Introduction: The receptor tyrosine kinases (RTKs) c-MET and RON and more importantly their crosstalk, play a crucial role in mediating local invasion, systemic dissemination and resistance in different types of cancer. Both RTKs are activated by the ligands HGF and MSP respectively and homo- or heterodimerize with each other. The RTK VEGFR-2 is the most important receptor for angiogenesis in solid epithelial tumors. CD44v6, a member of the CD44 family of transmembrane glycoproteins, was identified as an essential co-receptor for activation of c-MET, RON and VEGFR-2. AMC303, a peptide based allosteric and selective inhibitor of CD44v6, was investigated for its inhibitory effects on c-MET, RON and VEGFR-2 pathways in vitro and its effect on tumor growth and metastases in vivo. Methods: Affinity was determined using Microscale Thermophoresis. For in vitro blocking assays AMC303 was added 30 min prior to induction with the ligands. Analysis of RTK activation was carried out by western blot. Analysis of cell migration and invasion was performed with Boyden Chamber assays. VEGF-A induced tube formation was microscopically analyzed. For in vivo studies nude mice were orthotopically implanted with human tumor cells (L3.6pl). Animals were treated with AMC303 i.v. at 0.1, 1, 10 mg/kg QOD or QWK for 3 weeks. Regression of metastases was investigated at 1 mg/kg QOD. HPLC was used to detect the amount of AMC303 in the primary tumor lysates. Results: AMC303 binds to the ectodomain of CD44v6 with high affinity. In various pancreatic, breast, colon, lung and HNSCC tumor cell lines activation of c-MET and RON by their ligands was inhibited by AMC303 in vitro and consequently ligand induced cell scattering, migration and invasion was significantly reduced. In endothelial cells, activation of VEGFR-2 and VEGF-A induced formation of a tubular network was blocked by AMC303 treatment. In vivo treatment with AMC303 inhibited tumor growth in a dose dependent manner. The metastatic spreading of the primary tumor was prevented when animals were treated at early disease stage. Most strikingly, a marked regression of established liver metastases was observed at progressed disease state when animals were treated with AMC303 at 1 mg/kg QOD for 3 weeks. Conclusions: AMC303 inhibits activation of the RTKs c-MET and RON allosterically in different epithelial tumor cells and VEGFR-2 in endothelial cells by extracellular binding to CD44v6. This unique and novel mode of action results in a strong anti-tumor and anti-metastatic effect in vivo which together with its wide safety and tolerability window in preclinical toxicity studies is strongly supporting the clinical investigation. AMC303 is currently tested in a Phase I study in patients with solid epithelial tumors. Citation Format: Vanessa Al-Rawi, Thorsten Laeufer, Katrin Glocker, Yvonne Heneka, Alexandra Matzke-Ogi. Allosteric inhibition of the Receptor Tyrosine Kinases c-MET, RON and VEGFR-2 via the co-receptor CD44v6 by the novel compound AMC303 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4911. doi:10.1158/1538-7445.AM2017-4911

The tumorigenicity of cancer cells is highly influenced by the extracellular matrix (ECM) through mechanisms that are poorly understood. Here it is reported that a variety of 3D ECM microenvironments strongly induce expression of Id1 and Id3 in melanoma cells. Genetic ablation of Id1/Id3 impairs melanoma cell outgrowth in 3D Matrigel culture and inhibits melanoma initiation in vivo. Mechanistically, 3D ECM microenvironments hinder diffusion of endogenously produced bone morphogenetic proteins, thereby fostering autocrine signaling and Id1/Id3 expression. A compound screen identifies new coumarin derivatives that potently inhibit both Id1/Id3 expression and melanoma initiation in vivo. Together, the findings reveal a novel mechanism through which the ECM increases tumorigenicity, identify Id1/Id3 as melanoma-relevant therapeutic targets, and characterize inhibitors of Id1/Id3 expression with therapeutic potential.

Introduction: CD44v6, a member of the CD44 family, is an essential co-receptor for c-MET, RON and VEGFR-2. The receptor tyrosine kinases (RTKs) c-MET and RON, are key players in tumorigenic pathways, are activated by the ligands HGF and MSP, respectively and homo- or heterodimerize with each other or can be autoactivated due to amplification. The RTK VEGFR-2 is critical for angiogenesis in solid epithelial tumors. The peptidergic inhibitor AMC303, which allosterically and selectively binds to CD44v6, was investigated for its inhibitory effects on ligand-induced and autophosphorylated c-MET activation, and its impact on tumor growth and metastases in vivo. Methods: In vitro, tumor cells were incubated with AMC303 for 30 min prior to induction with an RTK-ligand. Analysis of RTK activation was carried out by Western blotting. For in vivo xenotransplantation human pancreatic tumor cells (L3.6pl) were orthotopically injected. Animals were treated with AMC303 i.p. at 0.1, 1, 10 mg/kg QOD or QWK for 3 weeks to address dose-dependency, the optimal treatment schedule was investigated with one, three or five times weekly administrations. Regression of metastases was investigated at 1 mg/kg QOD. Survival was evaluated with a dose of 1 mg/kg QOD. Immunohistochemical (IHC) analysis was performed for cleaved caspase 6, smooth muscle actin and CD31. Reduction of vessel permeability was assessed in Balb/C mice with the Miles assay and quantification of intra-tumoral FITC dextran after i.v. injection. Results: In vitro, AMC303 inhibited c-MET, VEGFR2 and RON phosphorylation induced by their respective ligands. Notably, AMC303 inhibited c-MET activation in cells with enhanced c-MET signaling caused by the exon 14 skipping mutation, but not c-MET auto-phosphorylation due to gene amplification. In vivo, AMC303 treatment attenuated primary xenograft tumor growth and metastasis and significantly prolonged survival. IHC on treated xenograft-tumors revealed an increase in apoptosis and necrosis along with a reduction in myofibroblast infiltration, angiogenesis and vessel permeability. Pharmacokinetic properties after i.p. administration of AMC303 revealed an terminal half-life of 6h. Administration schedules of AMC303 (1mg/kg) given one, three or five times per week had no significant impact on tumor growth attenuation. AMC303 treatment could be paused for 10 days without incurring significant tumor regrowth. Conclusions: AMC303 inhibits c-MET in vitro in a ligand-dependent fashion and in vivo significantly improved overall survival in an orthotopic pancreatic xenograft mouse tumor model. AMC303 effectively attenuated both primary and metastatic tumor growth by increasing apoptosis, reduction of angiogenesis and vascular normalization. These findings demonstrate the benefit of targeting multiple oncogenic RTK signaling pathways by AMC303. Citation Format: Tina Heumann, Vanessa Al-Rawi, Thorsten Läufer, Martin Augsten, Alexandra Matzke-Ogi, Oliver Coutelle. AMC303 inhibits tumor growth and metastasis in animal models by targeting CD44v6, a co-receptor of multiple oncogenic receptor tyrosine kinases [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 4855.