MYBL2SupportsDNADoubleStrandBreakRepair in Hematopoietic … · Molecular Cell Biology MYBL2SupportsDNADoubleStrandBreakRepair in Hematopoietic Stem Cells Rachel Bayley1, Daniel Blakemore1,

Molecular Cell Biology

MYBL2SupportsDNADouble StrandBreakRepairin Hematopoietic Stem CellsRachel Bayley1, Daniel Blakemore1, Laila Cancian1, Stephanie Dumon1,Giacomo Volpe1, Carl Ward1, Ruba Almaghrabi1, Jidnyasa Gujar1,Natasha Reeve1, Manoj Raghavan1,2, Martin R. Higgs1, Grant S. Stewart1,Eva Petermann1, and Paloma García1

Abstract

Myelodysplastic syndromes (MDS) are aheterogeneous group of diseases character-ized by blood cytopenias that occur as aresult of somaticmutations inhematopoieticstem cells (HSC). MDS leads to ineffectivehematopoiesis, and as many as 30% ofpatients progress to acute myeloid leukemia(AML). Themechanismsbywhichmutationsaccumulate in HSC during aging remainpoorly understood. Here we identify a novelrole for MYBL2 in DNA double-strand break(DSB) repair in HSC. In patients with MDS,low MYBL2 levels associated with and pre-ceded transcriptional deregulation of DNArepair genes. Stem/progenitor cells fromthese patients display dysfunctional DSB repair kinetics after exposure to ionizing radiation (IR). Haploinsufficiency ofMybl2 in mice also led to a defect in the repair of DSBs induced by IR in HSC and was characterized by unsustainedphosphorylation of the ATM substrate KAP1 and telomere fragility. Our study identifies MYBL2 as a crucial regulator ofDSB repair and identifies MYBL2 expression levels as a potential biomarker to predict cellular response to genotoxic treat-ments in MDS and to identify patients with defects in DNA repair. Such patients with worse prognosis may require a differenttherapeutic regimen to prevent progression to AML.

Significance: These findings suggest MYBL2 levels may be used as a biological biomarker to determine the DNA repaircapacity of hematopoietic stem cells from patients with MDS and as a clinical biomarker to inform decisions regardingpatient selection for treatments that target DNA repair.

GraphicalAbstract:http://cancerres.aacrjournals.org/content/canres/78/20/5767/F1.large.jpg.CancerRes;78(20);5767–79.�2018AACR.

IntroductionMyelodysplastic syndrome (MDS) is an age-associated

hematopoietic malignancy, characterized by abnormal blood

cell maturation and a high propensity for leukemic transforma-tion. It is a clonal disease thought to originate in the hemato-poietic stem cell (HSC; ref. 1). Effective management andtreatment of MDS is important, as early identification of patientswho are likely to progress to malignant disease allows for anoptimal therapeutic regime to be implemented. Genetic altera-tions are often present in MDS and a frequent chromosomeabnormality is del20q, whose common deleted region onlycontains 5 genes expressed in HSCs, one of which is MYBL2(2, 3). The MYBL2 gene encodes a ubiquitously expressedprotein belonging to the MYB family of transcription factorsand has been shown to form part of different protein complexessuch as the Myb-MuvB/DREAM complex (4–7), Myb–Clafi com-plex (8), and the MRN complex (9), through which it exerts itsvital role in cell-cycle regulation and maintenance of genomestability (10–14). Analysis of publicly available global geneexpression data from CD34þ-MDS patient cells (15) have

1Institute of Cancer and Genomic Sciences, College of Medical and DentalSciences, University of Birmingham, Birmingham, United Kingdom. 2Centre forClinical Haematology, University Hospitals Birmingham NHS Foundation Trust,Queen Elizabeth Hospital, Queen Elizabeth Medical Centre, Birmingham, UnitedKingdom.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Paloma García, University of Birmingham, Edgbaston,Birmingham B12 2TT, United Kingdom. Phone: 44-0-121-414-4093; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-18-0273

�2018 American Association for Cancer Research.

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confirmed that downregulation of MYBL2 expression correlateswith poor prognosis; even in patients with a normal karyotype(2, 3). This suggests that changes in MYBL2 expression couldhave significant consequences with regards to disease pathogen-esis. Furthermore, it has been demonstrated that mice with lowlevels of Mybl2 develop hematologic disorders during ageingthat closely resemble the human disease, implying that MYBL2functions as a haploinsufficient tumor suppressor gene (2, 3).However, how low MYBL2 expression contributes to MDSduring ageing remains unknown.

Given that HSCs must last for the entire lifetime of anindividual to guarantee continuous blood cell production, thisincreases the dependence of these cells on DNA repair tomaintain genomic integrity. Because HSCs are predominantlyquiescent, they are thought to primarily utilize nonhomolo-gous end joining (NHEJ) rather than homologous recombi-nation (HR) to repair DNA double-strand breaks (DSB; refs.16, 17). Although mainly error-free, NHEJ-dependent DSBrepair can also result in the generation of small genomicdeletions at the repaired break site, leading to the hypothesisthat HSCs accumulate somatic mutations over time. This isthought to precede the appearance of blood disorders such asMDS and acute myeloid leukemia (AML; refs. 18–20),although no direct link between DNA repair and the patho-genesis of MDS has been reported.

Because recent studies have shown that MDS originates fromHSCs and that MYBL2 is known to play a role in maintaininggenome stability (1, 21, 22), we hypothesized that low levelsof MYBL2 may compromise the DNA repair capacity of thecell, resulting in the accumulation of genetic alterations to asufficient level to induce HSC transformation. To test this, weused ionizing radiation (IR) in vitro to induce DNA damage inMDS patient's stem cells. Following treatment, we determinedthe ability of these cells to repair their DNA and correlated thiswith expression levels of MYBL2. To further study the role ofMYBL2 in DNA repair, we utilized a Mybl2 haploinsufficientmouse model, which is susceptible to MDS development. Ourfindings uncover a novel role for MYBL2 in regulating DSBrepair in the HSC population.

Materials and MethodsDifferential expression and reactome pathway enrichmentanalyses

To assess differential gene expression and pathway enrichmentbetween MDS samples displaying higher and lower levels ofMYBL2 expression, we used previously publishedmicroarray data(15) deposited in theNCBIGeoDataSets repository (GSE19429),and the BROAD Institute Gene Set Enrichment Analysis (GSEA)software (23, 24). Differential expression was assessed over1,000permutations and ranked according to signal-to-noise ratio.Aweighted enrichment statisticwas applied.Gene sets comprisingless than 15 geneswere excluded from the analysis (the list of geneset used in the analysis is presented as Supplementary Tables S1and S2). The adjustment of the FAB compositionwas done using amethod of random sample removal. To balance the compositionof the MYBL2hi and MYBL2lo sets, samples of specific diagnosis(for example RA or RAEB) were randomly removed when theywere found to be over-represented in a set. Four different permu-tations were performed to verify that the results were not affectedby the methodology.

Isolation and expansion of human peripheral blood cellsPeripheral blood samples from patients with MDS were

obtained in heparin-coated vacutainers. Peripheral blood mono-nuclear cells were isolated using Ficoll-Paque (GE Healthcare)and stored at�80�C. Cells were thawed and cultured for 8 days inexpansion medium as described previously (25), with the excep-tion that the base medium was StemSpan H3000 (Stem CellTechnologies). Medium was refreshed on day 3 and 6. On day 8,cells were harvested and CD34þ cells purified using microbeads(Miltenyi Biotec).

qRT-PCRFor human gene expression assays, qRT-PCR for MYBL2

(Hs00942543_m1 MYBL2, Applied Biosystems) was carriedout using TaqMan PCR Master Mix (Applied Biosystems)and qRT- PCR for b-glucuronidase (HsGusB QT00046046,Quantitect primer assay, Qiagen) was carried out usingSYBRGreen Master Mix (Thermo Fisher Scientific). For murinegene expression assays, qRT-PCR for p21 (Mm01303209_m1),Puma (Mm00519268_m1), Noxa (Mm00451763_m1), Bax(Mm00432051_m1), and b-2-microglobulin (Mm00437762_m1)were carried out using TaqMan PCR Master Mix (Applied Bio-systems). Reactions were carried out in a Stratagene Mx3000Pmachine and samples were run in duplicate. Relative gene expres-sion was calculated as 2�DDCt values relative to control genes(b-glucuronidase for human samples and b-2-microglobulin formurine samples).

MiceMice were maintained on a C57/BL6 background and geno-

typed by Transnetyx. For mouse studies, no specific random-ization or blinding protocol was used during experimentalprotocols. Mice of both genders were used. Age- and gender-matched mice were used per experiment. Seventy-week-oldhealthy mice were chosen to perform aging studies. Disease-free status of these animals was assessed on the basis ofbehavior and physical appearance of the mice, normal valuesof white blood cell, red blood cell, and platelets obtained fromperipheral blood counts, and by internal organ examinationafter dissection, in particular no signs of splenomegaly.

InhibitorsInhibitors were dissolved in DMSO; KU60019 (10 mmol/L)

was used for inhibition of ATM and NU7441 (1 mmol/L) wasused for inhibition of DNA-dependent protein kinase (TocrisBioscience).

Flow cytometry and cell sortingSingle-cell suspensions of bone marrow were prepared using

standard techniques and red blood cells were depleted by ACKlysis (0.15 mol/L NH4Cl, 1 mmol/L KHCO3, 0.1 mmol/L EDTA,pH 7.4). Nonspecific antibody binding to Fc receptors wasblocked using anti-CD16/CD32 (93, eBioscience) and the cellswere stained with a combination of fluorochrome-conjugatedantibodies including anti-mouse lineage; CD5, CD8a, CD11b,Gr-1, Ter119, B220 (APC or FITC), cKit (PeCy5 or e780; 2B8,eBioscience), Sca-1 PeCy7 (D7, eBioscience), Flk2 PE (A2F10,eBioscience), CD48 APC (HM48-1, eBioscience), and CD150PEcy7 (TC15-12F12.2, BioLegend), to allow identification ofFlk2� HSCs (lineage�cKitþ Sca-1þFlk2�) and long-term HSCs(SLAM staining; lineage�cKitþSca-1þCD48�CD150þ). Some

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cells were analyzed directly by flow cytometry using a CyAn ADPAnalyzer (Beckman Coulter) and some were sorted using aCytomation XDP MoFlo machine (Beckman Coulter). In bothcases, data were analyzed using either Summit software (Dako) orFlowJo software (FLOWJO, LLC). When cells were requiredfor sorting, a cKitþ enrichment using streptavidin microbeads(130-048-101, Miltenyi Biotec) and columns (130-042-201,Miltenyi Biotec) was performed prior staining.

Proliferation and apoptosis assaysFor proliferation assays in vivo using BrdU, mice were given

an intraperitoneal injection of 2 mg BrdU in PBS and 24 hourslater animals were sacrificed. cKit-enriched bone marrow cellswere isolated using anti-mouse cKit biotin (eBioscience) andstreptavidin microbeads (Miltenyi Biotec) as per the manufac-turer's instructions. For proliferation assays in vitro using BrdU,expanded CD34þ cells were labeled with 10 mmol/L BrdUfor 3.5 hours. Cells were stained using the BrdU Flow Kit(8811-6600, BD Biosciences) according to the manufacturer'sinstructions. For proliferation assays using Ki67, cKit-enrichedbone marrow cells were stained using the Ki67 flow kit (BDBiosciences) according to the manufacturer's instructions.For colony-forming assays, purified HSCs (lineage�cKitþSca-1þFlk2�) were obtained by sorting. Five-hundred HSCs wereplated in Methocult (Stem Cell Technologies) supplementedwith penicillin/streptomycin (Invitrogen) in 35-mm petridishes and incubated for 6 days at 37�C in an atmospherecontaining 5% CO2. Colonies were counted using a standardlight microscope with �10 objective. For G2–M checkpointstudies, cKit-enriched bone marrow cells were isolated asdescribed previously. A total of 1–2 � 106 cKit-enriched cellswere cultured for 18 hours in Iscove's modified Dulbecco'smedium (IMDM) containing 10% heat-inactivated FBS(HIFBS), 3% penicillin/streptomycin, 1 mmol/L sodium pyru-vate, 2 mmol/L L-glutamine, 0.1 mmol/L nonessential aminoacids, 50 mmol/L 2-mercaptoethanol, 25 ng/mL SCF, 10 ng/mLIL3, 25 ng/mL IL11, 25 ng/mL TPO, 4 U/mL EPO, 10 ng/mLGM-CSF, and 25 ng/mL FLT3L (complete cytokine medium).Medium was replaced and the cells were cultured for 1 hourwith DMSO or 10 mmol/L KU60019 prior to irradiation (IR) invitro (2 Gy). Cells were cultured for a further 5 hours andstained with (i) cell surface marker antibodies to identifysubpopulations; (ii) mouse anti-phospho histone H3 Ser10(2-hour staining; clone 6G3, Cell Signaling Technology) andgoat anti-mouse Alexa 488 (30-minute staining; A-11001,Molecular Probes); and (iii) Vybrant DyeCycle (MolecularProbes). Fixation and permeabilization were carried out usingbuffers from the BrdU Flow Kit (BD Biosciences) according tothe manufacturer's instructions. For apoptosis assays, wholebone marrow cells were stained with cell surface marker anti-bodies to identify subpopulations as well as mouse anti-cleaved PARP (Asp214; clone F21-852, BD Biosciences).

ImmunofluorescencePurified murine cells (Flk2� HSCs or SLAM HSC) or human

CD34þ cells were cytospun onto microscope slides for 5 minutesat 800 rpm. For 53BP1, gH2AX, and MRE11 staining, cells weretreated with CSK buffers (buffer 1 for 4 minutes at room tem-perature, buffer 2 for 1 minute at room temperature), washed inPBS, and fixed in 4% PFA/PBS for 15 minutes at room temper-ature. Fixation was quenched with 50 mmol/L ammonium chlo-

ride for 5 minutes at room temperature and the cells werepermeabilizedwith 0.3%Triton-X 100/PBS for 5minutes at roomtemperature. For pKap1, cells were fixed in 4% PFA/PBS for 10minutes at room temperature, washed in PBS, and permeabilizedwith ice-coldmethanol for 10minutes at room temperature. Cellswere blocked with 3% BSA/10% HIFBS/1% goat serum/0.3%Triton-X 100/PBS (blocking buffer) for 1 hour at room temper-ature. Cells were incubated with primary antibodies to 53BP1(NB-100-904, Novus Biologicals), pKAP1 (S824;A300-767A,Bethyl Laboratories Inc), gH2AX (JBW301, Merck), MRE11(4895S, Cell Signaling Technology), mouse IgG control (G3A1,Cell Signaling Technology), or rabbit IgG control (sc-2027, SantaCruz Biotechnology) and diluted in blocking buffer overnight at4�C. Cells were washed three times in 0.1% Tween 20/PBS andincubated with goat anti-rabbit Alexa 488 secondary antibodydiluted in blocking buffer for 1 hour at room temperature. Cellswere washed three times in 0.1% Tween 20/PBS, dipped in Milli-Q water, and mounted with ProLong Gold AntiFade Reagentcontaining DAPI (Invitrogen). Microscopy imaging was per-formed using a Zeiss LSM 510 Meta confocal microscope(100� objective NA 1.4 lens) and the images were analyzed usingImageJ software. For all immunofluorescence staining, 30–50cells were scored for each independent experiment. Experimentswere performed at least three times and results represent aminimum of 3 animals. For 53BP1 staining on CD34þ cells frompatients withMDS, aminimumof 25 cells were scored per patientper condition.

Comet assaysHSCs (Lin�/cKitþ/Sca1þ/Flk2�) were purified 0, 1, 5, and

24 hours after IR in vivo (2 Gy). Alkaline comet assays wereperformed as described previously (26). Cells were stained withSYBR Safe (Invitrogen) for 1 hour and imaged using a LeicaDM6000 microscope (20� objective). Analysis was performedusing ImageJ software using theOpenComet plugin and the headfinding was selected to the brightest region. Olive momentvalues were automatically generated by the software. Statisticalsignificance was calculated using GraphPad Prism software uti-lizing the Mann–Whitney test.

Peptide nucleic acid-FISHPurified HSCs were obtained by cell sorting, exposed to ion-

izing radiation in vitro (2 Gy), and cultured for further 7 daysin methylcellulose semi-solid medium containing cytokines(M3434). Colonies were dissociated and cultured with 100 ng/mLcolcemid for 3 hours at 37�C to arrest cells in metaphase. Peptidenucleic acid staining was performed as described previously (27).Briefly, cells were exposed to hypotonic solution (0.56% KCl) for16 minutes at 37�C and then fixed in methanol:acetic acid (3:1).After three changes offixative solution, the cellswere droppedon toslides that had been pretreated with 1 N HCl, followed by 100%ethanol and finally fixative solution. FISH with FITC-labeled(CCCTAA)3 peptide nucleic acid (F1009-5, Panagene) was per-formed followed by mounting with ProLong Gold AntiFadeReagent containing DAPI (Invitrogen). Microscopy imaging wasperformed using a Leica DM6000 microscope (100� objective)and images were analyzed blindly using ImageJ software. Anaverage of 20–30 metaphases were scored per condition.

Statistical analysisAll data shown are presented as mean � SEM. When com-

paring datasets between Mybl2þ/þ and Mybl2þ/D animals, two-

Low MYBL2 Expression in MDS Alters DNA DSB Repair in HSCs

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tailed unpaired Student t test was applied using GraphPadPrism software, unless indicated. No statistical method wasused to estimate the sample size. No specific randomization orblinding protocol was used. N indicates the numbers of inde-pendent experiments performed and was chosen to ensureadequate statistical power. P � 0.05 was considered statisticallysignificant. Significance tests were performed on all samplesand therefore graphs lacking P values indicate results were notstatistically significant.

Study approvalAll animal experiments were performed under an animal

project license in accordancewithUK legislation. Human patientswith MDS were recruited from the clinic held at the Centre forClinical Haematology, University Hospital Birmingham NHSFoundation Trust. All the subjects have read the patient informa-tion sheet and signed the consent form. The study was conductedas according to Good Clinical Practice guidelines, consistent withthe principles that have their origin in theDeclaration ofHelsinki.The studywas approved by theWestMidlands – Solihull ResearchEthics Committee (10/H1206//58).

ResultsLow MYBL2 expression associates with decreased expressionof DNA repair genes in human MDS

Given the role of MYBL2 in transcription and in maintaininggenome stability (10–14) and recent studies showing increasedDNA damage in MDS (28, 29), we decided to determinewhether MYBL2 levels were associated with altered expressionof DNA repair genes in patients with MDS. To do this, we firstsubdivided the patients into MYBL2lo and MYBL2hi popula-tions based on a differential gene expression analysis on HSCsfrom patients with MDS (15), using the 25th and 75th per-centiles of MYBL2 expression to define each group. Comparingthese two groups, genes were then ranked on the basis of thesignal-to-noise ratio for differential gene expression (DGE;Supplementary Table S1A). This DGE scoring was then ana-lyzed against a collection of reactome gene sets (SupplementaryTable S1B), to assess their individual enrichment, whichencompassed pathways with whichMYBL2 has previously beenassociated (cell cycle, DNA replication) and pathways relevantto this study (DNA damage, DNA repair, chromosome main-tenance, and apoptosis). Consistent with previously publishedobservations, the analysis (Supplementary Table S1C) con-firmed the previously published association between MYBL2levels and cell-cycle progression in the context of MDS (2).Remarkably, it also revealed a significant enrichment (NOMP ¼ 0.00065, FWER P ¼ 0.006) for the reactome gene set"DNA damage pathway" (R-HAS-73894), indicating an overalldownregulation of DNA repair pathways components in theMYBL2lo MDS cases (Fig. 1A). Upon further inspection of thetwo populations, and confirming our previous results (2), wefound that the MYBL2lo population was significantly enrichedin MDS with excess blasts type II (MDS-EB2) cases with worsediagnosis (Fig. 1B, top table). To exclude the possibility that theobserved link could primarily reflect an association between theDNA damage pathway and advanced disease states rather thanMYBL2 levels, we recurated the compared subsets to displaybalanced diagnosis composition (Fig. 1B, bottom table), andrepeated the gene expression analysis (Fig. 1C). This unbiased

analysis confirmed that expression of DNA repair genes cor-relates with MYBL2 levels in MDS (Fig. 1C; SupplementaryTable S2), independently of the disease state.

Low MYBL2 expression associates with poor DNA repair inhuman MDS

To determine whether the differential expression of DNArepair genes (Fig. 1) had a functional consequence, we nextassessed the DNA repair kinetics of CD34þ cells, a multipotentstem and progenitor cell population, from 5 patients with MDSwith differing prognoses (Table 1), without prior knowledge ofMYBL2 expression levels. CD34þ cells were irradiated in vitro (2Gy) and the prevalence of p53-binding protein 1 (53BP1) foci,a robust marker for DSBs, was measured at 1, 3, and 5 hourspost-irradiation (Fig. 2A and B). Cells from these patientsshowed a striking difference in their ability to repair IR-inducedDSBs, as evidence by their differing ability to resolve 53BP1foci. After MYBL2 expression levels were measured (Fig. 2C) inthe same patients, it was evident that DSB repair kineticsstrongly correlated with MYBL2 mRNA expression (Fig. 2D;R2 ¼ 0.83, P ¼ 0.0114). For example, a patient (patient 5)expressing similar MYBL2 levels to cells isolated from a healthyindividual could efficiently repair DSBs within 5 hours, whereasin contrast, patients expressing around 50% normal levels ofMYBL2 (patients 3 and 4), or even lower (patients 1 and 2)exhibited defective clearance of 53BP1 foci, suggesting thepersistence of unresolved DSBs. We also investigated whetherproliferation rates correlated with MYBL2 expression in MDSpatient CD34þ cells by performing BrdU incorporation assays.This revealed that MYBL2 expression in these patients did notcorrelate with proliferation, (Supplementary Fig. S1A and S1B),nor did this correlate with the cell's DNA repair capacity(Fig. 2E). These data suggest that reduced expression of DNAdamage genes in patients with compromisedMYBL2 expressionhas a functional impact on the ability of these cells to repairgenetic damage. Furthermore, we propose that MYBL2 mRNAexpression levels may also be used as a potential biomarkerpredicting the cellular response to DNA damage, which couldbe of use for patient stratification.

Slow DSB DNA repair kinetics in Mybl2þ/D HSCs afterin vivo IR

Following the observation that MYBL2 expression levelscorrelated with DSB repair kinetics in human MDS stem cells,we wanted to further investigate the involvement of MYBL2 inregulating the DNA damage response. To do this, we used aMybl2 haploinsufficient mouse model (Mybl2þ/D), known to besusceptible to MDS with ageing (30). We decided to focus ourstudies on the HSC population, the population in which MDSoriginates (1). Importantly, these animals did not show anymajor differences in the numbers of HSC/progenitor cells whencompared with wild-type mice prior to treatment (Supplemen-tary Fig. S2). To investigate the DNA damage response, weassessed the clearance of 53BP1 foci, as a measure of repairkinetics, in both wild-type and Mybl2þ/D HSCs [defined as(Lin�/cKitþ/Sca1þ/Flk2�)] followed by irradiation of the micewith 2 Gy of IR (Fig. 3A; Supplementary Fig. S3A). While we didnot observe any difference in the initial recruitment of 53BP11 hour post-irradiation between wild-type and Mybl2þ/D

HSCs, there were notable differences in the kinetics of 53BP1foci resolution over time between the two genotypes (Fig. 3B

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Figure 1.

Differential MYBL2 expression associates with a DNA repair gene signature in MDS. A, Heatmap of the reactome DNA repair gene set core signaturerepresented as a Blue-Pink O' Gram in the space of the analyzed gene set. B, Characteristics of the MYBL2hi and MYBL2lo subsets. A two-tailed Fisherexact test (right column) was applied to assess the dependence between the variables MYBL2 class and MDS World Health Organizationclassification. C, Enrichment plot for the DNA repair reactome gene set after adjustment of the FAB composition of the compared sample sets (left); heatmapof the reactome DNA repair gene set core signature represented as a Blue-Pink O' Gram in the space of the adjusted (unbiased) set (right; see alsoSupplementary Table S1; Supplementary Table S2).

Table 1. Clinical data for the patients with myelodysplastic syndrome used in this study

Patientnumber

Date ofdiagnosis

Age(years) Cytogenetics IPSSR

WHO 2016Diagnosis

Date of samplecollection

Disease progression(date)

1 01/02/2015 76 46, XY High risk MDS-EB1 29/09/2015 AML 27/08/20152 30/07/2014 67 46, XY (NPM1 mutation) High risk MDS-EB1 12/09/2014 —

3 12/09/2011 74 46, XX Low risk MDS-SLD 11/09/2015 —

4 08/11/2006 48 46, XY Low risk MDS-MD 14/11/2016 —

5 22/09/2014 57 46, XX Low risk MDS-MD 09/09/2016 —

NOTE: Description of the clinical data for the 5 patients used in this study.Abbreviations: IPSS-R, Revised International Prognostic Scoring System; MDS-EB, MDS with excess blasts; MDS-MD, MDS with multilineage dysplasia; MDS-SLD,MDS with single lineage dysplasia; NPM1, nucleophosmin.






and C). Wild-type HSCs showed a significant reduction in cellspositive for 53BP1 foci at 5 hours post-irradiation, whereas incontrast, more than 50% of Mybl2þ/D HSCs still retained asignificant number of 53BP1 foci–positive cells at this timepoint. Moreover, the absolute number of 53BP1 foci per cellwas also increased in the Mybl2þ/D HSCs (SupplementaryFig. S3B and S3C). To extend these observations to a morerefined HSC population, we repeated this experiment withHSCs purified from young animals (7 weeks) using SLAMstaining (KSL CD48�CD150þ). This revealed that the retentionof 53BP1 foci was also apparent inMybl2þ/D CD150þHSCs, butnot in their wild-type counterparts (Fig. 3D). Moreover, todetermine whether this effect was dose dependent, we mea-sured the percentage of 53BP1 foci 5 hours after 1 Gy of IR. Thisrevealed that at a lower dose of IR, Mybl2þ/D HSCs exhibited a"wild-type" 53BP1 response (Supplementary Fig. S3D), sug-gesting that there is an insufficient quantity of DNA repairproteins present in the Mybl2þ/D HSCs required to cope withrepairing high-level DNA damage. Because aged haploinsuffi-cient Mybl2 mice develop an MDS-like disease, we investigatedhow aging affected DSB repair capacity of wild-type andMybl2þ/D HSCs from young (7 weeks) and old mice (70 weeks).Unsurprisingly, 70-week-old HSCs exhibited a higher percent-age of 53BP1-positive cells than 7-week-old HSCs. However,in keeping with a role for MYBL2 in regulating DNA repair,Mybl2 haploinsufficiency exacerbated the age-associated

decrease in genome stability (Fig. 3B and C; SupplementaryFig. S3C). Interestingly, levels of 53BP1-positive cells in youngMybl2þ/D HSCs were comparable with aged wild-type HSCs,suggesting that these cells may demonstrate a premature agingphenotype. Together, these data indicate that Mybl2 haploin-sufficiency is associated with defective repair of IR-inducedDSBs in HSCs.

Because our data are only indicative of unrepaired DNADSBs, we performed comet assays to directly measure the totalamount of DNA damage remaining in these cells at differenttimes post-irradiation. In keeping with a failure to properlyrepair IR-induced damage, Mybl2þ/D HSCs displayed an increasein the olive tail moment 5 hours post-irradiation when compar-ed with wild-type HSCs (Fig. 3E). These differences in DNArepair kinetics were not the consequence of changes in cell-cycleprofile between wild-type and Mybl2þ/D HSCs, as HSCs fromboth genotypes showed a similar percentage of cells in G0–G1

prior to irradiation (Supplementary Fig. S4), and the samepercentage of cells in S-phase measured by in vivo BrdU incor-poration (Supplementary Fig. S5A and S5B). Importantly, wedid not observe any changes in the absolute numbers of HSCsafter in vivo IR (Supplementary Fig. S5C), nor apoptosis, mea-sured either by PARP1 cleavage (Supplementary Fig. S5D) orthe induction of p53-dependent apoptotic genes (Supplemen-tary Fig. S5E), which could account for our observations usingthe comet assay. Overall, these data demonstrate that Mybl2

Figure 2.

MYBL2 expression correlates with kinetics of 53BP1 clearance in MDS patient CD34þ cells after irradiation. A–E, Peripheral blood cells from a healthycontrol and patients with MDS were cultured for 8 days in cytokine containing medium to expand CD34þ cells. Cells were harvested on day 8 and CD34þ

cells were purified using microbeads for performing PCR, cell-cycle analysis, and immunofluorescence. Purified CD34þ cells were subject to 2 Gy irradiationin vitro and cultured in cytokine-containing medium. Cells were removed from culture at different time points (1, 3, and 5 hours), cytospun ontomicroscope slides, and immunofluorescence for 53BP1 was performed. A, Representative images of 53BP1 foci 5 hours after IR in MDS patient CD34þ

cells and CD34þ cells taken from a healthy individual. (Scale bar, 5mm). B, Average number of 53BP1 foci at different time points in patients with MDSand CD34þ cells taken from a healthy individual. Values are calculated as a percentage of the average number of 53BP1 foci present 1 hour after IR. (P, MDSpatient number; HC, healthy control). C, MYBL2 gene expression from purified CD34þ cells normalized using expression of GUSB. D, Negativecorrelation between number of 53BP1 foci 5 hours after IR and MYBL2 expression levels in MDS patient CD34þ cells. E, Purified CD34þ cells werelabeled with 10 mmol/L BrdU for 3.5 hours and stained with a fluorescent antibody directly conjugated to BrdU. No correlation was observed betweenthe number of 53BP1 foci 5 hours after IR and percentage of BrdUþ cells in MDS patient CD34þ cells (error bars, mean � SEM).

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haploinsufficient mice display a defect in the kinetics of DSBrepair in response to IR, which is heightened during aging, butthat has no impact on HSC survival.

Mybl2þ/D HSCs are highly dependent on DNA-dependentprotein kinase for DNA DSB repair

To gain a mechanistic understanding of the DNA repairdefect in Mybl2þ/D HSCs, we used small-molecule inhibitorsto investigate the relationship of Mybl2 haploinsufficiency withtwo key proteins involved in the DSB response, namely DNA-dependent protein kinase (DNA-PK) and ATM. Treatment ofwild-type HSCs with the DNA-PK inhibitor NU7441 (Fig. 4A;ref. 31) induced an increase in the percentage of cells positivefor 53BP1 foci, in line with an expected defect in DSB repair dueto inhibition of the NHEJ pathway (Fig. 4B–D). In contrast,Mybl2þ/D HSCs treated with the same inhibitor demonstrated a

dramatic loss of 53BP1 foci formation at 5 hours after IR (Fig.4B–D). These findings were recapitulated by analysis of theformation/retention of gH2AX foci, a pan-DNA damage marker(Fig. 4E–G). Nonetheless, 53BP1 foci were detected at 1 hourpost-irradiation in Mybl2þ/D HSCs (Fig. 4B–D), suggesting thatour observations did not reflect a global inability to form53BP1 foci. Furthermore, Mybl2þ/D HSCs treated with DNA-PK inhibitor were proficient at sensing DNA damage, becauseMRE11 foci formed 1 and 5 hours after IR (Fig. 4H and I).Moreover, the absence of 53BP1 and gH2AX foci 5 hours post-irradiation was not because DNA repair had been completed, asMybl2þ/D HSCs exhibited increased olive tail moments by cometassay (Fig. 4J). However, these breaks were eventually repaired, asby 24-hour levels of DNA damage in Mybl2þ/D HSCs was equalto that of wild-type cells (Fig. 4J). Interestingly, it has beenpreviously shown that DNA-PK and ATM are both required for

Figure 3.

MYBL2-deficient haematopoietic stem cells have altered kinetics of DNA DSB repair. Mybl2þ/þ and Mybl2þ/D animals aged 7 and 70 weeks were exposedto 2 Gy irradiation in vivo. Bone marrow cells were obtained at different time points after IR (1, 3, 5, and 24 hours) and HSCs were purified using cell sorting.Immunofluorescence was performed (53BP1 and DAPI) and alkaline comet assays. A, Experimental scheme for 53BP1 staining and comet assays usingpurified HSC subpopulations, including Flk2� HSC (KSL Flk2�) and SLAM HSC (KSL CD48�CD150þ). B, Representative images of 53BP1 staining of Flk2� HSCsfrom young and old animals 5 hours after IR. C, Left, percentage of Flk2� HSCs positive for 53BP1 foci at different time points in young animals (n ¼ 4). Right,comparison of the percentage of Flk2� HSCs positive for 53BP1 foci 5 hours after IR in young and old animals (n ¼ 4). D, Percentage of SLAM HSCs positive for53BP1 staining in young animals 5 hours after IR (n ¼ 3). E, Representative images of alkaline comets from Flk2� HSCs isolated from young animals5 hours after 2 Gy IR in vivo (left). Mean olive tail moment of alkaline comets of Flk2� HSCs isolated from young animals at different time points after2 Gy IR in vivo (right; Mybl2þ/þ, n ¼ 4 for 5 hours and n ¼ 3 for 0, 1, and 24 hours; Mybl2þ/D, n ¼ 5 for 5 hours and n ¼ 3 for 0, 1, and 24 hours). Error bars,mean � SEM; P values included in the figure when using a two-tailed unpaired Student t test. ns, nonsignificant.






Figure 4.

DNA-dependent protein kinase is required to maintain normal kinetics of fast DNA DSB repair in MYBL2-deficient HSCs. Bone marrow cells were obtainedfrom Mybl2þ/þ (n ¼ 3) and Mybl2þ/D (n ¼ 3) animals aged 7 weeks and enriched for cKit using microbeads. cKitþ-enriched cells were cultured for1 hour in medium containing cytokines and an inhibitor of DNA-PK (NU7441, 1 mmol/L). Cells were exposed to 2 Gy IR in vitro and cultured for a further 1, 5, or24 hours. Flk2� HSCs were purified using cell sorting and prepared for immunofluorescence and comet assays. A, Experimental scheme for isolation andculture of cKitþ cells with DNA-PK inhibitor for immunofluorescence and comet assays. B, Representative images of Flk2� HSCs stained with 53BP1. C,Percentage of Flk2� HSCs positive for 53BP1. D, Number of 53BP1 foci per cell. E, Representative images of Flk2� HSCs stained with gH2AX. F, Percentage ofFlk2� HSCs positive for gH2AX. G, Fluorescence intensity of gH2AX foci. H, Representative images of Flk2� HSCs stained with MRE11 (scale bar, 10 mm).I, Percentage of Flk2� HSCs positive for MRE11. J, Mean olive tail moment of alkaline comets of Flk2� HSCs at different time points after 2 Gy IR in vivo. �/þon axes indicate whether cells were treated with DNA-PK inhibitor. Error bars, mean � SEM. P values on the graphs obtained when using atwo-tailed unpaired Student t test.

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efficient H2AX phosphorylation and 53BP1 recruitment to DSBsand that ATM�/� B cells are completely dependent on DNA-PKto sustain the phosphorylation of H2AX after gamma irradia-tion (32). On the basis of this, these data suggest that reliancethat Mybl2þ/D HSCs have on DNA-PK to mediate DSB signalingis indicative of an underlying defect in the ATM-dependentDNA damage response.

The slow repair kinetics in Mybl2þ/D HSCs are epistatic withinhibition of ATM

While previously published data have been suggestiveof a link between MYBL2 and ATM signaling (9), we wantedto specifically determine whether Mybl2þ/D HSCs exhibitdefective ATM-dependent signaling in response to DSBs.To address this, we treated wild-type and Mybl2þ/D HSCswith the ATM inhibitor KU60019 (33), and analyzed 53BP1foci clearance (Fig. 5A). These analyses revealed that treatmentwith KU60019 delayed the clearance of 53BP1 foci in wild-typecells, with ATM inhibition causing a >2-fold increase in thenumber of cells still displaying 53BP1 foci 5 hours post-irra-diation (Fig. 5B–D), in line with the known requirementfor ATM in DSB repair. In contrast, ATM inhibition inMybl2þ/D HSCs had little effect on 53BP1 clearance afterIR (Fig. 5C; Supplementary Fig. S6A). These data further sup-port the prediction that the altered DSB repair kinetics observedin Mybl2þ/D HSCs are potentially due to a defect in ATM-dependent signaling.

Given these findings, it is tempting to speculate that becauseATM inhibition in wild-type HSCs mimics the 53BP1 fociclearance defect observed inMybl2þ/DHSCs, a similar treatmentwould also lead defective cell survival as seen in Mybl2þ/D cells.However, wild-type HSCs transiently treated with an ATMinhibitor did not display the same characteristics as untreatedMybl2þ/D HSCs when assessed by a colony-forming assay (Sup-plementary Fig. S6B), indicating that either short-term inhibi-tion of ATM pathway does not have the same overall effect asMybl2 haploinsufficiency or that Mybl2þ/D HSCs display addi-tional defects that are not mimicked by ATM inhibitor.

A subset of ATM function is impaired in Mybl2þ/D HSCsTo further investigate the defective ATM signaling inMybl2þ/D

HSCs, we next assessed the phosphorylation of KAP1. Ithas been previously reported that approximately 10%–15%of DSBs require ATM signaling to be repaired in G0–G1 phasesof the cell cycle (34) and that this repair requires the phos-phorylation of Kap1, which is known to be completelydependent on ATM (35). Therefore, we examined Kap1 phos-phorylation after exposure to IR in HSCs (Fig. 6A). Inline with previous reports, wild-type HSCs displayed robustpan-nuclear p-KAP1 staining within minutes following IR,which could be distinguished as discrete foci by 3 hourspost-irradiation (Fig. 6B and C; ref. 35). Interestingly, althoughMybl2þ/D HSCs also exhibited rapid KAP1 phosphorylationimmediately post-irradiation, they were unable to maintainthis phosphorylation at later time points (Fig. 6B and C). This

Figure 5.

ATM signaling is affected in MYBL2-deficient HSCs. Bone marrow cells were obtained from Mybl2þ/þ (n ¼ 3) and Mybl2þ/D (n ¼ 3) animals aged 7 weeksand enriched for cKit using microbeads. cKitþ-enriched cells were cultured for 1 hour in medium containing cytokines and an inhibitor of ATM(KU60019, 10 mmol/L). Cells were exposed to 2 Gy IR in vitro and cultured for a further 5 hours. Flk2� HSCs were purified using cell sorting andimmunofluorescence for 53BP1 and DAPI was performed. A, Experimental scheme for isolation and culture of cKitþ cells with ATM inhibitor forimmunofluorescence. B, Representative images of 53BP1 staining of Flk2� HSCs treated with the ATM inhibitor KU60019. C, Percentage of Flk2� HSCspositive for 53BP1 foci when treated with KU60019. D, Number of 53BP1 foci per cell when Flk2� HSCs were treated with KU60019. �/þ on axesindicates whether cells were treated with ATM inhibitor. Error bars, mean � SEM. Samples were not statistically significant when using a two-tailedunpaired Student t test. ns, nonsignificant.






further reinforced our findings that partial Mybl2 loss leads todefective ATM signaling, and also suggests that MYBL2 may berequired to maintain rather than initiate ATM-dependent sig-

naling during late-stage repair. To confirm this prediction, wenext stimulated HSCs to enter the cell cycle, and analyzedactivation and maintenance of the ATM-dependent G2–M

Figure 6.

MYBL2-deficient hematopoietic stem cells show a deficiency in the maintenance of ATM-dependent p-KAP1 and increased telomere fragility afterirradiation. A–C, Bone marrow cells were obtained from Mybl2þ/þ (n ¼ 2) and Mybl2þ/D (n ¼ 2) animals aged 7 weeks and enriched for cKit using microbeads.Cells were exposed to 2 Gy IR in vitro and cultured for a further 30 minutes, 3 hours, or 5 hours. Flk2� HSCs were purified using cell sorting andimmunofluorescence for p-KAP1 (S824) and DAPI was performed. A, Experimental scheme for isolation and culture of cKitþ cells for immunofluorescence.B, Representative images of p-KAP1 in Flk2� HSCs at different time points after IR. Scale bar, 5 mm. C, p-KAP1 corrected total cell fluorescence for eachHSC at different time points after IR. D, Purified Flk2� HSCs were obtained from Mybl2þ/þ (n ¼ 2) and Mybl2þ/D (n ¼ 2) animals aged 7 and 70 weeksby cell sorting. Cells were exposed to 2 Gy IR in vitro and cultured for a further 7 days in methylcellulose semi-solid medium containing cytokines.Colonies were dissociated and cultured with colcemid to arrest cells in metaphase. Metaphase preparations were performed and chromosomes stainedwith telomere peptide nucleic acid and DAPI. Shown are examples of fragile telomeres found in 7- and 70-week-old cells. Table shows the number ofchromatid ends scored and the percentage of chromatid ends with fragile telomeres. P values in the table were obtained using a Mann–Whitney testcomparing numbers of fragile telomeres in Mybl2þ/þ and Mybl2þ/D of the same age. Error bars on graphs, mean � SEM. P values obtained when using atwo-tailed unpaired Student t test are indicated.

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cell-cycle checkpoint after IR. Interestingly, Mybl2þ/D HSCsretained the ability to activate this checkpoint following expo-sure to IR (Supplementary Fig. S7A–S7C), suggesting that thesecells are not completely defective in ATM function. This sug-gests that MYBL2 is required to maintain, but not initiate,activation of a specific subset of ATM-dependent signalingpathways.

One of the characteristics of loss of ATM function is telomereinstability (36). We therefore postulated that lower MYBL2levels in HSCs might lead to telomere fragility as a result ofdefective ATM signaling. To examine this possibility, HSCsfrom young and old mice were irradiated and cultured for7 days in a colony-forming assay. Metaphase spreads wereprepared from these cells and stained with telomere probes.These investigations revealed that telomere instability (definedas sister chromatid fusion or loss of telomere signal) was twiceas frequent in the progeny derived from young Mybl2þ/D HSCscompared with controls (Fig. 6D). In fact, this percentage wassimilar between youngMybl2þ/D HSCs and old wild-type HSCs,in line with our earlier suggestion that Mybl2þ/D HSCs displayan aging phenotype that could lead to neoplastic lesions.

In conclusion, we demonstrate that Mybl2þ/D HSCs aredefective in the maintenance of ATM-dependent DNA damagesignaling at the sites of DSBs, leading to slower DSB repairkinetics and a higher dependency on DNA-PK in the survivingcells. Overall, these data suggest that correct MYBL2 proteinlevels are required for a proper DNA damage response andappropriate DSB repair in the HSC compartment. Deregulationof these levels leads to defective DSB repair, telomere instabil-ity, and likely contributes to the accumulation of geneticalterations in MDS.

DiscussionHSCs are the life-long pillars of continuous blood cell

production. Maintenance of their genetic integrity is para-mount to avert the accumulation of mutations that can con-tribute to the development of blood disorders such as MDSduring the aging process. Our work demonstrates a previouslyundescribed role for MYBL2 in promoting efficient DSB repairin HSCs, possibly via regulation of the ATM kinase. Further-more, our findings suggest that MYBL2 levels may be used as abiological biomarker to determine the DSB repair capacity ofCD34þ cells from patients with MDS. Furthermore, MYBL2levels could also be used as a clinical biomarker to informdecisions regarding patient selection for transplantation ortreatments that target DNA repair, highlighting the translation-al importance of this work.

In line with a role for MYBL2 in regulating ATM signaling, wehave shown that Mybl2 haploinsufficient HSCs display a delayin 53BP1 clearance after DNA damage induced by IR, which isexacerbated during ageing. In these cells, defective ATM signal-ing leads to loss of sustained KAP1 phosphorylation andtelomere instability. This renders these cells reliant on otherDNA repair pathways prevalent in noncycling cells. As a result,Mybl2þ/D HSCs are highly dependent on the NHEJ regulatorDNA-PK for DSB signaling, because inhibition of this kinaseleads to a failure to maintain gH2AX and 53BP1 at sites ofdamage. Moreover, because ATM-dependent phosphorylationof KAP1 has been suggested to be required for chromatinrelaxation and the repair of DSBs within heterochromatin

regions (35, 37), our data suggests a requirement for MYBL2in repairing a subset of DSBs associated with heterochromaticchromosomal regions.

In agreement with our conclusions, studies using human celllines have recently demonstrated that MYBL2 interacts withNbs1, which is required for the activation of ATM in response toDSBs and also ATM-dependent heterochromatic DSB repair inG0–G1 (9). While on face value, this may explain our observa-tions Mybl2þ/D HSCs, in stark contrast to the work of Henrichand colleagues, we were unable to detect a G2–M checkpointdefect in our Mybl2-haploinsufficient HSCs, suggesting that atleast some ATM-dependent signaling is intact in these cells.Moreover, Henrich and colleagues failed to observe any defectsin DNA repair, leading them to conclude that MYBL2 does nothave an essential role in the DNA repair response. In contrast,we have shown that MYBL2 does have a role for DSB repair inHSCs, and without sufficient MYBL2 expression cells showdefective repair kinetics of IR-induced DSBs. These differencescould be due to cell type differences, such as primary cellsversus cell lines, or the use of different DNA-damaging agents toinduce DSBs (IR vs. UV). Alternatively, the highly quiescentnature of HSCs in vivomay also account for these discrepancies,as this may make any defects in fast DSB repair by NHEJ morepronounced as they cannot utilize repair by HR.

The importance of an appropriate DNA damage response forthe maintenance and protection of the HSC pool againstfunctional decline during ageing has been well reported(38–41). Quiescent HSCs cannot use the HR pathway and thusrely on NHEJ-dependent mechanisms to repair their DNA(16, 17). A failure to repair DSBs by the canonical DNA repairpathways can be detrimental to the cell, as alternative pathwaysmay allow the potential for genome instability (42, 43). It hasalso been reported that reduction in or mutation of splicingfactors in MDS leads to altered splicing of DNA repair andtelomere maintenance genes (44). This then perturbs myeloiddifferentiation and contributes to disease development via amechanism not necessarily involving chromosomal rearrange-ments. Equally, abnormal DNA damage signaling in Mybl2þ/D

HSCs could increase the mutational burden by facilitating theuse of alternative, more error-prone signaling pathways.Indeed, after inducing irradiated Mybl2þ/D HSCs to proliferate,we found evidence of telomere instability in their progeny.In vivo, this prospect is likely to have a severe impact, and mayultimately lead to HSC malfunction and the accumulation ofcells that are primed for the development of blood disorderssuch as MDS. Recent work in the field of MDS suggests thattelomere dysfunction is a potent driver of the disease pheno-type (44) and telomere elongation using danazol treatmenthas been shown to improve hematologic responses includingreducing transfusion dependency (45).

Importantly, we show that the association between lowMYBL2levels and impaired DNA repair also holds true in patients withMDS. Thus, by directly measuring the DNA repair kinetics inCD34þ MDS cells, our data shows a correlation between MYBL2levels and functional DSB repair. Moreover, in CD34þ cells fromthese patients, low MYBL2 levels largely associate with lowexpression of DNA repair genes. Together, these data provide amolecular rationale for the accumulation of genetic anomalies inpatients deficient for MYBL2, which could play a role in theprogression of their disease. Importantly, this data represents thefirst direct study of repair kinetics in MDS patient CD34þ cells.






Current treatment options for patients with MDS are mostlybased on cytotoxic agents prior to autologous transplant, and arechallenged by the occurrence of clonogenic relapse, increasedresistance to therapy and, in some cases, leukemic transformation.Relapse of disease in these patients is typically driven by addi-tional genetic lesions. Of note, delq20 clones are not uncommonin patients following cytotoxic chemotherapy, with more than20% of these patients having a therapy-related myeloid neo-plasm, conferring a high mortality (46). Patients with therapy-related myeloid neoplasms more frequently have clonal hema-topoiesis with the originating mutation being present prior tochemotherapy treatment (47). We therefore hypothesize thatthese clones are susceptible to DNA-damaging agents and thatDNA repair defects may be involved in the etiology of theirsubsequent myeloid neoplasm. Moreover, our work supportsthe notion that Mybl2 haploinsufficiency results in changes inDNA repair kinetics and defective ATM signaling. Both ATMsignaling and NHEJ are known to be activated to repair DSBsinduced by doxorubicin (48, 49), indicating that our findingswith IR are also likely to be applicable to the use of anthracyclinesin chemotherapy.

In light of our findings, we propose that compromising theMYBL2-dependent DNA damage response in HSCs can facilitateMDS development by allowing inefficient repair of physiologicDSBs, and promoting telomere instability, two processes knownto contribute to the generation of oncogenic transformation in thesurviving HSC population. Furthermore, we suggest that sus-tained low levels of the MYBL2 protein in the premalignant cellsmay confer a susceptibility to disease progression through theaccumulation of incorrectly repaired DNA lesions.

Disclosure of Potential Conflicts of InterestR. Almaghrabi reports receiving a commercial research grant from The

Saudi Embassy. M. Raghavan has received speakers' bureau honoraria fromCelgene Ltd and Pfizer Ltd. No potential conflicts of interest were disclosedby the other authors.

Authors' ContributionsConception and design: R. Bayley, M.R. Higgs, E. Petermann, P. GarcíaDevelopment of methodology: R. Bayley, D. Blakemore, L. Cancian, P. GarciaAcquisition of data (provided animals, acquired and managed pati-ents, provided facilities, etc.): L. Cancian, G. Volpe, N. Reeve, M. Raghavan,P. GarcíaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Bayley, D. Blakemore, L. Cancian, S. Dumon,G. Volpe, C. Ward, P. GarcíaWriting, review, and/or revision of the manuscript: R. Bayley, L. Cancian,S. Dumon, G. Volpe, C. Ward, M. Raghavan, M.R. Higgs, GS Stewart,E. Petermann, P. GarcíaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R. Bayley, P. GarcíaStudy supervision: P. GarcíaOthers (contributed to the experiments): R. AlmaghrabiOthers (technical assistance to perform the experiments): J. GujarOthers (advised with experiments and contributed to writing/revision ofthe manuscript): M.R. Higgs

AcknowledgmentsThe authors wish to thank the members of the Petermann, Stewart,

Higgs, García, and Frampton laboratories for advice and constructivecriticisms, and Professor Frampton for covering the experiments throughhis animal license. The authors also wish to thank the animal facility andcell sorter facility at the University of Birmingham, and Donna Walsh forcollecting blood samples from myelodysplastic syndrome patients at theCentre for Clinical Haematology (Queen Elizabeth Hospital, Birmingham).R. Bayley and L. Cancian were supported by MRC grant (MR/K01076X/1).D. Blakemore was supported by a MRC PhD studentship (1632704).This work was funded by an MRC New Investigator Research Grant(MR/K01076X/1) and MRC Proximity to Discovery: Industry Engagementfund (MC_PC_14123; to P. García).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 1, 2018; revised June 22, 2018; accepted July 31, 2018;published first August 6, 2018.

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2018;78:5767-5779. Published OnlineFirst August 6, 2018.Cancer Res Rachel Bayley, Daniel Blakemore, Laila Cancian, et al. Hematopoietic Stem CellsMYBL2 Supports DNA Double Strand Break Repair in

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MYBL2SupportsDNADoubleStrandBreakRepair in Hematopoietic … · Molecular Cell Biology MYBL2SupportsDNADoubleStrandBreakRepair in Hematopoietic Stem Cells Rachel Bayley1, Daniel Blakemore1,