Supervisor: Dr Xavier Bisteau
Mesothelioma has been mainly associated with occupational exposure to mineral fibers present in nature. This led, since early 1980s, to implement strict regulations in developed countries to limit the use of 6 of 400 different mineral fibers, collectively called “asbestos”. Nevertheless, the remaining approximately 400 mineral fibers have not been regulated and can be used freely, although many of them are carcinogenic and have been associated with mesothelioma appearance. Despite these regulations, the rate of decrease in mesothelioma incidence is much lower than predicted. Instead, the number of new mesotheliomas per year and of deaths per year continue to increase both in high-resource countries and worldwide.
When asbestos and other fibers reach the pleura and peritoneum through lymphatics, they remain in place for months or years, triggering a chronic inflammatory process driven by HMGB1 secretion and related inflammasome activation, which in turn induces the activation of NF-κB and the phosphatidylinositol 3-kinase (PI3K) pathways in mesothelial cells. This environment favors the growth of mesothelial cells that accumulate mutations spontaneously or exposed to mutagenic ROS released by inflammatory cells around asbestos deposits.
To date, treatment of patients with pleural mesothelioma (PM) mainly consists on surgery and/or chemotherapy. Extensive chemotherapeutic treatment has then been the gold standard since almost 2 decades. This regimen however, only extend survival for few months. The addition of bevacizumab, a VEGF-A monoclonal antibody, improves survival over the use of the platinum-doublet alone, leading to its recommendation when bevacizumab is available and dual immune checkpoint inhibition with nivolumab plus ipilimumab has recently demonstrated superiority to platinum plus pemetrexed in PMs with non-epithelioid histologies. This could be explained due to differences in tumor-infiltrating immune cells in mesothelioma. A comprehensive investigation of the infiltration of immune cells within the tumor nest and tumor-associated stroma in 230 patients unveiled that the tumors with low infiltration of CD8+ T-lymphocyte and high infiltration CD163+ tumor-associated macrophages had the worst prognosis.
The poor response of patients with mesothelioma and the observed low survival rate led to the analysis of genomic alterations in this cancer to identify ‘druggable’ targets. Recurrent identified defects include mutations affecting NF2, LATS2, BAP1, TP53, CDKN2A and SETD2. Mutations of NF2, as well of LATS2 and MST1, affect the Hippo pathway involved in cell contact growth inhibition, that negatively regulates the expression of cyclin D1. In addition, frequent deletions (50–80%) of the CDKN2A/B locus (encoding the CDK4/6 inhibitors p16INK4A and p15INK4B) were also identified, which are associated with shorter overall survival.
PM is mainly driven by loss-of-function mutations in tumour-suppressor genes and mutations of unknown consequence. The paucity of recognizable driver mutations in well-characterized oncogenes underscore even more the lack of obvious ‘druggable’ targets that could be directly tested as alternative treatments against mesothelioma. It becomes clear that genomic analysis of PM has reached its limits to identify such targets. In this context, determining at the phosphoproteomics level which signaling pathways, and their associated proteins kinases, are overactivated will help to define alternative targets to be tested in functional assays. Protein kinases represent the easiest targets for which a battery of small inhibitory molecules already exist and that could directly be used.
In this idea, our group focused on the addiction of PM cells to the activity of CDK4/6. As both the deletion of CDKN2A/Band a defective NF2 oncosuppressor lead to an increased expression of cyclin D1, they deregulate the CDK4, likely rendering PMs to be addicted to its activity. Our recent report of CDK4/6i in MPM indicated the induction of a sustained proliferation arrest associated with senescence or apoptosis markers but also an up-regulation of genes involved in pathways that increase immunogenicity and/or facilitate anti-cancer immunotherapy in other models. We previously demonstrated that the activation of CDK4, which depends on its binding to cyclins D and phosphorylation on T172, is central for the cell cycle decision. The phosphorylation status of CDK4 successfully predicted the sensitivity to Palbociclib in 28 PM cell lines including 19 primary patient-derived ones. We further predicted the sensitivity to CDK4/6 inhibition of 80% of the tested frozen PM tumors. The insensitivity to palbociclib of some tested cell lines and tumors without T172-phosphorylation of CDK4 was associated with high p16CDKN2A expression and RB1 defect.
Interestingly, since few years, CDK4 expression has been shown to be associated with T cell exclusion and resistance to immunotherapy in melanoma, whereas CDK4/6i has been shown to overcome the primary resistance to PD1 blockade in PMs. Through this project we wonder how the primary infiltration in PM tumors is correlated to the expression of CDK4, RB1, p16CDKN2A and any other gene expression. We hypothesize that changes in immune cells infiltration in the tumor is affected by the genomic alterations found in the cancer cells affecting in fine the response of the tumor to treatments. The proposed project will therefore be aiming at evaluating which signaling pathways and especially which protein kinases are deregulated using phosphoproteomics analysis and whether there is a correlation between the defects found in tumors and the infiltration in immune cells using in silico analyses and multiplex immunoassay. The project will use clinical patients samples from international collaborations, requiring some bioinformatics analysis but also to generate and analyse proteomics data on various established and primary patient derived cell lines.
Techniques:
- Proteomics and phosphoproteomics by mass spectrometry
- Bioinformatics analysis
- Transcriptomics RNA-seq, genomic analysis
- Western blotting, qPCR, immunofluorescence, flow cytometry, …