c-Met Protein Research Articles

Uveal Melanoma and Paraneoplastic Perivascular Dermal Melanocytic Proliferation in the Setting of Bilateral Diffuse Uveal Melanocytic Proliferation: The Potential Role of the Hepatocyte Growth Factor/c-Met Axis in Their Pathogenesis

Two patients, with non-small cell lung carcinoma treated with pembrolizumab, developed bilateral diffuse uveal melanocytic proliferation (BDUMP) with interesting histopathological features. The first patient developed a right ciliary body mass concurrently with BDUMP. The globe was enucleated. The ciliary body mass was a mitotically active epithelioid uveal melanoma, invading the trabecular meshwork and peripheral corneal stroma, with over 90% of the cells expressing Cyclin D1 protein. The melanoma showed no chromosome 3 or 8 changes. The background uvea showed diffuse, bland spindle cell melanocytic proliferation with much lower Cyclin D1 expression (around 10%). In the choroid, this population was punctuated by islands of pigmented epithelioid cells, some of which were necrotic. All these islands expressed a high level of Cyclin D1, and some islands expressed nuclear preferentially expressed antigen in melanoma (PRAME). The ciliary body mass, epithelioid cell islands, and the BDUMP all expressed c-Met (the receptor for hepatocyte growth factor [HGF]). The features were those of ciliary body melanoma and choroidal melanoma “tumorlets,” developing on a background of BDUMP. The second patient developed bilateral periocular skin pigmentation following a diagnosis of BDUMP, which when biopsied, showed dermal islands of paraneoplastic perivascular melanocytic cell proliferation. These cells also expressed c-Met protein. These observations implicate the HGF/c-Met axis in the pathogenesis of BDUMP, the uveal melanomas in the ciliary body and choroid in the first patient and the paraneoplastic dermal melanocytic proliferation in the second patient.

1'-methylspiro[indoline-3,4'-piperidine] Derivatives: Design, Synthesis, Molecular Docking and Anti-tumor Activity Studies

Background: Spirocyclic indoline compounds widely exist in numerous natural products and synthetic molecules with significant biological activities. In recent years, these kinds of compounds have attracted extensive attention as potent anti-tumor agents in the fields of pharmacology and chemistry. Objective: In this study, we focused on designing and synthesizing novel 1'-methylspiro[indoline- 3,4'-piperidine] derivatives, which were evaluated by preliminary bioactivity experiment in vitro and molecular docking. Methods and Materials: The key intermediate 1'-methylspiro[indoline-3,4'-piperidine] (B4) reacted with benzenesulfonyl chloride with different substituents under alkaline condition to obtain its derivatives (B5-B10). We evaluated their antiproliferative activities against A549, BEL-7402 and HeLa cell lines by MTT assay. We performed the CDOCKER module in Accelrys Discovery Studio 2.5.5 for molecular docking of compound B5, and investigated the binding modes of compound B5 with three different target proteins. Results: The results indicated that compounds B4-B10 exhibited good antiproliferative activities against the above three types of cell lines, in which compound B5 with chloride atom as electronwithdrawing substituent on a phenyl ring showed the highest potency against BEL-7402 cell lines (IC50=30.03±0.43 μg/mL). The results of molecular docking showed that the binding energies of the prominent bioactive compound B5 with CDK, c-Met, and EGFR protein crystals are -44.3583 kcal/mol, -38.3292 kcal/mol, -33.3653 kcal/mol, respectively. Conclusion: 1'-methylspiro[indoline-3,4'-piperidine] and its six derivatives were synthesized and evaluated against BEL-7402, A 549, and Hela cell lines. Compound B5 showed significant inhibition on BEL-7402 cell lines. Molecular docking assays revealed that B5 as a ligand showed strong affinity and appropriate binding pose on the amino acid residues in active sites of the tested targets, which encourage us to conduct further evaluation such as the kinase experiment.

A Novel Fluorescent c-Met Targeted Imaging Agent for Intra-Operative Colonic Tumour Mapping: Translation from the Laboratory into a Clinical Trial

Background: Curative resection of colon cancer requires accurate intraoperative identification of tumour margins and lymph node (LN) burden. The c-Met protein is important for cell differentiation and migration and is overexpressed in many gastrointestinal cancers. We explored EMI-137, a novel c-Met targeting fluorescent probe, for application in fluorescence-guided colon surgery, in HT-29 colorectal cancer (CRC) cell line and an in vivo murine model. This informed a first-in-man clinical trial of EMI-137. Methods: HT-29 cells were used as a representative CRC model. SiRNA was used to demonstrate the specificity of EMI-137 for c-Met. A HT-29 CRC xenograft model was developed in female BALB/c mice. Tumours were cultured for 14 days to 10mm diameter. EMI-137 (0.18mg/kg) was injected into tail veins and biodistribution analysed through in vivo fluorescent imaging. Nine patients, aged 67-77 years, received a single intravenous dose of EMI-137 (0.13mg/kg), 1-3 hours before laparoscopic-assisted colon cancer resection surgery (NCT03360461). Tumour and LN fluorescence was assessed with a modified Karl Storz® laparoscope. Immunohistochemistry with a monoclonal antibody analysed c-Met expression in eight resected samples. Findings: c-Met expression in HT-29 cells was effectively silenced and imaged with EMI-137. Strong EMI-137 uptake in tumour xenografts was observed for up to 6 hours post-administration. At clinical trial, no serious adverse events related to EMI-137 were reported. Marked background fluorescence was observed in all trial participants, 4/9 showed an increase in tumour fluorescence over background; 5/9 had histological LN metastases; no fluorescent LN were detected intraoperatively. All primary tumours (8/8) and malignant LN (15/15) exhibited moderate to high c-Met protein expression. Interpretation: EMI-137, binds specifically to the human c-Met transmembrane protein, is safe in patients and with further refinement, shows potential for long-term application in fluorescence-guided colon surgery. Trial Registration: The trial was registered with ClinicalTrials.gov identifier NCT03360461. Funding: Research was supported by internal University of Leeds funding. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Ethical approval was granted by the Yorkshire and Humber Leeds West Research and Ethics Committee (Ref 17/YH/0263), on 19th October 2017 and Regulatory approval was granted by the Medicines and Healthcare products Regulatory Agency (MHRA), under The Medicines for Human Use (Clinical Trials) regulations on 29th August 2017.

Development of Antigen-specific Chimeric Antigen Receptor KHYG-1 Cells for Glioblastoma.

Glioblastoma is the most common cancer among primary brain tumors, however, its prognosis and treatment advances are very poor. Here, we investigated whether c-Met, FOLR1, and AXL proteins are promising targets for chimeric antigen receptor (CAR) T-cell therapy, for they are known to be over-expressed in a variety of solid tumors. CAR constructs were prepared and CAR KHYG-1 cells targeting c-Met, FOLR1, or AXL were made by lentiviral transduction. The activity of CAR KHYG-1 cells against cancer cells was measured by cytokine secretion and cell lysis assays. c-Met and AXL were over-expressed in most glioblastoma cell lines (11/13), but not in neuroblastoma cell lines (0/8). FOLR1 was over-expressed only in one among 16 glioblastoma cell lines. Our antigen-specific CAR KHYG-1 cells eradicated target positive glioblastoma cells selectively. Anti-c-Met and anti-AXL CAR NK or T cells could be effective in glioblastoma cells.

Near-infrared fluorescence-guided resection of micrometastases derived from esophageal squamous cell carcinoma using a c-Met-targeted probe in a preclinical xenograft model

The postoperative survival of esophageal squamous cell carcinoma (eSCC) is notably hindered by cancer recurrence due to difficulty in identifying occult metastases. Cellular mesenchymal-epithelial transition factor (c-Met), which is highly expressed in different cancers, including eSCC, has become a target for the development of imaging probes and therapeutic antibodies. In this study, we synthesized an optical probe (SHRmAb-IR800) containing a near-infrared fluorescence (NIRF) dye and c-Met antibody, which may help in NIRF-guided resection of micrometastases derived from eSCC. Cellular uptake of SHRmAb-IR800 was assessed by flow cytometry and confocal microscopy. In vivo accumulation of SHRmAb-IR800 and the potential application of NIRF-guided surgery were evaluated in eSCC xenograft tumor models. c-Met expression in human eSCC samples and lymph node metastases (LNMs) was analyzed via immunohistochemistry (IHC). Cellular accumulation of SHRmAb-IR800 was higher in c-Met-positive EC109 eSCC cells than in c-Met-negative A2780 cells. Infusion of SHRmAb-IR800 produced higher fluorescence intensity and a higher tumor-to-background ratio (TBR) than the control probe in EC109 subcutaneous tumors (P < 0.05). The TBRs of orthotopic EC109 tumors and LNMs were 3.01 ± 0.17 and 2.77 ± 0.56, respectively. The sensitivity and specificity of NIRF-guided resection of metastases derived from orthotopic cancers were 92.00% and 89.74%, respectively. IHC results demonstrated positive staining in 97.64% (124/127) of eSCC samples and 91.67% (55/60) of LNMs. Notably, increased c-Met expression was observed in LNMs compared to normal lymph nodes (P < 0.0001). Taken together, the results of this study indicated that SHRmAb-IR800 facilitated the resection of micrometastases of eSCC in the xenograft tumor model. This c-Met-targeted probe possesses translational potential in NIRF-guided surgery due to the high positive rate of c-Met protein in human eSCCs.

EGFR mutation is positively correlated with C-Met protein expression: a study of 446 resected lung adenocarcinoma.

BackgroundEpidermal growth factor receptor (EGFR) mutation and mesenchymal-epithelial transition factor (C-Met) amplification are known factors for primary resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in advanced primary lung adenocarcinoma. However, little is known about the relationship between high expression of C-Met protein and primary EGFR mutation. This research aims to investigate the correlation between EGFR mutation and C-Met protein expression in resected primary lung adenocarcinoma.MethodsFour hundred and forty-six surgically resected lung adenocarcinoma between 2013–2015 were collected for EGFR mutation analysis by real-time PCR (RT-PCR) and C-Met protein expression by immunohistochemistry (IHC). The relationship between the two biomarkers and clinicopathological features were analyzed.ResultsThe positive rate of EGFR mutation and C-Met protein expression were 66.4% (296/446) and 96.4% (430/446). EGFR mutation was significantly higher in female, mild to moderate differentiation, lepidic, acinar and papillary histological subtypes (P<0.05). C-Met expression was more prominent in female than male (201 vs. 123, 45.07% vs. 27.57%). EGFR mutation was found positively correlated with C-Met protein expression (P<0.05).ConclusionsEGFR mutation and C-Met protein expression are prone to have a female predominance, and are positively correlated with each other in surgically resected lung adenocarcinoma specimens. This finding may be beneficial in explaining some of the resistance mechanisms of EGFR-mutated cases, which is worth further study.

Incomplete thermal ablation of tumors promotes increased tumorigenesis

Purpose While systemic tumor-stimulating effects can occur following ablation of normal liver linked to the IL-6/HGF/VEGF cytokinetic pathway, the potential for tumor cells themselves to produce these unwanted effects is currently unknown. Here, we study whether partially treated tumors induce increased tumor growth post-radiofrequency thermal ablation (RFA). Methods Tumor growth was measured in three immunocompetent, syngeneic tumor models following partial RFA of the target tumor (in subcutaneous CT26 and MC38 mouse colorectal adenocarcinoma, N = 14 each); and in a distant untreated tumor following partial RFA of target subcutaneous R3230 rat breast adenocarcinoma (N = 12). Tumor cell proliferation (ki-67) and microvascular density (CD34) was assessed. In R3230 tumors, in vivo mechanism of action was assessed following partial RFA by measuring IL-6, HGF, and VEGF expression (ELISA) and c-Met protein (Western blot). Finally, RFA was performed in R3230 tumors with adjuvant c-Met kinase inhibitor or VEGF receptor inhibitor (at 3 days post-RFA, N = 3/arm, total N = 12). Results RFA stimulated tumor growth in vivo in residual, incompletely treated surrounding CT26 and MC38 tumor at 3–6 days (p < 0.01). In R3230, RFA increased tumor growth in distant tumor 7 days post treatment compared to controls (p < 0.001). For all models, Ki-67 and CD34 were elevated (p < 0.01, all comparisons). IL-6, HGF, and VEGF were also upregulated post incomplete tumor RFA (p < 0.01). These markers were suppressed to baseline levels with adjuvant c-MET kinase or VEGF receptor inhibition. Conclusion Incomplete RFA of a target tumor can sufficiently stimulate residual tumor cells to induce accelerated growth of distant tumors via the IL-6/c-Met/HGF pathway and VEGF production.

Design, Molecular Docking and Biological Evaluation of Fused Thienopyrimidines and Quinazoline

The anticancer activity of the condensed pyrimidine and quinazoline moieties are pronounced with a different pathway. Thienopyrimidine is considered as ring equivalent bioisosteres of quinazolines and present in other heterocyclic compounds including thienopyrimidine. The present investigation focused on the synthesis of thienopyrimidine and quinazoline derivatives for their anticancer activity against the human oral squamous carcinoma-3 (HSC-3) cell line. The synthesized compound confirmed for their structural characteristics from spectral analysis and tested for anti-proliferative activity from MTT assay. The electron-withdrawing group in 4-chloro thienopyrimidines and amino ester derivate/facilitate the inhibition of cancer cells. Further probing by docking studies revealed that the compounds exhibit possible interactions with VEGF, FGFR and c-Met proteins, which are known to have a role in the pathogenesis of oral squamous cell carcinoma. Among the derivatives a moderate activity demonstrated by substituted 4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]-pyrimidine (4a) and ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (1a) derivatives. Lack of chirality and the presence of bulky substituents in few of the compounds were found to be the cause for lower potency.

Analysis of the Protein-Protein Interaction Network Identifying c-Met as a Target of Gigantol in the Suppression of Lung Cancer Metastasis.

c-Met (mesenchymal-epithelial transition factor) facilitates cancer progression and is recognized as a promising drug target. The molecular target of gigantol from Dendrobium draconis in suppressing cancer metastasis is largely unknown. Proteins affected by gigantol treatment were subjected to proteomic and bioinformatic analysis. Protein-Protein interaction (PPI) networks were constructed by the Search Tool for the Retrieval of Interacting Genes (STRING). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database and hub gene were used to enrich the dominant pathways. Western blot analysis and immunofluorescence were used to validate the effect of gigantol on the target protein and signaling. Gigantol down-regulates 41 adhesion proteins and 39-migratory proteins, while it up-regulates 30 adhesion-related proteins and 22 proteins controlling cell migration. The key components of our constructed PPI network comprised 41 proteins of cell adhesion enriched in 40 nodes with 25 edges, 39 proteins of cell migration enriched in 39 nodes with 76 edges in down-regulated proteins, 30 proteins of cell adhesion enriched in 30 nodes with 21 edges, and 22 proteins of cell migration enriched in 22 nodes with 22 edges in up-regulated protein. c-Met was identified as a central protein of the PPI network in the largest degree. KEGG mapper further suggested that c-Met, PI3K, and AKT were the regulatory proteins affected by gigantol. To confirm, the effects of gigantol on c-Met, the p-PI3K, PI3K, p-AKT, and AKT proteins were investigated by western blotting and the results showed a consistent effect of gigantol in the suppression of the c-Met/PI3K/AKT signal. Next, immunofluorescence showed a dramatic decrease in c-Met, PI3K and AKT activation in response to gigantol. c-Met is an important target of gigantol treatment in lung cancer cells. Gigantol suppresses metastasis-related cell motility through decreasing c-Met resulting in PI3K/AKT signaling disruption.

Verticillin A inhibits colon cancer cell migration and invasion by targeting c-Met

Verticillin A is a diketopiperazine compound which was previously isolated from Amanita flavorubescens Alk (containing parasitic fungi Hypomyces hyalines (Schw.) Tul.). Here, we initially found, by wound healing assay and Transwell assay in vitro, that verticillin A possesses an inhibitory effect against the migration and invasion of the human colon cancer cell. Subsequently, c-mesenchymal-epithelial transition factor (c-Met) was identified as a molecular target of verticillin A by screening key genes related to cell migration. Verticillin A-mediated c-Met suppression is at the transcriptional level. Further study demonstrated that verticillin A suppressed c-MET phosphorylation and decreased c-MET protein level. In addition, verticillin A inhibited the phosphorylation of c-MET downstream molecules including rat sarcoma (Ras)-associated factor (Raf), extracellular signal-regulated kinase (ERK), and protein kinase B (AKT). Overexpression of Erk partially reversed the verticillin A-mediated anti-metastasis action in the human colon cancer cell. More importantly, verticillin A also inhibited cancer cell metastasis in vivo. Thus, verticillin A can significantly inhibit the migration and invasion of colon cancer cells by targeting c-Met and inhibiting Ras/Raf/mitogen-activated extracellular signal-regulated kinase (MEK)/ERK signaling pathways. Therefore, we determined that verticillin A is a natural compound that can be further developed as an anti-metastatic drug in human cancers.

1414TiP Evaluating telisotuzumab vedotin in combination with osimertinib in patients with advanced non-small cell lung cancer: A phase I/Ib study cohort

Acquired resistance to third-generation epidermal growth factor receptor (EGFR) inhibitors (eg, osimertinib) may result in MET gene amplification and/or c-MET protein overexpression. Telisotuzumab vedotin (ABBV-399; teliso-v), an anti–c-MET antibody-drug conjugate that can both disrupt c-MET signaling and deliver a cytotoxic payload into tumor cells, has shown antitumor activity in a phase 1/1b trial (NCT02099058) in patients (pts) with non-small cell lung cancer (NSCLC) with c-MET overexpression. Here we describe the teliso-v plus osimertinib cohort (arm E) of this trial. Arm E of this phase 1/1b open-label study evaluates safety, pharmacokinetics, and preliminary efficacy of teliso-v, administered intravenously once every 2 weeks, in combination with oral osimertinib (80 mg once a day) in pts with metastatic NSCLC whose disease progressed on osimertinib. Pts must have documented osimertinib-sensitive EGFR mutations, and c-MET overexpression (defined by central immunohistochemistry test) in tumor tissue obtained post-osimertinib progression. Arm E consists of safety lead-in (SLP; n=3–6), safety evaluation (SEP; n=3–12), and expansion (n=3–12) phases. Teliso-v will first be evaluated at 1.6 mg/kg and escalated to 1.9 mg/kg on the basis of safety signals in SLP and SEP phases. Radiographic tumor assessments will continue until disease progression, start of a new anticancer therapy, death, or consent withdrawal. Response evaluation will be based on Response Evaluation Criteria in Solid Tumors version 1.1. Planned enrollment is 3–24 pts; to date, 3 pts are enrolled. NCT02099058. Medical writing support was provided by Mary L. Smith, PhD, CMPP, from Aptitude Health, Atlanta, GA, and funded by AbbVie. AbbVie.

Tepotinib Inhibits the Epithelial-Mesenchymal Transition and Tumor Growth of Gastric Cancers by Increasing GSK3β, E-Cadherin, and Mucin 5AC and 6 Levels.

Aberrant expression of mucins (MUCs) can promote the epithelial–mesenchymal transition (EMT), which leads to enhanced tumorigenesis. Carcinogenesis-related pathways involving c-MET and β-catenin are associated with MUCs. In this study, we characterized the expression of EMT-relevant proteins including MET, β-catenin, and E-cadherin in human gastric cancer (GC) cell lines, and further characterized the differential susceptibility of these cell lines compared with the c-MET inhibitor tepotinib. We assessed the antitumor activity of tepotinib in GC cell lines. The effects of tepotinib on cell viability, apoptotic cell death, EMT, and c-MET and β-catenin signaling were evaluated by 3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium (MTS), flow cytometry, Western blotting, and qRT-PCR. The antitumor efficacy was assessed in MKN45 xenograft mice. Tepotinib treatment induced apoptosis in c-MET-amplified SNU620, MKN45, and KATO III cells, but had no effect on c-MET-reduced MKN28 or AGS cells. Tepotinib treatment also significantly reduced the protein levels of phosphorylated and total c-MET, phosphorylated and total ERK, β-catenin, and c-MYC in SNU620 and MKN45 cells. In contrast, this drug was only slightly active against KATO III cells. Notably, tepotinib significantly reduced the expression of EMT-promoting genes such as MMP7, COX-2, WNT1, MUC5B, and c-MYC in c-MET-amplified GC cells and increased the expression of EMT-suppressing genes such as MUC5AC, MUC6, GSK3β, and E-cadherin. In a mouse model, tepotinib exhibited good antitumor growth activity along with increased E-cadherin and decreased phosphorylated c-MET (phospho-c-MET) protein levels. Collectively, these results suggest that tepotinib suppresses tumor growth and migration by negatively regulating c-MET-induced EMT. These findings provide new insights into the mechanism by which MUC5AC and MUC6 contribute to GC progression.

Abstract 3046: Anti-tumor efficacy of CKD-702 in EGFR TKI-resistant patient-derived xenograft model

Abstract Background: c-MET is co-expressed with EGFR in approximately 70% of EGFR mutant-positive tumors. In addition, c-Met amplification was found in 10-20% of lung cancer patients with acquired tolerance to EGFR TKI, and c-Met overexpression was found in 14-69% of patients receiving EGFR TKI. CKD-702, a double antibody, is designed to simultaneously block both EGFR and c-MET expression. Purpose: We evaluated the anti-tumor efficacy of CKD-702 with PDX model derived from lung adenocarcinoma patients. Methods: We constructed the PDX model by transplanting surgical tissue from lung adenocarcinoma patient into NOD/SCID mice after the approval of research protocol in Seoul St. Mary's Hospital IRB. We confirmed the co-existence of activating EGFR mutation and c-MET amplification with PNAclamp and FISH methods. We also checked overexpression of EGFR and c-MET protein by immunohistochemistry. Results: The characteristics of tumor tissue transplanted for PDX model were: 1) Korean, lung cancer with EGFR TKI chemotherapy failure confirmed c-MET amplification (&amp;gt; 5 copies) and overexpression (IHC score 3+), 19del Positive EGFR mutation, 2) European lung adenocarcinoma, c-MET amplification (&amp;gt; 2.5 copy) and overexpression (IHC score 3+) were confirmed, and L858R EGFR mutation positive tumor tumor confirmed. The anti-cancer efficacy of CKD-702 was reduced by more than 80% compared to the tumor size of the control group regardless of the concentration. In the CKD-702 20mg/kg group, there was no significant difference in the change in the overall tumor size until the time of disappearing or ending during drug administration. The %TGI was 93.6 and 97.5 according to the concentration of CKD-702, and the % TGD was 118.5 and 311.8, respectively. CKD-702 has a significantly higher tumor growth inhibitory effect regardless of the concentration, but the tumor growth retardation effect is 2.6 times different depending on the concentration. % TGI of CKD-702 20mg / kg group was similar to Hu8c4 group or Hu8c4 + Vectibix group. However, % TGD was 1.4 times higher than Hu8c4 group efficacy and 3.1 times higher than Hu8c4 + Vectibix group% TGD. Even after the completion of drug administration, tumor still remained at 47 days and 67 days in the CKD-702 10 mg/kg and 20 mg/kg groups, respectively. Although c-MET protein expression decreased during the administration of CKD-702, it was confirmed that c-MET protein expression increased again in tumor tissue at the time of tumor growth. Although the tumor size was rather large (605.6 ~ 816.2mm3), CKD-702 showed twice the tumor growth inhibition effect (% TGI: 32.8 vs. 68.1) and the tumor growth delay effect was 4.4 times higher depending on the drug concentration (% TGI: 2.8 vs 12.3). Conclusion: Our data showed that CKD-702 have effective anti-tumor activity in EGFR TKI-resistant PDX model harboring both activating EGFR mutation and c-MET overexpression. Citation Format: Jung-Young Shin, Min-Young Kim, Mi-Ran Lee, Jeong-Oh Kim, Eun-Ju Jeon, Ji Hye Choi, Soon-Ki Hong, Kyu-Jin Park, Jin Hyoung Kang. Anti-tumor efficacy of CKD-702 in EGFR TKI-resistant patient-derived xenograft model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3046.

Abstract 6622: The identification of extranodal NK/T cell lymphoma-associated antigen for helper T cell-based immunotherapy

Abstract Extranodal natural killer/T-cell lymphoma, nasal type (NKTL) is a fatal destructive tumor that develops around the nasopharyngeal and nasal/paranasal areas. Although this disease was originally considered to be endemic to East Asia, Central and South America and was extremely rare in Europe and the United States, the incidence of NKTL has increased during the last several decades in countries where it was previously not epidemic. The combination of irradiation and chemotherapies such as SMILE and MPVIC-P are effective. However, these therapies cause severe and sometimes fatal adverse effects indicating that a novel strategy to treat NKTL is required. The positive results of clinical trials using immune checkpoint inhibitors in solid tumors proved that immune cells are capable of eliminating tumor. However, autoimmune diseases are inevitable adverse events in this type of therapy, which non-specifically activates T cells. Thus, the identification of epitope peptides in tumor has re-emerged as an attractive concept. I will describe our study to identify NKTL-derived T cell epitopes as a basis of cancer vaccine. The expression of c-Met and its ligand HGF plays a critical role in cell proliferation and is involved in numerous malignancies. Because c-Met expression and its role in NKTL remain unclear, we first studied the expression and function of c-Met in NKTL cells. As a result, we found that HGF and c-Met were expressed and contributed to the proliferation of cells in NK/T-cell lymphoma. Next, the helper epitopes were identified from c-Met protein using computer-based algorism. The identified epitope peptides could elicit peptide-specific helper CD4 T cells (HTLs), which showed antitumor responses in vitro. The addition of c-Met inhibitor suppressed TGF-β production from tumor followed by the augmentation of HTL responses. In summary, we identified novel c-Met HTL epitopes that can elicit effective antitumor responses against tumors expressing c-Met. Our results provide the rationale of combining c-Met targeting therapy and immunotherapy for NKTCLs and epithelial tumors. Citation Format: Takumi Kumai, Ryusuke Hayashi, Tatsuya Hayashi, Hiroya Kobayashi, Yasuaki Harabuchi. The identification of extranodal NK/T cell lymphoma-associated antigen for helper T cell-based immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6622.

A combined FAK, c-MET, and MST1R three-protein panel risk-stratifies colorectal cancer patients.

Focal adhesion kinase (FAK) is a key tyrosine kinase downstream of c-MET (or hepatocyte growth factor receptor, HGFR) and MST1R (macrophage-stimulating protein receptor or recepteur d'origine Nantais, RON) membrane receptors. The pathway plays an important role in cancer survival and invasion. In this study, we examined the protein expression of FAK, c-MET, and MST1R levels in a well-annotated cohort of 330 colorectal cancer patients. We found FAK to be overexpressed in colorectal adenocarcinomas (p = 0.0002), and FAK levels correlated positively with phospho-FAK levels (R2 = 0.81). In comparison, MST1R levels were not significantly different, and c-MET levels were slightly higher in the normal samples. We then developed a combined 3-protein panel of FAK, c-MET, and MST1R expression signatures that can robustly risk-stratify colorectal cancer across all stages into three clusters that differ in progression-free survival. The colorectal cancer subgroup with high FAK, low c-MET, and low MST1R protein levels showed the worst progression-free survival with particularly early progression of disease (p = 0.0053). Combined FAK, c-MET, and MST1R were independently prognostic for progression-free survival in stage II colorectal cancers in a multivariate model. The 3-protein panel provides a potentially clinically attractive method for risk-stratification and adjuvant therapy guidance, especially in stage II disease.

HDAC1/2 Inhibitor Romidepsin Suppresses DEN-Induced Hepatocellular Carcinogenesis in Mice.

BackgroundHepatocellular carcinoma (HCC) is a frequently diagnosed cancer and a leading cause of cancer-related death worldwide. Its rapid progression, combined with the limited treatment options at late stages, imposes the need for early detection and aggressive intervention. Based on the knowledge that hepatocarcinogenesis is significantly influenced by histone acetylation, we directed our search for novel HCC therapeutics among histone deacetylation inhibitors (HDACi). The aim of the present study was to investigate the effect of HDAC1/2 inhibitor Romidepsin in the well-established mouse model of diethylnitrosamine (DEN)-induced HCC.Materials and MethodsC56BL/6 mice were treated with Romidepsin at the critical point of 10 months after DEN challenge and their livers were examined 2 months later using histopathology and morphometry. Protein levels were assessed in serum using ELISA and in liver tissues using Western blot and immunohistochemistry (in-situ detection). Gene expression was quantified using real-time PCR.ResultsRomidepsin suppressed cancer progression. This effect was associated with decreased proliferation and increased apoptosis of cancer cells. The cell cycle regulator CK2a, the anti-inflammatory molecule PPAR-γ, and the tumor suppressors PTEN and CYLD were upregulated in treated HCC. By contrast, the expression of PI3K, NF-κB p65 and c-Jun was reduced. In line with this result, the levels of two major apoptosis regulators, ie, BAD and the multifunctional protein c-Met, were lower in the blood serum of treated mice compared to the untreated mice with HCC.ConclusionThese findings suggest that Romidepsin, a drug currently used in the treatment of lymphoma, could also be considered in the management of early-stage HCC.

Effect of silencing hepatocyte growth factor receptor c-Met expression on biological characteristics of colon cancer cells

Objective: To investigate the effect of silencing hepatocyte growth factor receptor (c-Met) expression on the biological characteristics of HCT116 colon cancer cells. Methods: Cellular model of c-Met transient transfection was established by using small interfering RNA (siRNA), the expression of c-Met in colon cancer cells was detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and western blot. The apoptosis assay, cell invasion assay, cell migration and other experiments were conducted to observe the effects of silencing c-Met on the biological characteristics of colon cancer cells. Results: RT-qPCR results showed that the relative expression levels of c-Met mRNA in siRNA-Met group, blank control group and siRNA negative control (siRNA-NC) group were 0.32±0.26, 1.01±0.03 and 1.05±0.23, respectively, and the difference was statistically significant (P<0.05). Western blot analysis showed that the expression level of c-Met protein in the siRNA-Met group was 0.24±0.03, significantly lower than 1.23±0.06 in the blank control group and 1.18±0.11 in the siRNA-NC group (P<0.05). The cell counting kit-8 (CCK8) results showed that the 72-hour absorbance (A) values of the siRNA-Met group, blank control group and the siRNA-NC group were 1.13±0.05, 1.48±0.08 and 1.53±0.07, respectively, and the difference was statistically significant (P<0.01). Cell cycle results showed that the proportion of cells in G(2)/M phase was (14.65±1.41)% in siRNA-Met group , (5.07±0.70)% in blank control group and (5.63±0.71)% in siRNA-NC group, and the difference was statistically significant (P<0.05). The expression levels of cell cycle regulatory proteins Cdc25c and cyclin B1 in siRNA-Met group were significantly decreased. The apoptotic rate in siRNA-Met group was (5.85±0.35)%, significantly higher than (1.00±0.17)% in blank control group and (0.91±1.14)% in siRNA-NC group (P<0.05). The expression level of apoptosis-related protein Bcl-2 in the siRNA-Met group was significantly decreased while Bcl-2 associated X protein (BAX) expression level was significantly increased. The cell scratching result showed that the cell migration abilities of the siRNA-Met group, blank control group and the siRNA-NC group were (51.33±8.62)%, (100.00±3.72)% and (102.33±6.43)%, respectively, and the difference was statistically significant (P<0.05). The number of cell penetrating into the basement membrane of the siRNA-Met group, blank control group and the siRNA-NC group were 47.50±10.60, 100.00±5.33 and 102.50±10.61, respectively, and the difference was statistically significant (P<0.05). The expressions of invasion related proteins including MMP-2 and MMP-9 in siRNA-Met group were decreased significantly. Conclusions: c-Met plays an important role in maintaining the biological characteristics of colon cancer cells. Inhibition of c-Met may have important values in the treatment of colon cancer.

A whole transcriptome sequencing (WTS)-based cMet dysregulation biomarker assay with correlates to cMet DNA mutations and cMet protein expression.

e15676 Background: Selective cMet kinase inhibitors (cMet-i) have demonstrated promising clinical activity as single-agents in non-small cell lung cancer (NSCLC) with cMet exon-14 skip mutations (MET∆14). APL-101 (PLB-1001) is a selective cMet-i and has demonstrated single-agent anti-tumor activity in secondary glioblastoma (GBM) harboring MET∆14 and/or MET fusion with evidence of blood-brain barrier permeability (Cell. 2018, 175: 1665-1678). A WTS-based cMet biomarker assay is being developed under Caris MI Transcriptome platform to predict clinical response to monotherapy APL-101 in tumors with cMet dysregulation (NCT03175224). Methods: In a pilot study, we used Caris WTS to analyze 202 patient samples of NSCLC (78%) and other tumor types (22%) mostly GBM and gastrointestinal tumors to establish key parameters of Caris WTS in detection of cMet dysregulation. These samples were also analyzed by DNA-seq and IHC to understand the correlations of Caris WTS to DNA-based cMet mutation detection and cMet protein expression. The samples included 63 (31%) MET∆14, 48 (24%) MET fusions based on WTS and covered a broad dynamic range in cMet expression and copy number variations. In order to establish a stable baseline against which to compare MET mRNA expression, 123 unique tissue samples from 93 unique non-tumor tissues covering 27 unique tissue types were analyzed for wild-type MET expression. Results: Concordance between WTS and DNA-seq in detection of MET∆14 was 84% based on 67 cases analyzed by both methods. WTS identified 45% positive MET∆14, compared to 35% by DNA-seq. Out of 11 discordant cases, 9 were positive by WTS and 2 positive by DNA-seq. The number of transcripts carrying MET∆14 variants correlated with cMet wild type mRNA expression (n = 63, r = 0.88, p&lt; 0.0001). cMet mRNA expression correlated with total cMet protein by IHC in MET fusion subset (n = 43, ρ = 0.65, p= &lt; 0.0001), MET∆14 subset (n = 55, ρ = 0.64, p&lt; 0.0001), and in all samples (n = 175, ρ = 0.75, p&lt; 0.0001). In 175 cases with both WTS and IHC results, 9 MET∆14 positive cases had no detectable cMet protein on plasma membrane albeit all showed a detectable level of total cMet protein. Conclusions: A cMet WTS assay has been established to identify patients with cMet dysregulation including MET∆14 and MET fusion. cMet IHC and DNA-seq may offer secondary utility to differentiate WTS-identified METΔ14 patients to further strengthen the prediction of clinical response to a cMet inhibitor.

Kinetic and thermodynamic study of c-Met interaction with single chain fragment variable (scFv) antibodies using phage based surface plasmon resonance

Mesenchymal epithelial transition factor (c-Met) has been recently regarded as an attractive target for the treatment of cancer. Our previous study showed that c-Met-specific single chain fragment variables (scFvs) can be considered as a promising therapy for cancer, however, their molecular interaction with c-Met protein have not been assessed. Accordingly, in the current study we aim to evaluate the kinetic and thermodynamic properties of c-Met interaction with these scFvs as anticancer agents by means of surface plasmon resonance (SPR) technique. Phage-scFvs were immobilized on the 11-mercaptoundecanoic acid gold chips after carboxylic groups activation by N-ethyl-N-(3-diethylaminopropyl) carbodiimide/N-hydroxysuccinimide and, then the c-Met binding to each scFvs (ES1, ES2, and ES3) at different concentrations (ranging from 20 to 665 μM) was explored. Kinetic studies revealed that ES1 has the highest affinity (KD = 3.36 × 10−8) toward its target at 25°C. Calculation of thermodynamic parameters also showed positive values for enthalpy and entropy changes, which was representative of hydrophobic forces between c-Met and ES1. Furthermore, the positive value of Gibbs free energy indicated that c-Met binding to ES1 was enthalpy-driven. Taken together, we concluded that produced ES1 can be applied as promising scFv-based therapy for diagnosis or targeting of c-Met in various cancers.

A novel non-agonist c-Met antibody drug conjugate with superior potency over a c-Met tyrosine kinase inhibitor in c-Met amplified and non-amplified cancers

ABSTRACTc-Met is a well-characterized oncogene that is associated with poor prognosis in many solid tumor types. While responses to c-Met inhibitors have been observed in clinical trials, activity appears to be limited to those with MET gene amplifications or mutations. We developed a c-Met targeted antibody-drug conjugate (ADC) with preclinical activity in the absence of MET gene amplification or mutation, and activity even in the context of moderate protein expression. The ADC utilized a high-affinity c-Met antibody (P3D12), that induced c-Met degradation with minimal activation of c-Met signaling, or mitogenic effect. P3D12 was conjugated to the tubulin inhibitor toxin MMAF via a cleavable linker (vc-MMAF). P3D12-vc-MMAF demonstrated potent in vitro activity in c-Met protein-expressing cell lines regardless of MET gene amplification or mutation status, and retained activity in cell lines with medium-low c-Met protein expression. In contrast, the c-Met tyrosine kinase inhibitor (TKI) PHA-665752 slowed tumor cell growth in vitro only in the context of MET gene amplification or very high protein expression. This differential activity was even more marked in vivo. P3D12-vc-MMAF demonstrated robust inhibition of tumor growth in the MET gene amplified MKN-45 xenograft model, and similar results in H1975, which expresses moderate levels of wild type c-Met without genomic amplification. By comparison, the c-Met TKI, PHA-665752, demonstrated modest tumor growth inhibition in MKN-45, and no inhibition at all in H1975. Taken together, these data suggest that P3D12-vc-MMAF may have a superior clinical profile in treating c-Met positive malignancies in contrast to c-Met pathway inhibitors.