Lung cancer is the leading cause of cancer in France and worldwide. Recent therapeutic advances have come from identification of molecular alterations associated with efficacy of targeted therapies. Thus, discovery of new actionable molecular alterations is a major challenge. Recently, mutations affecting the gene encoding the MET receptor have been detected in lung cancers, raising several clinical and scientific questions. These mutations affect splice sites of exon 14 and are highly heterogeneous which makes them difficult to detect. They induce exon 14 skipping, which functional consequences are still poorly understood. Importantly, these mutations are associated with efficacy of MET tyrosine kinase inhibitors (TKI) which represents a promising therapeutic opportunity. However, as for other targeted therapies, efficacy of MET TKI may be limited due to emergence of resistance which mechanisms are totally unknown so far. In the first part of this study, we will develop new diagnostic tools for detecting mutations leading to exon 14 skipping. We will develop techniques that can be easily transposed into routine practice, based on PCR fragment analysis and SnapShot technology, and high throughput techniques such as Next-Generation Sequencing (NGS). Moreover, we will develop an alternative to genomic techniques based on immunohistochemistry through the development of an exon 14 specific antibodies. We will compare these different methods of detection on several cohorts including unselected lung cancers, lung cancers with high MET overexpression and pulmonary sarcomatoid carcinomas. We will also have access to tumor samples from clinical trials evaluating MET TKI in patients with MET exon 14 mutations (MERCK phase II trial). In the second part of this study, we will address the functional consequences of exon 14 skipping. Exon 14 is known to harbor 3 different negative regulation sites. We will test the respective contribution of each of these regulation sites. To do so, we will use lentiviral infection with vectors harboring MET mutations affecting each of the regulation sites or all three of them. We will evaluate biological responses in the different cell lines along with sensitivity to MET TKI including tepotinib, a selective MET TKI, through collaboration with MERCK. We will also xenograft these cell lines in mice to evaluate tumor growth. In parallel, similar studies will be performed to investigate the effects of mutating exon 14 regulatory sites on the endogenous MET gene using CRISPR-barcoding technology. In the third part of this study, we will aim at identifying mechanisms of resistance to MET TKI emerging from MET exon 14 skipping cell lines and tumors. First we will generate resistant cell lines by exposing HS746T and H596 cell lines, known to harbor MET exon 14 skipping mutations, to increasing doses of MET TKI. We will also xenograft these cell lines and make them resistant to MET TKI through exposure to intermittent treatment cycles. We will compare parental and resistant cell lines regarding signaling pathways and genomic profile, including full-length MET sequencing, whole-exome sequencing and CGH array. Finally, we will aim at validating the identified mechanisms of resistance on tumor samples obtained from patients progressing upon MET TKI in the context of clinical trials (MERCK phase II trial). Overall, this study will improve detection of MET exon 14 mutations, understanding of functional consequences of exon 14 skipping and identification of mechanisms of resistance to MET TKI. This will facilitate selection of patients who may benefit from MET TKI and development of innovative therapeutic strategies to overcome resistance once progression has occurred.

One of the great challenges in the development of small protein drugs is the introduction of modifications which allow an increased the stability of the protein, its half-life in biological fluids, the yield of production and folding. The cyclization of small peptides is well developed, whereas the cyclization of large polypeptides or proteins is still in its infancy, primarily due to the difficulty in producing cyclic polypeptides or proteins using living systems. Given the fast growing importance of long polypeptides or proteins in the drug market, the development of processes enabling the efficient total synthesis of cyclic analogue libraries is of utmost importance. The aim of this project is to develop an innovative self-purifying process enabling the synthesis of large cyclic polypeptides or proteins by performing several sequential ligations of purified and unprotected peptide segments on a water compatible solid support. The method will be automated by adapting a commercially available multiple peptide synthesizer. The technology will be used for optimizing a lead cyclic protein (< 100 amino acids) currently developed in Lille for its capacity to act as a potent mimic of hepatocyte growth factor (HGF), which is the ligand of the MET receptor tyrosine kinase. Such potent MET agonists have a great potential in regenerative medicine. In particular, potent MET agonist can prevent the fulminant hepatic failure, an application which is mainly targeted in this project.