Investigation of the effects of pharmacological inhibition of proteins involved in cytoskeleton regulation and cell cycle progression.

Author(s)

Jorge Antonio Elias Godoy Carlos

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2020-12-10

Examining board members

João Agostinho Machado Neto; Mariana Lazarini; Luciana Biagini Lopes; Elvira Deolinda Rodrigues Pereira Velloso

• Advisor: João Agostinho Machado Neto

Abstract

Introduction: Acute leukemias (AL) are aggressive neoplasms characterized by clonal proliferation with replacement and accumulation of neoplastic hematopoietic cells in the bone marrow and other organs making hematopoiesis an ineffective process. In adult AL patients, therapeutic options are limited and remission rates decrease with increasing age. The aurora kinases (AURKs) and stathmin 1 (STMN1) proteins have been identified as potential therapeutic targets in hematological neoplasms due to the aberrant expression of these genes/proteins. AURKs are essential for the success of mitosis, acting in the organization of the mitotic spindle and cytokinesis (AURKA and AURKB). STMN1 protein participates in microtubule dynamics and cell cycle progression. Reversine is a drug that acts as a multikinase inhibitor with selectivity for AURKA and AURKB. GDP366 is described as a selective inhibitor for STMN1 and BIRC5, while AD80 was identified in that study as an analog of GDP366 by chemoinformatics. Methods: The expression of AURKA, AURKB, and STMN1 in AL patient samples was investigated in the Amazonia database! and/or on a panel of LA cell lines. Jurkat [acute lymphoblastic leukemia (ALL)-T], Namalwa (ALL-B), NB4 [acute myeloid leukemia (AML)], and/or U937 (AML) were used in cellular and molecular assays. Results: AURKB expression was higher in patients with ALL compared to normal lymphocytes (p<0.0001). LLA cell lines show aberrant AURKA and AURKB expression and activation. In ALL cellular models, reversine reduced cell viability (in concentration- and time-dependent manner), clonal growth and proliferation, and increased apoptosis, acidic vesicular organelles, and mitotic catastrophe (increase in cells in G2/M, cell size and damage in DNA) (p<0.05). In the molecular scenario, reversine treatment reduced the activity of AURKB, increased consumption of SQSTM1 p62, the levels of LC3BII and <font face = \"symbol\">gH2AX in cellular models of ALL. In Namalwa cells, reversine modulated 25 of 84 autophagy-related genes, including BCL2, BAD, ULK1, ATG10, IRGM, and MAP1LC3B indicating that reversine acts by initiating and maintaining the autophagic flow in ALL. STMN1 expression was higher in patients with AML and ALL compared to normal hematological cells (p<0.0001). GDP366 and AD80 reduced cell viability (in concentration- and time-dependent manner), clonogenicity and proliferation, increased apoptosis, and modulated cell cycle progression in ALL and AML models (p<0.05). Both drugs reduced phosphorylation of S6RP, increased levels of cleaved PARP1 and yH2AX. AD80 promoted a decrease in the expression of STMN1 and survivin, different from that observed in cells treated with GDP366. In Jurkat cells, GDP366 and AD80 modulated the cytoskeleton-related genes. Conclusion: Reversine inhibits the activity of aurora kinases and reduces cell viability through multiple mechanisms of cell death: apoptosis, mitotic catastrophe, and autophagy. GDP366 and AD80 reduce multiple characteristics related to the leukemia phenotype, including excessive proliferation and cell survival. Our findings indicate that drugs that act on aurora kinases and STMN1 may be promising in the treatment of acute leukemias. (AU)

• FAPESP's process: 18/19372-9 - Investigation of the effect of potential Stathmin 1 inhibitors obtained by chemoinformatics on the phenotype of acute leukemias
• Grantee: Jorge Antonio Elias Godoy Carlos
• Support Opportunities: Scholarships in Brazil - Master