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Vulnerability of esophageal cancer to their addiction to kinase activities: evaluation and prediction of eSCC tumors responsiveness to kinase inhibitors.

Periodic Reporting for period 1 - Kinaddict (Vulnerability of esophageal cancer to their addiction to kinase activities: evaluation and prediction of eSCC tumors responsiveness to kinase inhibitors.)

Reporting period: 2019-09-16 to 2021-09-15

Esophageal cancer (EC) ranks amongst the deadliest cancers worldwide with a 5-year survival ranging around 20%, that barely improved over the past two decades. The standard of care of the main EC disease, the squamous cell carcinoma (eSCC), consisting of surgery with neo-/adjuvant treatment combined or not with mitotic poisons has shown poor efficacy as such patients develop recurrences, causing death in a year. In this context, a deeper understanding of the pathogenesis is required to improve the treatment offered to patients with EC. Genomics analyses have pictured over the years a broad heterogenic genetic landscape between individuals with EC as well as inter- and intra-tumors, hampering in fine the approval of alternative treatments against EC. Reports of genomic alterations revealed the high prevalence of aberrations affecting the cell cycle regulation, especially on the Cyclin D - CDK4 – pRB – E2F axis in addition of other cascades originating from mutations and amplifications of RTKs (receptor tyrosine kinases), downstream mediators, or regulators of the oxidative stress, squamous cell differentiation, and WNT-ß Catenin signaling. To evaluate alternative therapies, the project aimed to identify kinase and their activity to which EC is addicted and use the broad arsenal of small inhibitor molecules to target specific kinases. By their critical role in the cell cycle progression, CDK1/2/4/7 are undeniably good target choices but only the inhibition of CDK4 (together with related CDK6) has been recently successful in cancer targeted therapy, due to its major effects on progression-free survival without major side effects. The understanding of the cell circuitry especially at kinase level and their associated pathways is necessary to determine the vulnerabilities of each tumor and propose an optimized treatment modality. In this idea, the present project aimed to define the activity of protein kinases in eSCC tumor cells alongside genetic alterations, and identify kinases to which tumor cells are addicted to. Because of the high probability of response of eSCC tumors to CDK4i, the verification of response of eSCC tumors cells to CDK4i is followed by the molecular analysis of potential acquired resistance and the assessment of combined therapies with CDK4i to identify biochemical markers predicting the response. Such novel therapies, although such would lead to significant benefits for many patients, are not deprived of flaws as it would not be the case for other patients. Furthermore, the cost of treatment using such novel drugs have shown to be a burden for patients and public health care systems. It pushes the need to deeper characterize the tumors from each patient and stratify those that would benefit of such targeted therapies.
In order to identify the addiction of eSCC to kinase activities, we profiled the abundance of protein and phosphopeptides in human eSCC cell lines. At this end, we constructed a large assay library for SWATH-DIA analysis using mass spectrometry that cover the peptides of thousands of proteins. A similar approach was performed after enrichment of phosphorylated peptides using TiO2 beads. The profiling of the protein and phosphorylated peptides level in all available eSCC cell lines revealed a high heterogeneity with a common activation of the Cyclin D - CDK4 – pRB – E2F axis. Parallel to this, the global analysis of genomic and transcriptomic data available for such cancer cells highlighted the alterations of various pathways including RTKs for which most of eSCC cells appeared to be addicted.
Because of the large prevalence of CCND1 amplification and loss of CDKN2A expression in eSCC, our experiments focused on characterizing the response of eSCC cancer cells to the inhibition of CDK4/6 (CDK4/6i). The evaluation of the phosphorylation status of CDK4 by bidimensional electrophoresis unveiled a strong phosphorylation that was parallel to a rapid decrease of the cell cycle in many of the cell lines parallel to the CDK4/6i. This analysis also unveiled a latent proliferation that led to the apparition of different nuclear aberrations that activated in cascade different phenotypes. To understand deeper, our analysis focused on the dynamics of the cell cycle and the appearance of such phenotype and challenged with combination of drugs. Parallel, we explored the benefits of the targeted inhibition of specific kinases on eSCC by collecting induced tumors in various genetics mouse models. Such tumors revealed similarly a heterogeneous behavior.
The results have been presented during a scientific conference and one science fair. Along the project, numerous methods have been implemented to assess the proteome characteristics and developed new models in the laboratory to study the interplay between eSCC cancer cells and their microenvironment
In collaboration with the group of Benjamin Beck, we published a study on the impact of the circadian rhythm on the response to genotoxic drug in eSCC cells. Our results published in “Biology” unveil the still existing circadian rhythm in eSCC cells despite the overall decreased expression of its regulatory genes. Interestingly, treating eSCC cells with cisplatin when PER2 expression is low enhances DNA damage and leads to a higher apoptosis rate. This process appeared to be conserved in a mouse model of chemically-induced eSCC ex vivo. Such results therefore suggest that response to therapy might be enhanced in esophageal cancers using chronotherapy.
Changes in kinase expression in eSCC cells