Reversine

Reversine exerts cytotoxic effects through multiple cell death mechanisms in acute lymphoblastic leukemia

Jorge Antonio Elias Godoy Carlos 1 & Keli Lima 1 & Juan Luiz Coelho-Silva 2 & Raquel de Melo Alves-Paiva 3 &
Natália Cestari Moreno 4 & Hugo Passos Vicari 1 & Fábio Pires de Souza Santos 3 & Nelson Hamerschlak 3 &
Leticia Veras Costa-Lotufo 1 & Fabiola Traina 2 & João Agostinho Machado-Neto 1

Accepted: 22 July 2020
# International Society for Cellular Oncology 2020

Abstract
Purpose Acute lymphoblastic leukemia (ALL) is an aggressive hematological cancer with limited therapeutic options for adult patients. Aurora kinases have drawn attention as potential targets in hematological neoplasms due to their high expression and biological functions. Aurora kinase A (AURKA) and AURKB are essential for a successful mitosis, acting in spindle mitotic organization and cytokinesis. Reversine is a synthetic purine analog that acts as a multi-kinase inhibitor with anti-neoplastic activity by targeting AURKA and AURKB.
Methods ALL patient gene expression data were retrieved from the Amazonia! database. For functional assays, Jurkat (T-ALL) and Namalwa (B-ALL) cells were exposed to increasing concentrations of reversine and submitted to various cellular and molecular assays.
Results We found that AURKB expression was higher in ALL patient samples compared to normal lymphocytes (p < 0.0001). The ALL cell lines tested displayed aberrant AURKA and AURKB expression. In Jurkat and Namalwa cells, reversine reduced cell viability in a dose- and time-dependent manner (p < 0.05). Reversine also significantly reduced the viability of primary ALL cells. Reversine induced apoptosis and autophagy, and reduced cell proliferation in both cell lines (p < 0.05). Mitotic catastrophe markers, including cell cycle arrest at G2/M, increased cell size and DNA damage, were observed upon reversine exposure. Short- and long-term treatment with reversine inhibited autonomous clonogenicity (p < 0.05). At the molecular level, reversine reduced AURKB activity, induced SQSTM1/p62 consumption, and increased LC3BII and γ-H2AX levels. In Namalwa cells, reversine modulated 25 out of 84 autophagy-related genes, including BCL2, BAD, ULK1, ATG10, IRGM and MAP1LC3B, which indicates that reversine acts by initiating and sustaining autophagy signals in ALL cells. Conclusions From our data we conclude that reversine reduces the viability of ALL cells by triggering multiple cell death mechanisms, including apoptosis, mitotic catastrophe, and autophagy. Our findings highlight reversine as a potential anticancer agent for ALL. Keywords Acute lymphoblastic leukemia . Cell death . Aurora kinases . Reversine Jorge Antonio Elias Godoy Carlos and Keli Lima contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13402-020-00551-3) contains supplementary material, which is available to authorized users. * João Agostinho Machado-Neto [email protected] 2 Department of Medical Images, Hematology and Clinical Oncology, University of São Paulo at Ribeirão Preto Medical School, Ribeirão Preto, São Paulo, SP, Brazil 1 Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 1524, São Paulo, SP CEP 05508-900, Brazil 3 4 Einstein’s Teaching and Research Institute, Albert Einstein Hospital, São Paulo, SP, Brazil Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil 1Introduction Acute lymphoblastic leukemia (ALL) is an aggressive hema- tological cancer characterized by aberrant proliferation of lymphoid progenitors in the bone marrow, peripheral blood and other extramedullary sites [1]. ALL is the most common neoplasia in childhood (2 to 5 years old) and presents relative- ly favorable outcomes [2]. On the other hand, in adult patients, ALL is a disease with a poor prognosis, and only 30–40% of the patients will achieve long-term remission [1]. Despite the great success obtained in treating childhood ALL patients, most adult and elderly ALL patients are not eligible for or do not respond properly to current therapies, what makes it imperative to develop new therapeutic options. Aurora kinases (AURKs) are overexpressed and asso- ciated with a poor prognosis in different types of cancer, including leukemia, which has drawn attention to this family of kinases as potential therapeutic targets [3–7]. AURKs are serine/threonine kinases that play crucial roles in cell cycle progression and proliferation. Aurora kinase A (AURKA) is mainly expressed during the G2/M phase of the cell cycle and is implicated in the formation of the mitotic spindle during mitosis [8–10]. Aurora ki- nase B (AURKB) is part of a chromosome passenger complex that controls the organization of the centromere-kinetochore region as well as microtubule- kinetochore attachments, and later in the cell cycle it plays a prominent role in cytokinesis [11, 12]. R e v e r s i n e ( 2 - ( 4 - m o r p h o l i n o a n i l i n o ) - 6 - cyclohexylaminopurine) was originally identified as a dedif- ferentiation agent for murine myoblasts into multipotent pro- genitor cells [13, 14]. Recent studies have indicated that reversine also presents anti-neoplastic activity in various ma- lignancies [15–18], including hematological malignancies [19–21]. Reversine is an ATP analog and has been reported to be a potent multi-kinase inhibitor selective for AURKs, MSP1 and JNK [22–24]. Among the cellular events triggered by this compound, apoptosis, mitotic catastrophe and autoph- agy have been described in cancer [16, 25]. In the present study, the cellular and molecular mechanisms underlying the suppressive effects of reversine on ALL were investigated. 2Material and methods 2.1Gene expression data AURKA (probe 208079_s_at) and AURKB (probe 209464_at) mRNA expression data from normal lymphocytes (n = 12), B- ALL (n = 82) and T-ALL (n = 54) samples were derived from a public access data portal of the Amazonia! database 2008 (http://amazonia.transcriptome.eu) [26]. Gene expression values were measured using Affymetrix HGU133 plus 2.0 arrays and data sets were cross-referenced using tumor- specific identification numbers. 2.2Cell culture, reagents and chemicals Daudi, Namalwa, MOLT4 and Jurkat cells were obtained from the ATCC (Philadelphia, PA, USA). Karpas 422 cells were kindly provided by Prof. Martin Dreyling (University Hospital Grosshadern/LMU, Munich, Germany), and CEM and RS4;11 cells were kindly provided by Prof. Carlos Alberto Scrideli (University of São Paulo, Ribeirão Preto, Brazil). The cells were cultured in RPMI-1640 medium con- taining 10% fetal bovine serum (FBS), glutamine and penicil- lin/streptomycin, and maintained at 37 °C, 5% CO2. Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll–Hypaque gradient centrifugation (Sigma-Aldrich, St Louis, MO, USA) from three healthy donors (1 female and 2 males, aged 30–35 years). Primary leukemia cells were obtained from four ALL patients (Supplementary Table 1). Informed consent was obtained from all healthy donors and ALL patients, and the Ethics Committee of the University of São Paulo at the Ribeirão Preto Medical School approved this study. Primary cells were cultured in RPMI-1640 medium containing 30% FBS, penicillin/streptomycin and recombi- nant cytokines (PeproTech, USA) (30 ng/ml IL3, 100 ng/ml IL7, 100 ng/ml FLT3-ligand, and 30 ng/ml SCF), at a density of 2 × 106 cells/ml. Reversine [2-(4-morpholinoanilino)-6- cyclohexylaminopurine] was obtained from TargetMol (Target Molecule Corp., Boston, MA, USA) and prepared as a 50 mM stock solution in dimethyl sulfoxide (Me2SO4; DMSO). 2.3Western blot analysis Equal amounts of protein were used from total extracts followed by SDS-PAGE and Western blot analysis with the indicated antibodies, and was carried out using a SuperSignal TM West Dura Extended Duration Substrate System (Thermo Fisher Scientific, San Jose, CA, USA) and a G:BOX Chemi XX6 gel doc system (Syngene, Cambridge, UK). Antibodies directed against AURKA (sc-25,425), AURKB (sc-25,426), phosphorylated (p)-Histone H3S10 (sc-8656-R), and α-tubulin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies directed against γH2AX (#9718), SQSTM1/p62 (#88588), LC3I/II (#2775), BCL2 (#15071) and GAPDH (#2118) were from Cell Signaling Technology (Danvers, MA, USA). 2.4Quantitative RT-PCR (qRT-PCR) Total RNA was obtained using TRIzol reagent (Thermo Fisher Scientific). cDNA was synthesized from 1 μg RNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed using an ABI 7500 Sequence Detector System in conjunction with a TaqMan system for the AURKA (Hs00269212.m1), AURKB (Hs00177782.m1), GAPDH (4326317E-0411007) and 18S rRNA (4332641) genes (Life Technologies) or a SybrGreen System for the BCL2, BAD, ULK1 , ATG10 , IRGM and MAP1LC3B genes (Supplementary Table 2). GAPDH, 18S rRNA, HPRT1 and ACTB were used as reference genes. Relative quantification 2.8 Immunofluorescence analysis Jurkat and Namalwa cells, treated or not with 5 μM reversine for 24 h, were attached on cover slips coated with poly-L-lisine (1 mg/ml), fixed with 3.7% formal- dehyde, permeabilized with 0.5% Triton X-PBS and blocked with 3% bovine serum albumin (BSA) PBS. Next, the cells were incubated with anti-α-tubulin Alexa Fluor® 488 conjugate (1:100 in 3% BSA PBS, -ΔΔCT values were calculated using the 2 equation. A negative Thermo Fisher Scientific) for 12 h, followed by a PBS ‘No Template Control’ was included for each primer pair. wash. Finally, the slides were mounted in ProLong Gold Anti-Fade Mounting Medium with DAPI (Thermo 2.5Cell viability assay In order to evaluate the effects of reversine on cell viability, methylthiazoletetrazolium (MTT) assays were performed. Briefly, 2 × 104 cells (ALL cell lines) or 2 × 105 primary cells per well were seeded in a 96-well plate in RPMI-1640 medi- um with 10% FBS in the presence, or not, of different con- centrations of reversine (1, 2.5, 5, 10, 25 and 50 μM) for 24, 48 and 72 h. Next, 10 μl MTT solution (5 mg/ml) was added and incubated at 37 °C, 5% CO2 for 4 h. The reaction was stopped using 100 μl 0.1 N HCl in anhydrous isopropanol. Cell viability was evaluated by measuring the absorbance at 570 nm. IC50 values were calculated using nonlinear regres- sion analysis in GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). 2.6Apoptosis assessment by annexin V staining A total of 1 × 105 cells per well were seeded in a 24-well plate in RPMI-1640 medium with 10% FBS in the presence, or not, of different concentrations of reversine (1, 2.5, 5, 10, 25 and 50 μM) for 24 h or reversine at 5 μM for 24, 48 and 72 h. Next, the cells were washed with ice-cold PBS and resuspend- ed in binding buffer containing 1 μg/ml 7AAD or propidium iodide (PI) and 1 μg/ml FITC- or APC-labeled annexin V. All specimens were analyzed by flow cytometry (FACSCalibur; Becton Dickinson) after incubation for 15 min at room tem- perature in a light-protected area. Ten thousand events were acquired for each sample. 2.7Cell cycle analysis A total of 6 × 105 cells per well were seeded in 6-well plates in the presence, or not, of reversine (1, 2.5, 5 and 10 μM), har- vested at 24 h, or reversine at 5 μM for 24, 48 and 72 h, fixed with 70% ethanol and stored at 4 °C for at least 2 h. Fixed cells were stained with 20 μg/ml propidium iodide (PI) containing 10 μg/ml RNase A for 30 min at room temperature in a light- protected area. DNA content distribution was acquired using cytometry (FACSCalibur) and analyzed using FlowJo soft- ware (Treestar, Inc.). Fisher Scientific). Images were captured using a Confocal Zeiss LSM-780-NLO microscope, and cell sizes were measured using ZEN 3.0 blue edition soft- ware (Carl Zeiss, Jena, Germany). 2.9Comet assay For alkaline Comet Assay, Jurkat and Namalwa cells (1.5 × 105) were treated with reversine 5 μM or vehicle for 24 h. The procedure used was as previously reported [27]. At least one hundred and fifty comets from two independent experiments were analyzed using LUCIA Comet Assay™ software (Laboratory Image, Prague, Czech Republic) and DNA dam- age was presented as tail moment value (head/tail DNA ratio) [28]. 2.10Proliferation assay using Ki-67 staining For Ki-67 staining, 2 × 105 cells/ml were treated, or not, with 1, 5 and 10 μM reversine for 24 h. Next, the cells were resus- pended in 70% ethanol and stored at -20 °C. The cells were stained with anti-Ki-67 according to the manufacturer’s in- structions (Ki-67 FITC clone B56; BD Bioscience, San Jose, CA, USA) and the mean of fluorescence intensity was mea- sured using flow cytometry (FACSCalibur). The IgG isotype was used as a negative control. Ten thousand events were acquired for each sample. 2.11Colony formation assay Colony formation assays were carried out in semisolid methyl cellulose medium (2 × 103/ml; MethoCult 4230; StemCell Technologies Inc., Vancouver, BC, Canada) in the presence, or not, of reversine (1, 2.5, 5 and 10 μM) for 24 h (short-term) or during the overall experiment time (long-term). Alternatively, cells were seeded at a high density (10 × 103/ml or 20 × 103/ml). Colonies were detected after 10 days of culture by adding 150 μl (5 mg/ml) MTT reagent and scored using Image J quan- tification software (U.S. National Institutes of Health, Bethesda, MD, USA). 2.12Assessment of autophagy by acridine orange staining For determining acidic vesicular organelle content, 5 × 105 cells/ml were cultured in the presence, or not, of reversine (1, 2.5, 5 and 10 μM) for 24 h. Cells were washed and incu- bated with an acridine orange solution (0.1 μg/ml; Sigma- Aldrich). All specimens were analyzed by flow cytometry (FACSCalibur) after incubation for 30 min at room tempera- ture in a light-protected area, according to published recom- mendations [29]. Ten thousand events were acquired for each sample. 2.13PCR array analysis Total RNA from Namalwa cells treated with reversine (10 μM) for 24 h was obtained using TRIzol reagent (Thermo Fisher Scientific). cDNA was synthesized from 2 μg RNA using a RT2 First Strand Kit (Qiagen Sciences Inc., Germantown, MD, USA). PCR array analysis was per- formed using a Human Autophagy RT2 Profiler PCR Array kit (#PAHS-084ZA; Qiagen Sciences Inc.) according to the manufacturer’s instructions. mRNA levels were normalized to those in untreated cells, and genes that presented a ≥ 2-fold change in expression in any direction were included in the heatmap using multiple experiment viewer (MeV) 4.9.0 soft- ware. Amplification was performed using an ABI 7500 Sequence Detector System (Thermo Fisher Scientific). 2.14Statistical analysis Statistical analyses were performed using GraphPad Instat 5 (GraphPad Software, Inc., San. Diego, CA, USA). For com- parisons, ANOVA and Bonferroni post-test, Kruskal-Wallis test and Dunn’s post-hoc test, or Student t-test were used. A p value < 0.05 was considered statistically significant. 3Results 3.1AURKB is highly expressed in acute lymphoblastic leukemia Firstly, we verified the expression of AURKA and AURKB in lymphocytes from healthy donors and lymphoblasts from ALL patients. We found that the AURKB, but not AURKA, mRNA levels were increased in malignant compared to nor- mal lymphocytes, especially in T-ALL (Fig. 1a-b, p < 0.05). In a panel containing B-ALL (Karpas 442, Daudi, Namalwa and RS4;11) and T-ALL (MOLT-4, Jurkat and CEM) cell lines, AURKA and AURKB mRNA and protein levels, as well as the AURKB activity marker p-Histone H3S10, were increased compared to a pool of normal lymphocytes (Fig. 1c-e). 3.2Reversine promotes apoptosis in Jurkat and Namalwa cells Next, we verified the effects of reversine on the viability in B- ALL (Namalwa) and T-ALL (Jurkat) cell models. We found that reversine reduced the viability in dose- and time- dependent manners in both cell models (Fig. 2a). Of note, reversine reduced the viability of all ALL cell lines (IC50 range 2.3–12.6 μM; Supplementary Table 3) and primary ALL cells (n = 4, IC50 range 5.5–24.4 μM; Supplementary Fig. 1) tested, but did not reduce the viability of normal lym- phocytes (IC50 > 50 μM; Supplementary Table 3). To define the events involved in the reduction of ALL cell viability upon reversine treatment, multiple cellular assays were performed. After 24 h of exposure to reversine, Jurkat and Namalwa cells exhibited significant levels of apoptosis only at higher con- centrations (p < 0.05, Fig. 2b-c). Increased exposure times to reversine (5 μM) significantly induced apoptosis in both ALL cell lines tested (Supplementary Fig. 2). 3.3Reversine induces cell cycle arrest at the G2/M phase and decreases AURKB activity We found that low concentrations of reversine induced cell cycle arrest at the G2/M phase (p < 0.05; Fig. 3a-b) and in- creased cell sizes (p < 0.05; Fig. 3c) in both ALL cell lines, which was followed by downregulation of AURKA expres- sion and AURKB activity (Fig. 3d). In Jurkat cells, an in- creased subG1 cell population was observed upon reversine treatment (10 μM), in agreement with the apoptosis data. Low concentrations of reversine upon increased time exposures (48 and 72 h) augmented the subG1 population in Jurkat cells and induced intense polyploidy in Namalwa cells, which suggests that mitotic catastrophe may be a time-dependent sub-route to apoptosis in this context (Supplementary Fig. 3). In addition, DNA damage (increased γH2AX expression) and comet cells (increased tail moment, p < 0.0001), were observed upon reversine exposure (Fig. 3d-e), underscoring mitotic catastro- phe in both ALL cell lines. 3.4Reversine decreases ALL cell proliferation and autonomous clonal growth In Jurkat and Namalwa cells, we found that short-term exposure to reversine significantly reduced cell prolifer- ation (p < 0.05; Fig. 4a-b). Next, autonomous clonal growth was assessed. Notably, short- (24 h) and long- term (10 days) reversine treatment strongly decreased a AURKA expression in ALL patients b AURKB expression in ALL patients 800 600 1000 800 *** *** *** 600 400 400 200 0 NL B-ALL T-ALL 200 0 NL B-ALL T-ALL n=12 n=82 n=54 n=12 n=82 n=54 c AURKA expression in ALL cell lines d AURKB expression in ALL cell lines 40 30 50 40 30 20 20 10 0 10 0 Normal PBMCKarpas-422 DaudiNamalwa RS4;11 MOLT4 Jurkat CEM B-ALL T-ALL Normal PBMCKarpas-422 DaudiNamalwa RS4;11 MOLT4 Jurkat CEM B-ALL T-ALL e IB: AURKA (46 kDa) IB: AURKB (39 kDa) IB: p-Histone H3S10 (15 kDa) IB: GAPDH (37 kDa) Normal B-ALL T-ALL Fig. 1 AURKB is highly expressed in ALL patient samples and cell lines. (a) AURKA (208079_s_at) and (b) AURKB (209464_at) mRNA levels in normal lymphocytes, B-ALL and T-ALL samples. The “y” axis represents gene expression data obtained from the Amazonia! database 2008 (http://amazonia.transcriptome.eu), which were measured using Affymetrix HGU133 plus 2.0 arrays. Data sets were cross-referenced using tumor-specific identification numbers. Numbers of subjects for each group are indicated. ***p < 0.0001; Kruskal-Wallis test and Dunn’s post-hoc test. AURKA (c) and AURKB (d) mRNA expression in normal peripheral blood mononuclear cells (PBMCs), and B-ALL (Karpas 442, Daudi, Namalwa and RS4;11) and T-ALL (MOLT-4, Jurkat and CEM) cells evaluated by qRT-PCR. PBMC was used as calibrator sample. GAPDH and 18S rRNA were used as reference genes. (e) Western blot analysis of AURKA, AURKB and phospho (p)-histone H3S10 in total extracts from PBMC, B- ALL and T-ALL cells, as indicated. The membranes were re-probed with an antibody directed against GAPDH, and developed using a SuperSignal™ West Dura Extended Duration Substrate system and a G: BOX Chemi XX6 gel doc system clonogenicity in both ALL cell lines (p < 0.001; Fig. 4c- d), even at high cell concentrations (Supplementary Fig. 4). These data indicate that reversine exposure during the first mitosis leads to an irreversible decrease in cell proliferation. 3.5Autophagic cell death is triggered by reversine in ALL cells Recent studies have reported that reversine may induce au- tophagy in solid tumors [15–18], but the molecular a 100 80 60 40 20 Jurkat cells 100 80 60 40 20 *** ** *** *** *** Namalwa cells *** *** *** *** *** *** 24hours 48 hours 72 hours *** *** *** *** *** *** IC50 8.4 ti M 5 ti M 5 ti M 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 b c Reversine Jurkat cells Namalwa cells log [Reversine (ti M)] ø 1 µM Annexin V Jurkat cells 2.5 µM 5 µM 10 µM log [Reversine (ti M)] 25µM Namalwa cells 50 µM 120 100 80 60 40 20 120 100 80 60 40 20 0 ti 1 2.5 5 10 25 50 Reversine (ti M) 0 ti 1 2.5 5 10 25 50 Reversine (ti M) Fig. 2 Reversine reduces the viability of Jurkat and Namalwa cells. a Dose- and time-response cytotoxicity analyses using a methylthiazoletetrazolium (MTT) assay for Jurkat and Namalwa cells treated with graded concentrations of reversine (1, 2.5, 5, 10, 25 and 50 μM) for 24, 48 and 72 h. Values are expressed as the percentage of viable cells for each condition relative to untreated controls. Results are shown as mean ± SD of at least four independent experiments. The p- values and cell lines are indicated in the graphs; *** p < 0.0001; ANOVA test and Bonferroni post-test. b Apoptosis detected by flow cytometry in Jurkat and Namalwa cells treated with graded concentrations of reversine (1, 2.5, 5, 10, 25 and 50 μM) for 24 h using an annexin V/ 7AAD staining method. Representative dot plots are shown for each condition. The upper and lower right quadrants (Q2 plus Q3) cumulative- ly contain the apoptotic population (annexin V+ cells). c Bar graphs representing the mean ± SD of at least four independent experiments quantifying apoptotic cell death. The p values and cell lines are indicated in the graphs; * p < 0.05, *** p < 0.0001; ANOVA test and Bonferroni post-test mechanisms involved have remained unclear. We found that in ALL cell lines reversine induced cellular and molecular markers of autophagy, including increased acidic vesicular organelle formation (p < 0.05, Fig. 5a-b) and LC3II expres- sion, as well as SQSTM1/p62 consumption (Fig. 5c). To in- vestigate the molecular mechanism involved in reversine- induced autophagy, a PCR array analysis covering key autophagy-related genes was performed. In Namalwa cells, reversine modulated 25 out of 84 autophagy-related genes (Fig. 5d, Supplementary Table 4). Based on the cellular effects observed, six genes (BCL2, BAD, ULK1, ATG10, IRGM and MAP1LC3B) were selected for validation in Jurkat and Namalwa cells (Fig. 5e). BCL2 mRNA expression was downregulated in Jurkat cells and upregulated in Namalwa cells (p < 0.05), and was confirmed by Western blotting (Fig. 5c). BAD, ULK1, IRGM and MAP1LC3B mRNA levels were found to be upregulated in both ALL cell lines (p < 0.05), whereas ATG10 mRNA expression was found to be increased in Namalwa cells (p < 0.05), but not in Jurkat cells (Fig. 5e). 4Discussion Here, we report AURKB upregulation in ALL cells and the cellular and molecular effects of reversine in ALL cell models. Fig. 3 Reversine induces G2/M phase cell cycle arrest and DNA damage, and inhibits AURKB activity in ALL cells. a Cell cycle progression determined by PI staining in Jurkat or Namalwa cells treated with reversine (1, 2.5, 5, 10 μM) for 24 h using a propidium iodide (PI) staining method. A representative histogram for each condi- tion is shown. b Bar graphs representing the mean ± SD of the percentage of cells in the G0/G1, S and G2/M phases upon reversine treatment of at least four independent experiments. The p values and cell lines are indi- cated in the graphs; * p < 0.05, ** p < 0.01, *** p < 0.001; ANOVA test and Bonferroni post-test. c Immunofluorescence analysis of Jurkat and Namalwa cells treated or not with 5 μM reversine for 24 h, showing α- tubulin (green) and DAPI (blue) staining. Scale bars are shown in the fig. (50 μm). Box plots represent the quantification of morphological findings of at least 15 cells for each condition. *** p < 0.001; Student t-test. d Western blot analysis of AURKA, AURKB, phospho(p)-histone H3S10 and p-histone H2A.XS139 (γ-H2A.X) in total cell extracts from Jurkat and Namalwa cells treated with graded concentrations of reversine (vehicle, 1, 2.5, 5 or 10 μM) for 24 h. The membranes were re-probed with antibody for the detection of the respective total proteins or α-tubulin, and devel- oped using a SuperSignal™ West Dura Extended Duration Substrate system and a G:BOX Chemi XX6 gel doc system. e Comet assay for Jurkat and Namalwa cells treated with 5 μM reversine or vehicle for 24 h. At least 150 cell comets for each condition were measured in two inde- pendent experiments. Dispersion graphs represent tail moment values (head/tail DNA ratio) obtained using LUCIA Comet Assay™ software. *** p < 0.001; Student t-test Previous studies by us and others have shown that reversine may induce apoptosis and mitotic catastrophe in myeloid neo- plasms, including acute myeloid leukemia [19], chronic mye- loid leukemia [20] and JAK2V617F-positive myeloproliferative neoplasms [21]. Reversine is known to target multiple kinases [19, 24, 30] including AURKs, which are upregulated during neoplasia, and associated with cancer development and pro- gression [ 31 ]. In ALL, AURKB was found to be overexpressed in both B-ALL and T-ALL, corroborating pre- vious findings [32] and suggesting that AURKB may be an attractive therapeutic target in this disease. Due to the importance of aurora kinases in malignant dis- orders, there is a growing interest in the development and biological testing of selective inhibitors [33, 34]. For instance alisertib, a selective AURKA inhibitor, has been tested in pediatric AML and ALL using in vitro and patient-derived xenograft models [35]. VX-680, a pan-aurora kinase inhibitor (also known as MK-0457), has been found to reduce the via- bility of the T-ALL cell lines MOLT-4 and CCRF-CEM [36]. Similarly, AZD1152, a selective AURKB inhibitor, has been found to reduce proliferation and induce G2/M cell cycle arrest in PALL-2 cells [37]. VX-680 and PHA-739358 reduced cell viability of BCR-ABL1- and BCR-ABL1T315I-expressing leu- kemia cells [38, 39], and VX-680 showed a clinical response in a Philadelphia chromosome-positive ALL patient [40]. In the current study, we found that reversine reduced via- bility without a strong induction of apoptosis in ALL cells, which differs from the myeloid neoplasms described above [20, 21]. On the other hand, similar to myeloid neoplasms, increased levels of mitotic catastrophe were observed in ALL a Jurkat cells c Jurkat cells Washout assay Namalwa cells IgG 1 5 10 120 100 80 60 40 20 *** 100 80 60 40 20 *** *** *** *** 100 80 60 40 20 b Ki-67 (M.F.I.) 0 1 5 10 Reversine ( M) Namalwa cells d 0 1 2.5 5 10 0 1 2.5 5 10 Reversine ( M) Reversine ( M) Long-term exposure assay Jurkat cells Namalwa cells IgG 1 5 10 120 100 80 60 * 100 80 60 40 100 80 60 40 40 *** 20 20 *** *** *** *** 20 *** *** *** Ki-67 (M.F.I.) 0 1 5 10 Reversine ( M) 0 1 2.5 5 10 Reversine ( M) 0 1 2.5 5 10 Reversine ( M) Fig. 4 Reversine inhibits proliferation and colony formation of Jurkat and Namalwa cells. Ki-67 mean fluorescence intensity (M.F.I.) was determined by flow cytometry after incubation of Jurkat (a) or Namalwa (b) cells with reversine (1, 5, 10 μM) for 24 h; histogram traces are illustrated. The bar graphs represent the Ki-67 M.F.I normalized to the respective untreated control cells, and results are shown as mean ± SD of at least four independent experiments. The p values and cell lines are indicated in the graphs; * p < 0.05, *** p < 0.001; ANOVA test and Bonferroni post-test. Colonies containing viable cells were detected by MTT assay after 10 days of culture of Jurkat and Namalwa cells treated with reversine (1, 2.5, 5, 10 μM) for 24 h (short-term exposure) (c) or for 10 days (long-term exposure) (d), and normalized to the corresponding DMSO-treated controls (Ø). Colony images are shown for one experi- ment and the bar graphs show the mean ± SD of at least four independent experiments. *** p < 0.0001; ANOVA test and Bonferroni post-test cells, as signified by increased polyploidy, extensive DNA damage and cell proliferation failure after the first mitotic cycle upon reversine treatment [41]. Importantly, short- or long-term exposure to reversine strongly reduced autonomous cell pro- liferation in ALL models, which is known to be associated with aggressiveness in hematological neoplasms [42, 43]. In addition, reversine did not strongly reduce the viability of lym- phocytes from healthy donors, corroborating previous data [19] and suggesting selectivity of the drug for leukemia cells. Recently, it was found that reversine may induce autoph- agic cell death in solid tumors, but the molecular mecha- nisms involved have remained poorly explored [15–18]. Indeed, doses of reversine that reduced cell viability, but did not induce apoptosis to the same extent, were able to induce high levels of autophagy in ALL cells. Through mo- lecular analysis, ULK1, IRGM and MAP1LC3B were found to be upregulated in Jurkat and Namalwa cells. ULK1 en- codes a serine/threonine-specific protein kinase that plays a critical role in the initiation stage of autophagy [44, 45], while MAP1LC3B encodes LC3B, which acts on the bio- genesis of autophagosomes [46]. IRGM interacts with ULK1 and Beclin 1, thereby promoting their co-assembly and formation of the autophagy initiation complex [47]. IRGM has been reported to act as a potent autophagy inductor in the context of intracellular pathogens [48], but its function in the autophagy of cancer cells remains poorly understood. Corroborating our results, Wang et al. [49] re- ported that IRGM may participate in the switch from apo- ptosis to autophagy in hepatocellular carcinoma cells. Taken together, these findings shed light on the molecular mechanisms involved in reversine-mediated autophagy in cancer cells, and show that reversine acts by initiating and sustaining autophagy in ALL cells. Of note, we found that BCL2 levels were reduced in Jurkat cells and increased in Namalwa cells upon reversine treat- ment, which underscores the greater sensitivity of Jurkat cells to reversine-induced apoptosis. Huang et al. [22] reported that reversine modulates the BCL2/BAX ratio to induce apoptosis in breast cancer cells. Similarly, we have reported that BCL2 downregulation is associated with sensitivity to reversine in myeloproliferative neoplasm models [21]. These data support our current findings that the BCL2 level in response to reversine predicts apoptotic response. In summary, we found that reversine reduces the viability of ALL cells by triggering multiple cell death mechanisms, including apoptosis, mitotic catastrophe and autophagy. Our findings further highlight reversine as a putative anticancer option for ALL. Fig. 5 Reversine triggers autophagy in ALL cells. a Acidic vesicular organelles detected by flow cytometry in Jurkat and Namalwa cells treated with graded reversine concentrations (vehicle [Ø], 1, 2.5, 5, 10 μM) for 24 h using acridine orange staining. Representative dot plots are shown for each condition. The gate “AVO” contains cells positive for the FL-3 channel, which are characterized by increased acidic vesicular organelle formation. b Bar graphs representing the mean ± SD of the percentage of “AVO” upon reversine treatment of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; ANOVA test and Bonferroni post-test. c Western blot analysis of LC3BI/II, p62/SQSTM1 and BCL2 in total extracts from Jurkat and Namalwa cells treated with graded concentrations of reversine for 24 h. The membranes were re-probed with antibody for the detection of α- tubulin, and developed using a SuperSignal™ West Dura Extended Duration Substrate system and a G:BOX Chemi XX6 gel doc system. d Expression of autophagy-related genes with ≥2-fold change in either di- rection in reversine-treated Namalwa cells (10 μM) compared to vehicle- treated cells in a heatmap. Two independent experiments of each condi- tion were used for the analysis. Green indicates decreased mRNA levels and red increased mRNA levels. e BCL2, BAD, ULK1, ATG10, IRGM and MAP1LC3B mRNA expression in Jurkat and Namalwa cells treated with reversine (10 μM; n = 4). Samples from vehicle-treated cells were used as calibrator and are represented by the dashed line. HPRT1 and ACTB were used as reference genes. The p values and cell lines are indicated in the graphs. ** p < 0.01, *** p < 0.001; Student t-test Acknowledgments The authors thank Prof. Carlos Frederico Martins Menck for providing assistance with the comet assay and Fernanda T. Udinal, from the Hemocentro Foundation of Ribeirão Preto, São Paulo, Brazil, for English language review. Author contributions J.A.E.G.C. and K.L. conception and design, data analysis and interpretation, and manuscript writing. J.L.C-S., R.M.A-P., N.C.M., H.P.V. and F.P.S.S. conception and design, data analysis and interpretation, and manuscript editing. N.H.,L.V.C-L. and F.T. data anal- ysis and interpretation, and manuscript writing. J.A.M-N. conception and design, data analysis and interpretation, manuscript writing and final ap- proval of the manuscript. All authors read and approved the final manu- script. 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