TARGETED PROTEOMIC ANALYSIS OF ACTIVATED KINASES IN CUTANEOUS MELANOMA

• Grant number: 23/08112-4
• Support Opportunities: Scholarships abroad - Research
• Start date: August 06, 2024
• End date: July 05, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: André Zelanis Palitot Pereira
• Grantee: André Zelanis Palitot Pereira
• Host Investigator: Gunnar Dittmar
• Host Institution: Instituto de Ciência e Tecnologia (ICT). Universidade Federal de São Paulo (UNIFESP). Campus São José dos Campos. São José dos Campos , SP, Brazil
• Institution abroad: Luxembourg Institute Of Health, Luxembourg

Abstract

Protein kinase-mediated phosphorylation is a ubiquitous post-translational modification (PTM) that regulates signal transduction pathways in metazoans. The biological effects of protein phosphorylation are diverse, ranging from subcellular localization and structural changes to turnover. With more than 550 kinases described so far and spanning almost 2% of the human genes, the protein kinase complement of the human genome (the 'kinome') encompasses proteins that participate in several cell signaling circuits, making kinases promising drug targets. On the other hand, the multiple reciprocal activations of kinase-specific pathways through signaling cross-talks make kinase signaling networks very challenging to study. An alternative approach is the targeted analysis only of the kinase activity which, in turn, allows an overview of the phospho-regulation in a cell or tissue. In this context, mass spectrometry has been used for targeting activated kinases, with the use of heavy isotope-labeled synthetic phosphopeptides mapping to the kinase activation loop, a region of ~20 to 30 amino acid residues within the kinase domain which is flanked by conserved regions. The impact of protein phosphorylation is of paramount importance in cancer development such as in melanoma, the deadliest form of skin cancer. From the molecular point of view, melanoma is characterized by constitutive activation of signaling pathways related to the cell cycle, such as mitogen-activated protein kinase (MAPK-) and phosphoinisitide 3-kinase (PI3K)-signaling pathways. In fact, almost 50% of the patients with melanoma bear the typical V600E mutation in BRAF gene leaving the RAF-MEK-ERK pathway constitutively activated. Thus, selective inhibitors targeting the V600E mutated form of B-Raf kinase are used in clinical practice. Therefore, due to the importance of protein phosphorylation in health and disease processes, the main goal of this project is to apply targeted proteomics to the analysis of the activation status of protein kinase network in human melanoma cell lines subjected to B-RAF and MEK inhibitors, as well as in melanoma organoids model to further validate the cell culture results. It is expected that the results obtained will provide valuable information on the underlying active pathways during the chemotherapeutic treatment of melanoma as well as implications for prognostics and tumor development.