Molecular biology-leading edge technologies for the characterization of resistance to asparaginase

• Grant number: 19/09953-7
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Effective date (Start): October 15, 2019
• Effective date (End): May 03, 2020
• Field of knowledge: Biological Sciences - Pharmacology - Biochemical and Molecular Pharmacology
• Principal Investigator: Gisele Monteiro
• Grantee: Iris Munhoz Costa
• Supervisor: Camila Oresco dos Santos
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Research place: Cold Spring Harbor Laboratory (CSHL), United States
• Associated to the scholarship: 16/25896-5 - Biochemical characterization and cytotoxic evaluation of mutant isoforms of L-Asparaginase II from Dickeya chrysanthemi (Erwinia chrysanthemi), BP.DR

Abstract

L-Asparaginase (ASNase) is a L-asparagine amidohydrolase used as first line of acute lymphoblastic leukemia (ALL) treatment. ASNase is able to catalyze the hydrolysis of asparagine, depleting this amino acid from the bloodstream. ALL cells depend on obtaining asparagine from the bloodstream for protein synthesis and cell proliferation, since they are not able to express, or express low levels of asparagine synthetase (ASNS) enzyme. Currently, the biopharmaceutical is obtained from bacteria Escherichia coli and Erwinia chrysanthemi, and despite its effectiveness, many adverse effects are observed in patients. Hypersensitivity and anti-ASNase antibody production can be observed in up to 60% of treated patients and these problems can result in drug resistance or discontinuation in ASNase use, which in turn results in increased levels of tumor relapse. Different ALL cell lines respond differently to ASNase treatment. The Reh ALL cell line is characterized as resistant by expressing high levels of ASNS; in addition, it constitutively expresses catheosin B (CTSB). However, how gene expression is modulated after ASNase exposure in this cell line and the influence of CTSB in this surveillance response are not known. Therefore, evaluating differences in gene expression of Reh cell line with and without CSTB expression may contribute to the elucidation of novel mechanisms of ASNase resistance and sensitivity, as well as the development of more efficient enzyme. For this, it will be constructed knockout of the CTSB gene in the Reh cell line through the CRISPR/Cas9 technique and the cells will be treated with different ASNase proteoforms. The RNA profile comparison can help to elucidate how Reh cell line modulate gene expression during exposure to asparaginase and if CTSB activity interferes with resistance of Reh to ASNase. (AU)