Leukemia (2013) 27, 629–634 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu

ORIGINAL ARTICLE BCR-ABL1 kinase inhibits uracil DNA UNG2 to enhance oxidative DNA damage and stimulate genomic instability

A Slupianek1, R Falinski1, P Znojek1, T Stoklosa1,2, S Flis1, V Doneddu3, D Pytel1,5, E Synowiec4, J Blasiak4, A Bellacosa3 and T Skorski1

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in chronic phase (CML-CP). Unfortunately, 25% of TKI-naive patients and 50–90% of patients developing TKI-resistance carry CML clones expressing TKI-resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen , which may result in accumulation of uracil derivatives in genomic DNA. Unfaithful and/or inefficient repair of these lesions generates TKI-resistant point mutations in BCR-ABL1 kinase. Using an array of specific substrates and inhibitors/blocking antibodies we found that uracil DNA glycosylase UNG2 were inhibited in BCR-ABL1-transformed cell lines and CD34 þ CML cells. The inhibitory effect was not accompanied by downregulation of nuclear expression and/or chromatin association of UNG2. The effect was BCR-ABL1 kinase-specific because several other fusion kinases did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI, we found that reduction of UNG2 activity increased the number of uracil derivatives in genomic DNA detected by modified comet assay and facilitated accumulation of ouabain-resistant point mutations in reporter Na þ /K þ ATPase. In conclusion, we postulate that BCR-ABL1 kinase-mediated inhibition of UNG2 contributes to accumulation of point mutations responsible for TKI resistance causing the disease relapse, and perhaps also other point mutations facilitating malignant progression of CML.

Leukemia (2013) 27, 629–634; doi:10.1038/leu.2012.294 Keywords: reactive oxygen species; genomic instability; chronic myeloid leukemia; BCR-ABL1; BER

INTRODUCTION mutations in BCR-ABL1 kinase causing the initial and persistent Chronic myeloid leukemia in chronic phase (CML-CP) is initiated resistance to TKIs. by t(9;22) encoding for BCR-ABL1 tyrosine kinase that transforms Point mutations may result from enhanced oxidative DNA 11,12 hematopoietic stem cells.1 CML-CP is leukemia stem cells (LSCs)- damage and/or deregulated mechanisms of DNA repair. derived disease, but deregulated growth of LSCs-derived leukemia Oxidative DNA damage arises from reactive oxygen species 13 progenitor cells (LPCs) leads to the manifestation of the disease.2 (ROS). The most important primary ROS is superoxide radical À Most CML-CP patients are currently treated with tyrosine kinase anion (O2 ), which may lead to the production of hydrogen 14 inhibitors (TKIs) such as imatinib, dasatinib and nilotinib, which peroxide (H2O2) and hydroxyl radical (OH). ROS can damage induce complete cytogenetic response or complete molecular DNA bases to produce numerous oxo-derivatives, among the most response in 60–70% and only 8% of cases, respectively.3,4 frequent are 5-hydroxy-cytosine (5-OH-C) generating 5-hydroxy- However, itt is unlikely that TKIs will ‘cure’ CML because CML-CP uracil (5-OH-U).14 Oxidative of cytosine generates cells are elusive targets even for the most advanced therapies highly mutagenic U:G mismatches that, if not repaired, leads to employing second- and third-generation TKIs.5 One of the major G:C-A:T transitions, the most frequent point mutations in human reasons of TKI resistance are BCR-ABL1 tyrosine kinase tumors.15 The potential role of ROS-induced oxidative DNA mutations.6,7 These TKI-resistant BCR-ABL1 forms usually result damage in accumulation of point mutations in CML-CP cells was from point mutations in the fragment encoding the kinase highlighted by our previous studies showing that BCR-ABL1- domain. In addition, accumulation of point mutations in other transformed cell lines, and CML-CP LSCs and also LPCs contained (for example, p53, Ras, p16) may lead to malignant 2–6 times more ROS and oxidized bases in comparison with their progression of the disease to the accelerated phase (CML-AP) normal counterparts.16 and blast phase (CML-BP).8 Point mutations in BCR-ABL1 and Oxidized bases in DNA are repaired by the chromosomal aberrations affecting different genes have been (BER) pathway.17 First step in this process is catalyzed by DNA detected in Lin-CD34 þ CD38 À LSCs and Lin-CD34 þ CD38 þ LPCs9 that recognize and excise damaged DNA base, and in freshly established 32D-BCR-ABL1 cells.10 This observation leaving an intact abasic site (AP site) followed by its removal by supports the notion that genomic instability in CML stem/ AP (APE1) or lyase activity of bifunctional DNA progenitor cells is responsible for accumulation of point glycosylases. Then, after the cleavage of the phosphodiester bond,

1Department of Microbiology and Immunology, School of Medicine, Temple University, Philadelphia, PA, USA; 2Department of Immunology, Center of Biostructure Research, Banacha 1A, Bldg F, Medical University of Warsaw, Warsaw, Poland; 3Cancer Biology Program and Epigenetics and Progenitor Cells Keystone Program, Fox Chase Cancer Center, Temple University, Philadelphia, PA, USA and 4Department of Molecular Genetics, University of Lodz, Lodz, Poland. Correspondence: Dr A Slupianek or Professor T Skorski, Department of Microbiology and Immunology, School of Medicine, Temple University, 3400N Broad Street, MRB 528A, Philadelphia, PA 19140, USA. E-mail: [email protected] (AS) or [email protected] (TS) 5Present address: Department of Cancer Biology, The Leonard and Madlyn Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia PA 19104, USA. Received 27 September 2012; revised 4 October 2012; accepted 5 October 2012; accepted article preview online 9 October 2012; advance online publication, 13 November 2012 Oxidative DNA damage repair in CML A Slupianek et al 630 BER may proceed by ‘short patch’ (characterized by the insertion Oligonucleotides marked by asterisk were radiolabeled with [g32P]ATP of one by polymerase b) or by ‘long patch’ using T4 polynucleotide kinase (Promega, Madison, WI, USA). When (characterized by the insertion of several by indicated, radiolabeled oligonucleotides were annealed to a complimen- o o polymerase b or the replicative polymerases d/e). Finally, DNA tary strand by heating to 95 C followed by slow cooling to 4 C to obtain ends are sealed by DNA ligase. double-stranded substrates. The following glycosylases are associated with the removal of uracil and its derivatives from DNA: nuclear uracil DNA glycosylase Nuclear cell lysates for glycosylase assay (UNG2) and single-strand selective monofunctional uracil DNA Nuclear cell lysates were prepared as described before.30 Briefly, cells were 18,19 glycosylase (SMUG1)—remove uracil and 5-OH-U; a homolog suspended in 10 mM Hepes buffer, pH 7.9, containing 1.5 mM MgCl2,10mM of Escherichia coli endonuclease III (NTH1) and a homolog of E. coli KCl, 0.5 mM dithiothreitol and protease inhibitors on ice and cells were endonuclease VIII-like 1 (NEIL1)—remove 5-OH-U, 5-OH-C, 5,6-OH- lysed by 10 strokes of Dounce homogenizer. The pellet was suspended in U;20 a homolog of E. coli endonuclease VIII-like 2 (NEIL2)—removes 20 mM Hepes, pH 7.9 containing 25% glycerol, 0.42 M NaCl, 2 mM MgCl2, 5-OH-U and 5-OH-C;21 finally, thymine glycosylase and methyl- 0.2 mM EDTA, 0.5 mM phenylmethanesulfonylfluoride, 0.5 mM dithiothreitol 22 and protease inhibitors, incubated on ice for 10 min and sonicated. CpG-binding domain protein 4 (MBD4)—remove U opposite G. Supernatants containing nuclear extracts were collected after Among the above glycosylases, UNG2 has by far the most centrifugation for 15 min, 15 000  g at 4 oC, and stored at À 80 oC. prevalent activity in mammalian extracts.23 In some cases, deficiencies in BER pathway can generate DNA mismatches that are repaired by mismatch repair mechanisms to Glycosylase assays prevent point mutations.24 As we found that mismatch repair is 5-OH-U incision reaction was performed following the protocol described 25 by Kavli et al.31 with modifications. Briefly, 10 mg of the nuclear extract inhibited in CML-CP, the activity of BER is critical for prevention o 32 of accumulation of point mutations. Here, we report that BCR- proteins were incubated in 37 C with 50fM of the [ P]-radiolabeled single-stranded or double-stranded substrate containing 5-OH-U or control ABL1 kinase inhibits the activity of uracil DNA glycosylase UNG2 in the reaction buffer (20 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA, 1 mM and propose that this reduced activity may contribute to dithiothreitol, 0.5 mg/ml bovine serum albumin, 2 mM ATP, 40 mM creatine accumulation of point mutations in CML-CP cells. phosphate, 2 U/ml creatine phosphokinase). Neutralizing antibodies (2 mg) and 2 U of uracil glycosylase inhibitor (UGI) (New England Biolabs, Inc., MA, USA)32 were added when indicated. The reactions were stopped by MATERIALS AND METHODS addition of equal volume of the solution containing 80% formamide, Cells 1xTris/Borate/EDTA and 10% bromophenol blue followed by heating in 90 oC for 2 min. Reactions were separated by electrophoresis in 18% Murine 32Dcl3 and human M07 parental hematopoietic cell lines, and their M 26–28 polyacrylamide gel containing 8 urea and visualized by autoradiography. counterparts transformed with p210BCR-ABL1 were used before and The products were quantified using Quantity One software (Bio-Rad, maintained in Iscove’s modified defined medium supplemented with 1 mM Hercules, CA, USA). glutamine, 10% fetal bovine serum and interleukin 3-conditioned medium (murine cells) or stem cell factor-conditioned medium and recombinant granulocyte–macrophage colony-stimulating factor (PeproTech Inc., Rocky Modified comet assay Hill, NJ, USA) (human cells) in the concentrations necessary to support To assess the level of uracil in DNA, the modified comet assay was parental cells proliferation. CML patient cells were obtained from the Stem conducted as described previously.33 Briefly, cells (approximately 5  104 Cell and Leukemia Core Facility of the University of Pennsylvania, per slide) were centrifuged and pellets were gently mixed with 50 mlof Philadelphia, PA, USA, after receiving informed consent. CD34 þ CML cells 0.75% low melting point agarose in phosphate-buffered saline and spread were obtained using human CD34 þ selection cocktail (StemCell Technol- onto a microscope slides precoated with 1% agarose. After solidifying, the þ ogies, Inc., Vancouver, BC, Canada). CD34 cells from healthy volunteers slides were treated with chilled lysis buffer containing 10 mM TRIS, 1% (normal bone marrow cells) were obtained from Cambrex Bio Science Triton X-100, 2.5 M NaCl and 0.1 M EDTA Na2 (pH 10.0) for 60 min at 4 1C, Walkersville (Walkersville, MD, USA). The research activities involving followed by three times washing with incubation buffer (60 mM human samples were approved by the Institutional Review Board. When Tris-HCl, 1 mM EDTA, 0.1 mg/ml bovine serum albumin; pH 8) at room indicated, cells were treated with 1 mM imatinib for 24 h. temperature. A 50 ml aliquot of UNG2 (1 U per gel; Roche Applied Science, Mannheim, Germany) or buffer alone, as control was subsequently placed onto gel surface and covered with a cover slip. Enzyme-treated samples Western blot analysis and controls were incubated in a moist chamber at 37 1C for 60 min. 29 Nuclear cell lysates were obtained as described before. In order to obtain Following incubation and removal of the cover slip, the slides were placed nuclear-soluble and chromatin-bound protein fractions cells were in an electrophoresis tank. DNA was allowed to unwind for 20 min in the processed using the Subcellular Protein Fractionation kit (Thermo electrophoresis buffer consisting of 300 mM NaOH and 1 mM EDTA, pH413. Scientific, Rockford, IL, USA) following manufacturer-recommended Electrophoresis was conducted in the same buffer at 4 1C for 20 min at procedures. Cell lysates were resolved by SDS-polyacrylamide gel 0.73 V/cm (300 mA). The slides were then washed in water, drained and electrophoresis and blotted with primary antibodies recognizing UNG, stained with 1 mg/ml 4’,6-diamidino-2-phenylindole and covered with SMUG1 and Lamin (Santa Cruz Biotechnology, Santa Cruz, CA, USA); APE1, cover slips. The slides were examined at  200 magnification in an Eclipse OGG1 (8-oxoguanine glycosylase), MBD4, thymine glycosylase and HDAC II fluorescence microscope (Nikon, Tokyo, Japan) attached to COHU 4910 (Abcam, Cambridge, MA, USA); NEIL1 and NEIL2 (Oncogene Research video camera (Cohu, San Diego, CA, USA) equipped with a UV-1 filter block Products, Cambridge, MA, USA); NTH1 (Novus Biologicals, Littleton, CO, consisting an excitation filter (359 nm) and a barrier filter (461 nm) and USA), followed by secondary antibodies conjugated with horseradish connected to a personal computer-based image analysis system, Lucia- peroxidase. Comet v. 5.41 (Laboratory Imaging, Praha, Czech Republic). Fifty images were randomly selected from each sample and the percentage of DNA in the tail of comets (% tail DNA) was measured. The mean value of the % tail Substrates for glycosylases DNA in a particular sample was taken as an index of the DNA damage in The following oligonucleotides (The Midland Certified Reagent Company, this sample. All experiments were performed in triplicate. The results Inc., Midland, TX, USA) were used as substrates: obtained for UNG2 were normalized by subtracting the level of DNA single-stranded: damage observed for the enzyme buffer alone. 50-*GCTTAGCTTGGAATCGTATCATGTA5-OH-UACTCGTGTGCCGTGTAGAC CGTGCC-30 and 50-*GGCACGGTCTACACGGCACACGAGTGTACATGATACGAT TCCAAGCTAAGC-30; double-stranded: 50-*GCTTAGCTTGGAATCGTATCATG Mutagenesis assay TA5-OH-UACTCGTGTGCCGTGTAGACCGTGCC-30 50-GGCACGGTCTACACGG Na þ /K þ ATPase mutagenesis leading to ouabain resistance was examined CACACGAGTATACATGATACGATTCCAAGCTAAGC-30, and 50-*GCTTAGCTTG as described before34 with modifications. Briefly, 32Dcl3 cells were infected GAATCGTATCATGTA-5-OH-U-ACTCGTGTGCCGTGTAGACCGTGCC-3050-GGC with UGI-expressing retrovirus carrying IRES-GFP (DNA construct kindly ACGGTCTACACGGCACACGAGTGTACATGATACGATTCCAAGCTAAG C-30. provided by Dr CJ. Jolly, The University of Sydney, Australia)35 or with

Leukemia (2013) 629 – 634 & 2013 Macmillan Publishers Limited Oxidative DNA damage repair in CML A Slupianek et al 631 IRES-GFP only. GFP þ cells were sorted and colonies growing in the APE1 endonuclease, which provides the lyase activity for presence of ouabain (concentration) were counted in methylcellulose. monofunctional UNG2 was not affected (Figure 1a). Elevated nuclear levels of UNG2 were associated with its enhanced Flow cytometry measurement for g-H2AX chromatin binding in 32D-BCR-ABL1 cells (Figure 1b). Nuclear DNA double-strand breaks were examined by immunofluorescent detec- expression of other glycosylases capable to recognize uracil tion of histone g-H2AX as described before16 with modifications. Briefly, derivatives in genomic DNA, such as SMUG1, NTH1, NEIL1, NEIL2, cells were fixed in 1.5% formaldehyde for 10 min at room temperature and thymine glycosylase and MBD4 were not changed in BCR-ABL1 permeabilized with cold methanol for 20 min at 4 oC. Then the cells were (Figure 1c). washed twice by adding phosphate-buffered saline supplemented with 0.5% bovine serum albumin followed by incubation with anti-g-H2AX antibody conjugated to Alexa Fluor 647 (Cell Signaling Technology, Removal of 5-OH-U from DNA is reduced in CML cells Danvers, MA, USA) for 1 h. After washing the cells were analyzed by flow To examine the activity of BER responsible for removal of cytometry. potentially mutagenic uracil derivatives from DNA, we analyzed the excision of 5-OH-U from single-stranded and double-stranded DNA substrates.36 Despite the enhanced expression levels of RESULTS UNG2 by BCR-ABL1, nuclear protein extracts derived from human BCR/ABL1 stimulates the expression of UNG2 CD34 þ CML-CP, CML-AP and CML-BP cells displayed three- to To determine the expression status of all uracil DNA glycosylases eight-fold reduction of 5-OH-U removal from a single-stranded þ in BCR-ABL1 positive cells we performed western blot analysis of DNA substrates when compared with CD34 cells obtained from nuclear fractions obtained from 32D-BCR-ABL1 cell line and its healthy donors; the excision capability diminished as the disease parental counterpart. We found that BCR-ABL1 kinase elevates the progressed (Figure 2a). 32D-BCR-ABL1 murine hematopoietic cell nuclear expression of UNG2 by Btwo-fold, whereas expression of line displayed similar reduction of 5-OH-U excision when compared with parental 32Dcl3 counterpart (Figure 2b). In addition, Btwo-fold reduction in 5-OH-U removal by 32D-BCR- ABL1 nuclear cell lysates was observed when double-stranded DNA substrates containing 5-OH-U: A or 5-OH-U: G were used (Figures 2c and d). The presence of BCR-ABL1 inhibited 5-OH-U incisions in a time-dependent manner (Figure 3). Interestingly, other fusion tyrosine kinases such as TEL-ABL1, TEL- PDGFRb and NPM-ALK were not associated with deregulation of 5-OH-U removal in BaF3 murine hematopoietic cells (Supplementary Figure 1), suggesting that this phenomenon is unique for CML cells.

Figure 1. Nuclear expression and chromatin binding of UNG2 is BCR-ABL1 kinase downregulates the activity of UNG2 enhanced in BCR-ABL1-positive cells. (a) Western blot analysis of Incubation of BCR-ABL1-positive cells with imatinib was able to UNG2 in the nuclear lysates obtained from 32Dcl3 parental (P) restore the 5-OH-U incision activity, indicating that it depends on and 32D-BCR-ABL1 (B/A) cell lines cultured in the presence of the kinase activity of BCR-ABL1 (Figure 4a). To identify the uracil threshold concentrations of interleukin-3. Lamin B was detected as DNA glycosylase(s) inhibited by BCR-ABL1 kinase we applied loading control. (b) UNG2 protein was detected in nuclear- neutralizing antibodies against a number of DNA glycosylases as and chromatin-bound cell lysates obtained from parental (P) and 31,37 32D-BCR-ABL1 (B/A) cell lines by western blot analysis using anti- described by others. We found that only UNG2-neutralizing UNG2 antibody. HDAC II served as the loading control. (c) Other antibody abrogated 5-OH-U incision activity in parental 32Dcl3 DNA glycosylases involved in removal of uracil derivatives from cells (Figure 4b). This observation indicated that UNG2 is the major genomic DNA were detected by western blotting in nuclear lysates uracil DNA glycosylase in hematopoietic cells and suggested that from parental (P) and 32D-BCR-ABL1 (B/A) cells. Lamin B served as UNG2 is affected by BCR-ABL1 kinase. To confirm this specula- loading controls. tion we showed that anti-UNG2 neutralizing antibody and UGI,

Figure 2. The incision of 5-OH-U lesions is inhibited in BCR-ABL1-positive cells. Nuclear extracts from (a) CD34 þ bone marrow cells from healthy donors (hBMC) and CML-CP or CML-BC patients (two donors per group), and from (b–d) parental (P) and 32D-BCR-ABL1 (B/A) cells were analyzed for an incision activity using single-stranded oligos containing 5-OH-U (U) or G (a, b), and double-stranded oligos containing 5-OH-U paired with A (U-A) (c) and with G (U-G) (d) as substrates. An asterisk (*) indicates the [g-32P]ATP-labeled strand. Bars represent mean±s.d. from two to three experiments; *Po0.01 in comparison with hBMC and P using Student’s t-test.

& 2013 Macmillan Publishers Limited Leukemia (2013) 629 – 634 Oxidative DNA damage repair in CML A Slupianek et al 632

Figure 3. BCR-ABL1-positive cells display time-dependent defect in the incision of 5-OH-U lesions. Nuclear extracts from (a) MO7 and MO7- BCR-ABL1 (MO7-B/A) cells and (b) 32Dcl3 and 32D-BCR-ABL1 (32Dcl3-B/A) were tested for an incision activity using the [g-32P]ATP-labeled single-stranded oligos containing 5-OH-U as a substrate as described in Figure 2. The reaction products were analyzed at the indicated time points. Results represent mean±s.d. from three experiments. Representative reaction panels are shown.

Figure 4. BCR-ABL1 kinase abrogates UNG2 activity. BER activity was measured using the [g-32P]ATP-labeled single-stranded oligomer containing 5-OH-U as a substrate as described in Figure 2. (a) When indicated, cells were treated with 1 mM imatinib (IM) for 24 h in the presence of growth factors before the reaction. UGI and anti-UNG neutralizing antibody were added to the reaction mixture in combination with IM. (b) The panel of neutralizing antibodies was added to the reaction to block DNA glycosylases activity. Bars represent mean±s.d. from three experiments; *Po0.01 and **Po0.01 in comparison with P and B/A þ IM, respectively, using Student’s t-test.

a specific peptide inhibitor of UNG2 (dissociates UNG2 from DNA), As oxidative DNA damage is a primary cause of spontaneous abrogated 5-OH-U incision activity, which was restored in imatinib- mutations, we tested whether reduced UNG2 activity may treated 32D-BCR-ABL1 cells (Figure 4). contribute to accumulation of mutations. The Na þ /K þ ATPase mutagenesis model was employed where specific amino-acid substitutions in the a1 subunit of the enzyme cause resistance to Inhibition of UNG2 activity is associated with accumulation of ouabain.34 We detected a modest, but statistically significant uracil in DNA and accumulation of point mutations increase of mutations in 32D-UGI cells when compared to parental The observation that BCR-ABL1 kinase significantly reduce the cells. removal of uracil derivatives from DNA through inhibition of UNG2 led us to hypothesize that accumulation of uracil in genomic DNA could be responsible for genomic instability of CML cells. To test DISCUSSION this hypothesis and mimic the situation present in CML cells, we Pre-existing and acquired point mutations in BCR-ABL1 kinase are generated 32D-UGI cells using UGI-expressing retrovirus carrying detected in B23% of imatinib-naive and 50–90% of imatinib- IRES-GFP; control cells expressed GFP only. Modified comet assay treated CML-CP patients.6 Moreover, BCR-ABL1 kinase mutants revealed that 32D-UGI cells displayed Beight-times higher levels resistant to second- and third-generation TKIs emerged, for of uracil derivatives in genomic DNA; conversely, accumulation of example due to new and/or compound mutations.38 Thus, CML DNA double-strand breaks measured by immunofluorescence of cells are elusive targets due to continuous accumulation of point histone g-H2AX was reduced in comparison with control cells mutations in BCR-ABL1 kinase, which encode the resistance to (Figures 5a, b, respectively). next generations of TKIs.5 The mechanisms responsible for the

Leukemia (2013) 629 – 634 & 2013 Macmillan Publishers Limited Oxidative DNA damage repair in CML A Slupianek et al 633

Figure 5. Inhibition of UNG2 increases the level of uracil derivatives in genomic DNA and stimulates mutagenesis. GFP þ 32Dcl3 cells (32D) and those expressing UGI (32D-UGI) were used to: (a) measure the level of uracil derivatives in genomic DNA by comet assay. Results represent percent of DNA in the tail of comets ±s.d.; *Po0.001 using Student’s t-test; (b) measure the level of double-strand breaks by immunostaining detection of g-H2AX. Relative g-H2AX levels represent the average of atleast three experiments (±s.d.); Po0.03 using Student’s t-test; and (c) detect ouabain-resistant clones containing mutations in Na þ /K þ ATPase. The frequency of ouabain-resistant cells was determined by clonogenic assay performed in the presence 0.5 mM ouabain. Results represent four independent experiments performed in duplicates; Po0.05 using Student’s t-test. appearance of TKI-resistant BCR-ABL1 kinase mutants have not been reported, implicating the involvement of additional DNA been fully characterized. repair mechanisms.50 For example, the role of DNA polymerase b in Point mutations are usually acquired due to the activity of unfaithful nucleotide excision repair and/or homologous unfaithful DNA polymerases during DNA replication and/or as a recombination repair in accumulation of point mutations in BCR- result of unfaithful/inefficient repair of oxidized DNA bases. We, ABL1-transformed cell lines was suggested.39,51–54 and others have shown that CML-CP LSCs (including quiescent In conclusion, we postulate that inhibition of BER activity mediated LSCs) and LPCs and also BCR-ABL1 -positive cell lines accumulate by UNG2 and perhaps also by OGG1 glycosylases contributes to high levels of ROS-induced oxidized DNA bases, which are accumulation of TKI-resistant BCR-ABL1 kinase mutations. responsible for accumulation of TKI-resistant mutations.16,34,39,40 Oxidized bases are excised during BER and, if not removed, mismatch repair corrects the mismatches to avoid point muta- CONFLICT OF INTEREST 24 25 tions. As we reported that mismatch repair is inhibited in CML, The authors declare no conflict of interest. BER become an essential mechanism to prevent accumulation of point mutations. We found that the activity of UNG2, a major uracil DNA ACKNOWLEDGEMENT glycosylase is inhibited by BCR-ABL1 kinase in CML primary cells This work was supported by NCI R01 CA123014 from the National Cancer Institute. and BCR-ABL1-transformed cell lines. The effect is highly specific for BCR-ABL1 kinase, because other fusion tyrosine kinases including TEL-ABL1 did not affect 5-OH-U incision (Supplementary Figure 1). REFERENCES Downregulation of UNG2 activity resulted in elevation of uracil 1 Melo JV, Barnes DJ. Chronic myeloid leukaemia as a model of disease evolution in derivatives in genomic DNA and accumulation of point mutations. human cancer. Nat Rev Cancer 2007; 7: 441–453. The molecular mechanism of BCR-ABL1 kinase-mediated 2 Marley SB, Gordon MY. Chronic myeloid leukaemia: stem cell derived but pro- inhibition of UNG2 is not known. Nuclear expression and genitor cell driven. 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