David Tulasne

Receptor tyrosine kinases (RTK) classically function as oncogenic drivers that promote survival and proliferation upon ligand binding. A subset of RTKs can also function as dependence receptors (DR), inducing apoptosis in the absence of their ligands. Genetic alterations that enhance RTK signaling are well characterized in cancer and can be targeted with kinase inhibitors, which show limited efficacy in some clinical settings. Elucidation of whether oncogenic mutations can promote tumorigenesis by directly abolishing the proapoptotic activity of DRs could help improve strategies to target RTKs. In this study, we identified MET exon 14 skipping (METex14Del) as a paradigmatic example of an oncogenic alteration that drives tumorigenesis through genetic inactivation of the DR function of an RTK. METex14Del removed both the caspase cleavage site and adjacent Casitas B-lineage lymphoma (CBL)-binding motif, preventing generation of the proapoptotic p40MET fragment while sustaining oncogenic MET signaling. Uncoupling regulatory functions of MET using genome editing showed that loss of apoptosis capacity is a critical determinant of METex14Del-driven tumorigenesis. Combined—but not individual—mutation of the caspase and CBL sites was sufficient to recapitulate resistance to apoptosis and tumor growth induced by METex14Del in hepatocyte growth factor–humanized mouse models. Importantly, inducible reexpression of p40MET in METex14Del-expressing cells restored apoptotic sensitivity, decreased tumor formation in vivo, and resensitized tumors to capmatinib. Together, these findings redefine RTKs as receptors with dual oncogenic and tumor-suppressive functions and show that disruption of DR-mediated apoptosis is an oncogenic mechanism. These results provide a conceptual framework explaining why therapies targeting only RTK signaling may fail and support strategies restoring DR function to achieve durable tumor suppression. Significance: MET exon 14 skipping drives tumorigenesis not only by sustaining signaling but by genetically disabling the dependence receptor–mediated apoptotic function of MET, uncovering a mechanism explaining resistance to kinase-targeted therapies.

The MET exon 14 skipping mutation (named METex14Del) described in lung cancer leads to prolonged activation of signaling pathways and aberrant cell responses, but the link between HGF signaling and cell responses remains unclear. A putative lung cancer regulatory network of influential transcription factors was constructed from the transcriptomes of lung cancer cell lines. Transcriptomic data from METex14Del-expressing cells, stimulated or not by HGF, were mapped onto this lung cancer reference network and revealed activation of a major regulatory node composed mainly by the highly influential transcription factors ETS1, FOSL1 and SMAD3. HGF activation of METex14Del receptor induced the expression and phosphorylation of these three master regulators and the expression of their predicted target genes involved in migration and invasion. All these molecular and biological effects were inhibited by trametinib, a MEK inhibitor, which was potentiated by combination with capmatinib, a MET inhibitor. New mapping with transcriptomic data from trametinib-treated METex14Del cells validated the key role of the RAS-ERK pathway signaling in the activation of ETS1, FOSL1 and SMAD3 regulators and the induction of their target genes in HGF-activated METex14Del receptor. Thus, we report an original and powerful strategy to uncover key regulators, including transcription factors that have not been widely described in METex14Del signaling, such as SMAD3. These factors are activated by specific signaling pathways and could provide a novel therapeutic strategy involving a combination of receptor and signaling inhibitors.

In hereditary papillary renal cell carcinoma (HPRCC), the hepatocyte growth factor receptor (MET) receptor tyrosine kinase (RTK) mutations recorded to date are located in the kinase domain and lead to constitutive MET activation. This contrasts with MET mutations identified in non‐small‐cell lung cancer (NSCLC), which lead to exon 14 skipping and deletion of a regulatory domain: In this latter case, the mutated receptor still requires ligand stimulation. Sequencing of MET in samples from 158 HPRCC and 2808 NSCLC patients revealed 10 uncharacterized mutations. Four of these, all found in HPRCC and leading to amino acid substitutions in the N‐lobe of the MET kinase, proved able to induce cell transformation, which was further enhanced by hepatocyte growth factor (HGF) stimulation: His1086Leu, Ile1102Thr, Leu1130Ser, and Cis1125Gly. Similar to the variant resulting in MET exon 14 skipping, the two N‐lobe MET variants His1086Leu and Ile1102Thr were found to require stimulation by HGF in order to strongly activate downstream signaling pathways and epithelial cell motility. The Ile1102Thr mutation also displayed transforming potential, promoting tumor growth in a xenograft model. In addition, the N‐lobe‐mutated MET variants were found to trigger a common HGF‐stimulation‐dependent transcriptional program, consistent with an observed increase in cell motility and invasion. Altogether, this functional characterization revealed that N‐lobe variants still require ligand stimulation, in contrast to other RTK variants. This suggests that HGF expression in the tumor microenvironment is important for tumor growth. The sensitivity of these variants to MET inhibitors opens the way for use of targeted therapies for patients harboring the corresponding mutations.

The MET exon 14 skipping mutation (named METex14del) described in lung cancer leads to prolonged activation of signaling pathways and aberrant cell responses, but the link between HGF signaling and cell responses remains unclear. A putative lung cancer regulatory network of influential transcription factors was constructed from the transcriptomes of lung cancer cell lines. Transcriptomic data from METex14del-expressing cells, stimulated or not by HGF, were mapped onto this lung cancer reference network and revealed activation of a major regulatory node composed mainly by the highly influential transcription factors ETS1, FOSL1 and SMAD3. HGF activation of METex14del receptor induced the expression and phosphorylation of these three master regulators and the expression of their predicted target genes involved in migration and invasion. All these molecular and biological effects were inhibited by trametinib, a MEK inhibitor, which was potentiated by combination with capmatinib, a MET inhibitor. New mapping with transcriptomic data from trametinib-treated METex14del cells validated the key role of the RAS-ERK pathway signaling in the activation of ETS1, FOSL1 and SMAD3 regulators and the induction of their target genes in HGF-activated METex14del receptor. Thus, we report an original and powerful strategy to uncover key regulators, essentially transcription factors, activated by specific signaling pathways, which can provide a novel therapeutic strategy using a combination of receptor and signaling inhibitors.

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.

The MET exon 14 skipping mutation (named METex14del) described in lung cancer leads to prolonged activation of signaling pathways and aberrant cell responses, but the link between HGF signaling and cell responses remains unclear. A putative regulatory network of influential regulators of target genes was constructed from the transcriptomes of lung cancer cell lines. Overlaying this reference network with transcriptomic data from METex14del-expressing cells, stimulated or not by HGF, revealed a major regulatory node consisting mainly of the transcription factors ETS1, FOSL1 and SMAD3. HGF activation of METex14del induced the phosphorylation of these master regulators and the expression of their predicted target genes in a RAS-ERK pathway-dependent manner. Furthermore, most of the transcription factors in the regulatory node are known regulators of epithelial-mesenchymal transition, consistent with their involvement in migration and invasion. New modeling with transcriptomic data from MEK inhibitor-treated METex14del cells validated the key role of RAS-ERK pathway regulators and their target genes in METex14del receptor activation. Thus, we report an original strategy to identify key transcriptional nodes associated with specific signaling pathways that may become novel therapeutic targets.

Tyrosine kinase inhibitors (TKI) directed against MET have been recently approved to treat advanced non-small cell lung cancer (NSCLC) harbouring activating MET mutations. This success is the consequence of a long characterization of MET mutations in cancers, which we propose to outline in this review. MET, a receptor tyrosine kinase (RTK), displays in a broad panel of cancers many deregulations liable to promote tumour progression. The first MET mutation was discovered in 1997, in hereditary papillary renal cancer (HPRC), providing the first direct link between MET mutations and cancer development. As in other RTKs, these mutations are located in the kinase domain, leading in most cases to ligand-independent MET activation. In 2014, novel MET mutations were identified in several advanced cancers, including lung cancers. These mutations alter splice sites of exon 14, causing in-frame exon 14 skipping and deletion of a regulatory domain. Because these mutations are not located in the kinase domain, they are original and their mode of action has yet to be fully elucidated. Less than five years after the discovery of such mutations, the efficacy of a MET TKI was evidenced in NSCLC patients displaying MET exon 14 skipping. Yet its use led to a resistance mechanism involving acquisition of novel and already characterized MET mutations. Furthermore, novel somatic MET mutations are constantly being discovered. The challenge is no longer to identify them but to characterize them in order to predict their transforming activity and their sensitivity or resistance to MET TKIs, in order to adapt treatment.

In hereditary papillary renal cell carcinoma (HPRCC), the hepatocyte growth factor receptor (MET) receptor tyrosine kinase (RTK) mutations recorded to date are located in the kinase domain and lead to constitutive MET activation. This contrasts with MET mutations identified in non-small cell lung cancer (NSCLC), which lead to exon 14 skipping and deletion of a regulatory domain: in this latter case, the mutated receptor still requires ligand stimulation. Sequencing of MET in samples from 158 HPRCC and 2808 NSCLC patients revealed ten uncharacterized mutations. Four of these, all found in HPRCC and leading to amino acid substitutions in the N-lobe of the MET kinase, proved able to induce cell transformation, which was further enhanced by hepatocyte growth factor (HGF) stimulation: His1086Leu, Ile1102Thr, Leu1130Ser and Cis1125Gly. Similar to the variant resulting in MET exon 14 skipping, the two N-lobe MET variants His1086Leu and Ile1102Thr were found to require stimulation by HGF in order to strongly activate downstream signaling pathways and epithelial cell motility. The Ile1102Thr mutation also displayed transforming potential, promoting tumor growth in a xenograft model. In addition, the N-lobe-mutated MET variants were found to trigger a common HGF-stimulation-dependent transcriptional program, consistent with an observed increase in cell motility and invasion. Altogether, this functional characterization revealed that N-lobe variants still require ligand stimulation, in contrast to other RTK variants. This suggests that HGF expression in the tumor microenvironment is important for tumor growth. The sensitivity of these variants to MET inhibitors opens the way for use of targeted therapies for patients harboring the corresponding mutations.

MET is a receptor tyrosine kinase that is activated in many cancers through various mechanisms. MET exon 14 (Ex14) skipping occurs in 3% of nonsmall cell lung tumors. However, the contribution of the regulatory sites lost upon this skipping, which include a phosphorylated serine (S985) and a binding site for the E3 ubiquitin ligase CBL (Y1003), remains elusive. Sequencing of 2808 lung tumors revealed 71 mutations leading to MET exon 14 skipping and three mutations affecting Y1003 or S985. In addition, MET exon 14 skipping and MET Y1003F induced similar transcriptional programs, increased the activation of downstream signaling pathways, and increased cell mobility. Therefore, the MET Y1003F mutation is able to fully recapitulate responses induced by MET exon 14 skipping, suggesting that loss of the CBL binding site is the main contributor of cell transformation induced by MET Ex14 mutations.