MET Receptor Tyrosine Kinase Research Articles

Functional interaction between receptor tyrosine kinase MET and ETS transcription factors promotes prostate cancer progression.

Prostate cancer, the most common malignancy in men, has a relatively favourable prognosis. However, when it spreads to the bone, the survival rate drops dramatically. The development of bone metastases leaves patients with aggressive prostate cancer, the leading cause of death in men. Moreover, bone metastases are incurable and very painful. Hepatocyte growth factor receptor (MET) and fusion of genes encoding E26 transformation-specific (ETS) transcription factors are both involved in the progression of the disease. ETS gene fusions, in particular, have the ability to induce the migratory and invasive properties of prostate cancer cells, whereas MET receptor, through its signalling cascades, is able to activate transcription factor expression. MET signalling and ETS gene fusions are intimately linked to high-grade prostate cancer. However, the collaboration of these factors in prostate cancer progression has not yet been investigated. Here, we show, using cell models of advanced prostate cancer, that ETS translocation variant 1 (ETV1) and transcriptional regulator ERG (ERG) transcription factors (members of the ETS family) promote tumour properties, and that activation of MET signalling enhances these effects. By using a specific MET tyrosine kinase inhibitor in a humanised hepatocyte growth factor (HGF) mouse model, we also establish that MET activity is required for ETV1/ERG-mediated tumour growth. Finally, by performing a comparative transcriptomic analysis, we identify target genes that could play a relevant role in these cellular processes. Thus, our results demonstrate for the first time in prostate cancer models a functional interaction between ETS transcription factors (ETV1 and ERG) and MET signalling that confers more aggressive properties and highlight a molecular signature characteristic of this combined action.

KRAS-mutant non-small cell lung cancer (NSCLC) therapy based on tepotinib and omeprazole combination

BackgroundKRAS-mutant non-small cell lung cancer (NSCLC) shows a relatively low response rate to chemotherapy, immunotherapy and KRAS-G12C selective inhibitors, leading to short median progression-free survival, and overall survival. The MET receptor tyrosine kinase (c-MET), the cognate receptor of hepatocyte growth factor (HGF), was reported to be overexpressed in KRAS-mutant lung cancer cells leading to tumor-growth in anchorage-independent conditions.MethodsCell viability assay and synergy analysis were carried out in native, sotorasib and trametinib-resistant KRAS-mutant NSCLC cell lines. Colony formation assays and Western blot analysis were also performed. RNA isolation from tumors of KRAS-mutant NSCLC patients was performed and KRAS and MET mRNA expression was determined by real-time RT-qPCR. In vivo studies were conducted in NSCLC (NCI-H358) cell-derived tumor xenograft model.ResultsOur research has shown promising activity of omeprazole, a V-ATPase-driven proton pump inhibitor with potential anti-cancer properties, in combination with the MET inhibitor tepotinib in KRAS-mutant G12C and non-G12C NSCLC cell lines, as well as in G12C inhibitor (AMG510, sotorasib) and MEK inhibitor (trametinib)-resistant cell lines. Moreover, in a xenograft mouse model, combination of omeprazole plus tepotinib caused tumor growth regression. We observed that the combination of these two drugs downregulates phosphorylation of the glycolytic enzyme enolase 1 (ENO1) and the low-density lipoprotein receptor-related protein (LRP) 5/6 in the H358 KRAS G12C cell line, but not in the H358 sotorasib resistant, indicating that the effect of the combination could be independent of ENO1. In addition, we examined the probability of recurrence-free survival and overall survival in 40 early lung adenocarcinoma patients with KRAS G12C mutation stratified by KRAS and MET mRNA levels. Significant differences were observed in recurrence-free survival according to high levels of KRAS mRNA expression. Hazard ratio (HR) of recurrence-free survival was 7.291 (p = 0.014) for high levels of KRAS mRNA expression and 3.742 (p = 0.052) for high MET mRNA expression.ConclusionsWe posit that the combination of the V-ATPase inhibitor omeprazole plus tepotinib warrants further assessment in KRAS-mutant G12C and non G12C cell lines, including those resistant to the covalent KRAS G12C inhibitors.

Abstract 5101: Comparative genomic analysis reveals distinctive immune profiles in MET exon 14 mutated and MET amplified lung cancer

Abstract Background: In non-small cell lung cancer (NSCLC), the MET tyrosine kinase receptor can be mutated or amplified resulting in dysregulation of receptor function leading to tumor proliferation. MET exon 14 (METEx14) mutated and MET amplified NSCLC tumors have shown varied response to immunotherapy. Therefore, we sought to compare the genomic and immune landscape of MET-altered NSCLC. Methods: The genomic profiles of 3,821 NSCLC tumors sequenced using the Strata Select assay on the Strata Oncology Platform were analyzed. Tumors were sorted based on METex14 mutations, MET amplification (METamp) defined as copy number gain (CNG) ≥6 and MET wild-type (METwt). The RNA expression of 19 immune regulatory genes, tumor mutation burden (TMB), Strata Immunotherapy Response Signature (IRS) score and the frequency of gene co-alterations were compared across groups. Statistical comparisons of medians were done with Kruskal-Wallis testing and categorical comparisons with Chi square using R Studio version 4.3.2. Results: 122 (3.2%) METex14, 70 (1.8%) METamp, and 3,629 (95.0%) METwt were identified. Patients with METex14 were older and more frequently female. MET gene expression was found to be higher in METamp (median 14.4) compared to METex14 (median 12.3) (P<0.01). TMB was lowest in METex14 (median 2.7) group compared to METamp (median 7.1) and METwt (median 5.1) (P<0.001). High PD-L1 expression occurred in 71.4% of METamp, 68.0% of METex14 and 41.3% of METwt (P<0.001). Overall, METamp tumors had a greater proportion of high IRS scores (52.9%) compared to METex14 (35.2%) and METwt (45.4%) (P<0.01). mRNA expression of immune checkpoints CTLA4, PD-1, LAG3, IDO1, TIGIT and HAVCR2 were all higher in METex14 compared to METamp and METwt (P<0.001 for all). METex14 tumors also exhibited higher gene expression of CD4, CD8A and FOXP3 compared to METamp and METwt (p<0.001). METex14 had higher prevalence of MDM2 amplification (34% vs 3% vs 4%) and lower prevalence of alterations in EGFR (0.8% vs 22% vs 21%), KRAS (0.8% vs 10% vs 26%), CDKN2A (17% vs 29% v 28%) and TP53 (34% vs 87% vs 64%) compared with METamp and METwt tumors, respectively (p<0.05). Conclusion: METex14 and METamp tumors harbor distinct immune-gene expression profiles and co-occurring gene alterations that distinguish themselves from each other and METwt tumors. Further analysis must be conducted to determine how these variations impact immunotherapy in MET-altered NSCLC. Citation Format: Saahil Javeri, Rachel Minne, Shrey Ramesh, Andrew Baschnagel, Randy Kimple, Laura Lamb, Scott Tomlins, Bryan Johnson, Nickolay Khazanov. Comparative genomic analysis reveals distinctive immune profiles in MET exon 14 mutated and MET amplified lung cancer [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 5101.

Abstract 1914: Amivantamab efficacy in wildtype EGFR NSCLC tumors correlates with levels of ligand expression

Abstract Amivantamab (RYBREVANT™) is an FDA-approved, low-fucose IgG1 bispecific antibody targeting EGF and Met receptor tyrosine kinases, with proven clinical activity against EGFR mutant non-small cell lung cancer (NSCLC). Preclinical studies demonstrated that three context-dependent mechanisms of action (MOA) contribute to amivantamab efficacy: immune cell-mediated killing by antibody-dependent cellular cytotoxicity and trogocytosis, receptor internalization and degradation, and inhibition of ligand binding to both EGF and Met receptors. In tumors lacking known cancer-driver genetic aberrations, ligand binding to wildtype EGFR (EGFRWT) and/or MetWT may activate downstream signaling pathways, inducing pro-tumorigenic processes. Among the EGFR ligands, we demonstrated that amphiregulin (AREG) is highly expressed in EGFRWT NSCLC primary tumors, with significantly higher circulating protein levels in NSCLC patients than in healthy volunteers, and induces EGFR phosphorylation, promoting downstream signaling and increasing cell proliferation. Importantly, treatment of AREG-stimulated EGFRWT cells/tumors with amivantamab, or an AREG-targeting antibody, inhibits ligand-induced signaling and cell/tumor proliferation/growth. Across 10 EGFRWT NSCLC patient-derived xenograft mouse models, amivantamab efficacy correlated with AREG RNA levels, suggesting AREG as a potential predictive marker for amivantamab activity in this population. Interestingly, in these xenograft models, amivantamab anti-tumor activity was independent of amivantamab's Fc engagement with immune cells, suggesting that, in this context, the amivantamab’s ligand-blocking function is sufficient for its efficacy. Juxtaposedly, we previously demonstrated that low fucose amivantamab, active Fc domain, was necessary for maximal anti-tumor activity against tumors with EGFR or Met activating mutations. Altogether, these results demonstrated amivantamab context-dependent MOA for efficacy. In conclusion, these data 1) highlight EGFR ligand AREG as a driver of tumor growth in some EGFRWT NSCLC models, 2) illustrate the preclinical efficacy of amivantamab in ligand-driven EGFRWT NSCLC, and 3) identify AREG as a potential predictive biomarker for amivantamab against anti-EGFRWT therapies. Citation Format: Ricardo Rivera-Soto, Benjamin Henley, Marian Pulgar, Stacey Lehman-Tenuto, Himanshu Gupta, Megan Weindorfer, Smruthi Vijayaraghavan, Tsun-Wen Yao, Sylvie Laquerre, Sheri Moores. Amivantamab efficacy in wildtype EGFR NSCLC tumors correlates with levels of ligand expression [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 1914.

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.

Biophysical and structural characterization of the impacts of MET phosphorylation on tepotinib binding

The receptor tyrosine kinase MET is activated by hepatocyte growth factor binding, followed by phosphorylation of the intracellular kinase domain (KD) mainly within the activation loop (A-loop) on Y1234 and Y1235. Dysregulation of MET can lead to both tumor growth and metastatic progression of cancer cells. Tepotinib is a highly selective, potent type Ib MET inhibitor and approved for treatment of non-small cell lung cancer harboring METex14 skipping alterations. Tepotinib binds to the ATP site of unphosphorylated MET with critical π-stacking contacts to Y1230 of the A-loop, resulting in a high residence time. In our study, we combined protein crystallography, biophysical methods (surface plasmon resonance, differential scanning fluorimetry), and mass spectrometry to clarify the impacts of A-loop conformation on tepotinib binding using different recombinant MET KD protein variants. We solved the first crystal structures of MET mutants Y1235D, Y1234E/1235E, and F1200I in complex with tepotinib. Our biophysical and structural data indicated a linkage between reduced residence times for tepotinib and modulation of A-loop conformation either by mutation (Y1235D), by affecting the overall Y1234/Y1235 phosphorylation status (L1195V and F1200I) or by disturbing critical π-stacking interactions with tepotinib (Y1230C). We corroborated these data with target engagement studies by fluorescence cross-correlation spectroscopy using KD constructs in cell lysates or full-length receptors from solubilized cellular membranes as WT or activated mutants (Y1235D and Y1234E/1235E). Collectively, our results provide further insight into the MET A-loop structural determinants that affect the binding of the selective inhibitor tepotinib.

43154 Prognostic and predictive value of MACC1 and the Met receptor tyrosine kinase in cutaneous melanoma

43154 Prognostic and predictive value of MACC1 and the Met receptor tyrosine kinase in cutaneous melanoma

Circular RNA encoded MET variant promotes glioblastoma tumorigenesis.

Activated by its single ligand, hepatocyte growth factor (HGF), the receptor tyrosine kinase MET is pivotal in promoting glioblastoma (GBM) stem cell self-renewal, invasiveness and tumorigenicity. Nevertheless, HGF/MET-targeted therapy has shown limited clinical benefits in GBM patients, suggesting hidden mechanisms of MET signalling in GBM. Here, we show that circular MET RNA (circMET) encodes a 404-amino-acid MET variant (MET404) facilitated by the N6-methyladenosine (m6A) reader YTHDF2. Genetic ablation of circMET inhibits MET404 expression in mice and attenuates MET signalling. Conversely, MET404 knock-in (KI) plus P53 knock-out (KO) in mouse astrocytes initiates GBM tumorigenesis and shortens the overall survival. MET404 directly interacts with the MET β subunit and forms a constitutively activated MET receptor whose activity does not require HGF stimulation. High MET404 expression predicts poor prognosis in GBM patients, indicating its clinical relevance. Targeting MET404 through a neutralizing antibody or genetic ablation reduces GBM tumorigenicity in vitro and in vivo, and combinatorial benefits are obtained with the addition of a traditional MET inhibitor. Overall, we identify a MET variant that promotes GBM tumorigenicity, offering a potential therapeutic strategy for GBM patients, especially those with MET hyperactivation.

Genomic comparison of MET exon 14 skipping and MET amplified non-small cell lung cancer.

8553 Background: In non-small cell lung cancer (NSCLC), the MET tyrosine kinase receptor can be dysregulated by mutations and/or gene amplification. The most common MET mutation is in exon 14 (METex14), leading to impaired receptor degradation and increased MET-mediated signaling causing sustained tumor proliferation. MET amplification also leads to continued MET signaling and oncogenesis and can be a mechanism of resistance to targeted therapy. We sought to compare the genomic landscape of METex14 and high-level MET amplified tumors, both of which can be targeted with tyrosine kinase inhibitors. Methods: We analyzed 18,047 NSCLC tumors (any stage/subtype) sequenced with Tempus xT assay (DNA-seq of 648 genes at 500x coverage, full transcriptome RNA-seq). Tumors were queried for METex14 mutations, high MET amplification (METamp), defined as copy number gain (CNG) ≥10, and other MET mutations (METother). Immuno-oncology (IO) biomarkers were compared across MET-altered and MET wild-type (METwt) groups. The prevalence of somatic gene alterations was compared similarly with the false-discovery rate (FDR) adjustment. Results: A total of 276 (1.53%) METex14, 138 (0.76%) METamp, 27 (0.15%) METother, and 17,606 (97.56%) METwt tumors were identified. Across groups, patients with METex14 were older, more likely to be female, and nonsmokers. METex14 exhibited the lowest tumor mutational burden (TMB) and lowest neoantigen tumor burden (NTB). PD-L1 positivity rates were higher in METex14 tumors compared to METamp and METwt tumors. MET log10 gene expression was highest in METamp. METamp exhibited the lowest proportion of CD4 T cells and the highest proportion of NK cells. Compared to METex14, METamp exhibited increased prevalence of TP53 (83% vs 37%), TFEC (46% vs 1.1%), CFTR (37% vs 1.4%), EGFR (31% vs. 7.6%), WNT2 (11% vs 0.4%), KEAP1 (14% vs 0.7%), and STK11 (8% vs 1.4%), and decreased prevalence of MDM2 (4.3% vs. 14%), and FRS2 (2.9% vs. 12%). Conclusions: In this large population-based analysis of MET-altered NSCLC, METex14 tumors exhibited differences in IO biomarkers and the somatic landscape compared to non-METex14 NSCLC tumors. Variations in immune profiles may affect immunotherapy selection in MET-altered NSCLC and require further exploration. [Table: see text]

Abstract 5745: Proposing the best MET inhibitor to improve anti-PD-1 efficacy in HCC

Abstract Despite recently approved immunotherapy combinations, Hepatocellular carcinoma (HCC) remains among the most therapeutically intractable cancers with a 5-year survival rate of only 18%. Underlying chronic liver disease due to hepatitis B/C infection, alcohol abuse, hemochromatosis, or nonalcoholic steatohepatitis promotes HCC development. Current therapeutic strategies include surgery, radiotherapy and multikinase inhibitors alone or with immune-checkpoint blockade (ICB). In particular, the receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF) are known drivers of HCC through promotion of tumor growth, survival, tissue invasion, and angiogenesis. Previously, our collaborators showed that the limited clinical benefit of MET kinase inhibition was partially a result of induced upregulation PD-L1 leading to local T cell suppression. We hypothesized that combining MET inhibition with blockade of the PD-1 immune checkpoint would overcome the limitations of each individual approach and act synergistically to promote HCC tumor regression. We first validated that both MET selective (Capmatinib and Tivantinib) and non-selective (Cabozantinib) inhibitors all induced PD-L1 on HCC cell lines in vitro. Next, we compared Capmatinib and Cabozantinib alone and with PD-1 ICB in vivo in the ICB-sensitive Hepa1-6 and HCA-1 models of HCC. In HCA-1, statistically significant benefit of the MET inhibitor and αPD-1 combination was evident in limitation of tumor growth and extension of survival. In each case, the Capmatinib combination trended toward better outcomes with PD-1 blockade. Mechanistically, the Capmatinib and αPD-1 combination decreased tumor Treg cells while promoting increased accumulation of activated, non-exhausted, proliferating CD8 T cells. Central memory CD8 T cell frequencies were increased by the combination during the effector phase, and combination treated animals were better protected against subsequent rechallenge than those cured by αPD-1 alone. In the myeloid compartment, M1 macrophage frequencies were increased while PMN-MDSC both decreased in frequency and in Arginase 1 levels. Finally, we studied a serially-passaged, αPD-1 refractory DEN HCC model. Despite lack of efficacy of either component therapy, the MET inhibitor and αPD-1 combination significantly extended survival and inhibited tumor growth. As in the αPD-1 sensitive setting, Capmatinib appeared to offer the most therapeutic benefit with αPD-1. Ongoing studies are focused on characterizing changes mediated by the combination therapy which confer aPD-1 sensitivity to the otherwise refractory DEN HCC tumor microenvironment. Molecular studies are focused on understanding the superior ICB potentiating capacity of Type I MET inhibitors versus the other classes. Given the clinical availability of numerous MET and PDL-1 inhibitors, we hope to inform near-term optimization of MET and αPD-1 clinical trial design for HCC. Citation Format: Ricardo A. de Azevedo, Broderick Turner, Priyamvada Jayaprakash, Krithikaa Rajkumar Bhanu, Anupallavi Srinivasamani, Brittany Morrow, Michelle Winkler, Shweta Mahendra Hedge, Arthur Liu, Ravaen Slay, Michael A. Curran. Proposing the best MET inhibitor to improve anti-PD-1 efficacy in HCC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5745.

Blocking the HGF-MET pathway induces resolution of neutrophilic inflammation by promoting neutrophil apoptosis and efferocytosis

Inflammation resolution is an active process that involves cellular events such as apoptosis and efferocytosis, which are key steps in the restoration of tissue homeostasis. Hepatocyte growth factor (HGF) is a growth factor mostly produced by mesenchymal-origin cells and has been described to act via MET receptor tyrosine kinase. The HGF/MET axis is essential for determining the progression and severity of inflammatory and immune-mediated disorders. Here, we investigated the effect of blocking the HGF/MET signalling pathway by PF-04217903 on the resolution of established models of neutrophilic inflammation. In a self-resolving model of gout induced by MSU crystals, HGF expression on periarticular tissue peaked at 12 h, the same time point that neutrophils reach their maximal accumulation in the joints. The HGF/MET axis was activated in this model, as demonstrated by increased levels of MET phosphorylation in neutrophils (Ly6G+ cells). In addition, the number of neutrophils was reduced in the knee exudate after PF-04217903 treatment, an effect accompanied by increased neutrophil apoptosis and efferocytosis and enhanced expression of Annexin A1, a key molecule for inflammation resolution. Reduced MPO activity, IL-1β and CXCL1 levels were also observed in periarticular tissue. Importantly, PF-04217903 reduced the histopathological score and hypernociceptive response. Similar findings were obtained in LPS-induced neutrophilic pleurisy. In human neutrophils, the combined use of LPS and HGF increased MET phosphorylation and provided a prosurvival signal, whereas blocking MET with PF-04217903 induced caspase-dependent neutrophil apoptosis. Taken together, these data demonstrate that blocking HGF/MET signalling may be a potential therapeutic strategy for inducing the resolution of neutrophilic inflammatory responses.

Abstract 655: Macrophage mediated resistance to TKI therapy in ALK fusion positive non-small cell lung cancer

Abstract Patients with lung cancer bearing driver oncogenes such as epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) fusions, often experience long term disease control on targeted therapy. Despite excellent initial responses, ultimately these tumors develop resistance to targeted therapy. While several cancer cell intrinsic mechanisms of targeted therapy resistance are well known, they do not account for all cases of resistance. Based on our previous observations, we hypothesized components of the tumor microenvironment, specifically monocytes and macrophages, also contribute to the development of targeted therapy resistance. We generated bone marrow derived macrophages from C57BL/6 mice and polarized them to an M0, M1, or M2 phenotype over the course of 6 days. At day 7, cells were plated in fresh media and after 2 days, the conditioned media (CM) was collected. Macrophage polarization was verified via qPCR. Murine cell lines, EA-1, EA-2, and EA-3 are unique, murine lung adenocarcinomas bearing an oncogenic ALK fusion derived from C57BL/6 mice. These murine cell lines were then cultured in the macrophage CM prior to exposure to ALK fusion specific tyrosine kinase inhibitors (TKI), alectinib or crizotinib. We observed that culture in M2-CM resulted in at least a 10-fold increase in alectinib resistance across all 3 murine cell lines when compared to the same cell lines cultured in M1 or M0-CM. We next tested for the activity of known, clinically relevant bypass signaling pathways as mediator of M2-CM protection. Aftatinib, an inhibitor of EGFR and human epidermal growth factor receptor 2 (HER2) family of proteins, did not alter M2-CM protection in the presence of alectinib. M2-CM did not impart TKI resistance to the murine cell lines in the presence of crizotinib, a TKI which inhibits ALK fusion activity and MET receptor tyrosine kinase activity, suggesting bypass signaling through MET drives the M2-CM resistance. Ongoing animal model studies demonstrate a clear role for innate immune cells in decreasing tumor responsiveness to TKI. In summary, our work demonstrates TKI therapy may be modulated by the presence of and interaction with specific immune cells within the tumor microenvironment. These data support exploring macrophage targeting therapies to improve TKI responsiveness. Citation Format: Melanie Mandell, Katherine Priest, Anh Le, Lynn Heasley, Raphael Nemenoff, Erin L. Schenk. Macrophage mediated resistance to TKI therapy in ALK fusion positive non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 655.

Investigating Susceptibility of ꞵ‐catenin‐mutated Hepatocellular Carcinoma to Checkpoint Inhibitors

Hepatocellular Carcinoma (HCC) is the 5th most common cause of cancer‐related death in the United States with an estimated 32,000 annual deaths. The standard of care for HCC as established by the IMBrave150 trial is a combination of the PD‐L1 inhibitor, Atezolizumab, and an antiangiogenic, Bevacizumab. While this combination extends overall survival to 19.2 months in unresectable HCC, only 52% of patients survive to 18 months. Furthermore, objective response rates are around 32% and hence clarifying underlying mechanisms regulating the immune response in HCC oncogenesis might strengthen immunotherapy efficacy. Especially important is to identify response rates of these agents in specific molecular subclasses of HCC may help in better patient selection. The oncogene ꞵ‐catenin is consistently mutated and activated among 30% of HCC patients. The Wnt/ꞵ‐catenin pathway has been approached as a target for precision medicine in HCC. We recently modeled HCCs with ꞵ‐catenin mutations in mice up to 69% genetic similarity. Here, we combine hydrodynamic tail vein injections with the Sleeping Beauty Transposase which is a non‐viral method of expressing genes of interest. Our model co‐expresses an S45Y point mutant of ꞵ‐catenin with the tyrosine kinase receptor MET (B+M). This combination represents ~10% of patient HCCs and provides a clinically relevant tool for the investigation of personalized HCC therapeutics.A recent study demonstrated HCCs driven by a Δ90‐truncation mutant of ꞵ‐catenin in combination with the Myc oncogene are shown to lack sufficient immune cell surveillance. These HCCs have poor cytotoxic T cell and dendritic cell recruitment while responding poorly to checkpoint inhibitors. Since activating point mutations in CTNNB1 gene are common in patient HCCs, we asked whether poor immune surveillance extends to HCC models harboring point mutations. We first checked and found the presence of CD45+, F4/80+, and CD11b+ cells both 2 and 4 weeks after B+M tumor induction. This indicates the presence of both adaptive and innate immune cells in the B+M tumor microenvironment. We next asked whether checkpoint inhibition will effectively reduce tumor burden in the B+M model. Therefore, we treated B+M mice with either IgG or anti‐PD1 monoclonal antibodies 4 weeks after tumor induction. Tissues were harvested 3.5 weeks after treatment. We found a modest 20% reduction in liver weight to body weight ratio, a measure of tumor burden, in B+M mice treated with anti‐PD1 compared to IgG controls. Our results demonstrate that HCCs with missense ꞵ‐catenin mutations do respond albeit marginally to checkpoint inhibitors. In the future, combination of checkpoint inhibitors with other inhibitors will be tested in these models to determine their efficacy in treating ꞵ‐catenin‐mutated HCCs.

Deletion of Neuropilin‐1 in the Epidermis Delays Wound Healing

When cutaneous tissue is exposed to trauma it undergoes complex biological processes to achieve healing. Our goal is to better understand the cellular pathways and molecular mechanisms that control wound healing. Upon injury to the skin, cells in the epidermis and dermis secrete a number of cytokines and growth factors to promote cell survival, proliferation, and migration. Hepatocyte growth factor (HGF) is a protein secreted from fibroblasts that has been shown to function in several different stages of wound healing, including re‐epithelialization. HGF and its tyrosine kinase receptor MET are upregulated in various phases of the healing cascade such as in the wound epidermis as well as in granulation tissue and blood vessels. Previous studies have shown that the transmembrane glycoprotein Neuropilin‐1 (Nrp1) acts as a co‐receptor for HGF (in addition to VEGF) in endothelial cells, while our laboratory has shown that Nrp1 is constitutively expressed in keratinocytes and its expression is up‐regulated following stress (such as after UV‐irradiation). However, the role of Nrp1 in epithelial cells or in the wound healing process, and its contribution to epidermal HGF/MET signaling is unclear. We hypothesized that Nrp1 cooperates with MET to signal via HGF to promote epithelial proliferation and improve wound healing. Adult male and female transgenic mice (K14‐creERT;Nrp1f/f) were either untreated (Controls; n=10 mice/group) or treated with tamoxifen to delete Nrp1 in keratinocytes (K14‐iNrp1‐KO; n=10 mice/group). Two weeks later, mice received full‐thickness dermal punch biopsies on their shaved dorsum. Wounds were measured daily and wound areas were graphed and compared. At select time points, additional wounded mice were euthanized and tissues were embedded for histologic examination. Our results indicate that under basal conditions, Nrp1 deletion in the epidermis does not affect the number or differentiation status of keratinocytes as K14‐iNrp1‐KO mice skin was equal in thickness and morphology to control littermates. However, upon injury, wound closure rates were significantly slower in male K14‐iNrp1‐KO mice compared to female K14‐iNrp1‐KO mice or Controls. In summary, absence of Nrp1 in the epidermis causes delays in wound healing indicating that Nrp1 plays a significant role in re‐epithelialization. Ongoing studies are investigating the activation of MET by HGF following Nrp1 silencing in epithelial cells to decipher the co‐receptor dependency of MET on Nrp1. These findings may lead to an improved understanding of the wound healing process and suggest targets for future drug development.

The Occurrence of MET Ectodomain Shedding in Oral Cancer and Its Potential Impact on the Use of Targeted Therapies.

Simple SummaryHead and neck cancer is the sixth most common cancer type worldwide, comprising tumors of the upper aero/digestive tract. Approximately 50% of these cancers originate in the oral cavity. Depending on disease stage, oral cancer patients are treated with single-modality surgery, or in combination with radiotherapy with or without chemotherapy. Despite advances in these modalities, the 5-year survival rate is merely 50%. Therefore, implementation of targeted therapies, directed against signaling molecules, has gained attention. One potential target is the MET protein, which can be present on the surface of cancer cells, orchestrating aggressive behavior. As cancer cells can shed the extracellular part of MET from their surface, it is important to identify for MET positive patients whether they possess the entire and/or only the intracellular part of the receptor to assess whether targeted therapies directed against the extracellular, intracellular, or both parts of MET need to be implemented.The receptor tyrosine kinase MET has gained attention as a therapeutic target. Although MET immunoreactivity is associated with progressive disease, use of targeted therapies has not yet led to major survival benefits. A possible explanation is the lack of companion diagnostics (CDx) that account for proteolytic processing. During presenilin-regulated intramembrane proteolysis, MET’s ectodomain is shed into the extracellular space, which is followed by γ-secretase-mediated cleavage of the residual membranous C-terminal fragment. The resulting intracellular fragment is degraded by the proteasome, leading to downregulation of MET signaling. Conversely, a membrane-bound MET fragment lacking the ectodomain (MET-EC-) can confer malignant potential. Use of C- and N-terminal MET monoclonal antibodies (moAbs) has illustrated that MET-EC- occurs in transmembranous C-terminal MET-positive oral squamous cell carcinoma (OSCC). Here, we propose that ectodomain shedding, resulting from G-protein-coupled receptor transactivation of epidermal growth factor receptor signaling, and/or overexpression of ADAM10/17 and/or MET, stabilizes and possibly activates MET-EC- in OSCC. As MET-EC- is associated with poor prognosis in OSCC, it potentially has impact on the use of targeted therapies. Therefore, MET-EC- should be incorporated in the design of CDx to improve patient stratification and ultimately prolong survival. Hence, MET-EC- requires further investigation seen its oncogenic and predictive properties.

The Role of Decorin Proteoglycan in Mitophagy.

Simple SummaryThe eminent rise of extracellular matrix constituents, chiefly hailing from the proteoglycan gene family, has revolutionized our understanding of how intracellular catabolism is regulated at the intersection of autophagy and breast cancer. In this review, we examine the mechanisms of decorin, a small leucine-rich proteoglycan, as it relates to autophagy and mitochondrial autophagy (mitophagy). In each case, decorin signals via a unique cell surface receptor tyrosine kinase to evoke autophagy (VEGFR2) or mitophagy (MET receptor) that converges on a novel tumor suppressor gene. The downstream function of either Peg3 or mitostatin in response to decorin manifests as potent means to subdue breast cancer development and progression.Proteoglycans are emerging as critical regulators of intracellular catabolism. This rise in prominence has transformed our basic understanding and alerted us to the existence of non-canonical pathways, independent of nutrient deprivation, that potently control the autophagy downstream of a cell surface receptor. As a member of the small leucine-rich proteoglycan gene family, decorin has single-handedly pioneered the connection between extracellular matrix signaling and autophagy regulation. Soluble decorin evokes protracted endothelial cell autophagy via Peg3 and breast carcinoma cell mitophagy via mitostatin by interacting with VEGFR2 or the MET receptor tyrosine kinase, respectively. In this paper, we give a mechanistic perspective of the vital factors underlying the nutrient-independent, SLRP-dependent programs utilized for autophagic and/or mitophagic progression in breast cancer. Future protein therapies based on decorin (or fellow proteoglycan members) will represent a quantum leap forward in transforming autophagic progression into a powerful tool to control intracellular cell catabolism from the outside.

Abstract 1387: MET inhibition radiosensitizes KRAS-mutant rectal cancer

Abstract Rectal carcinoma, representing about a third of all newly diagnosed colorectal cancers, is one of the most common malignant tumors. The standard of care for locally advanced rectal cancer (LARC), consisting of neoadjuvant chemo/radiotherapy prior to surgical resection, is poorly effective, leading to complete tumor regression only in 10-30% of the cases. Approximately 40% of LARC harbor activating KRAS mutations, which have been extensively associated with primary resistance to targeted EGFR therapy and with radioresistance as well. Previous work showed that the MET receptor tyrosine kinase supports radioresistance and can be inhibited to radiosensitize tumor cell subpopulations retaining stem-like properties. Here, we show that LARC often express high levels of MET and can be successfully radiosensitized by MET inhibition. This was assessed in rectal stem-like cells isolated from human tumors (rectospheres), and transplanted in the mouse to regenerate tumors that faithfully reproduce the phenotype, the genotype and the therapeutic response of the original tumor. Mechanistically, we found that radioresistant KRAS-mutant rectospheres display significantly higher basal levels of RAD51, a master regulator of DNA homologous recombination repair, and increased RAD51 recruitment to irradiation-induced DNA double-strand breaks, as compared with KRAS-wild type rectospheres. Importantly, we showed that MET pharmacological inhibition by small-molecule kinase inhibitors, combined with radiotherapy, impairs RAD51 expression and function. This leads to DNA damage accumulation and results in effective radiosensitization of K-RAS-mutant rectal stem-like cells in vitro and in vivo, and significant inhibition of experimental tumors. Therefore, preclinical evidence is provided that MET can be exploited as a therapeutic target to radiosensitize KRAS-mutant rectal cancer at stem-like cell level. Citation Format: Antonio D'Ambrosio, Federica Verginelli, Francesca Orzan, Raffaella Albano, Elena Casanova, Paolo Luraghi, Francesca De Bacco, Andrea Bertotti, Livio Trusolino, Rebecca Senetta, Anna Sapino, Elena Del Mastro, Marco Gatti, Paolo M. Comoglio, Carla Boccaccio. MET inhibition radiosensitizes KRAS-mutant rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1387.

State of the structure address on MET receptor activation by HGF.

The MET receptor tyrosine kinase (RTK) and its cognate ligand hepatocyte growth factor (HGF) comprise a signaling axis essential for development, wound healing and tissue homeostasis. Aberrant HGF/MET signaling is a driver of many cancers and contributes to drug resistance to several approved therapeutics targeting other RTKs, making MET itself an important drug target. In RTKs, homeostatic receptor signaling is dependent on autoinhibition in the absence of ligand binding and orchestrated set of conformational changes induced by ligand-mediated receptor dimerization that result in activation of the intracellular kinase domains. A fundamental understanding of these mechanisms in the MET receptor remains incomplete, despite decades of research. This is due in part to the complex structure of the HGF ligand, which remains unknown in its full-length form, and a lack of high-resolution structures of the complete MET extracellular portion in an apo or ligand-bound state. A current view of HGF-dependent MET activation has evolved from biochemical and structural studies of HGF and MET fragments and here we review what these findings have thus far revealed.

TAMI-32. TEMPOROSPATIAL INVASION AND GENETIC EVOLUTION FROM INFRATENTORIAL TO SUPRATENTORIAL COMPARTMENT IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas (DMGs) are very aggressive pediatric brain tumors with dismal prognosis due to therapy-resistant tumor growth and invasion. We performed the first integrated histologic/genomic/proteomic analysis of 21 tumor foci from three pontine DMG cases with supratentorial dissemination. Histone H3.3 K27M was the driver mutation, usually at high variant allele fraction due to recurrent chromosome 1q copy number gain, in combination with germline variants in ATM, FANCM and MYCN genes. Both previously reported and novel recurrent copy number variations and somatic pathogenic mutations in chromatin remodeling, DNA damage response and PI3K/MAPK growth pathways were variably detected, either in multiple or isolated foci. Proteomic analysis showed global upregulation of histone H3, lack of K27 tri-methylation, and further impairment of polycomb repressive complex 2 by ASXL1 downregulation. Activation of oncogenic pathways resulted from combined upregulation of N-Myc, SOX2, p65/p50 NF-kB and STAT3 transcription factors, EGFR, FGFR2, PDGFRa/b and MET receptor tyrosine kinases, and downregulation of PHLPP1/2, PTEN and p16/INK4A tumor suppressors. Upregulation of SMAD4, PAI-1, CD44, and c-Src in multiple foci most likely contributed to invasiveness. This integrated comprehensive analysis allowed spatiotemporal modeling of tumor progression and identified two general pathways of supratentorial invasion, and a multitude of migratory subpopulations within the infratentorial compartment. It also delineated common signaling pathways and potential therapeutic targets, revealing an unsuspected activation of a multitude of oncogenic pathways that may explain the resistance of DMG to current therapies.

Abstract 6082: Uveal melanoma exosomes express MET and re-program the recipient cells

Abstract Uveal melanoma (UM) is a rare melanoma subtype with an estimated annual incidence of approximately 2000 in the United States. Despite excellent rates of local disease control with surgery or radiotherapy, nearly 50% of patients will develop metastatic disease, mostly to the liver, with a life expectancy of 12 months. The receptor tyrosine kinase MET is overexpressed in 60-86% of cases and, together with its ligand HGF, it plays a significant role in UM migration and metastasis. In recent years, it has been demonstrated that tumor cells communicate with the microenvironment at distant organs by secreting exosomes, extracellular small vesicles released into body fluids, to support cancer development, immune evasion and metastatic progression. Exosomes are able to stably transfer their unique cargos of proteins and RNA from the primary tumor to distant sites, such as the pre-metastatic “niche”. The functional role of UM exosomes in the tumor microenvironment cross-talk is largely unknown. To get deeper insight into UM exosome biology, we have isolated exosomes from UM cells before and after treatment with the MET inhibitor crizotinib and examined their content by proteomic analysis. Approximately 600 proteins were identified from each of these samples, with 444 proteins in common and 145 and 167 proteins uniquely detected in exosomes from untreated and treated cells, respectively. The expression of several exosome-associated proteins, including MET, were suppressed by crizotinib, suggesting that the exosomal cargo can be pharmacologically manipulated. Exposure of normal hepatocytes to UM-derived exosomes results in activation of MET in a dose dependent fashion, with induction of both the MAPK and AKT/PI3K pathways. Exosomes derived from serum of patients with advanced uveal melanoma also express MET and induced similar effects in hepatocytes. Furthermore, UM-derived exosomes activate the expression of pro-angiogenic and pro-inflammatory factors in normal hepatocytes, while exosomes isolated from crizotinib-treated UM cells reduced these effects. UM-derived exosomes also stimulated immune cells to release cytokines and chemokines, including IL-6, IL-8 CCL2, CCL3, CCL5 and CXCL1. Functionally, we demonstrated that conditioned media from exosome-treated hepatocyte highly stimulated UM cell migration, which could be repressed by the combination of MET and cytokine receptor inhibitors. These findings provide evidence that UM derived exosomes play a role in reprogramming the recipient cells to facilitate the establishment of metastasis, and the inhibition of exosome-mediated signaling may provide a potential therapeutic strategy in the early stages of metastasis. Citation Format: Grazia Ambrosini, Alex J. Rai, Blake Ebert, Gary K. Schwartz. Uveal melanoma exosomes express MET and re-program the recipient cells [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 6082.