MYBL2 Supports DNA Double Strand Break Repair in Hematopoietic

Published OnlineFirst August 6, 2018; DOI: 10.1158/0008-5472.CAN-18-0273

Cancer Molecular Cell Biology Research

MYBL2 Supports DNA Double Strand Break Repair in Hematopoietic Stem Cells Rachel 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 a heterogeneous group of diseases characterized by blood cytopenias that occur as a result of somatic mutations in hematopoietic stem cells (HSC). MDS leads to ineffective hematopoiesis, and as many as 30% of patients progress to acute myeloid leukemia (AML). The mechanisms by which mutations accumulate in HSC during aging remain poorly understood. Here we identify a novel role 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 DNA repair genes. Stem/progenitor cells from these patients display dysfunctional DSB repair kinetics after exposure to ionizing radiation (IR). Haploinsufficiency of Mybl2 in mice also led to a defect in the repair of DSBs induced by IR in HSC and was characterized by unsustained phosphorylation of the ATM substrate KAP1 and telomere fragility. Our study identifies MYBL2 as a crucial regulator of DSB repair and identifies MYBL2 expression levels as a potential biomarker to predict cellular response to genotoxic treatments in MDS and to identify patients with defects in DNA repair. Such patients with worse prognosis may require a different therapeutic regimen to prevent progression to AML.

Signiﬁcance: These ﬁndings suggest MYBL2 levels may be used as a biological biomarker to determine the DNA repair capacity of hematopoietic stem cells from patients with MDS and as a clinical biomarker to inform decisions regarding patient selection for treatments that target DNA repair. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/20/5767/F1.large.jpg. Cancer Res; 78(20); 5767–79. 2018 AACR.

Introduction cell maturation and a high propensity for leukemic transformation. It is a clonal disease thought to originate in the hemato- Myelodysplastic syndrome (MDS) is an age-associated poietic stem cell (HSC; ref. 1). Effective management and hematopoietic malignancy, characterized by abnormal blood treatment of MDS is important, as early identiﬁcation of patients who are likely to progress to malignant disease allows for an 1Institute of Cancer and Genomic Sciences, College of Medical and Dental optimal therapeutic regime to be implemented. Genetic altera- Sciences, University of Birmingham, Birmingham, United Kingdom. 2Centre for tions are often present in MDS and a frequent chromosome Clinical Haematology, University Hospitals Birmingham NHS Foundation Trust, abnormality is del20q, whose common deleted region only Queen Elizabeth Hospital, Queen Elizabeth Medical Centre, Birmingham, United contains 5 genes expressed in HSCs, one of which is MYBL2 Kingdom. (2, 3). The MYBL2 gene encodes a ubiquitously expressed Note: Supplementary data for this article are available at Cancer Research protein belonging to the MYB family of transcription factors Online (http://cancerres.aacrjournals.org/). and has been shown to form part of different protein complexes Corresponding Author: Paloma García, University of Birmingham, Edgbaston, such as the Myb-MuvB/DREAM complex (4–7), Myb–Claﬁ com- Birmingham B12 2TT, United Kingdom. Phone: 44-0-121-414-4093; E-mail: plex (8), and the MRN complex (9), through which it exerts its [email protected] vital role in cell-cycle regulation and maintenance of genome doi: 10.1158/0008-5472.CAN-18-0273 stability (10–14). Analysis of publicly available global gene þ 2018 American Association for Cancer Research. expression data from CD34 -MDS patient cells (15) have

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confirmed that downregulation of MYBL2 expression correlates Isolation and expansion of human peripheral blood cells with poor prognosis; even in patients with a normal karyotype Peripheral blood samples from patients with MDS were (2, 3). This suggests that changes in MYBL2 expression could obtained in heparin-coated vacutainers. Peripheral blood mono- have significant consequences with regards to disease pathogen- nuclear cells were isolated using Ficoll-Paque (GE Healthcare) esis. Furthermore, it has been demonstrated that mice with low and stored at 80C. Cells were thawed and cultured for 8 days in levels of Mybl2 develop hematologic disorders during ageing expansion medium as described previously (25), with the excep- that closely resemble the human disease, implying that MYBL2 tion that the base medium was StemSpan H3000 (Stem Cell functions as a haploinsufficient tumor suppressor gene (2, 3). Technologies). Medium was refreshed on day 3 and 6. On day 8, þ However, how low MYBL2 expression contributes to MDS cells were harvested and CD34 cells purified using microbeads during ageing remains unknown. (Miltenyi Biotec). Given that HSCs must last for the entire lifetime of an individual to guarantee continuous blood cell production, this qRT-PCR increases the dependence of these cells on DNA repair to For human gene expression assays, qRT-PCR for MYBL2 maintain genomic integrity. Because HSCs are predominantly (Hs00942543_m1 MYBL2, Applied Biosystems) was carried quiescent, they are thought to primarily utilize nonhomolo- out using TaqMan PCR Master Mix (Applied Biosystems) gous end joining (NHEJ) rather than homologous recombi- and qRT- PCR for b-glucuronidase (HsGusB QT00046046, nation (HR) to repair DNA double-strand breaks (DSB; refs. Quantitect primer assay, Qiagen) was carried out using 16, 17). Although mainly error-free, NHEJ-dependent DSB SYBRGreen Master Mix (Thermo Fisher Scientific). For murine repair can also result in the generation of small genomic gene expression assays, qRT-PCR for p21 (Mm01303209_m1), deletions at the repaired break site, leading to the hypothesis Puma (Mm00519268_m1), Noxa (Mm00451763_m1), Bax that HSCs accumulate somatic mutations over time. This is (Mm00432051_m1), and b-2-microglobulin (Mm00437762_m1) thought to precede the appearance of blood disorders such as were carried out using TaqMan PCR Master Mix (Applied Bio- MDS and acute myeloid leukemia (AML; refs. 18–20), systems). Reactions were carried out in a Stratagene Mx3000P although no direct link between DNA repair and the patho- machine and samples were run in duplicate. Relative gene expres- genesis of MDS has been reported. sion was calculated as 2 DDCt values relative to control genes Because recent studies have shown that MDS originates from (b-glucuronidase for human samples and b-2-microglobulin for HSCs and that MYBL2 is known to play a role in maintaining murine samples). genome stability (1, 21, 22), we hypothesized that low levels of MYBL2 may compromise the DNA repair capacity of the Mice cell, resulting in the accumulation of genetic alterations to a Mice were maintained on a C57/BL6 background and geno- sufficient level to induce HSC transformation. To test this, we typed by Transnetyx. For mouse studies, no specificrandom- used ionizing radiation (IR) in vitro to induce DNA damage in ization or blinding protocol was used during experimental MDS patient's stem cells. Following treatment, we determined protocols. Mice of both genders were used. Age- and gender- the ability of these cells to repair their DNA and correlated this matched mice were used per experiment. Seventy-week-old with expression levels of MYBL2. To further study the role of healthy mice were chosen to perform aging studies. Disease- MYBL2 in DNA repair, we utilized a Mybl2 haploinsufficient free status of these animals was assessed on the basis of mouse model, which is susceptible to MDS development. Our behavior and physical appearance of the mice, normal values findings uncover a novel role for MYBL2 in regulating DSB of white blood cell, red blood cell, and platelets obtained from repair in the HSC population. peripheral blood counts, and by internal organ examination after dissection, in particular no signs of splenomegaly.

Materials and Methods Inhibitors Differential expression and reactome pathway enrichment Inhibitors were dissolved in DMSO; KU60019 (10 mmol/L) analyses was used for inhibition of ATM and NU7441 (1 mmol/L) was To assess differential gene expression and pathway enrichment used for inhibition of DNA-dependent protein kinase (Tocris between MDS samples displaying higher and lower levels of Bioscience). MYBL2 expression, we used previously published microarray data (15) deposited in the NCBI Geo DataSets repository (GSE19429), Flow cytometry and cell sorting and the BROAD Institute Gene Set Enrichment Analysis (GSEA) Single-cell suspensions of bone marrow were prepared using software (23, 24). Differential expression was assessed over standard techniques and red blood cells were depleted by ACK 1,000 permutations and ranked according to signal-to-noise ratio. lysis (0.15 mol/L NH4Cl, 1 mmol/L KHCO3, 0.1 mmol/L EDTA, A weighted enrichment statistic was applied. Gene sets comprising pH 7.4). Nonspecific antibody binding to Fc receptors was less than 15 genes were excluded from the analysis (the list of gene blocked using anti-CD16/CD32 (93, eBioscience) and the cells set used in the analysis is presented as Supplementary Tables S1 were stained with a combination of fluorochrome-conjugated and S2). The adjustment of the FAB composition was done using a antibodies including anti-mouse lineage; CD5, CD8a, CD11b, method of random sample removal. To balance the composition Gr-1, Ter119, B220 (APC or FITC), cKit (PeCy5 or e780; 2B8, of the MYBL2hi and MYBL2lo sets, samples of specific diagnosis eBioscience), Sca-1 PeCy7 (D7, eBioscience), Flk2 PE (A2F10, (for example RA or RAEB) were randomly removed when they eBioscience), CD48 APC (HM48-1, eBioscience), and CD150 were found to be over-represented in a set. Four different permu- PEcy7 (TC15-12F12.2, BioLegend), to allow identification of þ þ tations were performed to verify that the results were not affected Flk2 HSCs (lineage cKit Sca-1 Flk2 ) and long-term HSCs þ þ þ by the methodology. (SLAM staining; lineage cKit Sca-1 CD48 CD150 ). Some

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cells were analyzed directly by flow cytometry using a CyAn ADP ride for 5 minutes at room temperature and the cells were Analyzer (Beckman Coulter) and some were sorted using a permeabilized with 0.3% Triton-X 100/PBS for 5 minutes at room Cytomation XDP MoFlo machine (Beckman Coulter). In both temperature. For pKap1, cells were fixed in 4% PFA/PBS for 10 cases, data were analyzed using either Summit software (Dako) or minutes at room temperature, washed in PBS, and permeabilized FlowJo software (FLOWJO, LLC). When cells were required with ice-cold methanol for 10 minutes at room temperature. Cells þ for sorting, a cKit enrichment using streptavidin microbeads were blocked with 3% BSA/10% HIFBS/1% goat serum/0.3% (130-048-101, Miltenyi Biotec) and columns (130-042-201, Triton-X 100/PBS (blocking buffer) for 1 hour at room temper- Miltenyi Biotec) was performed prior staining. ature. Cells were incubated with primary antibodies to 53BP1 (NB-100-904, Novus Biologicals), pKAP1 (S824;A300-767A, Proliferation and apoptosis assays Bethyl Laboratories Inc), gH2AX (JBW301, Merck), MRE11 For proliferation assays in vivo using BrdU, mice were given (4895S, Cell Signaling Technology), mouse IgG control (G3A1, an intraperitoneal injection of 2 mg BrdU in PBS and 24 hours Cell Signaling Technology), or rabbit IgG control (sc-2027, Santa later animals were sacrificed. cKit-enriched bone marrow cells Cruz Biotechnology) and diluted in blocking buffer overnight at were isolated using anti-mouse cKit biotin (eBioscience) and 4C. Cells were washed three times in 0.1% Tween 20/PBS and streptavidin microbeads (Miltenyi Biotec) as per the manufac- incubated with goat anti-rabbit Alexa 488 secondary antibody turer's instructions. For proliferation assays in vitro using BrdU, diluted in blocking buffer for 1 hour at room temperature. Cells þ expanded CD34 cells were labeled with 10 mmol/L BrdU were washed three times in 0.1% Tween 20/PBS, dipped in Milli- for 3.5 hours. Cells were stained using the BrdU Flow Kit Q water, and mounted with ProLong Gold AntiFade Reagent (8811-6600, BD Biosciences) according to the manufacturer's containing DAPI (Invitrogen). Microscopy imaging was per- instructions. For proliferation assays using Ki67, cKit-enriched formed using a Zeiss LSM 510 Meta confocal microscope bone marrow cells were stained using the Ki67 flow kit (BD (100 objective NA 1.4 lens) and the images were analyzed using Biosciences) according to the manufacturer's instructions. ImageJ software. For all immunofluorescence staining, 30–50 þ For colony-forming assays, purified HSCs (lineage cKit Sca- cells were scored for each independent experiment. Experiments þ 1 Flk2 ) were obtained by sorting. Five-hundred HSCs were were performed at least three times and results represent a þ plated in Methocult (Stem Cell Technologies) supplemented minimum of 3 animals. For 53BP1 staining on CD34 cells from with penicillin/streptomycin (Invitrogen) in 35-mm petri patients with MDS, a minimum of 25 cells were scored per patient dishes and incubated for 6 days at 37 Cinanatmosphere per condition. containing 5% CO2. Colonies were counted using a standard light microscope with 10 objective. For G –Mcheckpoint Comet assays 2 þ þ fi studies, cKit-enriched bone marrow cells were isolated as HSCs (Lin /cKit /Sca1 /Flk2 ) were puri ed 0, 1, 5, and described previously. A total of 1–2 106 cKit-enriched cells 24 hours after IR in vivo (2 Gy). Alkaline comet assays were were cultured for 18 hours in Iscove's modified Dulbecco's performed as described previously (26). Cells were stained with medium (IMDM) containing 10% heat-inactivated FBS SYBR Safe (Invitrogen) for 1 hour and imaged using a Leica (HIFBS), 3% penicillin/streptomycin, 1 mmol/L sodium pyru- DM6000 microscope (20 objective). Analysis was performed using ImageJ software using the Open Comet plugin and the head vate, 2 mmol/L L-glutamine, 0.1 mmol/L nonessential amino fi acids, 50 mmol/L 2-mercaptoethanol, 25 ng/mL SCF, 10 ng/mL nding was selected to the brightest region. Olive moment IL3, 25 ng/mL IL11, 25 ng/mL TPO, 4 U/mL EPO, 10 ng/mL values were automatically generated by the software. Statistical fi GM-CSF, and 25 ng/mL FLT3L (complete cytokine medium). signi cance was calculated using GraphPad Prism software uti- – Medium was replaced and the cells were cultured for 1 hour lizing the Mann Whitney test. with DMSO or 10 mmol/L KU60019 prior to irradiation (IR) in Peptide nucleic acid-FISH vitro (2 Gy). Cells were cultured for a further 5 hours and Purified HSCs were obtained by cell sorting, exposed to ion- stained with (i) cell surface marker antibodies to identify izing radiation in vitro (2 Gy), and cultured for further 7 days subpopulations; (ii) mouse anti-phospho histone H3 Ser10 in methylcellulose semi-solid medium containing cytokines (2-hour staining; clone 6G3, Cell Signaling Technology) and (M3434). Colonies were dissociated and cultured with 100 ng/mL goat anti-mouse Alexa 488 (30-minute staining; A-11001, colcemid for 3 hours at 37C to arrest cells in metaphase. Peptide Molecular Probes); and (iii) Vybrant DyeCycle (Molecular nucleic acid staining was performed as described previously (27). Probes). Fixation and permeabilization were carried out using Briefly, cells were exposed to hypotonic solution (0.56% KCl) for buffers from the BrdU Flow Kit (BD Biosciences) according to 16 minutes at 37Candthenfixed in methanol:acetic acid (3:1). the manufacturer's instructions. For apoptosis assays, whole After three changes of fixative solution, the cells were dropped on to bone marrow cells were stained with cell surface marker anti- slides that had been pretreated with 1 N HCl, followed by 100% bodies to identify subpopulations as well as mouse anti- ethanol and finally fixative solution. FISH with FITC-labeled cleaved PARP (Asp214; clone F21-852, BD Biosciences). (CCCTAA)3 peptide nucleic acid (F1009-5, Panagene) was performed followed by mounting with ProLong Gold AntiFade Immunofluorescence Reagent containing DAPI (Invitrogen). Microscopy imaging was Purified murine cells (Flk2 HSCs or SLAM HSC) or human þ performed using a Leica DM6000 microscope (100 objective) CD34 cells were cytospun onto microscope slides for 5 minutes and images were analyzed blindly using ImageJ software. An at 800 rpm. For 53BP1, gH2AX, and MRE11 staining, cells were average of 20–30 metaphases were scored per condition. treated with CSK buffers (buffer 1 for 4 minutes at room temperature, buffer 2 for 1 minute at room temperature), washed in Statistical analysis PBS, and fixed in 4% PFA/PBS for 15 minutes at room temper- All data shown are presented as mean SEM. When com- þ þ þ ature. Fixation was quenched with 50 mmol/L ammonium chlo- paring datasets between Mybl2 / and Mybl2 /D animals, two-

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tailedunpairedStudentt test was applied using GraphPad analysis confirmed that expression of DNA repair genes cor- Prism software, unless indicated. No statistical method was relates with MYBL2 levels in MDS (Fig. 1C; Supplementary used to estimate the sample size. No specific randomization or Table S2), independently of the disease state. blinding protocol was used. N indicates the numbers of independent experiments performed and was chosen to ensure Low MYBL2 expression associates with poor DNA repair in adequate statistical power. P 0.05 was considered statistically human MDS significant. Significance tests were performed on all samples To determine whether the differential expression of DNA and therefore graphs lacking P values indicate results were not repair genes (Fig. 1) had a functional consequence, we next þ statistically significant. assessed the DNA repair kinetics of CD34 cells, a multipotent stem and progenitor cell population, from 5 patients with MDS with differing prognoses (Table 1), without prior knowledge of Study approval þ All animal experiments were performed under an animal MYBL2 expression levels. CD34 cells were irradiated in vitro (2 project license in accordance with UK legislation. Human patients Gy) and the prevalence of p53-binding protein 1 (53BP1) foci, with MDS were recruited from the clinic held at the Centre for a robust marker for DSBs, was measured at 1, 3, and 5 hours Clinical Haematology, University Hospital Birmingham NHS post-irradiation (Fig. 2A and B). Cells from these patients Foundation Trust. All the subjects have read the patient informa- showed a striking difference in their ability to repair IR-induced tion sheet and signed the consent form. The study was conducted DSBs, as evidence by their differing ability to resolve 53BP1 as according to Good Clinical Practice guidelines, consistent with foci. After MYBL2 expression levels were measured (Fig. 2C) in the principles that have their origin in the Declaration of Helsinki. the same patients, it was evident that DSB repair kinetics The study was approved by the West Midlands – Solihull Research strongly correlated with MYBL2 mRNA expression (Fig. 2D; 2 Ethics Committee (10/H1206//58). R ¼ 0.83, P ¼ 0.0114). For example, a patient (patient 5) expressing similar MYBL2 levels to cells isolated from a healthy individual could efficiently repair DSBs within 5 hours, whereas Results in contrast, patients expressing around 50% normal levels of Low MYBL2 expression associates with decreased expression MYBL2 (patients 3 and 4), or even lower (patients 1 and 2) of DNA repair genes in human MDS exhibited defective clearance of 53BP1 foci, suggesting the Given the role of MYBL2 in transcription and in maintaining persistence of unresolved DSBs. We also investigated whether genome stability (10–14) and recent studies showing increased proliferation rates correlated with MYBL2 expressioninMDS þ DNA damage in MDS (28, 29), we decided to determine patient CD34 cells by performing BrdU incorporation assays. whether MYBL2 levels were associated with altered expression This revealed that MYBL2 expression in these patients did not of DNA repair genes in patients with MDS. To do this, we first correlate with proliferation, (Supplementary Fig. S1A and S1B), subdivided the patients into MYBL2lo and MYBL2hi popula- nor did this correlate with the cell's DNA repair capacity tions based on a differential gene expression analysis on HSCs (Fig. 2E). These data suggest that reduced expression of DNA from patients with MDS (15), using the 25th and 75th per- damage genes in patients with compromised MYBL2 expression centiles of MYBL2 expression to define each group. Comparing has a functional impact on the ability of these cells to repair these two groups, genes were then ranked on the basis of the genetic damage. Furthermore, we propose that MYBL2 mRNA signal-to-noise ratio for differential gene expression (DGE; expression levels may also be used as a potential biomarker Supplementary Table S1A). This DGE scoring was then ana- predicting the cellular response to DNA damage, which could lyzed against a collection of reactome gene sets (Supplementary be of use for patient stratification. Table S1B), to assess their individual enrichment, which þ/ encompassed pathways with which MYBL2 has previously been Slow DSB DNA repair kinetics in Mybl2 D HSCs after associated (cell cycle, DNA replication) and pathways relevant in vivo IR to this study (DNA damage, DNA repair, chromosome main- Following the observation that MYBL2 expression levels tenance, and apoptosis). Consistent with previously published correlated with DSB repair kinetics in human MDS stem cells, observations, the analysis (Supplementary Table S1C) con- we wanted to further investigate the involvement of MYBL2 in firmed the previously published association between MYBL2 regulating the DNA damage response. To do this, we used a þ levels and cell-cycle progression in the context of MDS (2). Mybl2 haploinsufficient mouse model (Mybl2 /D), known to be Remarkably, it also revealed a significant enrichment (NOM susceptible to MDS with ageing (30). We decided to focus our P ¼ 0.00065, FWER P ¼ 0.006) for the reactome gene set studies on the HSC population, the population in which MDS "DNA damage pathway" (R-HAS-73894), indicating an overall originates (1). Importantly, these animals did not show any downregulation of DNA repair pathways components in the major differences in the numbers of HSC/progenitor cells when MYBL2lo MDS cases (Fig. 1A). Upon further inspection of the compared with wild-type mice prior to treatment (Supplemen- two populations, and confirming our previous results (2), we tary Fig. S2). To investigate the DNA damage response, we found that the MYBL2lo population was significantly enriched assessed the clearance of 53BP1 foci, as a measure of repair þ in MDS with excess blasts type II (MDS-EB2) cases with worse kinetics,inbothwild-typeandMybl2 /D HSCs [defined as þ þ diagnosis (Fig. 1B, top table). To exclude the possibility that the (Lin /cKit /Sca1 /Flk2 )] followed by irradiation of the mice observed link could primarily reflect an association between the with 2 Gy of IR (Fig. 3A; Supplementary Fig. S3A). While we did DNA damage pathway and advanced disease states rather than not observe any difference in the initial recruitment of 53BP1 þ MYBL2 levels, we recurated the compared subsets to display 1 hour post-irradiation between wild-type and Mybl2 /D balanced diagnosis composition (Fig. 1B, bottom table), and HSCs, there were notable differences in the kinetics of 53BP1 repeated the gene expression analysis (Fig. 1C). This unbiased foci 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 signature represented 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 Fisher exact test (right column) was applied to assess the dependence between the variables MYBL2 class and MDS World Health Organization classiﬁcation. C, Enrichment plot for the DNA repair reactome gene set after adjustment of the FAB composition of the compared sample sets (left); heatmap of 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 also Supplementary Table S1; Supplementary Table S2).

Table 1. Clinical data for the patients with myelodysplastic syndrome used in this study Patient Date of Age WHO 2016 Date of sample Disease progression number diagnosis (years) Cytogenetics IPSSR Diagnosis collection (date) 1 01/02/2015 76 46, XY High risk MDS-EB1 29/09/2015 AML 27/08/2015 2 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.

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Figure 2. MYBL2 expression correlates with kinetics of 53BP1 clearance in MDS patient CD34þ cells after irradiation. A–E, Peripheral blood cells from a healthy control 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 irradiation in vitro and cultured in cytokine-containing medium. Cells were removed from culture at different time points (1, 3, and 5 hours), cytospun onto microscope slides, and immunofluorescence for 53BP1 was performed. A, Representative images of 53BP1 foci 5 hours after IR in MDS patient CD34þ þ cells and CD34 cells taken from a healthy individual. (Scale bar, 5mm). B, Average number of 53BP1 foci at different time points in patients with MDS and 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, MDS patient number; HC, healthy control). C, MYBL2 gene expression from purified CD34þ cells normalized using expression of GUSB. D, Negative correlation between number of 53BP1 foci 5 hours after IR and MYBL2 expression levels in MDS patient CD34þ cells. E, Purified CD34þ cells were labeled with 10 mmol/L BrdU for 3.5 hours and stained with a fluorescent antibody directly conjugated to BrdU. No correlation was observed between the number of 53BP1 foci 5 hours after IR and percentage of BrdUþ cells in MDS patient CD34þ cells (error bars, mean SEM).

and C). Wild-type HSCs showed a significant reduction in cells decrease in genome stability (Fig. 3B and C; Supplementary positive for 53BP1 foci at 5 hours post-irradiation, whereas in Fig. S3C). Interestingly, levels of 53BP1-positive cells in young þ þ contrast, more than 50% of Mybl2 /D HSCs still retained a Mybl2 /D HSCs were comparable with aged wild-type HSCs, significant number of 53BP1 foci–positivecellsatthistime suggesting that these cells may demonstrate a premature aging point. Moreover, the absolute number of 53BP1 foci per cell phenotype. Together, these data indicate that Mybl2 haploin- þ was also increased in the Mybl2 /D HSCs (Supplementary sufficiency is associated with defective repair of IR-induced Fig. S3B and S3C). To extend these observations to a more DSBs in HSCs. refined HSC population, we repeated this experiment with Because our data are only indicative of unrepaired DNA HSCs purified from young animals (7 weeks) using SLAM DSBs, we performed comet assays to directly measure the total þ staining (KSL CD48 CD150 ). This revealed that the retention amount of DNA damage remaining in these cells at different þ þ of 53BP1 foci was also apparent in Mybl2 /D CD150 HSCs, but times post-irradiation. In keeping with a failure to properly þ not in their wild-type counterparts (Fig. 3D). Moreover, to repair IR-induced damage, Mybl2 /D HSCs displayed an increase determine whether this effect was dose dependent, we mea- in the olive tail moment 5 hours post-irradiation when compar- sured the percentage of 53BP1 foci 5 hours after 1 Gy of IR. This ed with wild-type HSCs (Fig. 3E). These differences in DNA þ revealed that at a lower dose of IR, Mybl2 /D HSCs exhibited a repair kinetics were not the consequence of changes in cell-cycle þ "wild-type" 53BP1 response (Supplementary Fig. S3D), sug- profile between wild-type and Mybl2 /D HSCs, as HSCs from gesting that there is an insufficient quantity of DNA repair both genotypes showed a similar percentage of cells in G0–G1 þ proteins present in the Mybl2 /D HSCs required to cope with prior to irradiation (Supplementary Fig. S4), and the same repairing high-level DNA damage. Because aged haploinsuffi- percentage of cells in S-phase measured by in vivo BrdU incor- cient Mybl2 mice develop an MDS-like disease, we investigated poration (Supplementary Fig. S5A and S5B). Importantly, we how aging affected DSB repair capacity of wild-type and did not observe any changes in the absolute numbers of HSCs þ Mybl2 /D HSCs from young (7 weeks) and old mice (70 weeks). after in vivo IR (Supplementary Fig. S5C), nor apoptosis, mea- Unsurprisingly, 70-week-old HSCs exhibited a higher percent- sured either by PARP1 cleavage (Supplementary Fig. S5D) or age of 53BP1-positive cells than 7-week-old HSCs. However, the induction of p53-dependent apoptotic genes (Supplemen- in keeping with a role for MYBL2 in regulating DNA repair, tary Fig. S5E), which could account for our observations using Mybl2 haploinsufficiency exacerbated the age-associated the comet assay. Overall, these data demonstrate that Mybl2

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Low MYBL2 Expression in MDS Alters DNA DSB Repair in HSCs

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 exposed to 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 using purified HSC subpopulations, including Flk2 HSC (KSL Flk2) and SLAM HSC (KSL CD48CD150þ). B, Representative images of 53BP1 staining of Flk2 HSCs from 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 for 53BP1 staining in young animals 5 hours after IR (n ¼ 3). E, Representative images of alkaline comets from Flk2 HSCs isolated from young animals 5 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 after 2GyIRin vivo (right; Mybl2þ/þ, n ¼ 4for5hoursandn ¼ 3 for 0, 1, and 24 hours; Mybl2þ/D,n¼ 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.

haploinsufficient mice display a defect in the kinetics of DSB dramatic loss of 53BP1 foci formation at 5 hours after IR (Fig. repair in response to IR, which is heightened during aging, but 4B–D). These findings were recapitulated by analysis of the that has no impact on HSC survival. formation/retention of gH2AX foci, a pan-DNA damage marker (Fig. 4E–G). Nonetheless, 53BP1 foci were detected at 1 hour þ/ þ Mybl2 D HSCs are highly dependent on DNA-dependent post-irradiation in Mybl2 /D HSCs (Fig. 4B–D), suggesting that protein kinase for DNA DSB repair our observations did not reflect a global inability to form þ To gain a mechanistic understanding of the DNA repair 53BP1 foci. Furthermore, Mybl2 /D HSCs treated with DNA- þ defect in Mybl2 /D HSCs, we used small-molecule inhibitors PK inhibitor were proficientatsensingDNAdamage,because to investigate the relationship of Mybl2 haploinsufficiency with MRE11 foci formed 1 and 5 hours after IR (Fig. 4H and I). two key proteins involved in the DSB response, namely DNA- Moreover, the absence of 53BP1 and gH2AX foci 5 hours post- dependent protein kinase (DNA-PK) and ATM. Treatment of irradiation was not because DNA repair had been completed, as þ wild-type HSCs with the DNA-PK inhibitor NU7441 (Fig. 4A; Mybl2 /D HSCs exhibited increased olive tail moments by comet ref. 31) induced an increase in the percentage of cells positive assay (Fig. 4J). However, these breaks were eventually repaired, as þ for 53BP1 foci, in line with an expected defect in DSB repair due by 24-hour levels of DNA damage in Mybl2 /D HSCs was equal to inhibition of the NHEJ pathway (Fig. 4B–D). In contrast, to that of wild-type cells (Fig. 4J). Interestingly, it has been þ Mybl2 /D HSCs treated with the same inhibitor demonstrated a previously shown that DNA-PK and ATM are both required for

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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 obtained from Mybl2þ/þ (n ¼ 3) and Mybl2þ/D (n ¼ 3) animals aged 7 weeks and enriched forcKitusingmicrobeads.cKitþ-enriched cells were cultured for 1 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, or 24 hours. Flk2 HSCs were purified using cell sorting and prepared for immunofluorescence and comet assays. A, Experimental scheme for isolation and culture 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 of Flk2 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. /þ on axes indicate whether cells were treated with DNA-PK inhibitor. Error bars, mean SEM. P values on the graphs obtained when using a two-tailed unpaired Student t test.

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Low MYBL2 Expression in MDS Alters DNA DSB Repair in HSCs

efficient H2AX phosphorylation and 53BP1 recruitment to DSBs Given these findings, it is tempting to speculate that because and that ATM / B cells are completely dependent on DNA-PK ATM inhibition in wild-type HSCs mimics the 53BP1 foci þ to sustain the phosphorylation of H2AX after gamma irradia- clearance defect observed in Mybl2 /D HSCs, a similar treatment þ tion (32). On the basis of this, these data suggest that reliance would also lead defective cell survival as seen in Mybl2 /D cells. þ that Mybl2 /D HSCs have on DNA-PK to mediate DSB signaling However, wild-type HSCs transiently treated with an ATM is indicative of an underlying defect in the ATM-dependent inhibitor did not display the same characteristics as untreated þ DNA damage response. Mybl2 /D HSCs when assessed by a colony-forming assay (Sup- plementary Fig. S6B), indicating that either short-term inhibition of ATM pathway does not have the same overall effect as þ þ The slow repair kinetics in Mybl2 /D HSCs are epistatic with Mybl2 haploinsufficiency or that Mybl2 /D HSCs display addi- inhibition of ATM tional defects that are not mimicked by ATM inhibitor. While previously published data have been suggestive þ/ of a link between MYBL2 and ATM signaling (9), we wanted A subset of ATM function is impaired in Mybl2 D HSCs þ þ to specifically determine whether Mybl2 /D HSCs exhibit To further investigate the defective ATM signaling in Mybl2 /D defective ATM-dependent signaling in response to DSBs. HSCs, we next assessed the phosphorylation of KAP1. It þ To address this, we treated wild-type and Mybl2 /D HSCs has been previously reported that approximately 10%–15% with the ATM inhibitor KU60019 (33), and analyzed 53BP1 of DSBs require ATM signaling to be repaired in G0–G1 phases foci clearance (Fig. 5A). These analyses revealed that treatment of the cell cycle (34) and that this repair requires the phos- with KU60019 delayed the clearance of 53BP1 foci in wild-type phorylation of Kap1, which is known to be completely cells, with ATM inhibition causing a >2-fold increase in the dependent on ATM (35). Therefore, we examined Kap1 phos- number of cells still displaying 53BP1 foci 5 hours post-irra- phorylation after exposure to IR in HSCs (Fig. 6A). In diation (Fig. 5B–D),inlinewiththeknownrequirement line with previous reports, wild-type HSCs displayed robust for ATM in DSB repair. In contrast, ATM inhibition in pan-nuclear p-KAP1 staining within minutes following IR, þ Mybl2 /D HSCs had little effect on 53BP1 clearance after which could be distinguished as discrete foci by 3 hours IR (Fig. 5C; Supplementary Fig. S6A). These data further sup- post-irradiation (Fig. 6B and C; ref. 35). Interestingly, although þ port the prediction that the altered DSB repair kinetics observed Mybl2 /D HSCs also exhibited rapid KAP1 phosphorylation þ in Mybl2 /D HSCs are potentially due to a defect in ATM- immediately post-irradiation, they were unable to maintain dependent signaling. this 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 weeks and 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 andculturedforafurther5hours.Flk2 HSCs were purified using cell sorting and immunofluorescence for 53BP1 and DAPI was performed. A, Experimental scheme for isolation and culture of cKitþ cells with ATM inhibitor for immunofluorescence. B, Representative images of 53BP1 staining of Flk2 HSCs treated with the ATM inhibitor KU60019. C, Percentage of Flk2 HSCs positive for 53BP1 foci when treated with KU60019. D, Number of 53BP1 foci per cell when Flk2 HSCs were treated with KU60019. /þ on axes indicates whether cells were treated with ATM inhibitor. Error bars, mean SEM. Samples were not statistically significant when using a two-tailed unpaired Student t test. ns, nonsignificant.

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Figure 6. MYBL2-deficient hematopoietic stem cells show a deficiency in the maintenance of ATM-dependent p-KAP1 and increased telomere fragility after irradiation. 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 and immunofluorescence 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 each HSC 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 weeks by 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 stained with telomere peptide nucleic acid and DAPI. Shown are examples of fragile telomeres found in 7- and 70-week-old cells. Table shows the number of chromatid ends scored and the percentage of chromatid ends with fragile telomeres. P values in the table were obtained using a Mann–Whitney test comparing 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 a two-tailed unpaired Student t test are indicated.

further reinforced our ﬁndings that partial Mybl2 loss leads to naling during late-stage repair. To conﬁrm this prediction, we defective ATM signaling, and also suggests that MYBL2 may be next stimulated HSCs to enter the cell cycle, and analyzed required to maintain rather than initiate ATM-dependent sig- activation and maintenance of the ATM-dependent G2–M

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þ cell-cycle checkpoint after IR. Interestingly, Mybl2 /D HSCs regions (35, 37), our data suggests a requirement for MYBL2 retained the ability to activate this checkpoint following expo- in repairing a subset of DSBs associated with heterochromatic sure to IR (Supplementary Fig. S7A–S7C), suggesting that these chromosomal regions. cells are not completely defective in ATM function. This sug- In agreement with our conclusions, studies using human cell gests that MYBL2 is required to maintain, but not initiate, lines have recently demonstrated that MYBL2 interacts with activation of a specific subset of ATM-dependent signaling Nbs1, which is required for the activation of ATM in response to pathways. DSBs and also ATM-dependent heterochromatic DSB repair in OneofthecharacteristicsoflossofATMfunctionistelomere G0–G1 (9). While on face value, this may explain our observa- þ instability (36). We therefore postulated that lower MYBL2 tions Mybl2 /D HSCs, in stark contrast to the work of Henrich levels in HSCs might lead to telomere fragility as a result of and colleagues, we were unable to detect a G2–Mcheckpoint defective ATM signaling. To examine this possibility, HSCs defect in our Mybl2-haploinsufficient HSCs, suggesting that at from young and old mice were irradiated and cultured for least some ATM-dependent signaling is intact in these cells. 7 days in a colony-forming assay. Metaphase spreads were Moreover, Henrich and colleagues failed to observe any defects prepared from these cells and stained with telomere probes. in DNA repair, leading them to conclude that MYBL2 does not These investigations revealed that telomere instability (defined have an essential role in the DNA repair response. In contrast, as sister chromatid fusion or loss of telomere signal) was twice we have shown that MYBL2 does have a role for DSB repair in þ as frequent in the progeny derived from young Mybl2 /D HSCs HSCs, and without sufficient MYBL2 expression cells show compared with controls (Fig. 6D). In fact, this percentage was defective repair kinetics of IR-induced DSBs. These differences þ similar between young Mybl2 /D HSCs and old wild-type HSCs, could be due to cell type differences, such as primary cells þ in line with our earlier suggestion that Mybl2 /D HSCs display versus cell lines, or the use of different DNA-damaging agents to an aging phenotype that could lead to neoplastic lesions. induce DSBs (IR vs. UV). Alternatively, the highly quiescent þ In conclusion, we demonstrate that Mybl2 /D HSCs are nature of HSCs in vivo may also account for these discrepancies, defective in the maintenance of ATM-dependent DNA damage as this may make any defects in fast DSB repair by NHEJ more signaling at the sites of DSBs, leading to slower DSB repair pronounced as they cannot utilize repair by HR. kinetics and a higher dependency on DNA-PK in the surviving The importance of an appropriate DNA damage response for cells. Overall, these data suggest that correct MYBL2 protein the maintenance and protection of the HSC pool against levels are required for a proper DNA damage response and functional decline during ageing has been well reported appropriate DSB repair in the HSC compartment. Deregulation (38–41). Quiescent HSCs cannot use the HR pathway and thus of these levels leads to defective DSB repair, telomere instabil- rely on NHEJ-dependent mechanisms to repair their DNA ity, and likely contributes to the accumulation of genetic (16, 17). A failure to repair DSBs by the canonical DNA repair alterations in MDS. pathways can be detrimental to the cell, as alternative pathways may allow the potential for genome instability (42, 43). It has also been reported that reduction in or mutation of splicing Discussion factors in MDS leads to altered splicing of DNA repair and HSCs are the life-long pillars of continuous blood cell telomere maintenance genes (44). This then perturbs myeloid production. Maintenance of their genetic integrity is para- differentiation and contributes to disease development via a mount to avert the accumulation of mutations that can con- mechanism not necessarily involving chromosomal rearrange- þ tribute to the development of blood disorders such as MDS ments. Equally, abnormal DNA damage signaling in Mybl2 /D during the aging process. Our work demonstrates a previously HSCs could increase the mutational burden by facilitating the undescribed role for MYBL2 in promoting efficient DSB repair use of alternative, more error-prone signaling pathways. þ in HSCs, possibly via regulation of the ATM kinase. Further- Indeed, after inducing irradiated Mybl2 /D HSCs to proliferate, more, our findings suggest that MYBL2 levels may be used as a we found evidence of telomere instability in their progeny. biological biomarker to determine the DSB repair capacity of In vivo,thisprospectislikelytohaveasevereimpact,andmay þ CD34 cells from patients with MDS. Furthermore, MYBL2 ultimately lead to HSC malfunction and the accumulation of levels could also be used as a clinical biomarker to inform cells that are primed for the development of blood disorders decisions regarding patient selection for transplantation or such as MDS. Recent work in the field of MDS suggests that treatments that target DNA repair, highlighting the translation- telomere dysfunction is a potent driver of the disease pheno- al importance of this work. type (44) and telomere elongation using danazol treatment In line with a role for MYBL2 in regulating ATM signaling, we has been shown to improve hematologic responses including have shown that Mybl2 haploinsufficient HSCs display a delay reducing transfusion dependency (45). in 53BP1 clearance after DNA damage induced by IR, which is Importantly, we show that the association between low MYBL2 exacerbated during ageing. In these cells, defective ATM signal- levels and impaired DNA repair also holds true in patients with ing leads to loss of sustained KAP1 phosphorylation and MDS. Thus, by directly measuring the DNA repair kinetics in þ telomere instability. This renders these cells reliant on other CD34 MDS cells, our data shows a correlation between MYBL2 þ DNA repair pathways prevalent in noncycling cells. As a result, levels and functional DSB repair. Moreover, in CD34 cells from þ Mybl2 /D HSCs are highly dependent on the NHEJ regulator these patients, low MYBL2 levels largely associate with low DNA-PK for DSB signaling, because inhibition of this kinase expression of DNA repair genes. Together, these data provide a leads to a failure to maintain gH2AX and 53BP1 at sites of molecular rationale for the accumulation of genetic anomalies in damage. Moreover, because ATM-dependent phosphorylation patients deficient for MYBL2, which could play a role in the of KAP1 has been suggested to be required for chromatin progression of their disease. Importantly, this data represents the þ relaxation and the repair of DSBs within heterochromatin first direct study of repair kinetics in MDS patient CD34 cells.

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Current treatment options for patients with MDS are mostly Authors' Contributions based on cytotoxic agents prior to autologous transplant, and are Conception and design: R. Bayley, M.R. Higgs, E. Petermann, P. García challenged by the occurrence of clonogenic relapse, increased Development of methodology: R. Bayley, D. Blakemore, L. Cancian, P. Garcia resistance to therapy and, in some cases, leukemic transformation. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Cancian, G. Volpe, N. Reeve, M. Raghavan, Relapse of disease in these patients is typically driven by addi- P. García tional genetic lesions. Of note, delq20 clones are not uncommon Analysis and interpretation of data (e.g., statistical analysis, biostatistics, in patients following cytotoxic chemotherapy, with more than computational analysis): R. Bayley, D. Blakemore, L. Cancian, S. Dumon, 20% of these patients having a therapy-related myeloid neo- G. Volpe, C. Ward, P. García plasm, conferring a high mortality (46). Patients with therapy- Writing, review, and/or revision of the manuscript: R. Bayley, L. Cancian, related myeloid neoplasms more frequently have clonal hema- S. Dumon, G. Volpe, C. Ward, M. Raghavan, M.R. Higgs, GS Stewart, E. Petermann, P. García topoiesis with the originating mutation being present prior to Administrative, technical, or material support (i.e., reporting or organizing chemotherapy treatment (47). We therefore hypothesize that data, constructing databases): R. Bayley, P. García these clones are susceptible to DNA-damaging agents and that Study supervision: P. García DNA repair defects may be involved in the etiology of their Others (contributed to the experiments): R. Almaghrabi subsequent myeloid neoplasm. Moreover, our work supports Others (technical assistance to perform the experiments): J. Gujar the notion that Mybl2 haploinsufficiency results in changes in Others (advised with experiments and contributed to writing/revision of the manuscript): M.R. Higgs DNA repair kinetics and defective ATM signaling. Both ATM signaling and NHEJ are known to be activated to repair DSBs Acknowledgments fi induced by doxorubicin (48, 49), indicating that our ndings The authors wish to thank the members of the Petermann, Stewart, with IR are also likely to be applicable to the use of anthracyclines Higgs, García, and Frampton laboratories for advice and constructive in chemotherapy. criticisms, and Professor Frampton for covering the experiments through In light of our findings, we propose that compromising the his animal license. The authors also wish to thank the animal facility and MYBL2-dependent DNA damage response in HSCs can facilitate cell sorter facility at the University of Birmingham, and Donna Walsh for MDS development by allowing inefficient repair of physiologic collecting blood samples from myelodysplastic syndrome patients at the Centre for Clinical Haematology (Queen Elizabeth Hospital, Birmingham). DSBs, and promoting telomere instability, two processes known R. Bayley and L. Cancian were supported by MRC grant (MR/K01076X/1). to contribute to the generation of oncogenic transformation in the D. Blakemore was supported by a MRC PhD studentship (1632704). surviving HSC population. Furthermore, we suggest that sus- This work was funded by an MRC New Investigator Research Grant tained low levels of the MYBL2 protein in the premalignant cells (MR/K01076X/1) and MRC Proximity to Discovery: Industry Engagement may confer a susceptibility to disease progression through the fund (MC_PC_14123; to P. García). accumulation of incorrectly repaired DNA lesions. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Disclosure of Potential Conflicts of Interest advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate R. Almaghrabi reports receiving a commercial research grant from The this fact. Saudi Embassy. M. Raghavan has received speakers' bureau honoraria from Celgene Ltd and Pfizer Ltd. No potential conflicts of interest were disclosed Received February 1, 2018; revised June 22, 2018; accepted July 31, 2018; by the other authors. published first August 6, 2018.

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www.aacrjournals.org Cancer Res; 78(20) October 15, 2018 5779

MYBL2 Supports DNA Double Strand Break Repair in Hematopoietic Stem Cells

Rachel Bayley, Daniel Blakemore, Laila Cancian, et al.

Cancer Res 2018;78:5767-5779. Published OnlineFirst August 6, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-0273

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