BIRC5 (median of probes 202,094_at and 202,095_s_at) and XIAP (median of probes 206,536_s_at, 206,537_at, 225,858_s_at, 225,859_at, 235,222_x_at, and 235,222_x_at) mRNA expression data from blood samples of healthy donors (n = 21), and ET (n = 19), PV (n = 41), and PMF patients (n = 9) were obtained from GEOR2 (https://www.ncbi.nlm.nih.gov/geo/geo2r/; GEO accession GSE26049).

HEL cells were kindly provided by Prof. Dr. Sara Teresinha Olalla Saad (Hemocentro, University of Campinas, Campinas, Brazil). Additionally, SET2 cells were kindly provided by Prof. Dr. Fabíola Attié de Castro (School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil). SET2 and HEL cells harboring the JAK2V617F mutation were tested and authenticated by short tandem repeat (STR) matching analysis using the PowerPlex® 16 HS system (Promega, Madison, WI, USA) and the ABI 3500 Sequence Detector System (Life Technologies, Foster City, CA, USA). Cell culture conditions were created following the recommendations of the American Type Culture Collection (ATCC) and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). All cell lines were mycoplasma free. Primary CD34+ cells were obtained from the peripheral blood of a healthy donor using MIDI-MACS immunoaffinity columns (Miltenyi Biotec, Auburn, CA, USA). Samples were obtained after obtaining informed consent and the study protocol was approved by the Ethics Committee of the Institute of Biomedical Sciences of the University of São Paulo (CAAE:39,510,920.1.0000.5467) following the Declaration of Helsinki. YM155 was obtained from Cayman Chemical (Ann Arbor, MI, USA) and ruxolitinib was obtained from InvivoGen (San Diego, CA, USA).

Total protein extraction was achieved using a buffer containing 100 mM Tris (pH 7.6), 1 % Triton X-100, 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM Na3VO4, 100 mM NaF, 10 mM Na4P2O7, and 4 mM ethylenediaminetetraacetic acid (EDTA). Equal amounts of protein (30 μg) from the samples were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in an electrophoresis device, followed by electrotransfer of the proteins to nitrocellulose membranes. The membranes were blocked with 5 % non-fat dry milk and incubated with specific primary antibodies diluted in blocking buffer, followed by secondary antibodies conjugated to horseradish peroxidase (HRP). Western blot analysis was performed using a SuperSignalTM West Dura extended Duration Substrate system (Thermo Fisher Scientific) and a G:BOX Chemi XX6 gel document system (Syngene). Antibodies against survivin/BIRC5 (#2808S), phospho(p)-ribosomal protein S6 (RPS6; #4858), RPS6 (#2217), p-ULK1 (#14,202), ULK1 (#8054), SQSTM1/p62 (#88,588), LC3B (#2775), PARP1 (#9542), γH2AX (#9718), actin (#8456), and α-tubulin (#2144) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against XIAP (ab28151) were obtained from Abcam (Cambridge, MA, USA). Band intensities were determined using UN-SCAN-IT gel 6.1 software (Silk Scientific; Orem, UT, USA).

Cell viability was measured using the methylthiazoletetrazolium (MTT) assay. HEL (2  ×  104 cells/well) and SET2 cells (4  ×  104 cells/well) were cultured in a 96-well plates in Roswell Park Memorial Institute (RPMI) medium containing 10 % or 20 % fetal bovine serum (FBS), respectively, in the presence of a vehicle or graded concentrations of YM155 (0.0032, 0.016, 0.08, 0.4, 2, 10, and 50 μM) for 24, 48, and 72 hours. Half-maximal inhibitory concentration (IC50) values were calculated using the nonlinear regression analysis of GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). For combined treatment analysis, HEL and SET2 cells were exposed to graded concentrations of YM155 (0.08, 0.16, 0.32, 0.64, 1.25, 2.5, and/or 5  μM) and ruxolitinib (3, 10, 30,100, 300, and 1000 nM) alone or in combination for 48 hours. Data are illustrated using multiple experiment viewer (MeV) 4.9.0 software (http://www.tm4.org/mev/).

Colony formation assays were performed in semi-solid methylcellulose medium (1  ×  103 cells/mL in MethoCult 4230: StemCell Technologies Inc., Vancouver, BC, Canada) in the presence of a vehicle or YM155 (0.04, 0.08, 0.15, 0.3, and 0.6 μM). After eight days (HEL cells) or twelve days (SET2 cells) of culture, colonies were detected by adding a MTT solution (5 mg/mL). Images were acquired using the G:BOX Chemi XRQ (Syngene, Cambridge, UK) and analyzed using ImageJ software (US National Institutes of Health, Bethesda, MD, USA).

Total RNA was extracted using the TRIzol reagent (Thermo Fisher Scientific) and DNA was synthesized from 1 μg RNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed using a QuantStudio 3 Real-Time PCR System in conjunction with a SybrGreen System (Thermo Fisher Scientific). HPRT1 and ACTB were used as reference genes. Relative quantification values were calculated using the 2-ΔΔCT equation.19 Expression of cell cycle-, apoptosis-, DNA-damage-, and autophagy-related genes (Supplementary Table 1) were investigated in HEL and SET2 cells upon a vehicle or YM155 (2.5 and 5 μM, respectively) exposure for 24 h. A Heatmap was drawn using the multiple experiment viewer (MeV) 4.9.0 software (http://mev.tm4.org). Differentially expressed genes that presented p-values <0.05 were used for network construction by the GeneMANIA database (https://genemania.org/).

In total, HEL and SET2 cells (1 × 105 per well) were seeded in 24-well plates in the presence of a vehicle or YM155 (0.32, 0.64, 1.25, 2.5, 5, and 10 μM) for 48 h. Next, the cells were washed with ice-cold phosphate-buffered saline (PBS) and resuspended in a binding buffer containing 1 μg/mL propidium iodide (PI) and 1 μg/mL APC-labeled annexin V. All specimens were analyzed by flow cytometry (FACSCalibur; Becton Dickinson, Franklin Lakes, New Jersey, USA) after incubation for 15 min at room temperature in a light-protected area. Ten thousand events were recorded for each sample.

In total, HEL and SET2 cells (1 × 105 per well) were seeded in 12-well plates in the presence of a vehicle or YM155 (0.32, 0.64, 1.25, 2.5, 5, and 10 μM), harvested at 48 h, fixed with 70 % ethanol, and stored at 4 °C for at least 4 h. Next, the fixed cells were stained with PBS containing 20 μg/mL propidium iodide (PI) and 10 μg/mL RNase A for 30 min at room temperature in a light-protected area. The DNA content distribution was determined using flow cytometry (FACSCalibur; Becton Dickinson, FranklinLakes, NJ, USA) and analyzed using FlowJo software (Treestar, Inc. San Carlos, CA, USA).

Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software Inc.). The Kruskal-Wallis test and Dunn post-hoc or ANOVA and Bonferroni post-test, or Student t-test, were used, as appropriate. All p-values were two-sided, and the significance level was set to 5 %.

Taking advantage of publicly available gene expression data, the expression of BIRC5 and XIAP were investigated in samples from healthy donors and from patients with ET, PV, and PMF. BIRC5 expression was significantly higher in PMF patients compared to healthy donors (p-value <0.05, Figure 1A). On the other hand, XIAP expression was reduced in MPN patients (all p-values <0.05, Figure 1A). Next, the expression of BIRC5 and XIAP were investigated in cellular models with JAK2V617F mutations. BIRC5 expression was higher in HEL and SET2 cell lines when compared to normal hematopoietic cells (10- to 20-fold), whereas XIAP expression was similar between tested samples, being slightly lower in HEL cells (Figure 1B-C). The JAK2V617F cell models used differ in respect to the disease of origin, proliferation rate, and sensitivity to JAK2 inhibitors. HEL cells are more malignant, being derived from erythroleukemia, have rapid proliferation and intrinsic resistance to JAK2 inhibitors20, while SET2 cells are derived from an AML secondary to ET, which have a lower proliferation rate and sensitivity to JAK2 inhibitors.21,22 To test whether this differential expression of BIRC5 and XIAP could be a vulnerability in MPN models, YM155, a pharmacological suppressant of survivin and XIAP (Figure 1D), was used in JAK2V617F-mutated cell lines.

Figure 1.

Survivin and XIAP are differently expressed in myeloproliferative neoplasm patients and cellular models. (A) Gene expression data was obtained from GEOR2 (https://www.ncbi.nlm.nih.gov/geo/geo2r/; GEO accession GSE26049) for blood samples from polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) patients. The median of BIRC5 (202,094_at and 202,095_s_at) and XIAP (206,536_s_at, 206,537_at, 225,858_s_at, 225,859_at, 235,222_x_at, and 235,222_x_at) data obtained for each patient was used and number of patients are indicated; *p-value <0.05, **p-value <0.01, ***p-value <0.001; Kruskal-Wallis test and Dunn's post-test. (B) qPCR analysis of BIRC5 and XIAP mRNA expression in normal CD34+, HEL, and SET2 cells. (C) Western blot analysis for survivin and XIAP in total cell extracts from normal CD34+, HEL, and SET2 cells; membranes were reprobed with the antibody for the detection of actin, and developed with the SuperSignal™ West Dura Extended Duration Substrate system using a G:BOX Chemi XX6 gel document system. (D) Chemical structure of the pharmacological survivin/XIAP suppressant YM155.

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First, the concentration- and time-dependent effects of YM155 on cell viability in JAK2V617F cells were determined. HEL cells showed greater sensitivity to YM155 when compared to SET2 cells. IC50 values for HEL cells were 48.4, 1.0, and 0.6 µM, and for SET2 cells they were 31.4, 3.8, and 3.3 µM for 24, 48, and 72 h, respectively (Figure 2A). Of note, long-term exposure to YM155 strongly reduced the capacity for autonomous clonal growth, with a > 90 % reduction at the 0.3 µM concentration in both cell lines evaluated (p-value <0.05, Figure 2B). The effects of combined treatment with YM155 and ruxolitinib on HEL and SET2 cells were also evaluated. In both cell lines, synergic, additive, or antagonistic effects were not observed in the combination of drugs, indicating that the drugs can be used together without gains or losses in individual effects (Figure 2C).

Figure 2.

YM155 reduces cell viability and autonomous clonal growth in JAK2V617F cellular models. (A) Dose- and time-response cytotoxicity was analyzed by methylthiazoletetrazolium (MTT) assay for HEL and SET2 cells treated with graded concentrations of YM155 (ranging from 0.0032 to 50 µM) for 24, 48, and 72 h. Values are expressed as the percentage of viable cells for each condition relative to vehicle-treated controls. Results are shown as the mean ± standard deviation (SD) of at least three independent experiments. (B) Colonies containing viable cells were detected by adding an MTT reagent after 8–12 days of culturing the cells in presence of vehicle or YM155 (0.04, 0.08, 0.16, 0.3, and 0.6 µM). Colony images are shown for one experiment and bar graphs show the mean ± SD of at least three independent experiments. *p-value <0.05, **p-value <0.01, ***p-value <0.001; ANOVA test and Bonferroni post-test. (C) Dose-response cytotoxicity for combined treatment was analyzed by methylthiazoletetrazolium (MTT) assay for JAK2V617F cells treated with graded concentrations of YM155 (HEL: ranged from 0.08 to 2.5 μM, SET2: ranged from 0.16 to 5 μM) and ruxolitinib (ranged from 3 to 1000 nM) alone or in combination for 48 hours, as indicated. Values are expressed as the percentage of viable cells for each condition relative to vehicle-treated controls. Results are shown as the mean of at least three independent experiments.

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Next, a panel of genes related to apoptosis, cell cycle, DNA damage, and autophagy were investigated to understand the mechanisms involved in YM155-induced cell viability reduction in JAK2V617F cells. In HEL cells, 13 of out 22 investigated genes were modulated (BCL2, BAX, PMAIP1, CCND1, CCNE1, CCNB1, CDKN1A, CDKN1B, GADD45A, MAP1LC3B, SQSTM1, BECN1, and BIRC5; all p-values <0.05; Figure 3A), whereas in SET2 cells 8 of out 22 investigated genes were modulated (CCND1, BIRC5, CCNE1, SQSTM1, BCL2, ATG5, GADD45A, and BBC3; all p-values <0.05; Figure 3A). Among the cellular and molecular processes associated with genes modulated by YM155 in HEL and SET2 cells, the most noteworthy are G1/S transition of mitotic cell cycle, G1 DNA damage checkpoint, intrinsic apoptotic signaling pathway, outer organelle membrane, serine/threonine protein kinase complex, transferring phosphorus-containing groups, signal transduction by p53 class mediator, and serine/threonine protein kinase complex (all false discovery rates (FDRs) <0.05; Figure 3B).

Figure 3.

YM155 modulates apoptosis-, cell cycle-, DNA damage- and autophagy-related genes in HEL and SET2 cells. (A) Heatmap depicting the gene expression profile of JAK2V617F cells treated with a vehicle or YM155 (HEL: 2.5 μM, SET2: 5 μM) for 24 h. The blue color on the heatmap indicates decreased mRNA levels while red indicates induced mRNA levels, which were normalized by vehicle-treated cells (n = 4). The fold-change (FC) to vehicle-treated cells, standard deviation (SD) and p-values are described, Student t-test. (B) Network for YM155 modulated genes constructed using the GeneMANIA database (https://genemania.org/) for HEL and SET2 cells. Genes significantly modulated are illustrated as crosshatched circles; the interacting genes included by modeling the software are indicated by circles without crosshatched. The main biological interactions, associated functions, and false discovery rate (FDR) q values are described in the Figure.

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Given the results obtained in the gene expression analysis, the effects of YM155 on the induction of apoptosis and cell cycle progression were investigated. In HEL and SET2 cells, YM155 induced apoptosis in a dose-dependent manner (p-value <0.05, Figure 4A). At lower concentrations, YM155 induced a cell cycle delay as seen by the increase in the percentage of cells in the G0/G1 phases (p-value <0.05). At higher concentrations, the drug-induced cell death and/or DNA damage as evidenced by the increase in cells in subG1 phase (p-value <0.05, Figure 4B). Consistent with the cell viability results, these phenotypic cellular events were more pronounced in HEL cells than in SET2 cells.

Figure 4.

YM155 induces cell death and cell cycle delay in HEL and SET2 cells. (A) Cell death was detected by flow cytometry in JAK2V617F cells treated with vehicle or with increasing concentrations of YM155 (0.32, 0.64, 1.25, 2.5, 5, and 10 μM) for 48 h using an APC-annexin V/ propidium iodide (PI) staining method. Representative dot plots are shown for each condition. The upper and lower right quadrants (Q2 plus Q3) cumulatively contain the cell death population (annexin V+ cells). Bar graphs represent the mean ± standard deviation (SD) of at least three independent experiments. The p-values and cell lines are indicated in the graphs; *** p-value <0.0001; ANOVA and Bonferroni post-test. (B) Phases of the cell cycle were determined by analyzing the DNA content by staining with propidium iodide and acquiring the data by flow cytometry following exposure of JAK2V617F cells to the vehicle or YM155 (0.32, 0.64, 1.25, and 2.5 μM) for 48 h. A representative histogram for each condition is illustrated. The mean ± SD of at least three independent experiments is represented in the bar graph. The p-values and cell lines are indicated in the graphs; * p-value <0.05; ** p-value <0.01; *** p-value <0.001, ANOVA and Bonferroni post-test.

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Finally, the effects of YM155 on drug targets (survivin/BIRC5 and XIAP) and key molecular markers of related pathways (RPS6), autophagy (ULK1, SQSTM1/p62, and LC3B), apoptosis (cleaved-PARP1) and DNA damage (γH2AX) were evaluated. In HEL, but not in SET2 cells, YM155 reduced XIAP expression. On the other hand, survivin and p-RPS6 were reduced in SET2, but not in HEL cells. In both JAK2V617F cells, YM155 induced autophagy markers, including reductions of phosho-ULK1 and SQSTM1/p62, and increases of LC3BII, which was more prominent in SET2 cells. Apoptosis and DNA damage markers were strongly induced in both cell lines (Figure 5). Together these molecular findings corroborate our exploratory data on gene expression and the observed cellular events, in addition to providing insights for the interpretation of differences in observed YM155 potency between the cellular models used.

Figure 5.

YM155 induces autophagy, apoptosis, and DNA damage markers in JAK2V617F cells. Western blot analysis for survivin/BIRC5, XIAP, phospho(p)-ribosomal protein S6 (RPS6), RPS6, p-ULK1, ULK1, SQSTM1/p62, LC3BI/II, PARP1 (total and cleaved), and γH2AX in total extracts from HEL and SET2 cells treated with a vehicle or with increasing doses of YM155 (0.62, 1.25, and 2.5 µM) for 24 h. Membranes were re-incubated with α-tubulin antibody and developed with the SuperSignal™ West Dura Extended Duration Substrate system and GBox. Relative quantification was performed using band intensities determined by UN-SCAN-IT gel 6.1 software. The expression of XIAP, survivin/BIRC5, SQSTM1/p62, and γH2AX was normalized by α-tubulin. Ratios for p-RPS6/RPS6, p-ULK1/ULK1, LC3BII/LCBI, and cleaved PARP1/full-length PARP1 are also described.

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