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MYBL2 Supports DNA Double Strand Break Repair in Hematopoietic

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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 character- ized 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 treat- ments 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. Ó2018 AACR. Introduction cell maturation and a high propensity for leukemic transforma- tion. 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 Ó2018 American Association for Cancer Research. expression data from CD34 -MDS patient cells (15) have www.aacrjournals.org 5767 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst August 6, 2018; DOI: 10.1158/0008-5472.CAN-18-0273 Bayley et al. 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 À80C. 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- À 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
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