[New gene editing products launch] MET Ex14 skipping new members

date:2023-02-15 source:

Background


MET receptor tyrosine kinase is a proto-oncogene, and its abnormal activation is related to tumorigenesis. Various underlying mechanisms, including alterations in transcripts, amplification, gene rearrangements, and exon skipping, are responsible for abnormal MET signaling. One of the key MET alterations leading to non-small cell lung cancer (NSCLC) is MET exon 14 (MET Ex 14) skipping, a driver mutation that accounts for about 3-4% of lung adenocarcinomas. MET Ex14 hopping leads to the formation of a functionally active and stable truncated receptor that lacks the juxtamembrane regulatory domain responsible for MET ubiquitination. To date, several MET kinase inhibitors targeting the MET receptor have been developed worldwide, many of which are in clinical trials.



The structure of MET


The MET gene is located on chromosome 7q31 of the human genome, spans about 125kb DNA, and contains 21 exons and 20 introns. MET is encoded as a precursor that is modified into a mature protein by proteolytic cleavage between its a and b subunits. The mature MET protein consists of a small a subunit (50kDa) and a larger b (145kDa) subunit, linked together by disulfide bonds. One a subunit and part of the b subunit together constitute the extracellular region of the heterodimeric protein, while the rest of the b subunit constitutes the transmembrane and intracellular regions.


The extracellular component of MET contains three domains. The N-terminal Sema (Sema-phorin) is the largest domain, comprising 500 residues, including part of the a and b subunits. This domain is critical for ligand binding, dimerization and activation of MET. The Sema domain is followed by the plexin-signalin-integrin (PSI) domain, which contains four disulfide bridges, which are critical for the correct positioning of the ligand-bound receptor. The PSI domain is linked to the transmembrane helix of MET by an immunoglobulin-plexin-transcription factor domain. The intracellular portion of the receptor includes the juxtamembrane (JM) domain, the tyrosine kinase (TK) catalytic domain, and the C-terminal multifunctional docking site. Binding of its ligand, hepatocyte growth factor (HGF), also known as scatter factor, is critical for activation of the kinase activity. HGF is the hitherto known MET receptor ligand and binds to the receptor with high affinity.



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Fig1. The structure of MET protein

 

Mechanism of MET Ex14 jumping


Binding of HGF to MET leads to dimerization of the receptor, leading to autophosphorylation of intracellular residues Y1234 and Y1235 in the kinase domain, followed by phosphorylation of two additional tyrosine residues Y1349 and Y1356 at the C-terminus outside the kinase domain. Phosphorylation of C-terminal residues leads to the formation of docking sites, followed by adapter and effector proteins such as GRB2 (growth factor receptor binding protein 2), GAB1 (GRB2-related binding protein 1), and SHC (containing Src homology 2 structures domain), binds to the docking site and triggers downstream signaling.


Jumping of MET Ex14 in NSCLC was reported in 2005. Replacement or deletion of the 3' splice site of intron 13 or the 5' end splice site of intron 14 resulted in skipping of MET Ex14. This somatic alteration at or near the splice junction of MET Ex14 results in loss of exon 14 in the transcript and synthesis of MET protein and an in-frame deletion of 47 amino acids in the JM domain (including residue Y1003) Ablation of CBL-mediated receptor ubiquitination and degradation. Thus, MET Ex14 jumping leads to increased MET protein levels, which can drive the activation of downstream signaling pathways that promote tumor development.

 

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Fig 2. Mechanism of MET Ex14 jumping

 

MET Ex14 jumping mutations


According to literature reports, the common mutations leading to MET Ex14 skipping in non-small cell lung cancer are as follows:


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Fig 3. Common mutations leading to MET Ex14 skipping

 

It is also found in numerous other cancer types, with approximately 3–4% of NSCLC having MET Ex14 alterations. Among histologic subtypes, MET Ex14 skipping alterations were common in sarcomatoid carcinoma (4.9–31%),69–72 adenosquamous carcinoma (4–8%), adenocarcinoma (3–4%), and squamous cell carcinoma ( 2%). Furthermore, in adenocarcinoma, the predominant subtypes were acinar (35-52.9%) or solid (35.3-53%). Clinically, abnormal jumping of METEx14 is more common in elderly patients.


Detection of MET Ex14 jumps


In general, DNA- and RNA-based molecular assays are the methods to detect METex14 alterations. DNA-based sequencing analysis can detect MET alterations, such as splice site insertions, deletions, point mutations, or duplications, that may lead to exon 14 skipping. However, METEx14 jumping is associated with more than 120 reported sequence variations in splice sites, making it challenging to detect these mutations using only DNA-based assays. Therefore, analysis of RNA transcripts could validate the fusion between exons 13 and 15. Ideally, DNA- and RNA-based assays would complement each other to reliably detect METEx14 alterations.


 

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Fig 4. Common methods for DNA and RNA detection techniques to identify MET Ex14 skipping

 

Standard for MET Ex14 jumps


Using the method of gene editing, we successfully obtained a variety of MET Ex14 jumping standard products, showing splicing mutations at the DNA level and deletions of exon 14 at the RNA level. Some products are listed below:

 

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Table 1. Some products of MET Ex14 skipping mutation

 

The detection data is as follows (take c.3028+1_c.3028+9del as an example)

 

AI-Edigene® MET Splice Site Mutation(c.3028+1_c.3028+9del)Reference Standard

 

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Fig 5. Sanger sequencing of DNA shows c.3028+1_c.3028+9del


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Fig 6. The deletion of exon 14 in RNA sanger sequencing

 

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Fig 7. ddPCR detection of DNA, showing a mutation frequency of 100%

 

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Fig 8. ddPCR detection of RNA shows that the copy number is 1315.07 copies/ng

 

As can be seen from the above data, not only splicing mutations occurred at the DNA level, but also at the RNA level, and according to the copy number data of ddPCR, the expression of MET was also greatly increased.

 

We have extensive experience in using gene editing technology to customize site mutations, insertions, deletions, and fusions. Welcome to communicate and discuss with us.


 


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