Ethical approval
The institutional review board from the School of Pharmaceutical Sciences of Ribeirão Preto (FCFRP-USP) and Ribeirão Preto Medical School Hospital (HC-FMRP-USP) at University of São Paulo approved the study protocol (CAAE 30901714.3.3001.5440). All patients and healthy subjects signed an informed written consent form that complied with the Brazilian National Health Council guidelines (law 466/2012).
Subjects
This study enrolled ET, PMF, and PV patients followed at HC-FMRP-USP and at Euryclides de Jesus Zerbini Transplant Hospital, São Paulo-SP, Brazil. For peripheral blood, ET patients’ group was composed of 35 individuals (9 males, 26 females) with mean age of 59 years: 15 patients harbored JAK2V617F mutation and 9 patients harbored CALR mutation. The PMF patients’ group was composed of 22 individuals (15 males, 7 females) with mean age of 62 years: 12 patients harbored the JAK2V617F mutation and 5 harbored the CALR mutation. The PV patients’ group was composed of 31 individuals (14 males, 17 females) with mean age of 65 years: 30 harbored the JAK2V617F mutation and one was negative for all the three mutations. The control group comprised 60 healthy individuals (21 males, 39 females) with mean age of 56 years, who lived in Ribeirão Preto, State of São Paulo, Brazil (Table S1).
CD34+ gene expression in bone marrow cells was analyzed in some samples from patients who also donated peripheral blood. The ET group was composed of 20 patients (7 males, 13 females) with mean age of 58 years; the PMF group was composed of 7 patients (6 males, 1 female) with mean age of 65 years; the PV group was composed of 15 patients (8 males, 7 females) with mean age of 64 years; and the control group was composed of 15 healthy bone marrow donors (8 males, 7 females) with mean age of 54 years (Table S2).
Blood samples
The patients’ and healthy subjects’ peripheral blood was collected using EDTA tubes (Vacutainer®; Becton, Dickinson, and Company). The patients’ blood samples were collected at the time of diagnosis, based on the WHO 2016 criteria; therefore, none of them were under therapy at the time of blood collection. Exclusion criteria for controls were the presence of neoplasms, autoimmune diseases, viral infections, HIV or diabetes.
Clinic-hematological data
The red blood cell count (RBC), white blood cell count (WBC), platelet number, hemoglobin concentration (Hb), and hematocrit percentage (Ht) were determined using the automatic equipment ABX Micros 60 (HORIBA ABX SAS). Data from hematological parameters are reported in Table S1. Clinical data as LDH, thrombotic events, blasts percentage and spleen size were obtained from the medical record.
Isolation of CD34+ hematopoietic precursor cells
Five mL of bone marrow samples were collected from patients and healthy donors. The bone marrow mononuclear cells were isolated using the Ficoll-Hypaque (Sigma-Aldrich) method. After washing with 0.05% albumin (Sigma-Aldrich) in PBS-ACD buffer, the cells were suspended in the same buffer and centrifuged. Then, the cell pellets were sequentially treated with Fc-blocking antibody (MidiMACS® - Miltenyi Biotech) and the primary anti-CD34-hapten antibody (MidiMACS® - Miltenyi Biotech). The cells were incubated at 4 °C for 20 minutes in the dark, diluted with 30 mL of PBS-ACD-albumin buffer, and centrifuged at 240 ×g, for 10 minutes, at 4 °C.
Next, the secondary anti-hapten antibody (MidiMACS® - Miltenyi Biotech) conjugated with microbeads was added to the cell pellet, followed by incubation at 4 °C for 20 minutes in the dark. The suspension was diluted with 20 mL of PBS-ACD-albumin buffer and centrifuged at 240 ×g, for 10 minutes, at 4 °C. The cell pellet was suspended in 10 mL of PBS-ACD-albumin buffer, and applied on the magnetic column. The column was washed twice with the same buffer, far from the magnetic support, to release the CD34+ cells. The eluate was centrifuged at 240 ×g, for 10 minutes, at 4 °C, the cell pellet was suspended in RPMI 1640 medium (Gibco), and the cells were counted in a Neubauer chamber. The purity of the CD34+ population was analyzed by flow cytometry; samples with purity above 85% were considered suitable for use. The concentration of bone marrow hematopoietic cells was adjusted to 2×105 in 500 µL of Trizol® (Invitrogen Life Technologies®) and the suspension was frozen for further RNA extraction.
Isolation of peripheral blood leukocytes from patients and control subjects
Isolation of peripheral blood leukocytes from patients and control subjects relied on the principle of density difference among leukocytes, red blood cells, and platelets using Voluven (Fresenius) [19]. The protocol consists of adding four parts of whole blood to one part of Voluven, followed by 90 minutes of incubation. The supernatant was collected and centrifuged at 400 ×g for 15 minutes. Cells were suspended in phosphate-buffer saline (PBS; pH = 7.4), diluted in 0.4% trypan blue (Sigma-Aldrich), and counted in Neubauer chamber. Leukocyte suspension at 1×107 cells/mL was frozen in Trizol® (Invitrogen Life Technologies®) for further RNA extraction.
Cell culture
The human cell line HEL.92.1.7 (erythroblastic cell line - HEL.92.1.7 - ATCC® TIB-180™) harbors the JAK2V617F mutation. HEL.92.1.7 cells were cultured in RPMI 1640 medium (Invitrogen Life Technologies®) supplemented with 10% fetal bovine serum, 1% glutamine, 1% penicillin, and 1% streptomycin (Gibco®) under an atmosphere of 5% CO2, at 37 °C.
Cell treatment with JAK inhibitor I
HEL.92.1.7 cells (5×106/mL) were treated with 10 µM of JAK Inhibitor I – an ATP-competitive inhibitor of JAK1, JAK2, JAK3, and TyK2 (Calbiochem, EMD Millipore Corp, Billerica, MA, USA) – for (i) 6, 12 and 24 hours for Hippo pathway and apoptosis gene expression assays; (ii) 48 hours for annexin-V assay; and (iii) 24, 48 and 72 hours for cell proliferation assay. Next, part of the cell suspension was used in cell death and proliferation assays, and part was stored in Trizol® for RNA extraction and qPCR assays.
Cell death quantification
After JAK Inhibitor I treatment, 1×105 HEL.92.1.7 cells per tube were diluted with 500 µL of PBS and centrifuged at 240 ×g for 10 minutes, at 4 ºC. The supernatant was discarded, the cell pellets were suspended in 100 µL of annexin-binding buffer (250 µg/mL), and incubated for 20 minutes, in the dark, at 4 °C. The cells were washed three times with PBS and suspended in 200 µL of binding buffer containing 1 µL of propidium iodide. The percentage of apoptosis (annexin-V-FITC+ cells) was determined in a flow cytometer (FACSCanto, BD), based on acquisition of 10,000 events. The cell death rate was expressed as percentage of annexin-V-FITC-labeled cells.
Analysis of cell proliferation
To quantify cell proliferation rate, HEL.92.1.7 cells were labeled using the CellTrace Violet Cell Proliferation Kit (CTV - ThermoFisher Scientific). Cells (1×107 in 2 mL of PBS) were labeled with 1 µL of 5 µM CTV staining solution and incubated for 30 minutes, at 37 °C, under 5% CO2. The cell suspension was diluted with 5 mL of RPMI and incubated for 1 minute, at 37 °C, to inactivate unbound CTV. Then, cells were washed three times with RPMI, centrifuged (1500 ×g, 4 °C, 5 minutes), suspended in 1 mL of RPMI, distributed into 6-well plates, and treated with JAK inhibitor I for 24, 48, and 72 hours. Finally, the fluorescence intensity of CTV-labeled cells was measured in a flow cytometer (FACSCanto, BD), based on acquisition of 10,000 events to quantify cell proliferation. The results were expressed as fluorescence intensity and plotted as histograms. Cell proliferation was inversely proportional to CTV labeling.
Transient expression of LATS2 gene
The plasmid pClneoMyc-LATS2 (Addgene catalog number: #66852) was used for LATS2 expression (Bao et al., 2011). Hel 92.1.7 cells were cultured in 75 cm2 flasks (as described above), harvested, centrifuged (400 ×g, 5 minutes, 4 °C), and washed with PBS. The cells were counted in Neubauer chamber and normalized to 1×107/mL. The cells were pelleted (1×107/plasmid) and suspended in 5 mg of pClneoMyc-LATS2 in RPMI. The cell suspensions were transferred to 4-mm sterile gap cuvettes and pulsed (250 V; 950 mF) in a BioRad GenePulser Xcell [20,21]. As control, cells were transfected with pcDNA3.1 (ThermoFisher, catalog number: #V790-20) under the same conditions. After transfection, the cells were transferred to 75 cm2 flasks and maintained in RPMI supplemented with 10% bovine fetal serum for 72 hours, at 37 ˚C, and under 5% CO2. The overexpression of LATS2 was confirmed by RT-PCR (Figure S1). After 72 hours of culture, the transfected cells were harvested and treated with 10 µM of JAK inhibitor I for (i) 48 hours to analyze cell death rate; and (i) 24, 48, and 72 hours to examine whether LATS2 overexpression affected cell proliferation.
RNA extraction and cDNA synthesis
The lysed cells in Trizol were treated with 10 μL glycogen (20 μg/μL) for 5 minutes, at room temperature. Then, 300 μL of cold chloroform was added to the mixture and the sample was shaken for 15 seconds. After centrifugation (12,000 ×g, 15 minutes, 4 °C), the aqueous phase was transferred to a new tube and mixed with 500 μL of cold isopropanol. The mixture was stored at - 20 °C for 18 hours and centrifuged (12,000 ×g, 15 minutes, 4 °C). The supernatant was discarded and the precipitate was washed with 70% cold ethanol and centrifuged (12,000 ×g, 10 minutes, 4 °C). The supernatant was discarded and the pellet (RNA) was suspended in DNAse- and RNAse- free water. RNA was quantified using the NanoVue Spectrophotometer (GE Healthcare) at 260 nM.
Reverse transcription reactions were performed using 1 μg of RNA and the High Capacity cDNA reverse transcription (Applied Biosystems) assay kit. Polymerization of the novel complementary DNA strands (cDNA) was performed in a thermocycler apparatus (Mastercycler, Eppendorf) at 25 ºC, for 10 minutes, to promote primer annealing, followed by a 2-hour step at 37 ºC for the extension of the novel DNA strands.
Quantification of target genes by real-time PCR
Real-time PCR assays were performed on the StepOnePlus (Thermo Fisher) using the TaqMan gene expression assay kit (Applied Biosystems®). The Hippo pathway probes used are described in Table S3. As the TaqMan probe (Hs00371735_m1) did not detect the YAP1 gene, we used the Sybr green master mix kit (Thermo Fisher) and the primer pair sequence employed for RNA quantification described in Table S4. The Sybr green master mix kit (Thermo Fisher) was also used to analyze pro- and anti-apoptotic gene expression. The oligonucleotide sequences employed in amplification of the target genes are described in Table S4. The gene expression results were reported as relative expression unit (REU = 10000 / 2DCt) for patients or Fold Change for cell line. The DCT = CT of the target gene - geometric mean of the reference genes CTs. REU = 10000 / 2deltaCt.
Statistical analyses
The groups were compared using analysis of variance (ANOVA) followed by Tukey's post-test for multiple comparisons. These models assume that their residuals have a normal distribution with a mean of 0 and a constant σ² variance. Transformation in the response variable were used when this assumption was not observed. All the graphs presented were plotted using the R software, version 4.0.4, while data analysis was performed using the SAS 9.4 software. For all comparisons, a significance level of 5% was adopted and correlation analyses were performed using the Pearson’s correlation test. Wild type (WT), MOCK, and LATS2 differences were compared using ANOVA, with the aid of the GraphPad Prism 8.0 software (Graph-Pad Software, San Diego, CA, USA). Statistical differences were considered significant when p < 0.05.