Dados do Trabalho

Título

Targeting aurora kinases in CSF3RT618I cells

Introdução

Chronic neutrophilic leukemia (CNL) emerges as a type of rare cancer, involving dysfunctions in myeloid lineages and can be diagnosed by genetic tests. Among the markers used for its identification, there is the CSF3RT618I mutation, reflected by the constitutive activation of the CSF3R receptor. In this scenario, the participation of aurora kinases proteins in mitotic entry, spindle assembly, and cytokinesis, stimulated by the active receptor, stands out.

Objetivo

The present project proposes to analyze the impact of the aforementioned mutation on the expression of AURKA and AURKB in Ba/F3 cells, as well as the potency and efficiency of its inhibitors in transformed cells.

Métodos

Ba/F3 CSF3RT618I cells and p-MIG CSF3RT618I vectors were provided by Prof. Brian J. Druker (Oregon Health & Science University, USA). The effect of drugs on cell viability was measured by the MTT reduction assay, placing cells under increasing concentrations (0-10μM) of aurora A inhibitor I, AZD1152-HQPA, and reversine for an interval of 24, 48, and 72 h. Experiments were carried out in sequence to verify the clonogenic capacity and the apoptosis rate by marking with annexin V-APC and propidium iodide. Changes in cell cycle progression were monitored by flow cytometry. Molecular markers of CSF3R receptor-associated pathways (AURKA, AURKB, STAT3, STAT5, Histone H3, and p-Histone H3Ser10), apoptosis (PARP1) and DNA damage (γH2AX) were investigated by Western Blot. Assays to verify the proliferative capacity (Ki-67) and gene expression profile (quantitative PCR) were also performed. ANOVA tests and Bonferroni's post-test or t-Student test were applied for statistical analysis (p<0.05).

Resultados

The IC50 ranged from 20 to 15.8 µM for aurora A inhibitor I, from >100 to 0.15 µM for AZD1152-HQPA, and from 2.41 to 0.06 µM for reversine. AZD1152-HQPA, and reversine completely inhibited the autonomous clonal growth rate at concentrations from 0.12 μM and all aurora kinase inhibitors significantly induced apoptosis in a dose-dependent manner. It was reported a decrease in the proliferation marker at 0.8 μM of aurora A inhibitor I, 0.032 μM of AZD1152-HQPA, and 0.16 μM of reversine. In the molecular scenario, all inhibitors reduced histone H3 phosphorylation, aurora A inhibitor I and reversine reduced STAT5 phosphorylation, and AZD1152-HQPA and reversine induced PARP1 cleavage and γH2AX expression. Cell cycle analysis revealed an increase in subG1 cell population and in ploidy. Aurora A inhibitor I downregulated Nfkb1 and Bcl2 expression, which was also observed for reversine, that modulated cell cycle and apoptosis-related genes too. AZD1152-HQPA reduced Mcl1 expression.

Conclusões

In summary, our data indicate that AURKB inhibitors, AZD1152–HQPA and reversine, showed antineoplastic potential in Ba/F3 CSF3RT618I, causing a decrease in cell viability, clonogenicity, and proliferation, and favoring a tumor suppressor molecular network, shedding new insights into the use of aurora kinase inhibitors in the context of CNL.

Palavras-chave

Myeloproliferative neoplasms; Ba/F3 CSF3RT618I; Aurora kinase inhibitors.

Financiador do resumo

N.S.P., K.L., J.A.E.G.C., N.P.F., L.B.L.M., and B.O.A. received fellowships from FAPESP (grants #2022/11039-4, #2020/12842-0, #2021/06138-0, #2019/05722-0, #2022/03316-8, and #2022/11038-8). This study was supported by grant #2021/11606-3 from the São Paulo Research Foundation (FAPESP). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil (CAPES), Finance Code 001.

Área

Pesquisa básica / translacional