET results in increased levels of surface-bound MET and ligand-dependent MET signaling. In both cases, the FusionPlex CTL kit can be used to determine relative expression levels of MET. Furthermore, the VariantPlex CTL or VariantPlex Solid Tumor kit in conjunction with the expression data obtained by the FusionPlex CTL kit can be used to distinguish between overexpression caused by gene amplification versus transcriptional upregulation.
Activation of the MET receptor tyrosine kinase is deregulated in an array of human cancers of epithelial origin (10, 11). Constitutive or prolonged MET activation leads to the induction of several signaling pathways that promote tumorigenesis (7). Aside from dysregulated ligand-dependent autocrine and paracrine signaling, several types of genomic aberrations have been identified that drive MET deregulation, including gene fusions, activating point mutations, expression of the isoform METex14 and gene amplification (17). Transcriptional upregulation, which typically occurs in response to the unique tumor microenvironment, also leads to constitutive MET activation and subsequent promotion of tumorigenesis. ArcherDX provides VariantPlex and FusionPlex kits that enable detection of all known types of genetic lesions leading to MET deregulation as well as determine relative mRNA expression levels. Please see Table 1 below for a summary of known MET alterations, their downstream effect on MET proteins and the Archer kits that can be used to detect them.
|Aberration type||Result of aberration||FusionPlex Kit||VariantPlex Kit|
|Activating point mutation||Constitutive activation||N/A|
|Exon 14 skipping (METex14)||Prolonged stability||N/A|
|Gene amplification||Increased protein level||CTL|
|Transcriptional upregulation||Increased protein level||CTL||N/A|
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