Small Molecule Inhibitor Of c-Met Research Articles

Phase 2 trial of crizotinib in Japanese patients with advanced NSCLC harboring a MET gene alteration: a Co-MET study.

MET exon 14 skipping mutations occur in 3-4% and MET high amplifications occur in < 1% of patients with non-small-cell lung cancer (NSCLC). Crizotinib, a selective ATP-competitive small-molecule inhibitor of c-Met, ALK, and ROS1 tyrosine kinases, has shown activity in cancer models with various types of MET activation. The Co-MET study is a single-arm phase 2 trial to assess the safety and efficacy of crizotinib in MET inhibitor-naïve patients with advanced NSCLC harboring MET exon 14 skipping mutation (cohort 1) or high MET gene copy number of ≥ 7 (cohort 2). The primary endpoint was the objective response rate (ORR) per RECIST v1.1 by independent radiology review in cohort 1. The key secondary endpoints were the duration of response (DoR), progression-free survival (PFS), overall survival (OS), and safety. A total of 28 patients (23 in cohort 1 and 5 in cohort 2) were enrolled between March 2018 and February 2020. The primary endpoint was met as the ORR (90% confidence interval: CI) in cohort 1 was 38.1% (20.6-58.3). Median DoR, PFS, and OS (95% CI) were 7.6 (1.9-NE), 5.7 (2.1-11.3), 9.1 (4.0-19.9) months, respectively, in cohort 1. ORR in cohort 2 was 40.0% (18.9-92.4). The safety signals were generally consistent with the known safety profile of crizotinib. Crizotinib showed a clinical activity similar to that of tepotinib and capmatinib in patients with NSCLC harboring MET exon 14 skipping mutations. UMIN000031623.

Abstract 2116: Telisotuzumab vedotin (Teliso-V)-mediated c-Met internalization, endolysosomal trafficking, and potent cytotoxic activity are unaffected by MET exon-14 skipping mutation

Abstract Background: Telisotuzumab vedotin (Teliso-V) is an antibody-drug conjugate (ADC) composed of a c-Met (MET protein) antibody coupled to the microtubule inhibitor monomethyl auristatin E (MMAE). Teliso-V was granted FDA Breakthrough Therapy Designation for advanced/metastatic EGFR WT, NSQ NSCLC with high c-MET overexpression. For MMAE drug delivery, Teliso-V must first undergo receptor internalization and endolysosomal (EL) trafficking. Hepatocyte growth factor (HGF)-dependent MET EL trafficking and degradation depend on the E3 ubiquitin ligase Casitas B-lineage lymphoma (CBL). METex14 skipping and CBL-binding site mutations, which delete the binding site for CBL, occur in ~ 3% of NSCLC. The role of CBL in Teliso-V-MMAE delivery and activity was assessed in this work. Methods: WT-human c-Met, c-Met-Y1003F/N (CBL binding site mutations in MET), and c-Met-ex14∆ (47 aa deletion to mimic ex14 skipping) or CBL-L620f, G807*, and W802* (CBL inactivating mutants) were expressed in NIH3T3, Ba/F3 cells, or A549 tumor cells using retroviral transduction. Genetically modified cells were then used to define the role of CBL in c-Met surface expression, intracellular signaling, EL trafficking (using pHrodo Red-labeled Teliso-V), and Teliso-V sensitivity in 2D/3D cell cultures. Results: c-Met expression was required for Teliso-V cytotoxicity and intracellular payload delivery in all cell lines. While minimal changes in cell surface c-Met levels were observed, CBL binding mutants had increased phosphorylated c-Met in the absence of HGF, and ex14∆-MET and CBL binding site mutants showed increased sensitivity to small molecule c-Met inhibitors. Compared to WT c-Met, ex14∆ and CBL-binding mutants showed similar EL trafficking and no change in sensitivity to Teliso-V. Co-treatment with a pan-CBL inhibitor also failed to alter Teliso-V EL trafficking and anti-tumor activity. Like most ADCs, EL trafficking was dependent on a dynamin-mediated process with reduced EL trafficking by co-treatment with Dyngo4a. To further understand the role of CBL in Teliso-V sensitivity, we expressed CBL inactivating mutations in A549 tumor cells. While the L602F and W802* mutants increased spheroid size, as well as activated p-MET, there was no change in sensitivity to Teliso-V. Conclusions: Loss of CBL-binding sites on c-Met via ex14 deletion or Y1003F/N leads to increased MET-targeted TKI sensitivity and increased phospho-c-Met levels but does not impact Teliso-V sensitivity or EL trafficking. Our results indicate that Teliso-V traffics to the EL via a dynamin-mediated process. Therefore, Teliso-V delivers MMAE through a robust ADC-mediated EL-dependent mechanism that is independent of CBL, suggesting that high c-Met expression in Ex14skipping or CBL binding site mutant NSCLC patients should be further investigated for response to Teliso-V. Citation Format: Izhar Batth, Ronald Pandoy, Shamim Khaja, Dolonchampa Maji, Tamar Uziel, Athan Vasilopoulos, Peter Ansell, PK Epling-Burnette. Telisotuzumab vedotin (Teliso-V)-mediated c-Met internalization, endolysosomal trafficking, and potent cytotoxic activity are unaffected by MET exon-14 skipping mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2116.

Candida albicans stimulates formation of a multi-receptor complex that mediates epithelial cell invasion during oropharyngeal infection.

Fungal invasion of the oral epithelium is central to the pathogenesis of oropharyngeal candidiasis (OPC). Candida albicans invades the oral epithelium by receptor-induced endocytosis but this process is incompletely understood. We found that C. albicans infection of oral epithelial cells induces c-Met to form a multi-protein complex with E-cadherin and the epidermal growth factor receptor (EGFR). E-cadherin is necessary for C. albicans to activate both c-Met and EGFR and to induce the endocytosis of C. albicans. Proteomics analysis revealed that c-Met interacts with C. albicans Hyr1, Als3 and Ssa1. Both Hyr1 and Als3 are required for C. albicans to stimulate c-Met and EGFR in oral epithelial cells in vitro and for full virulence during OPC in mice. Treating mice with small molecule inhibitors of c-Met and EGFR ameliorates OPC, demonstrating the potential therapeutic efficacy of blocking these host receptors for C. albicans.

Enhancing nanoparticle paclitaxel antitumor response through targeted inhibition of c-Met pathway by merestinib in gastric cancer models.

372 Background: Gastric adenocarcinoma (GAC) is the fifth most common malignancy and the third most common cause of cancer-related deaths worldwide. Combination chemotherapy regimens are commonly used to treat GAC, but the median survival time remains less than one year. Nanoparticle albumin-bound paclitaxel ( nab-paclitaxel, NPT), an approved treatment for breast cancer, non-small cell lung cancer and pancreatic cancer, has demonstrated high antitumor activity in previous GAC studies. Many growth factors and their receptors are overexpressed in GAC and have been implicated in its pathophysiology. Aberrant activation of the c-Met pathway has been reported in up to 50% of GAC patients. We hypothesize that merestinib, a small-molecule inhibitor of c-Met, with additional inhibition of Axl and DDR1/2 pathways, will have significant antitumor effects and will enhance nab-paclitaxel chemotherapy response in GAC preclinical models. Methods: GAC subcutaneous xenografts were established by injecting MKN-45 and SNU-1 cells in NOD/SCID mice to study treatment effects on tumor volume, tumor weight, net tumor growth, and mouse body weight. Immunohistochemistry analyses of xenograft tissues were performed using Ki67 and endomucin to examine tumor cell proliferation and microvessel density, respectively. Results: In vitro assays showed that nab-paclitaxel and merestinib decreased cell proliferation in all three GAC-associated cell lines tested, with an additive effect in combination. Reduction in cell proliferation at low doses of nab-paclitaxel (10 nM), merestinib (100 nM), and their combination was 87%, 82%, and 94% (MKN-45, high phospho-c-Met expression), 59%, 50%, and 82% (SNU-1, low phospho-c-Met expression), and 53%, 19%, and 66% in gastric fibroblasts. Immunoblot analysis of merestinib treated MKN-45 cells revealed increased expression of pro-apoptotic proteins and decreased expression of phospho-c-Met, phospho-EGFR, phospho-IGF-1R, phospho-ERK, and phospho-AKT. In gastric fibroblasts, merestinib decreased phospho-ERK and increased pro-apoptotic protein expression. Phospho-c-Met and phospho-EGFR were not detected in SNU-1 immunoblots; however, phospho-ERK, phospho-VEGFR, and apoptotic protein expression increased after treatment. In MKN-45 xenografts, net tumor growth in control, nab-paclitaxel, merestinib, and combination groups was 503 mm3, 115 mm3, 91 mm3, and -9.7 mm3. Immunohistochemistry analysis of tumor cell proliferation and microvessel density corroborated tumor growth study results. Conclusions: These findings suggest that merestinib has strong antitumor activity in GAC and exhibits an additive effect when administered with nab-paclitaxel. These results provide compelling evidence that this therapeutic approach may lead to a clinically relevant combination to improve GAC patients’ survival.

Synthesis of ethyl 4-(2-fluoro-4-nitrophenoxy) picolinate

Cancer has seriously affected people's production and life. The appearance of anti-cancer drugs has brought good news to people. Ethyl 4-(2-fluoro-4-nitrophenoxy) picolinate is an important basic skeleton of a small molecule inhibitor of c-Met and a major intermediate in cancer therapy. A rapid and efficient method for the synthesis of compound 8 was established. Compound 8 was synthesized from picolinic acid by acylation and substitution. These steps were weight gain reaction. The synthesis method was optimized and the structure was confirmed by hydrogen NMR spectroscopy.

O13-5 Phase 2 trial of crizotinib in Japanese patients with advanced NSCLC harboring a MET gene alteration: Co-MET study

Among patients with non-small-cell lung cancer (NSCLC), MET exon 14 skipping mutations occur in 3-4% and MET high amplifications occur in <1%. Crizotinib, a selective ATP-competitive small-molecule inhibitor of c-Met, ALK, and ROS1 tyrosine kinases showed activity in cancer models with various types of MET activation.

An updated patent review of small-molecule c-Met kinase inhibitors (2018-present)

Introduction c-Met tyrosine kinase receptor is a high-affinity ligand of hepatocyte growth factor (HGF). c-Met is widely expressed in a variety of normal human tissues, but it exhibits abnormally high expression, amplification or mutation in tumor tissues such as lung cancer, gastric cancer, and breast cancer. Therefore, the use of c-Met as a target can achieve the inhibition of a series of abnormal physiological processes such as tumourigenesis, development and metastasis. A number of small molecule tyrosine kinase inhibitors targeting c-Met have been successfully marketed. Areas covered This article reviews recent advances in patented c-Met small molecule inhibitors and their inhibitory activity against various cancer cells from 2018 to date. Expert opinion To date, small molecule inhibitors targeting c-Met have demonstrated impressive therapeutic efficacy in the clinical setting. Most recent patents have focused on addressing the direction of c-Met amplification and overexpression. Despite the great success in the development of selective c-Met inhibitors, the effects of bypass secretion and mutagenesis have led to a need for new c-Met small molecule inhibitors that are safe, efficient, selective and less toxic with novel structures and effective against other targets.

Radiosensitizing effect of c-Met kinase inhibitor BPI-9016M in esophageal squamous cell carcinoma cells in vitro and in vivo.

Backgroundc-Met is the receptor of hepatocyte growth factor (HGF) which plays a key role in inhibiting apoptosis. BPI-9016M is a small-molecule c-Met inhibitor that can promote apoptosis and enhance the cytotoxicity of various DNA-damaging agents. Here, we evaluated the radiosensitizing potential of BPI-9016M in Eca109 human esophageal squamous cell carcinoma (ESCC) cells in vitro and in vivo.MethodsCell Counting Kit-8 (CCK-8) assay was used to measure cell viability. Clonogenic survival assay and a murine tumor xenograft in male nude mice were used to evaluate the radiosensitizing effect of BPI-9016M. Apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and flow cytometry experiment. Apoptosis-related proteins were detected by western blot. By evaluating the activation of the ATR-Chk1/ATM-Chk2 pathway to detect radiation-induced DNA double-strand break and homologous recombination repair. BPI-9016M induced a radiosensitizing effect in Eca109 cells and reduced the survival rate of clone formation in vitro.ResultsThe combination of BPI-9016M with irradiation (IR) significantly delayed the growth of ESCC tumor xenografts than treatment alone (P<0.05). The radiosensitizing effects of BPI-9016M were due to increased apoptosis, such as the up-regulation of cleaved-caspase 3 and 9, down-regulation of mutant P53 and Bcl-2, the decreased of phosphorylation of ATR and ATM, and the inhibition of γ-H2AX accumulation in vitro and in vivo.ConclusionsThese findings indicated that BPI-9016M exerts a radiosensitizing effect and enhances apoptosis by inhibiting homologous recombination DNA repair in irradiated ESCC cells.

A selective small-molecule inhibitor of c-Met suppresses keloid fibroblast growth in vitro and in a mouse model

Keloids, tumor-like lesions that result from excessive scar formation, have no definitive treatment modality. Activation of c-mesenchymal-epithelial transition factor (c-Met) promotes cell proliferation and survival. Selective c-Met inhibitors, such as PHA-665752, may attenuate the activity of keloid fibroblasts and reduce keloid formation. Here, we aimed to evaluate the effect of PHA-665752, a second-generation selective small-molecule inhibitor of c-Met, on human keloid fibroblasts in vitro and in a mouse model. We performed in vitro cytotoxicity assays, scratch tests, western blotting, and immunofluorescence on human keloid fibroblasts. We also injected human fibroblasts into severe combined immunodeficient mice and measured the degree of nodule formation and skin histologic characteristics. We found that keloid fibroblast migration was inhibited by PHA-665752. Inhibitor treatment was also associated with lower expression of members of the hepatocyte growth factor/c-Met pathway, and lower fibroblast activity and collagen synthesis. In the in vivo experiments, PHA-665752—treated mice had lower nodule volumes and weights, accompanied by less inflammatory cell infiltration and collagen deposition, than those in control mice. These findings showed that although an in vivo model may not accurately represent the pathophysiology of human keloid development, PHA-665752 suppressed keloid fibroblast activity by inhibiting the c-Met—related tyrosine kinase pathway.

Abstract PO045: Impact of fibroblast mediated stromal protection on evolution of resistance to targeted therapies in NSCLC

Abstract BACKGROUND: Clinical introduction of tyrosine kinases inhibitors (TKIs) for ALK+ non-small cell lung cancer (NSCLC) has improved patient survival, but it did not translate into cures as acquired resistance inevitably emerges in patients with advanced and metastatic cancers. Cancer associated fibroblasts (CAFs) have emerged as the major determinant of environmental protection. While the ability of stroma to protect tumor cells from TKI is well documented in-vitro, whether the impact of stromal protection is relevant in 3D context and in-vivo remains unresolved. Thus, we hypothesized that stromal protection impacts the initial response of tumor cells to targeted therapies. METHODS: Cancer cell lines were cultured in presence of CAF conditioned media (CM) and treated with TKIs to monitor cell viability. To interrogate molecular mechanism, loss and gain of function experiments were performed using pharmacological inhibitors and recombinant human protein respectively. To address the relevance of stromal protection in-vivo, we worked with tumor xenograft mouse model. We also used mathematical modelling to understand the tumor dynamics, using inferences from xenograft mouse models to build agent-based models. RESULTS: We observed that patient derived matched normal tissue associated fibroblast (NAFs) and CAFs, both confers various level of protection to tumor cells against TKIs. Addition of rh Hepatocyte growth factor (HGF) protein mimicked the effect of CAF CM and addition of small molecule inhibitors of cMET abolished the CAF-mediated protection to tumor cells against TKIs. Our xenograft experiment showed the significant increase in stroma/tumor ratio during the initial tumor response to targeted therapy and we observed increase in tumor proliferating cells in proximity to stroma. Our math model for stroma and pre-existing resistant population showed delay in development of resistance to TKI. CONCLUSION: Fibroblast-mediated stromal protection is critically dependent on HGF-cMET axis. Dual inhibition of ALK and MET signaling abolished the fibroblast-mediated protection and increased the cell sensitivity to ALK TKIs, suggesting the crucial role of microenvironment-mediated resistance in NSCLC. To study tumor dynamics using mathematical models, we addressed that space could be key to understand the impact of stromal protection in-vivo. Our study shows that understanding the signaling changes carried by fibroblast in response to therapy may lead to potential better combination therapy. Citation Format: Bina Desai. Impact of fibroblast mediated stromal protection on evolution of resistance to targeted therapies in NSCLC [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO045.

HGF/c-Met Axis: The Advanced Development in Digestive System Cancer.

Numerous studies have indicated that abnormal activation of the HGF/c-Met signaling pathway can lead to cell proliferation, invasiveness, and metastasis of cancers of the digestive system. Moreover, overexpression of c-Met has been implicated in poor prognosis of patients with these forms of cancer, suggesting the possibility for HGF/c-Met axis as a potential therapeutic target. Despite the large number of clinical and preclinical trials worldwide, no significant positive success in the use of anti-HGF/c-Met treatments on cancers of the digestive system has been achieved. In this review, we summarize advanced development of clinical research on HGF/c-Met antibody and small-molecule c-Met inhibitors of cancers of the digestive system and provide a possible direction for future research.

A common MET polymorphism harnesses HER2 signaling to drive aggressive squamous cell carcinoma

c-MET receptors are activated in cancers through genomic events like tyrosine kinase domain mutations, juxtamembrane splicing mutation and amplified copy numbers, which can be inhibited by c-MET small molecule inhibitors. Here, we discover that the most common polymorphism known to affect MET gene (N375S), involving the semaphorin domain, confers exquisite binding affinity for HER2 and enables METN375S to interact with HER2 in a ligand-independent fashion. The resultant METN375S/HER2 dimer transduces potent proliferative, pro-invasive and pro-metastatic cues through the HER2 signaling axis to drive aggressive squamous cell carcinomas of the head and neck (HNSCC) and lung (LUSC), and is associated with poor prognosis. Accordingly, HER2 blockers, but not c-MET inhibitors, are paradoxically effective at restraining in vivo and in vitro models expressing METN375S. These results establish METN375S as a biologically distinct and clinically actionable molecular subset of SCCs that are uniquely amenable to HER2 blocking therapies.

Synthesis of Pyridinium Pyrimidines C-Met Small Molecule Inhibitors

Cancer has gradually become a serious threat to human health, and molecular targeted therapy has largely broken through the shortcomings of traditional treatment methods. C-met has been found to be overexpressed in a variety of cancer cells, and inhibition of its signaling pathway can effectively inhibit the occurrence of cancer. To explore the structure-activity relationship of the pyridinium pyrimidine cmet small molecule inhibitors, we synthesized the final compound A.

1959P - HER2 inhibition in aggressive squamous cell carcinomas driven by a common MET sema domain polymorphism

BackgroundOncogenic MET signaling is often deployed by tumors for malignant transformation through genomic events like tyrosine kinase domain mutations, juxtamembrane splicing mutation and amplified copy numbers, which can be inhibited by c-MET small molecule inhibitors. We present biochemical and clinical findings on a polymorphism of MET, which is common in the East Asian population. MethodsGermline DNA (gDNA) of lung, nasopharyngeal, liver, breast, gastric and colon cancer cohorts among East Asians (n=1,076) were genotyped for MET (p.N375S) polymorphism using digital droplet PCR (ddPCR). Relapse free survival (RFS) with curative intent was compared using log-rank test. Screening for protein-protein interaction was performed with SILAC. Clinical study is now enrolling MET-N375S+ squamous cell (SCC) head and neck patients to be given 30mg afatinib daily. Treatment outcome was determined with F-FDG PET/CT scan. Baseline (BL) and post-treatment biopsies were analyzed with immunohistochemistry for response biomarkers and in situ hybridization-proximity ligation assay (ISH-PLA) for receptor interaction. ResultsFraction of the MET-N375S+ cases is comparable across cancer patients and cancer-free controls (9-18%). METN375S was associated with significantly shorter relapse-free survival (RFS) in SCC of the head and neck (HNSCC; P=0.005) and lung (LUSC; P=0.029). SILAC revealed interaction of MET-N375S with HER2 (P<0.05). Two patients with refractory HNSCC who completed 1 cycle of afatinib, a pan-HER inhibitor, showed radiological tumour shrinkage and reduction in metabolic activity, with partial response by RECIST 1.1 measurement observed in one patient. Evaluable BL biopsy showed strong MET-HER2 interaction (ISH-PLA) with strong staining of phosphorylated MET and HER2 (IHC), which were significantly reduced post-treatment, indicative of successive intervention of MET-N375S/HER2 signaling. ConclusionsWe established MET-N375S as a biologically-distinct molecular subset of SCC that drives malignancy through HER2 signaling, and could serve as a predictive biomarker to select refractory patients with dismal prognosis for afatinib treatment. Clinical trial identificationNCT03938012. Actual study start date: October 3, 2017; Estimated primary completion date: October 2021; Estimated study completion date: October 2022. Legal entity responsible for the studyNational University Cancer Institute, Singapore. FundingNational Medical Research Council, Singapore. DisclosureAll authors have declared no conflicts of interest.

The effects of crizotinib in a transgenic Drosophila model expressing the human TPM4-ALK fusion gene or TPM4.

ABSTRACTAnaplastic lymphoma kinase (ALK) fusion events lead to constitutive activation of the ALK kinase domain, thereby functioning as oncogenic drivers. These fusion proteins have been identified in numerous cancers. Crizotinib, a small molecule inhibitor of c-Met and ALK, is a Food and Drug Administration-approved drug with reported efficacy in the treatment of cancer. Tropomyosins (TPMs) are a family of actin filament-binding proteins. Altered TPM expression has been found in a variety of human tumors. Inhibitors of cancer-associated TPMs and actin-targeting compounds have been developed, but anti-actin agents have cardiac and respiratory muscle toxicities. In this study, we investigated the sensitivities of human TPM4 (hTPM4), human ALK (hALK), and their fusion gene (hTPM4-hALK) to crizotinib by measuring the lifespan of transgenic Drosophila. Flies overexpressing hTPM4-hALK, hTPM4 and hALK showed decreased lifespans compared with controls. Although crizotinib is an inhibitor of ALK, treatment with crizotinib significantly extended the lifespans of Drosophila expressing hTPM4 and hTPM4-hALK but had no effect on hALK-expressing flies. Autophosphorylation of Tyr1278 is necessary for full activation of the ALK domain. We confirmed that hTPM4-hALK was phosphorylated at Tyr1278 in a ligand-independent manner, and hTPM4-hALK-expressing flies treated with crizotinib showed a decreased level of Tyr1278 phosphorylation compared with untreated hTPM4-hALK-expressing flies, with a greater decrease induced by 1 µM compared with 200 nM crizotinib. Taken together, the results suggest that crizotinib is effective for treating ALK-driven cancer and might be a new therapeutic drug, without cardiac or respiratory muscle toxic effects, for TPM4-expressing cancers.

Abstract 2351: AXL and c-MET cross-talk in the regulation of PD-L1 expression on renal cancer cells

Abstract Purpose: The purpose of this research is to study the cross-talk between receptor tyrosine kinases (RTKs) c-MET and AXL in renal cancer cells; and to explore if the blockade of AXL-c-MET pathways can restrict PD-L1-mediated growth and immune escape of renal cancer cells. Background: c-MET and AXL are overexpressed in renal cell carcinoma (RCC). Upon binding with their respective ligands hepatocyte growth factor (HGF) and GAS6, c-MET and AXL activate a wide range of pathways, including tumor promoting events. Renal tumors show increased T cell infiltration, yet the T cells are rendered incapable of tumor rejection. The co-inhibitory molecules PD-1 and PD-L1 belong to cell surface glycoprotein B7 family that governs T cell function and proliferation. In RCC, tumor-infiltrating T cells express PD-1 and are associated with poor outcomes for patients. Similarly, PD-L1 expression on renal cancer cells hinders prognosis and indicates tumor aggressiveness. Therefore, PD-L1 in renal tumors may play a significant role in down-regulating host anti-tumor immune responses. Our recent report suggests that c-MET activation induces overexpression of PD-L1 in renal cancer cells; and the blockade of PD-L1 promotes immune-mediated killing of renal cancer cells. However, the cross talk between AXL and c-MET in the regulation of PD-L1 has not been studied in RCC. Results: We have confirmed that both AXL and c-MET are overexpressed in human renal cancer cells (786-O and ACHN) when compared with normal renal tubular epithelial cells. We performed co-immunoprecipitation assays with c-MET antibody and found that c-MET forms a complex with AXL in renal cancer cells. We utilized siRNA-mediated gene silencing approach and found that the knock-down of AXL increased the sensitivity of 786-O and ACHN cells to apoptosis following treatment with XL-184 (a small molecule inhibitor of c-MET, AXL and VGFRs, obtained from Exelixis) as measured by flow cytometry in Annexin V/PI stained cells. We also found that GAS6 treatment enhanced HGF-induced expression of cytoprotective molecule Heme oxygenase-1 (HO-1) and anti-apoptotic molecules Bcl-2 and Bcl-xL in renal cancer cells. In both 786-O and ACHN cells, c-MET activation increased the cell surface expression of PD-L1 as measured by flow cytometry. Interestingly, GAS6 treatment further increased the expression of PD-L1, suggesting a synergistic action of RTKs AXL and c-MET in renal cancer cells. We also utilized a PD-L1 promoter-luciferase plasmid to measure PD-L1 promoter activity and found that GAS6 treatment enhanced HGF-induced effects on PD-L1 transcription. In mouse renal cancer cells (RENCA), which express very high levels of PD-L1, we observed a significant decrease in the expression of PD-L1 when AXL was knocked down in cells. Conclusion: Together, our results demonstrate that AXL-mediated signaling enhances c MET-induced cytoprotection and PD-L1 expression on renal cancer cells. Citation Format: Murugabaskar Balan, Toni Choueiri, Soumitro Pal. AXL and c-MET cross-talk in the regulation of PD-L1 expression on renal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2351.

Abstract 1039: c-Met activation promotes epithelial-to-mesenchymal transition (EMT) in renal cancer cells through the upregulation of the chemokine receptor CXCR7

Abstract Purpose: The purpose of this study is to explore how the receptor tyrosine kinase (RTK) c-Met modulates the expression of the pro-tumorigenic molecule CXCR7, a chemokine receptor, in renal cancer cells; and to investigate the role of c-Met and CXCR7 in promoting the epithelial-mesenchymal transition (EMT) of renal cancer cells. Background: c-Met, a pro-oncogenic RTK, can play a major role in the survival and progression of cancer cells; and it is often overexpressed in both clear cell and papillary renal cell carcinoma (RCC). c-Met, upon activation by its specific ligand hepatocyte growth factor (HGF), can induce a broad spectrum of biological pathways involved in the growth of cancer cells. CXCR family of chemokine receptors and their chemokine ligands were shown to exhibit decisive roles in tumor initiation, promotion, progression, and metastasis. CXCR7 is upregulated in several types of cancers, including renal cancer, and found to be involved in tumor cell development, survival, and metastasis. A global process integral to the cancer metastasis is EMT, which results in the disruption of cell-to-cell junctions and endows epithelial cells with the ability to migrate and invade surrounding tissue. EMT process is associated with an increase of mesenchymal markers like vimentin and a decrease of epithelial markers like E-cadherin in cancer cells. Sustained activation of c-Met induces EMT in renal cancer cells, however, the cross-talk between CXCR7 and c-Met signaling in the promotion of renal cancer EMT remains unclear. Results: We assessed the expression levels of CXCR7 by Western blot analysis and found that CXCR7 is overexpressed in human renal cancer cells (both 786-O and ACHN) when compared to normal renal epithelial cells (RPTEC). Also, in both 786-O and ACHN cells, HGF-induced activation of c-Met further increased the expression of CXCR7 at both RNA and protein levels. In renal cancer cells (both 786-O and ACHN), siRNA-mediated knockdown of c-Met or treatment with XL-184 (a small molecule inhibitor of c-Met) inhibited c-Met-induced upregulation of CXCR7. c-Met activation increased the expression of pro-EMT markers such as vimentin, N-cadherin, and MMP-9 and suppressed the expression of anti-EMT molecule E-cadherin, as measured by Western blot and flow cytometry. On the other hand, siRNA-mediated silencing of CXCR7 attenuated the induction of EMT, suggesting that CXCR7 plays a major role in c-Met-induced EMT in renal cancer cells. Conclusion: Together, our findings demonstrate that the c-Met-CXCR7 axis can trigger the EMT, which can play an important role(s) in renal cancer proliferation and migration. Citation Format: Samik Chakraborty, Murugabaskar Balan, Soumitro Pal. c-Met activation promotes epithelial-to-mesenchymal transition (EMT) in renal cancer cells through the upregulation of the chemokine receptor CXCR7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1039.

Safety and Tolerability of c-MET Inhibitors in Cancer.

The role of aberrant hepatocyte growth factor receptor (c-MET, also known as tyrosine-protein kinase MET)/hepatocyte growth factor (HGF) signaling in cancer progression and invasion has been extensively studied. c-MET inhibitors have shown promising pre-clinical and early phase clinical trial anti-tumor activity in several tumor types, although results of most phase III trials with these agents have been negative. To date, two small molecule c-MET inhibitors, cabozantinib and crizotinib, have been approved by regulatory authorities for the treatment of selected cancer types, but several novel c-MET inhibitors (either monoclonal antibodies or small molecule c-MET tyrosine kinase inhibitors) and treatment combinations are currently under study in different settings. Here we provide an overview of the mechanism of action and rationale of c-MET inhibition in cancer, the efficacy of approved agents, and novel promising c-MET-inhibitors and novel targeted combination strategies under development in different cancer types, with a focus on the safety profile and tolerability of these compounds.

Enhancing the Radiation Response in KRAS Mutant Colorectal Cancers Using the c-Met Inhibitor Crizotinib

INTRODUCTION: C-Met plays important roles in treatment resistance, tumor invasion, and metastasis. In this study, we used a small molecule inhibitor of c-Met, crizotinib, in cetuximab-resistant, mutant KRAS-driven colorectal cancer cell lines and assessed radiosensitization. MATERIALS AND METHODS: A tissue microarray containing colorectal tumors was used to study the relationship between KRAS mutations and c-Met expression. For in vivo studies, we used the KRAS mutant cell lines HCT116, DLD1, and LoVo. Colony formation assays were performed to assess the effects of crizotinib and cetuximab. Immunoblot analysis was used to determine the effect of crizotinib on c-Met and downstream pathways and DNA damage response. We then selected noncytotoxic doses of crizotinib to assess clonogenic survival with radiation. To study potential mechanisms of radiosensitization, cell cycle analysis was performed using flow cytometry. RESULTS: Analysis of the tissue microarray revealed that KRAS mutant tumors had active c-Met signaling. KRAS mutant cell lines LoVo, HCT116, and DLD1 were resistant to cetuximab but sensitive to crizotinib. Pretreatment with crizotinib for 24 hours radiosensitized LoVo, DLD1, and HCT116 cell lines with enhancement ratios of 1.54, 1.23, and 1.30, respectively. Immunoblot analysis showed that crizotinib blocked radiation-induced c-Met phosphorylation and attenuated downstream signaling pathways. Cell cycle analysis revealed minimal G1 arrest with crizotinib. Additionally, crizotinib completely blocked HGF induced cell migration. CONCLUSIONS: Inhibition of c-Met with crizotinib effectively sensitizes cetuximab-resistant KRAS mutant colorectal cancer cell lines to radiation. Crizotinib has the potential to improve outcomes in locally advanced rectal cancer patients undergoing chemoradiation.

MicroRNA-198 inhibition of HGF/c-MET signaling pathway overcomes resistance to radiotherapy and induces apoptosis in human non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) is the most common cause of death from cancer worldwide. MicroRNAs (miRNAs) are a group of important regulators in NSCLC, including miR-198. However, the underlying molecular mechanisms of miR-198 involvement in intrinsic resistance to radiotherapy in NSCLC remain to be elucidated. In this study, to investigate the clinical significance of miR-198 in NSCLC in relation to the response to radiotherapy, we determined the expression patterns of miR-198 between responders and nonresponders after 2 months of radiotherapy and found that decreased expressions of miR-198 were associated with radiotherapy resistance. In addition, we altered the endogenous miR-198 using mimics or inhibitors to examine the effects of miR-198 on 4-Gy-irradiated A549 and SPCA-1 cells in vitro. Upregulating miR-198 was shown to inhibit cell proliferation, migration, and invasion and induce apoptosis. MiR-198 inhibition produced a reciprocal result. PHA665752, a selective small-molecule c-Met inhibitor, potently inhibited hepatocyte growth factor (HGF)-stimulated and constitutive c-Met phosphorylation and rescued 4-Gy-irradiated A549 and SPCA-1 cells from miR-198 inhibition. Most importantly, we established tumor xenografts of 4-Gy-irradiated A549 and SPCA-1 cells in nude mice and found that miR-198 could suppress tumor formation. Hence, our data delineates the molecular pathway by which miR-198 inhibits NSCLC cellular proliferation and induces apoptosis following radiotherapy, providing a novel target aimed at improving the radiotherapeutic response in NSCLC.