Recording and classifying MET receptor mutations in cancers.

Tyrosine kinase inhibitors (TKI) directed against MET have been recently approved to treat advanced non-small cell lung cancer (NSCLC) harbouring activating MET mutations. This success is the consequence of a long characterization of MET mutations in cancers, which we propose to outline in this review. MET, a receptor tyrosine kinase (RTK), displays in a broad panel of cancers many deregulations liable to promote tumour progression. The first MET mutation was discovered in 1997, in hereditary papillary renal cancer (HPRC), providing the first direct link between MET mutations and cancer development. As in other RTKs, these mutations are located in the kinase domain, leading in most cases to ligand-independent MET activation. In 2014, novel MET mutations were identified in several advanced cancers, including lung cancers. These mutations alter splice sites of exon 14, causing in-frame exon 14 skipping and deletion of a regulatory domain. Because these mutations are not located in the kinase domain, they are original and their mode of action has yet to be fully elucidated. Less than five years after the discovery of such mutations, the efficacy of a MET TKI was evidenced in NSCLC patients displaying MET exon 14 skipping. Yet its use led to a resistance mechanism involving acquisition of novel and already characterized MET mutations. Furthermore, novel somatic MET mutations are constantly being discovered. The challenge is no longer to identify them but to characterize them in order to predict their transforming activity and their sensitivity or resistance to MET TKIs, in order to adapt treatment.

Deficiency of the HGF/Met pathway leads to thyroid dysgenesis by impeding late thyroid expansion.

The mechanisms of bifurcation, a key step in thyroid development, are largely unknown. Here we find three zebrafish lines from a forward genetic screening with similar thyroid dysgenesis phenotypes and identify a stop-gain mutation in hgfa and two missense mutations in met by positional cloning from these zebrafish lines. The elongation of the thyroid primordium along the pharyngeal midline was dramatically disrupted in these zebrafish lines carrying a mutation in hgfa or met. Further studies show that MAPK inhibitor U0126 could mimic thyroid dysgenesis in zebrafish, and the phenotypes are rescued by overexpression of constitutively active MEK or Snail, downstream molecules of the HGF/Met pathway, in thyrocytes. Moreover, HGF promotes thyrocyte migration, which is probably mediated by downregulation of E-cadherin expression. The delayed bifurcation of the thyroid primordium is also observed in thyroid-specific Met knockout mice. Together, our findings reveal that HGF/Met is indispensable for the bifurcation of the thyroid primordium during thyroid development mediated by downregulation of E-cadherin in thyrocytes via MAPK-snail pathway.

Development of an Engineered Single-Domain Antibody for Targeting MET in Non-Small Cell Lung Cancer.

The Mesenchymal Epithelial Transition (MET) receptor tyrosine kinase is upregulated or mutated in 5% of non-small-cell lung cancer (NSCLC) patients and overexpressed in multiple other cancers. We sought to develop a novel single-domain camelid antibody with high affinity for MET that could be used to deliver conjugated payloads to MET expressing cancers. From a naïve camelid variable-heavy-heavy (VHH) domain phage display library, we identified a VHH clone termed 1E7 that displayed high affinity for human MET and was cross-reactive with MET across multiple species. When expressed as a bivalent human Fc fusion protein, 1E7-Fc was found to selectively bind to EBC-1 (MET amplified) and UW-Lung 21 (MET exon 14 mutated) cell lines by flow cytometry and immunofluorescence imaging. Next, we investigated the ability of [89Zr]Zr-1E7-Fc to detect MET expression in vivo by PET/CT imaging. [89Zr]Zr-1E7-Fc demonstrated rapid localization and high tumor uptake in both xenografts with a %ID/g of 6.4 and 5.8 for EBC-1 and UW-Lung 21 at 24 h, respectively. At the 24 h time point, clearance from secondary and nontarget tissues was also observed. Altogether, our data suggest that 1E7-Fc represents a platform technology that can be employed to potentially both image and treat MET-altered NSCLC.

Unraveling the Significance of MET Focal Amplification in Lung Cancer: Integrative NGS, FISH, and IHC Investigation.

MET amplification (METamp) represents a promising therapeutic target in non-small cell lung cancer, but no consensus has been established to identify METamp-dependent tumors that could potentially benefit from MET inhibitors. In this study, an analysis of MET amplification/overexpression status was performed in a retrospectively recruited cohort comprising 231 patients with non-small cell lung cancer from Shanghai Chest Hospital (SCH cohort) using 3 methods: fluorescence in situ hybridization (FISH), hybrid capture-based next-generation sequencing, and immunohistochemistry for c-MET and phospho-MET. The SCH cohort included 130 cases known to be METamp positive by FISH and 101 negative controls. The clinical relevance of these approaches in predicting the efficacy of MET inhibitors was evaluated. Additionally, next-generation sequencing data from another 2 cohorts including 22,010 lung cancer cases were utilized to examine the biological characteristics of different METamp subtypes. Of the 231 cases, 145 showed MET amplification/overexpression using at least 1 method, whereas only half of them could be identified by all 3 methods. METamp can occur as focal amplification or polysomy. Our study revealed that the inconsistency between next-generation sequencing and FISH primarily occurred in the polysomy subtype. Further investigations indicated that compared with polysomy, focal amplification correlated with fewer co-occurring driver mutations, higher protein expressions of c-MET and phospho-MET, and higher incidence in acquired resistance than in de novo setting. Moreover, patients with focal amplification presented a more robust response to MET inhibitors compared with those with polysomy. Notably, a strong correlation was observed between focal amplification and programmed cell death ligand-1 expression, indicating potential therapeutic implications with combined MET inhibitor and immunotherapy for patients with both alterations. Our findings provide insights into the molecular complexity and clinical relevance of METamp in lung cancer, highlighting the role of MET focal amplification as an oncogenic driver and its feasibility as a primary biomarker to further investigate the clinical activity of MET inhibitors in future studies.

Comparison of Tepotinib, Paclitaxel, or Ramucirumab Efficacy According to the Copy Number or Phosphorylation Status of the MET Gene: Doublet Treatment versus Single Agent Treatment.

Although conventional combination chemotherapies for advanced gastric cancer (GC) increase survival, such therapies are associated with major adverse effects; more effective and less toxic treatments are required. Combinations of different anti-cancer drugs, for example, paclitaxel plus ramucirumab, have recently been used as second-line treatments for advanced GC. This study evaluated how copy number variations of the MET gene, MET mutations, and MET gene and protein expression levels in human GC cells modulate the susceptibility of such cells to single-agent (tepotinib, ramucirumab, or paclitaxel) and doublet (tepotinib-plus-paclitaxel or ramucirumab-plus-paclitaxel treatment regimens. Compared with ramucirumab-plus-paclitaxel, tepotinib-plus-paclitaxel better inhibited the growth of GC cells with MET exon 14 skipping mutations and those lacking MET amplification but containing phosphorylated MET; such inhibition was dose-dependent and associated with cell death. Tepotinib-plus-paclitaxel and ramucirumab-plus-paclitaxel similarly inhibited the growth of GC cells lacking MET amplification or MET phosphorylation, again in a dose-dependent manner, but without induction of cell death. However, tepotinib alone or tepotinib-plus-ramucirumab was more effective against c-MET-positive GC cells (>30 copy number variations) than was ramucirumab or paclitaxel alone or ramucirumab-plus-paclitaxel. These in vitro findings suggest that compared with ramucirumab-plus-paclitaxel, tepotinib-plus-paclitaxel better inhibits the growth of c-MET-positive GC cells, cells lacking MET amplification but containing phosphorylated MET, and cells containing MET mutations. Clinical studies are required to confirm the therapeutic effects of these regimens.

Activity of Tepotinib in Hepatocellular Carcinoma With High-Level MET Amplification: Preclinical and Clinical Evidence.

PURPOSE: MET amplification (METamp) has been reported in 1%-5% of patients with hepatocellular carcinoma (HCC) and may be sensitive to MET inhibition. Tepotinib, a selective MET inhibitor, has shown promising activity in HCC with MET overexpression. We investigated the preclinical and clinical activity of tepotinib in HCC with METamp (MET gene copy number [GCN] ≥5), including high-level METamp (MET GCN ≥10). METHODS: Preclinical antitumor activity of tepotinib 100 mg/kg (orally, days 1-5, every 7 days, 3-5 weeks; 3-12 replicates) was evaluated according to METamp status, as determined using the nCounter platform (NanoString), in 37 HCC patient-derived xenografts (PDXs) in immunodeficient mice. Clinical outcomes were evaluated in patients with METamp by fluorescence in situ hybridization who received tepotinib 500 mg (450 mg active moiety) in two phase Ib/II trials in HCC with MET overexpression. RESULTS: Across the PDX models, tepotinib induced complete or near-complete tumor regression in the only two models with high-level METamp. Median tumor volume reductions were 100% and 99.8% in models with MET GCN 47.1 and 44.0, respectively. Across the two clinical trials, 15/121 patients had METamp. Disease control was achieved by 11/15 patients with METamp (complete response [CR], n = 1; partial response [PR], n = 4; stable disease [SD], n = 6) and 4/4 with high-level METamp (CR, n = 1; PR, n = 2; SD, n = 1). All three patients with high-level METamp and objective response received treatment for >1 year, including one patient who received first-line tepotinib for >6 years. CONCLUSION: High-level METamp may be an oncogenic driver in HCC that is sensitive to MET inhibitors such as tepotinib.

Aberrant MET Receptor Tyrosine Kinase Signaling in Glioblastoma: Targeted Therapy and Future Directions.

Brain tumors represent a heterogeneous group of neoplasms characterized by a high degree of aggressiveness and a poor prognosis. Despite recent therapeutic advances, the treatment of brain tumors, including glioblastoma (GBM), an aggressive primary brain tumor associated with poor prognosis and resistance to therapy, remains a significant challenge. Receptor tyrosine kinases (RTKs) are critical during development and in adulthood. Dysregulation of RTKs through activating mutations and gene amplification contributes to many human cancers and provides attractive therapeutic targets for treatment. Under physiological conditions, the Met RTK, the hepatocyte growth factor/scatter factor (HGF/SF) receptor, promotes fundamental signaling cascades that modulate epithelial-to-mesenchymal transition (EMT) involved in tissue repair and embryogenesis. In cancer, increased Met activity promotes tumor growth and metastasis by providing signals for proliferation, survival, and migration/invasion. Recent clinical genomic studies have unveiled multiple mechanisms by which MET is genetically altered in GBM, including focal amplification, chromosomal rearrangements generating gene fusions, and a splicing variant mutation (exon 14 skipping, METex14del). Notably, MET overexpression contributes to chemotherapy resistance in GBM by promoting the survival of cancer stem-like cells. This is linked to distinctive Met-induced pathways, such as the upregulation of DNA repair mechanisms, which can protect tumor cells from the cytotoxic effects of chemotherapy. The development of MET-targeted therapies represents a major step forward in the treatment of brain tumours. Preclinical studies have shown that MET-targeted therapies (monoclonal antibodies or small molecule inhibitors) can suppress growth and invasion, enhancing the efficacy of conventional therapies. Early-phase clinical trials have demonstrated promising results with MET-targeted therapies in improving overall survival for patients with recurrent GBM. However, challenges remain, including the need for patient stratification, the optimization of treatment regimens, and the identification of mechanisms of resistance. This review aims to highlight the current understanding of mechanisms underlying MET dysregulation in GBM. In addition, it will focus on the ongoing preclinical and clinical assessment of therapies targeting MET dysregulation in GBM.

Ningetinib plus gefitinib in EGFR-mutant non-small-cell lung cancer with MET and AXL dysregulations: A phase 1b clinical trial and biomarker analysis.

BACKGROUND: MET and AXL dysregulations are implicated in acquired resistance to EGFR-TKIs in NSCLC. But consensus on the optimal definition for MET/AXL dysregulations in EGFR-mutant NSCLC is lacking. Here, we investigated the efficacy and tolerability of ningetinib (a MET/AXL inhibitor) plus gefitinib in EGFR-mutant NSCLC, and evaluated the clinical relevance of MET/AXL dysregulations by different definitions. METHODS: Patients in this phase 1b dose-escalation/dose-expansion trial received ningetinib 30 mg/40 mg/60 mg plus gefitinib 250 mg once daily. Primary endpoints were tolerability (dose-escalation) and objective response rate (dose-expansion). MET/AXL status were analyzed using FISH and IHC. RESULTS: Between March 2017 and January 2021, 108 patients were enrolled. The proportion of MET focal amplification, MET polysomy, MET overexpression, AXL amplification and AXL overexpression is 18.1 %, 5.6 %, 55.8 %, 8.1 % and 45.3 %, respectively. 6.8 % patients have concurrent MET amplification and AXL overexpression. ORR is 30.8 % for tumors with MET amplification, 0 % for MET polysomy, 24.1 % for MET overexpression, 20 % for AXL amplification and 27.6 % for AXL overexpression. For patients with concurrent MET amplification and AXL overexpression, ningetinib plus gefitinib provides an ORR of 80 %, DCR of 100 % and median PFS of 4.7 months. Tumors with higher MET copy number and AXL expression tend to have higher likelihood of response. Biomarker analyses show that MET focal amplification and overexpression are complementary in predicting clinical benefit from MET inhibition, while AXL dysregulations defined by an arbitrary level may dilute the efficacy of AXL blockade. CONCLUSIONS: This study demonstrates that combined blockade of MET, AXL and EGFR is a feasible strategy for a subset of EGFR-mutant NSCLC. TRIAL REGISTRATION: Chinadrugtrials.org.cn, CTR20160875.

Functional Heterogeneity in MET Pathway Activation in PDX Models of Osimertinib-resistant EGFR-driven Lung Cancer.

MET pathway activation is one of the most common mechanisms of resistance to osimertinib in EGFR-mutant non-small cell lung cancer (NSCLC). We previously demonstrated spatial and temporal heterogeneity in MET pathway activation upon osimertinib resistance in EGFR-mutant NSCLC; however, the functional relevance of these findings is unclear. Here, we generated 19 patient-derived xenografts (PDX) from 9 patients with multi-region and temporal sampling of osimertinib-resistant tumor tissue from patients with EGFR-mutant NSCLC. MET pathway activation was a putative mechanism of osimertinib resistance in 66% (n = 6/9) patients from whom PDXs were generated. Significant spatial and temporal heterogeneity in MET pathway activation was evident. Osimertinib-resistant PDXs with MET amplification by FISH (defined as MET/CEP7 ratio ≥2.0 or mean MET ≥ 6.0 copies/cell) and high-level phospho-MET, but not c-MET expression, had better responses to osimertinib and savolitinib combination than to osimertinib alone. MET polysomy tumors by FISH from both PDXs and patients had evidence of subclonal phospho-MET expression. Select MET polysomy PDX tumors with phospho-MET expression responded better to osimertinib and savolitinib combination than MET polysomy PDX tumors without phospho-MET expression. Our results suggest osimertinib and savolitinib combination is most effective for osimertinib-resistant EGFR-mutant tumors with MET pathway activation as evidenced by phospho-MET. As subclonal MET amplification may be evident in MET polysomy tumor progression, MET polysomy warrants close clinical follow-up with phospho-MET IHC in parallel with FISH diagnostic. SIGNIFICANCE: Using a novel cohort of in vivo PDX models of MET pathway activation with acquired resistance to osimertinib in EGFR-mutant lung cancer, we demonstrate that phospho-MET may be a clinically relevant assay to guide treatment selection with osimertinib and savolitinib combination. In addition, our work shows that patients with MET polysomy tumors may have subclonal MET amplification and therefore require close follow up for the use of osimertinib and savolitinib combination.

Inhibition of autocrine HGF maturation overcomes cetuximab resistance in colorectal cancer.

Although amplifications and mutations in receptor tyrosine kinases (RTKs) act as bona fide oncogenes, in most cancers, RTKs maintain moderate expression and remain wild-type. Consequently, cognate ligands control many facets of tumorigenesis, including resistance to anti-RTK therapies. Herein, we show that the ligands for the RTKs MET and RON, HGF and HGFL, respectively, are synthesized as inactive precursors that are activated by cellular proteases. Our newly generated HGF/HGFL protease inhibitors could overcome both de novo and acquired cetuximab resistance in colorectal cancer (CRC). Conversely, HGF overexpression was necessary and sufficient to induce cetuximab resistance and loss of polarity. Moreover, HGF-induced cetuximab resistance could be overcome by the downstream MET inhibitor, crizotinib, and upstream protease inhibitors. Additionally, HAI-1, an endogenous inhibitor of HGF proteases, (i) was downregulated in CRC, (ii) exhibited increased genomic methylation that correlated with poor prognosis, (iii) HAI-1 expression correlated with cetuximab response in a panel of cancer cell lines, and (iv) exogenous addition of recombinant HAI-1 overcame cetuximab resistance in CC-HGF cells. Thus, we describe a targetable, autocrine HAI-1/Protease/HGF/MET axis in cetuximab resistance in CRC.

Receptor tyrosine kinase gene expression profiling of orbital rhabdomyosarcoma unveils MET as a potential biomarker and therapeutic target.

Receptor tyrosine kinases (RTKs) serve as molecular targets for the development of novel personalized therapies in many malignancies. In the present study, expression pattern of receptor tyrosine kinases and its clinical significance in orbital RMS has been explored. Eighteen patients with histopathologically confirmed orbital RMS formed part of this study. Comprehensive q-PCR gene expression profiles of 19 RTKs were generated in the cases and controls. The patients were followed up for 59.53 ± 20.93 years. Clustering and statistical analysis tools were applied to identify the significant combination of RTKs associated with orbital rhabdomyosarcoma patients. mRNA overexpression of RTKs which included MET, AXL, EGFR was seen in 60-80% of cases; EGFR3, IGFR2, FGFR1, RET, PDGFR1, VEGFR2, PDGFR2 in 30-60% of cases; and EGFR4, FGFR3,VEGFR3 and ROS,IGFR1, EGFR1, FGFR2, VEGFR1 in 10-30% of cases. Immunoexpression of MET was seen in 89% of cases. A significant association was seen between MET mRNA and its protein expression. In all the cases MET gene expression was associated with worst overall survival (P = 0.03).There was a significant correlation of MET mRNA expression with RET, ROS, AXL, FGFR1, FGFR3, PDGFR1, IGFR1, VEGFR2, and EGFR3 genes. Association between MET gene and collective expression of RTKs was further evaluated by semi-supervised gene cluster analysis and Principal component analysis, which showed well-separated tumor clusters. MET gene overexpression could be a useful biomarker for identifying high risk orbital rhabdomyosarcoma patients. Well-separated tumor clusters confirmed the association between MET gene and collective expression of RTK genes. Therefore, the therapeutic potential of multi-kinase inhibitors targeting MET and the 9 other significant RTKs needs to be explored.

HGF facilitates methylation of MEG3, potentially implicated in vemurafenib resistance in melanoma.

BACKGROUND: Melanoma, a frequently encountered cutaneous malignancy characterized by a poor prognosis, persists in presenting formidable challenges despite the advancement in molecularly targeted drugs designed to improve survival rates significantly. Unfortunately, as more therapeutic choices have developed over time, the gradual emergence of drug resistance has become a notable impediment to the effectiveness of these therapeutic interventions. The hepatocyte growth factor (HGF)/c-met signaling pathway has attracted considerable attention, associated with drug resistance stemming from multiple potential mutations within the c-met gene. The activation of the HGF/c-met pathway operates in an autocrine manner in melanoma. Notably, a key player in the regulatory orchestration of HGF/c-met activation is the long non-coding RNA MEG3. METHODS: Melanoma tissues were collected to measure MEG3 expression. In vitro validation was performed on MEG3 to prove its oncogenic roles. Bioinformatic analyses were conducted on the TCGA database to build the MEG3-related score. The immune characteristics and mutation features of the MEG3-related score were explored. RESULTS: We revealed a negative correlation between HGF and MEG3. In melanoma cells, HGF inhibited MEG3 expression by augmenting the methylation of the MEG3 promoter. Significantly, MEG3 exhibits a suppressive impact on the proliferation and migration of melanoma cells, concurrently inhibiting c-met expression. Moreover, a predictive model centered around MEG3 demonstrates notable efficacy in forecasting critical prognostic indicators, immunological profiles, and mutation statuses among melanoma patients. CONCLUSIONS: The present study highlights the potential of MEG3 as a pivotal regulator of c-met, establishing it as a promising candidate for targeted drug development in the ongoing pursuit of effective therapeutic interventions.

MET exon 14 skipping mutation drives cancer progression and recurrence via activation of SMAD2 signalling.

BACKGROUND: c-Met encoded by the proto-oncogene MET, also known as hepatocyte growth factor (HGF) receptor, plays a crucial role in cellular processes. MET exon 14 skipping alteration (METΔ14EX) is a newly discovered MET mutation. SMAD2 is an important downstream transcription factor in TGF-ß pathway. Unfortunately, the mechanisms by which METΔ14EX leads to oncogenic transformation are scarcely understood. The relationship between METΔ14EX and SMAD2 has not been studied yet. METHODS: We generate METΔ14EX models by CRISPR-Cas9. In vitro transwell, wound-healing, soft-agar assay, in vivo metastasis and subcutaneous recurrence assay were used to study the role of METΔ14EX in tumour progression. RNA-seq, Western blotting, co-immunoprecipitation (CO-IP) and immunofluorescent were performed to explore the interaction between c-Met and SMAD2. RESULTS: Our results demonstrated that METΔ14EX, independent of HGF, can prolong the constitutive activation of c-Met downstream signalling pathways by impeding c-Met degradation and facilitating tumour metastasis and recurrence. Meanwhile, METΔ14EX strengthens the interaction between c-Met and SMAD2, promoting SMAD2 phosphorylation. Therapeutically, MET inhibitor crizotinib impedes METΔ14EX-mediated tumour metastasis by decreasing SMAD2 phosphorylation. CONCLUSIONS: These data elucidated the previously unrecognised role of METΔ14EX in cancer progression via activation of SMAD2 independent of TGF-ß, which helps to develop more effective therapies for such patients. METΔ14EX alteration significantly triggers tumour progression via activation of SMAD2 signalling that are involved in activating tumour invasion, metastasis and recurrence. On the left, in the MET wild-type (METWT), the juxtamembrane (JM) domain is involved in the regulation of tyrosine kinase activity, receptor degradation, and caspase cleavage. On the right, the METΔ14EX mutation leads to the loss of the juxtamembrane domain, resulting in an abnormal MET protein lacking a CBL-binding site. This causes the accumulation of truncated MET receptors followed by constitutive activation of the MET signalling pathway. Thus, the METΔ14EX-mutated protein has strong binding and phosphorylation to SMAD2, which results in the phosphorylation of a large number of SMAD2/3 proteins that combine with SMAD4 to form a complex in the nucleus, activating downstream signalling pathways, such as EMT and ECM remodelling, resulting in tumour progression and recurrence. TF transcription factor.

Hereditary papillary renal cell carcinoma.

Hereditary papillary renal cell carcinoma (HPRCC) is an autosomal dominant syndrome characterized by the occurrence of bilateral and multifocal, classic type papillary renal cell carcinomas. In the recent decades, extensive molecular studies have narrowed the molecular underpinnings of this syndrome to missense mutations in tyrosine kinase domain of MET proto-oncogene. Although MET mutations are specific to HPRCC, it has been found in sporadic papillary renal cell carcinomas and as recently reported, in biphasic squamoid alveolar variant of papillary renal cell carcinoma. Dual MET/VEGFR2 kinase inhibitor and tyrosine kinase inhibitors have shown promising results in systemic therapy for HPRCC.

Transformable DNA Nanorobots Reversibly Regulating Cell Membrane Receptors for Modulation of Cellular Migrations.

Oligomerization of cellular membrane receptors plays crucial roles in activating intracellular downstream signaling cascades for controlling cellular behaviors in physiological and pathological processes. However, the reversible and controllable regulation of receptors in a user-defined manner remains challenging. Herein, we developed a versatile DNA nanorobot (nR) with installed aptamers and hairpin structures to reversibly and controllably regulate cell migration. This was achieved by dimerization and de-dimerization of mesenchymal-epithelial transition (Met) receptors through DNA strand displacement reactions. The functionalized DNA nR not only plays similar roles as hepatocyte growth factor (HGF) in inducing cell migration but also allows a downgrade to the original state of cell migration. The advanced DNA nanomachines can be flexibly designed to target other receptors for manipulating cellular behaviors and thus represent a powerful tool for the future of biological and medical engineering.

The Combined Therapy of Cabozantinib, Crizotinib, and Osimertinib in a Lung Cancer Patient with Acquired MET Amplification and Resistance Mutations.

EGFR-mutant lung cancers develop a wide range of potential resistance alterations under therapy with the third-generation EGFR tyrosine kinase inhibitor osimertinib. MET amplification ranks among the most common acquired resistance alterations and is currently being investigated as a therapeutic target in several studies. Nevertheless, targeted therapy of MET might similarly result in acquired resistance by point mutations in MET, which further expands therapeutic and diagnostic challenges. Here, we report a 50-year-old male patient with EGFR-mutant lung adenocarcinoma and stepwise acquired resistance by a focal amplification of MET followed by D1246N (D1228N), D1246H (D1228H), and L1213V (L1195V) point mutations in MET, all detected by NGS. The patient successfully responded to the combined and sequential treatment of osimertinib, osimertinib/crizotinib, and third-line osimertinib/cabozantinib. This case highlights the importance of well-designed, sequential molecular diagnostic analyses and the personalized treatment of patients with acquired resistance.

Real-World Data on Combined EGFR-TKI and Crizotinib Treatment for Acquired and De Novo MET Amplification in Patients with Metastatic EGFR-Mutated NSCLC.

Amplification of the mesenchymal epithelial transition (MET) gene is a mechanism of acquired resistance to epidermal growth factor receptor (EGFR)-tyrosine-kinase-inhibitors (TKIs) in over 20% of patients with advanced EGFR-mutated (EGFRm+) non-small lung cancer (NSCLC). However, it may also occur de novo in 2-8% of EGFRm+ NSCLC cases as a potential mechanism of intrinsic resistance. These patients represent a group with unmet needs, since there is no standard therapy currently approved. Several new MET inhibitors are being investigated in clinical trials, but the results are awaited. Meanwhile, as an alternative strategy, combinations of EGFR-TKIs with the MET/ALK/ROS1-TKI Crizotinib may be used in this setting, despite this use is principally off-label. Thus, we studied five of these MET amplified cases receiving EGFR-TKI and Crizotinib doublet after progression on EGFR-TKI treatment to assess the benefits and challenges related to this combination and the possible occurrence of genomic and phenotypic co-alterations. Furthermore, we compared our cases with other real-world reports on Crizotinib/EGFR-TKI combinations, which appeared effective, especially in patients with high-level MET amplification. Yet, we observed that the co-occurrence of other genomic and phenotypical alterations may affect the response to combined EGFR-TKI and Crizotinib. Finally, given the heterogeneity of MET amplification, the diagnostic methods for assessing it may be discrepant. In this respect, we observed that for optimal detection, immunohistochemistry, fluorescence in situ hybridization, and next-generation sequencing should be used together, as these methods possess different sensitivities and complement each other in characterizing MET amplification. Additionally, we addressed the issue of managing EGFR-mutated NSCLC patients with de novo MET amplification causing primary EGFR-TKI resistance. We conclude that, while data from clinical trials with new MET inhibitors are still pending, adding Crizotinib to EGFR-TKI in NSCLC patients acquiring MET amplification at progression on EGFR-TKI monotherapy is a reasonable approach, with a progression-free survival of 3-19 months.

Knockout of c-Cbl/Cbl-b slows c-Met trafficking resulting in enhanced signaling in corneal epithelial cells.

In many cell types, the E3 ubiquitin ligases c-Cbl and Cbl-b induce ligand-dependent ubiquitylation of the hepatocyte growth factor (HGF)-stimulated c-Met receptor and target it for lysosomal degradation. This study determines whether c-Cbl/Cbl-b are negative regulators of c-Met in the corneal epithelium (CE) and if their inhibition can augment c-Met-mediated CE homeostasis. Immortalized human corneal epithelial cells were transfected with Cas9 only (Cas9, control cells) or with Cas9 and c-Cbl/Cbl-b guide RNAs to knockout each gene singularly (-c-Cbl or -Cbl-b cells) or both genes (double KO [DKO] cells) and monitored for their responses to HGF. Cells were assessed for ligand-dependent c-Met ubiquitylation via immunoprecipitation, magnitude, and duration of c-Met receptor signaling via immunoblot and receptor trafficking by immunofluorescence. Single KO cells displayed a decrease in receptor ubiquitylation and an increase in phosphorylation compared to control. DKO cells had no detectable ubiquitylation, had delayed receptor trafficking, and a 2.3-fold increase in c-Met phosphorylation. Based on the observed changes in receptor trafficking and signaling, we examined HGF-dependent in vitro wound healing via live-cell time-lapse microscopy in control and DKO cells. HGF-treated DKO cells healed at approximately twice the rate of untreated cells. From these data, we have generated a model in which c-Cbl/Cbl-b mediate the ubiquitylation of c-Met, which targets the receptor through the endocytic pathway toward lysosomal degradation. In the absence of ubiquitylation, the stimulated receptor stays phosphorylated longer and enhances in vitro wound healing. We propose that c-Cbl and Cbl-b are promising pharmacologic targets for enhancing c-Met-mediated CE re-epithelialization.

Improved Survival Outcomes in Patients With MET-Dysregulated Advanced NSCLC Treated With MET Inhibitors: Results of a Multinational Retrospective Chart Review.

BACKGROUND: We evaluated the disease and patient characteristics, treatment, and MET testing patterns, predictive biomarkers and survival outcomes in patients with MET-dysregulated metastatic non-small-cell lung cancer (NSCLC) in a real-world setting. PATIENTS AND METHODS: This was a multinational, retrospective, noninterventional chart review study. Data from medical records of patients with advanced/metastatic EGFR wild-type, MET-dysregulated NSCLC (December 2017-September 2018) were abstracted into electronic data collection forms. RESULTS: Overall, 211 patient charts were included in this analysis; 157 patients had MET exon 14 skipping mutations (METex14; with or without concomitant MET amplification) and 54 had MET amplification only. All patients were tested for METex14, whereas MET amplification was evaluated in 168 patients. No overlap was reported between MET dysregulation and ALK, ROS1 or RET rearrangements, or HER2 exon 20 insertions. Overall, 56 of 211 patients (26.5%) received MET inhibitor (METi) therapy in any treatment-line setting (31.2% in the METex14 cohort; 13% in the MET-amplified only cohort). In the METex14 cohort, median OS in patients receiving METi was 25.4 months versus 10.7 months in patients who did not (HR [95% CI]: 0.532 [0.340-0.832]; P = .0055). In the MET-amplified only cohort, median OS was 20.6 months in patients treated with METi compared with 7.6 months in those without METi (HR [95% CI]: 0.388 [0.152-0.991]; P = .0479). CONCLUSIONS: MET alterations in NSCLC typically occur in the absence of other oncogenic driver mutations and are associated with poor survival outcomes. Notably, METi therapies are associated with improved survival outcomes in patients with MET-dysregulated NSCLC.