Unraveling the oncogenic mechanisms of mutations described in A.C.Camargo Cancer Center using gene editing tools

• Grant number: 19/25453-4
• Support Opportunities: Scholarships in Brazil - Master
• Effective date (Start): June 01, 2020
• Effective date (End): January 02, 2022
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Martín Roffé
• Grantee: Dimas Pontes Café Filho
• Host Institution: A C Camargo Cancer Center. Fundação Antonio Prudente (FAP). São Paulo , SP, Brazil

Abstract

At the A.C.Camargo Cancer Center (ACCCC) we often come across rare or undescribed gene mutations/alterations that could be associated with tumor development in patients. A variant of uncertain (unknown) significance (VUS) is a variation in a genetic sequence for which the association with disease risk is unclear, posing a major challenge for genetic counseling and clinical management of the families in which they are identified. Thus, specific approaches to each individual alteration may be required to determine its relevance. In this project, we will use variations of the CRISPR/Cas9 technique to construct cell models bearing VUS of two tumor suppressor genes, TP53 and MUTYH, found in ACCCC patients who performed germline genetic testing. Pathogenic alterations in TP53 are associated with the hereditary cancer syndrome called Li-Fraumeni. MUTYH is a DNA repair enzyme and alterations in its gene are associated with the recessive inherited syndrome called MUTYH-associated polyposis, characterized by a high predisposition to polyp development and colorectal cancer. Moreover, it is not yet fully established what are the tumorigenic mechanisms associated with monoallelic alterations in the MUTYH gene. We will use the CRISPR/Cas9 technique to generate mono and biallelic knockout cells for MUTYH in order to establish their different behaviors. Finally, it has been described that TP53 activation regulates MUTYH expression and thus we will verify whether TP53 mutations are determinant to potentiate the oncogenic effect of monoallelic alterations in the MUTYH gene. Through specific assays for the function of both genes in the generated models, we hope to establish the pathogenic mechanisms of these VUS, which may be useful for the management and counseling of carrier families. (AU)