Differential expression of hydroxyurea transporters in normal and polycythemia vera hematopoietic stem and progenitor cell subpopulations

•Hydroxyurea downregulates proliferation gene sets in PV HSC/MPPs but not CMP/MEPs.•Twenty-nine of 57 solute carrier transporters are expressed and CMP/MEPs.•The HU transporter OCTN1 is upregulated untreated patients.•The diffusion facilitator UTB downregulated compared with CMP/MEPs. Polycythemia vera (PV) a myeloproliferative neoplasm marked by hyperproliferation the myeloid lineages presence an activating JAK2 mutation. Hydroxyurea (HU) standard treatment for high-risk patients PV. Because disease-driving mechanisms thought to arise stem cells, effective treatments should target primarily cell compartment. We tested antiproliferative effect patient fluorescence-activated sorting-isolated hematopoietic stem/multipotent progenitor cells (HSC/MPPs) more committed erythroid progenitors (common myeloid/megakaryocyte–erythrocyte [CMP/MEPs]) using RNA-sequencing set enrichment analysis. led significant downregulation associated HSCs/MPPs, To explore mechanism underlying this finding, we assessed expression membrane transporters, which mediate transmembrane movement drugs such as into cells. The active uptake was CMP/MEPs PV, urea B (UTB) all control groups tested. 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Fresh peripheral samples were collected treated HU, controls phlebotomy-requiring hemochromatosis during clinical routine appointments (Department Medical Oncology Hematology, University Hospital Zurich, Switzerland) (Table 1). All informed consent, approved responsible local ethics committee (Kanton Switzerland).Table 1Characteristics controlsGroupSubgroups combinedPatient IDAgeSexDiagnosisNo. collectedJAK2 V617F allele burden HSC/MPPs/ based RNA-seq dataJAK2 granulocytes ddPCRTherapyPVPVchronUTPV5*Patient previously (stopped because side effects).59MPV220%/70%43%ASAPVPVchronUTPV653MPV570%/100%93%ASAPVPVchronUTPV868FPV10%/80%ASA, BP, statinPVPVchronUTPV1355MPV5100%/100%78%ASA, BPPVPVchronUTPV1444FPV1n.a./100%97%ASA, anti-depressantPVPVchronUTPV1650MPV60%/50%43%ASAPVPVprogUTPV1764MPost-PV MF180%/100%BP, anti-uric, prednisonePVPVprogUTPV1872FPV transforming MF2100%/100%98%ASA, anti-uricPVPVchronHUPV265MPV80%/0% (GMPs: 20%)HU, ASA, statinPVPVchronHUPV377MPV980%/50%95%HU, BPPVPVchronHUPV457MPV70%/n.a.4%HU, BPPVPVchronHUPV560MPV545%/25%43%HU, ASAPVPVprogHUPV1065MPV AML575%/70%HU, ASAPVPVprogHUPV1161MPost-PV MF160%/100%99%HU, ASAPVPVprogHUPV1582MPost-PV MF1100%/50%HU, phenprocoumon, anti-uricControlControlCON171MHemochromatosis80%/0%BPControlControlCON242MHemochromatosis30%/0%NoneControlControlCON266MHemochromatosis30%/0%ASA, anti-depressantControlControlCON261FHemochromatosis10%/0%InhalControlControlCON240MHemochromatosis20%/0%noneControlControlCON354MHemochromatosis90%/0%ASA, anti-diabeticControlControlCON445MHemochromatosis30%/0%ThyroxineControlControlCON460FHemochromatosis30%/0%BP, HRTControlControlCON563MHemochromatosis80%/0%StatinAML=Acute leukemia; anti-uric=uric acid-lowering medication; ASA=acetylsalicylic acid; BP=blood pressure F=female; GMP=granulocyte–macrophage progenitors; HRT=hormone replacement therapy; HU=under hydroxyurea; Inhal=inhalative M=male; MF=myelofibrosis; n.a.=not available; UT=untreated. Patient effects). Open table new tab AML=Acute obtain required numbers different subpopulations, 0.4–3.6 L had individual consecutive settings periods few months. volumes cannot retrieved donors, recruited physically receive regular phlebotomies subjects. Human cell-enriched subfractions isolated sorting (FACS) described [22Amon Meier-Abt Gillet LC al.Sensitive proteomics data-independent acquisition mass spectrometry.Mol Cell Proteomics. 18: 1454-1467Abstract (20) Scholar] depicted Figure 1. brief, mononuclear Ficoll gradient centrifugation (VWR, Dietikon, Switzerland). CD34+ then enriched immunomagnetic beads according manufacturer's instructions (CD34 MicroBead Kit; Miltenyi Biotec, Bergisch Gladbach, Germany) viably frozen. After thawing, FACS CD34+-enriched anti-hCD34, anti-hCD38, anti-hCD123, anti-hCD45RA, 12 antibodies markers (anti-hCD2, anti-hCD3, anti-hCD4, anti-hCD7, anti-hCD8, anti-hCD10, anti-hCD11b, anti-hCD14, anti-hCD19, anti-hCD20, anti-hCD56, anti-hCD235a). complete list used provided Supplementary Table E1 (online only, available www.exphem.org). Up 10,000 sorted RNeasy lysis buffer (Qiagen, Hilden, β-mercaptoethanol subsequent experiments.Supplementary 1AntibodyCloneProviderOrder numberTricolor/phycoerythrin (PE)-Cy5-conjugated anti-hCD2S5.5ThermoFisherScientific-InvitrogenCD0206Tricolor/phycoerythrin anti-hCD37D6ThermoFisherScientific-InvitrogenMHCD03065Tricolor/phycoerythrin anti-hCD4S3.5ThermoFisherScientific-InvitrogenMHCD0406Tricolor/phycoerythrin anti-hCD7CD7-6B7ThermoFisherScientific-InvitrogenMHCD0706Tricolor/phycoerythrin anti-hCD83B5ThermoFisherScientific-InvitrogenMHCD0806Tricolor/phycoerythrin anti-hCD14TuK4ThermoFisherScientific-InvitrogenMHCD1406Tricolor/phycoerythrin anti-hCD19SJ25-C1ThermoFisherScientific-InvitrogenMHCD1906Tricolor/phycoerythrin anti-hCD56MEM-188ThermoFisherScientific-InvitrogenMHCD5606Phycoerythrin (PE)-Cy5 anti-hCD10HI10aBioLegend312206Phycoerythrin anti-hCD11bICRF44BioLegend301308Phycoerythrin anti-hCD202H7BioLegend302308Phycoerythrin anti-hCD235aGA-R2BD Biosciences559944PE-Cy7-conjugated anti-hCD348G12BD Biosciences348811FITC-conjugated anti-hCD38HIT2BD Biosciences555459APC-conjugated anti-hCD1236H6ThermoFisherScientific-Invitrogen17-1239-42APC780-conjugated anti-hCD45RAHI100ThermoFisher Scientific-Invitrogen47-0458-41 Total RNA purified Plus Micro Kit Germany). sequencing delineated Picelli al. [23Picelli Faridani OR Björklund Winberg Sagasser Sandberg Full-length Smart-seq2.Nat Protoc. 9: 171-181Crossref (1334) NovaSeq platform (Illumina, San Diego, CA). Adapters low-quality tails trimmed reads before mapped transcriptome. STAR aligner (Version 2.6.1.c) [24Dobin Davis CA Schlesinger al.STAR: ultrafast universal aligner.Bioinformatics. 29: 15-21Crossref (12267) employed align data Ensembl Release 91 reference genome build GRCh38.p10. Gene values determined featureCounts Bioconductor package Rsubread 1.32.4) [25Liao Y Smyth GK Shi Subread aligner: fast, accurate scalable read mapping seed-and-vote.Nucleic Acids 41: e108Crossref (1148) Differential calculated DESeq2 1.22.2) [26Love MI Huber Anders Moderated estimation fold change dispersion DESeq2.Genome Biol. 15: 550Crossref (19332) followed GATK Best Practices Workflow [27Van der Auwera GA Carneiro MO Hartl al.From FastQ high confidence variant calls: Genome Analysis Toolkit best practices pipeline.Curr Protoc Bioinformatics. 43 (11.10.1–11.10.33)Crossref (2524) Mapped duplicates marked, split, base quality recalibrated. Variants called HaplotypeCaller 4.0.8.1) annotated VEP [28McLaren Gil Hunt SE predictor.Genome 17: 122Crossref (1761) estimated its burden. Granulocytes 150 mM NH4Cl/10 KHCO3/0.1 Na2EDTA remove contaminating erythrocytes. Genomic DNA extracted QIAamp Mini (Qiagen) instructions. Droplet digital PCR (ddPCR) JAK2-V617F frequency determination BioRad QX200 ddPCR system assay dHsaMDV2010061 (BioRad, Hercules, CA) protocol. analysis Ontology Consortium database (www.geneontology.org) May 26, 2019. Ranked lists built normalized filtered transcriptome log2 (fold change) ranking criterion. Only RNAs least half replicates both comparison considered. run preranked Version 4.1.0 software (http://www.broadinstitute.org/gsea) default [29Subramanian Tamayo Mootha VK al.Gene analysis: knowledge-based approach interpreting genome-wide profiles.Proc 102: 15545-15550Crossref (19712) Significance defined stringently false discovery rate (FDR) <0.05. Isolated mRNA reverse transcribed SuperScript IV VILO Master Mix ezDNase enzyme (SuperScript Vilo Enzyme, ThermoFisher Scientific, Waltham, MA) Quantitative cDNA employing Taqman probes master mix (TaqMan Fast Advanced Mix, Scientific). Individual included Hs00998199_m1 (UTB, Hs00268200_m1 (OCTN1, Hs99999903_m1 (ACTB) (ThermoFisher ACTB housekeeping gene. Expression derived ΔCt approach. Statistical significance between two-way variance fitted mixed model analyses, correcting multiple testing Tukey method, t tests Holm-Sidak method correction testing. Regression analyses ordinary least-squares estimation. Principal component FPKM (fragments per kilobase transcript million reads) prcomp function R. transcriptomics have deposited omnibus (GEO) repository accession number GSE145802. study molecular undifferentiated transcriptomic profiles 99 8 7 9 controls. Consecutive same pooled ensure adequate FACS-isolated two instances, individuals maintain constant input (10,000 cells). An average 14,272 sample identified. detailed overview characteristics distributions majority studied male (80% 78% controls). ages times collection similar (62 years 56 If diagnosed myelofibrosis leukemia 1 year after transition state time sampling, they referred (PVprog) opposed (PVchron). overall profile severe (Supplementary E1, online At collection, 15 received treatment. Thereby, only clinically responded study. (but no controls) carried Allele burdens varied 4% 100%. Different observed HSC/MPPs, CMP/MEPs, granulocytes, consistently seen differentiated examine impact pathways, ontologies G1/S mitotic cycle. Significant upregulation pathways (Figure 2), suggesting increased resulted strong highly cycle HSC/MPPs. contrast stringent cutoff FDR = 0.05, 2). summary, negative (CMP/MEPs). Possible reasons include transporters. 3). Among studied, 29 normal samples. Most equally Exceptions monocarboxylate MCT4, peptide PEPT2, organic OCTN1, PEPT2 being MCT4 known OCTN2, UTB, and, reduced extent, neither nor (Figures 3 4). Detailed explanations E2 www.exphem.org) http://slc.bioparadigms.org/.Figure 4Expression patient/control As correlated hematocrit, examined PVchron.UT ≥10% above lower limit. independently patients. validated qPCR (see E2). *Adjusted p < 0.05. **Adjusted 0.01. ***Adjusted 0.001. Error bars represent errors.View Large Image ViewerDownload Hi-res image Download (PPT)Supplementary 2Suppl 2Gene nameFull nameProtein protein nameFunction (as UniProtKB/Swiss-Prot)SLC2A4Solute Carrier Family 2 Member 4GLUT4Glucose Transporter Type 4Insulin-regulated facilitative glucose transporterSLC2A5Solute 5GLUT5Glucose 5Fructose transporterSLC5A8Solute 5 8SMCT1Sodium-Coupled Monocarboxylate 1Electrogenic sodium-coupled transport monocarboxylatesSLC5A12Solute 12SMCT2Sodium-Coupled 2Electroneutral monocarboxylatesSLC6A6Solute 6 6TAUTTaurine TransporterSodium-dependent taurine beta-alanine transporterSLC7A5Solute 5LAT1L-Type Amino Acid 1Sodium-independent large neutral amino acidsSLC10A1Solute 10 1NTCPNa+/Taurocholate Cotransporting PolypeptideHepatic sodium/bile-acid transporterSLC10A2Solute 2ASBTApical Sodium-Dependent Bile reabsorption bile acids small intestineSLC14A1Solute 14 1UTBUrea 1Urea channel facilitates down concentration gradientSLC14A2Solute 2UTAUrea 2Specialized low-affinity vasopressin-regulated transporterSLC15A1Solute 1PEPT1Peptide 1Proton-coupled intake oligopeptides 4 acidsSLC15A2Solute 2PEPT2Peptide 2Proton-coupled acidsSLC15A3Solute 3PHT2Peptide/Histidine 2Proton oligopeptide cotransporterSLC15A4Solute 4PHT1Peptide/Histidine 1Proton cotransporterSLC16A1Solute 16 1MCT1Monocarboxylate transporterSLC16A3Solute 3MCT4Monocarboxylate 3Proton-linked transporterSLC16A7Solute 7MCT2Monocarboxylate transporterSLC19A1Solute 19 1RFCReduced Folate ProteinTransporter mediates import folates subset cyclic dinucleotidesSLC22A1Solute 22 1OCT1Organic Cation 1Translocates broad array cations wi