Cristina Basilico

Mutant Met-mediated transformation is ligand-dependent and can be inhibited by HGF antagonists

Mutations in the genes encoding for Met, Ret and Kit receptor tyrosine kinases invariably result in increased kinase activity and in the acquisition of transforming potential. However, the requirement of receptor ligands for the transformation process is still unclear. We have investigated the role of hepatocyte growth factor (HGF), the high-affinity ligand for Met, in mutant Met-mediated cell transformation. We provide evidence that the transforming potential displayed by mutant forms of Met found in human cancer is not only sensitive but entirely dependent on the presence of HGF, by showing that mutant Met transforms NIH3T3 fibroblasts, which produce endogenous HGF, but is not able to transform epithelial cells, unless exogenous HGF is supplied. Accordingly, mutant Met-induced transformation of NIH3T3 cells can be inhibited by HGF antagonists and increased by HGF stimulation. We also show that an engineered Met receptor which contains an oncogenic mutation but is impaired in its ability to bind HGF completely loses its transforming activity, which can be rescued by causing receptor dimerization using a monoclonal antibody. These results indicate that point mutations resulting in Met kinase activation are necessary but not sufficient to cause cell transformation, the latter being dependent on ligand-induced receptor dimerization. They also suggest that mutant Met-driven tumour growth depends on the availability and tissue distribution of active HGF, and provide proof-of-concept for the treatment of mutant-Met related pathologies by HGF-antagonizing drugs.

A High Affinity Hepatocyte Growth Factor-binding Site in the Immunoglobulin-like Region of Met

Hepatocyte growth factor (HGF) and its high affinity receptor, the tyrosine kinase Met, play a key role in embryo development and tumor invasion. Both HGF and Met are established targets for cancer therapy. However, the mechanism of their interaction is complex and remains elusive. HGF is secreted as a monomeric precursor (pro-HGF) that binds to but does not activate Met. Mature HGF is a α/β heterodimer containing a high affinity Met-binding site in the α-chain (HGF-α) and a low affinity Met-binding site in the β-chain (HGF-β). The extracellular portion of Met contains a semaphorin (Sema) domain, a cysteine-rich hinge (plexin-semaphorin-integrin), and four immunoglobulin-like domains (immunoglobulin-like regions in plexins and transcription factors (IPT) 1-4). HGF-β binds to Sema through a low affinity contact. The domain of Met responsible for high affinity binding to HGF-α has not been identified yet. Here we show that this long sought after binding site lies in the immunoglobulin-like region of Met and more precisely in IPT 3 and 4. We also show that IPT 3 and 4 are sufficient to transmit the signal for kinase activation to the cytoplasm, although the lack of Sema makes the receptor equally sensitive to mature HGF and pro-HGF. Finally, we provide evidence that soluble Met-derived proteins containing either the low affinity or high affinity HGF-binding site antagonize HGF-induced invasive growth both in vitro and in xenografts. These data suggest that the immunoglobulin-like region of Met cooperates with the Sema domain in binding to HGF and in controlling Met kinase activity. Although the IPT-HGF-α interaction provides binding strength, the Sema-HGF-β contact confers selective sensitivity to the active form of the ligand.

An uncleavable form of pro–scatter factor suppresses tumor growth and dissemination in mice

Scatter factor (SF), also known as hepatocyte growth factor, is ubiquitously present in the extracellular matrix of tissues in the form of an inactive precursor (pro-SF). In order to acquire biological activity, pro-SF must be cleaved by specific proteases present on the cell surface. The mature form of SF controls invasive cues in both physiological and pathological processes through activation of its receptor, the Met tyrosine kinase. By substituting a single amino acid in the proteolytic site, we engineered an unprocessable form of pro-SF (uncleavable SF). Using lentivirus vector technology, we achieved local or systemic delivery of uncleavable SF in mice. We provide evidence that (a) uncleavable SF inhibits both protease-mediated pro-SF conversion and active SF-induced Met activation; (b) local expression of uncleavable SF in tumors suppresses tumor growth, impairs tumor angiogenesis, and prevents metastatic dissemination; and (c) systemic expression of uncleavable SF dramatically inhibits the growth of transplanted tumors and abolishes the formation of spontaneous metastases without perturbing vital physiological functions. These data show that proteolytic activation of pro-SF is a limiting step in tumor progression, thus suggesting a new strategy for the treatment or prevention of the malignant conversion of neoplastic lesions.

Different point mutations in the met oncogene elicit distinct biological properties

The MET proto‐oncogene, encoding the tyrosine kinase receptor for HGF, controls genetic programs leading to cell growth, invasiveness, and protection from apoptosis. Recently, MET mutations have been identified in hereditary and sporadic forms of papillary renal carcinoma (PRC). Introduction of different naturally occurring mutations into the MET cDNA results in the acquisition of distinct biochemical and biological properties of transfected cells. Some mutations result in a high increase in tyrosine kinase activity and confer transforming ability in focus forming assays. These mutants hyperactivate the Ras signaling pathway. Other mutations are devoid of transforming potential but are effective in inducing protection from apoptosis and sustaining anchorage‐independent growth. These MetPRC receptors interact more efficiently with the intracellular transducer Pi3Kinase. The reported results show that METPRC mutations can be responsible for malignant transformation through different mechanisms, either by increasing the growth ability of cells or by protecting cells from apoptosis and allowing accumulation of other genetic lesions.—Giordano, S., Maffe, A., Williams, T. A., Artigiani, S., Gual, P., Bardelli, A., Basilico, C., Michieli, P., Comoglio, P. M. Different point mutations in the met oncogene elicit distinct biological properties. FASEB J. 14, 401–408 (2000)

An HGF-MSP chimera disassociates the trophic properties of scatter factors from their pro-invasive activity

Hepatocyte growth factor (HGF) and macrophage-stimulating protein (MSP) have an intrinsic dual nature: they are trophic cytokines preventing apoptosis on one side and scatter factors promoting invasion on the other. For therapeutic use, their anti-apoptotic activity must be separated from their pro-invasive activity. To this end, we engineered chimeric factors containing selected functional domains of HGF and/or MSP in different combinations, and tested their biological activity. Here we present a chimeric cytokine derived from the α-chains of HGF and MSP, named Metron factor 1 for its ability to concomitantly activate the HGF receptor (Met) and the MSP receptor (Ron). We provide evidence that Metron factor 1 prevents apoptosis and stimulates cell proliferation at nanomolar concentrations, but is devoid of any pro-invasive activity. In an in vivo murine model of drug-induced nephrotoxicity, intravenous injection of recombinant Metron factor 1 prevented renal damage and preserved tubular integrity.

Magic-factor 1, a partial agonist of Met, induces muscle hypertrophy by protecting myogenic progenitors from apoptosis.

Background Hepatocyte Growth Factor (HGF) is a pleiotropic cytokine of mesenchymal origin that mediates a characteristic array of biological activities including cell proliferation, survival, motility and morphogenesis. Its high affinity receptor, the tyrosine kinase Met, is expressed by a wide range of tissues and can be activated by either paracrine or autocrine stimulation. Adult myogenic precursor cells, the so called satellite cells, express both HGF and Met. Following muscle injury, autocrine HGF-Met stimulation plays a key role in promoting activation and early division of satellite cells, but is shut off in a second phase to allow myogenic differentiation. In culture, HGF stimulation promotes proliferation of muscle precursors thereby inhibiting their differentiation. Methodology/Principal Findings Magic-Factor 1 (Met-Activating Genetically Improved Chimeric Factor-1 or Magic-F1) is an HGF-derived, engineered protein that contains two Met-binding domains repeated in tandem. It has a reduced affinity for Met and, in contrast to HGF it elicits activation of the AKT but not the ERK signaling pathway. As a result, Magic-F1 is not mitogenic but conserves the ability to promote cell survival. Here we show that Magic-F1 protects myogenic precursors against apoptosis, thus increasing their fusion ability and enhancing muscular differentiation. Electrotransfer of Magic-F1 gene into adult mice promoted muscular hypertrophy and decreased myocyte apoptosis. Magic-F1 transgenic mice displayed constitutive muscular hypertrophy, improved running performance and accelerated muscle regeneration following injury. Crossing of Magic-F1 transgenic mice with α-sarcoglycan knock-out mice –a mouse model of muscular dystrophy– or adenovirus-mediated Magic-F1 gene delivery resulted in amelioration of the dystrophic phenotype as measured by both anatomical/histological analysis and functional tests. Conclusions/Significance Because of these features Magic-F1 represents a novel molecular tool to counteract muscle wasting in major muscular diseases such as cachexia or muscular dystrophy.

Four individually druggable MET hotspots mediate HGF-driven tumor progression.

Activation of MET by HGF plays a key role in tumor progression. Using a recently developed llama platform that generates human-like immunoglobulins, we selected 68 different antibodies that compete with HGF for binding to MET. HGF-competing antibodies recognized 4 distinct hotspots localized in different MET domains. We identified 1 hotspot that coincides with the known HGF β chain binding site on blades 2-3 of the SEMA domain β-propeller. We determined that a second and a third hotspot lie within blade 5 of the SEMA domain and IPT domains 2-3, both of which are thought to bind to HGF α chain. Characterization of the fourth hotspot revealed a region across the PSI-IPT 1 domains not previously associated with HGF binding. Individual or combined targeting of these hotspots effectively interrupted HGF/MET signaling in multiple cell-based biochemical and biological assays. Selected antibodies directed against SEMA blades 2-3 and the PSI-IPT 1 region inhibited brain invasion and prolonged survival in a glioblastoma multiforme model, prevented metastatic disease following neoadjuvant therapy in a triple-negative mammary carcinoma model, and suppressed cancer cell dissemination to the liver in a KRAS-mutant metastatic colorectal cancer model. These results identify multiple regions of MET responsible for HGF-mediated tumor progression, unraveling the complexity of HGF-MET interaction, and provide selective molecular tools for targeting MET activity in cancer.

Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy

Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo.

Inhibition of ligand-independent constitutive activation of the Met oncogenic receptor by the engineered chemically-modified antibody DN30

An awesome number of experimental and clinical evidences indicate that constitutive activation of the Met oncogenic receptor plays a critical role in the progression of cancer toward metastasis and/or resistance to targeted therapies. While mutations are rare, the common mechanism of Met activation is overexpression, either by gene amplification (‘addiction’) or transcriptional activation (‘expedience’). In the first instance ligand‐independent kinase activation plays a major role in sustaining the transformed phenotype. Anti‐Met antibodies directed against the receptor binding site behave essentially as ligand (Hepatocyte Growth Factor, HGF) antagonists and are ineffective to counteract ligand‐independent activation. The monovalent chimeric MvDN30 antibody fragment, PEGylated to extend its half‐life, binds the fourth IPT domain and induces ‘shedding’ of the Met extracellular domain, dramatically reducing both the number of receptors on the surface and their phosphorylation. Downstream signaling is thus inhibited, both in the absence or in the presence of the ligand. In vitro, MvDN30 is a strong inhibitor not only of ligand‐dependent invasive growth, sustained by both paracrine and autocrine HGF, but notably, also of ligand‐independent growth of ‘Met‐addicted’ cells. In immunocompromised mice, lacking expression of Hepatocyte Growth Factor cross‐reacting with the human receptor – thus providing, by definition, a model of ‘ligand‐independent’ Met activation – PEGylated MvDN30 impairs growth of Met ‘addicted’ human gastric carcinoma cells. In a Met‐amplified patient‐derived colo‐rectal tumor (xenopatient) MvDN30‐PEG overcomes the resistance to EGFR targeted therapy (Cetuximab). The PEGylated MvDN30 is thus a strong candidate for targeting tumors sustained by ligand‐independent Met oncogenic activation.

Antihistaminic/antiallergic activity of 2-dialkylaminoalkylthio(oxy)-1-substituted benzimidazoles: Evaluation “in vitro” and “in vivo”

A new series of 2-dialkylamino-alkylthio(oxy)-1-substituted benzimidazoles synthesized in our laboratories was found to have promising antihistaminic activity. The results of pharmacological screening (“in vitro”: radioreceptor binding and isolated organs; “in vivo”: protection against mortality induced by histamine or by compound 48/80, passive cutaneous anaphylaxis, and prolongation of barbiturate-induced sleeping-time) gave clear-cut structure-activity relationships. This series of products has a general selectivity towards H1 receptors, weak antiallergic properties and negligible central effects. DF 10967 (1-ethoxyethyl-2-dimethyl-aminoethylthiobenzimidazole) was the most interesting compound, being very potent both “in vitro” (Ki=3.2±0.8 nM) and “in vivo” (ID50 11 μg/kg, i.p. and 8μg/kg, i.p. against histamine- and 48/80-induced mortality), with no central effects. The last finding is probably due to poor penetration into the brain (as confirmed by “in vivo” binding test with [3H]-mepyramine) and to lack of interaction with other central receptors.