Oncogenic mechanism-based pharmaceutical validation of therapeutics targeting MET receptor tyrosine kinase

Abstract

Aberrant expression and/or activation of the MET receptor tyrosine kinase is characterized by genomic recombination, gene amplification, activating mutation, alternative exon-splicing, increased transcription, and their different combinations. These dysregulations serve as oncogenic determinants contributing to cancerous initiation, progression, malignancy, and stemness. Moreover, integration of the MET pathway into the cellular signaling network as an addiction mechanism for survival has made this receptor an attractive pharmaceutical target for oncological intervention. For the last 20 years, MET-targeting small-molecule kinase inhibitors (SMKIs), conventional therapeutic monoclonal antibodies (TMABs), and antibody-based biotherapeutics such as bispecific antibodies, antibody-drug conjugates (ADC), and dual-targeting ADCs have been under intensive investigation. Outcomes from preclinical studies and clinical trials are mixed with certain successes but also various setbacks. Due to the complex nature of MET dysregulation with multiple facets and underlying mechanisms, mechanism-based validation of MET-targeting therapeutics is crucial for the selection and validation of lead candidates for clinical trials. In this review, we discuss the importance of various types of mechanism-based pharmaceutical models in evaluation of different types of MET-targeting therapeutics. The advantages and disadvantages of these mechanism-based strategies for SMKIs, conventional TMABs, and antibody-based biotherapeutics are analyzed. The demand for establishing new strategies suitable for validating novel biotherapeutics is also discussed. The information summarized should provide a pharmaceutical guideline for selection and validation of MET-targeting therapeutics for clinical application in the future.

Keywords: MET receptor tyrosine kinase; antibody–drug conjugates; bispecific antibody; dual-targeting ADC; pharmaceutical validation; small-molecule kinase inhibitor; therapeutic monoclonal antibody; tumorigenic mechanism.

Figure 1.

Schematic representation of structures of the MET gene, MET, and its ligand hepatocyte growth factor (HGF). (a) The MET gene is located in the 7p31 locus of chromosome 7. It contains 21 exons separated by 21 introns. The classical promoter contains two transcription factors including specificity protein 1 (SP1) and activating protein-2 (AP2)-binding elements and is responsible for the transcription of full-length MET with 1408 amino acids. (b) MET is first synthesized as a biologically inactive single-chain precursor (pro-MET). Proteolytic conversion is required to activate MET. Mature MET is composed of a 45 KDa α-chain and a 145 kDa β-chain linked by a disulfide bond. Structurally, the MET α-chain is an extracellular component containing a portion of the semaphorin (SEMA) domain. The extracellular sequence of the MET β-chain contains a large portion of the SEMA domain, followed by a plexin-semaphorin-integrin (PSI) domain, and 4 immunoglobulin-like plexin and transcription (IPT) motifs. The intracellular sequence harbors a short transmembrane (TM) segment followed by a juxtamembrane domain (JM), a tyrosine kinase (TK) domain, and a C-terminal tail. Regulatory tyrosine residues, Y1003 in the JM domain and Tyr¹²³⁴ and Tyr¹²³⁵ in the TK domain are indicated. Also, Tyr¹³⁴⁹ and Tyr¹³⁵⁶ in the MET C-terminal tail, which form the functional docking site, respectively, are marked. (c) HGF is first synthesized as a biologically inactive single-chain precursor known as pro-HGF. Proteolytic cleavage results in a biologically active two-chain form of mature HGF. The HGF α-chain contains a hairpin loop (HPL) followed by four kringle domains (K1 to K4). The HGF β-chain contains a serine protease-like domain with substation of amino acids in the active site. The high-affinity MET-binding site is in the HGF α-chain and the low-affinity MET-binding site is in the HGF β-chain.

Figure 2.

Dysregulated MET activation, signaling pathway, and tumorigenic consequence. Activation of MET in cancer cells, in general, is mediated through multiple mechanisms including ligand binding, activating mutation, receptor overexpression, aberrant splicing/alternative initiation, and transactivation through other receptor tyrosine kinases such as EGFR, IGF-1R, and RON. HGF-induced MET activation, a classical model, is functional through phosphorylation of several critical tyrosine residues and creates the C-terminal functional docking site, which recruits cytoplasmic molecules such as SOS and GRB2. The negative modulator c-CBL, a ubiquitin ligase, also binds the docking site and mediates MET endocytosis and degradation. Multiple signaling cascades, such as RAS/MAP kinase, PI3K/AKT, Wnt/β-catenin, and TGF-β/SMAD pathways, are activated upon MET phosphorylation in cancer cells, which creates a complex intracellular signaling network. The biological consequence is to induce cell proliferation with a malignant phenotype known as EMT, which leads to increased cellular survival, invasiveness, chemoresistance, and tumorigenic stemness. AKT, BCL-2, B cell lymphoma-2; Cbl, protein kinase B; EMT, epithelial to mesenchymal transition; GRB2, growth factor receptor-bound protein-2; MAP, mitogen-activated protein kinase; PI3K, phosphatidyl-inositol 3 kinase; RAS, reticular activating system; Smad, small mothers against decapentaplegic; SOS, son of sevenless; TGF-β, transforming growth factor-β.

Figure 3.

MET dysregulations observed in cancer cells from different tissues and therapeutics suitable for the targeted therapy. Different types of cancerous MET dysregulation are depicted in red oval circles. Various forms of therapeutics specific to MET that are suitable for targeting MET-expressing cancer cells are indicated in yellow boxes.

Figure 4.

Activating mutations in the different functional domains of MET. (a) Various mutations in the tyrosine kinase domain of MET. Point mutations in more than 16 amino acid residues in the kinase domain have been documented in different types of primary cancer samples. These mutations often result in a conformational change that facilitates the kinase domain to convert into an active mode with increased kinase activity. (b) Missense mutations in the exon 14 ubiquitination site. The JM domain is encoded by MET exon-4. The tyrosine residue Tyr¹⁰⁰³ in the JM domain is responsible for the interaction with the ubiquitin E3 ligase, which promotes MET degradation, a negative feedback mechanism for controlling levels of MET activation. The mutation results in the inability of Tyr¹⁰⁰³ to interact with ubiquitin E3 ligase, leading to an increase in stability of MET. (c) Alterations in the exon-14 splice site often results in exon-14 skipping, leading to formation of a MET slicing variant known as MET exon-14 skipping. The consequence is that this MET variant is resistant to ubiquitin-mediated protein degradation with increased stability and kinase activity. (d) Various mutations are documented in the SEMA domain of MET. Since the SEMA domain contains the MET-binding pocket; it is speculated that these mutations will affect the ability of HGF binding to MET with reduced affinity. However, pathological implication of these mutations in association with clinical oncological events currently are largely unknown.