Regular Article

LYMPHOID NEOPLASIA Opposing regulation of BIM and BCL2 controls glucocorticoid-induced apoptosis of pediatric acute lymphoblastic leukemia cells

Duohui Jing,1,2 Vivek A. Bhadri,1,2 Dominik Beck,2,3 Julie A. I. Thoms,2,3 Nurul A. Yakob,1,2 Jason W. H. Wong,2,3 Kathy Knezevic,2,3 John E. Pimanda,2,3,4 and Richard B. Lock1,2

1Children’s Cancer Institute Australia for Medical Research, 2Lowy Cancer Research Centre, and the 3Prince of Wales Clinical School, University of New South Wales, Sydney, Australia; and 4Department of Haematology, Prince of Wales Hospital, Sydney, Australia

Key Points Glucocorticoids are critical components of combination chemotherapy regimens in pediatric acute lymphoblastic leukemia (ALL). The proapoptotic BIM protein is an important • The glucocorticoid receptor mediator of glucocorticoid-induced apoptosis in normal and malignant lymphocytes, coordinately regulates the whereas the antiapoptotic BCL2 confers resistance. The signaling pathways regulating BIM antiapoptotic BCL2 and and BCL2 expression in glucocorticoid-treated lymphoid cells remain unclear. In this study, proapoptotic BIM genes in pediatric ALL patient-derived xenografts (PDXs) inherently sensitive or resistant to pediatric ALL cells in vivo. glucocorticoids were exposed to dexamethasone in vivo. Microarray analysis showed KLF13 MYB • GR binding at a novel intronic that and gene expression changes were significantly greater in dexamethasone- sensitive than -resistant PDXs. Chromatin immunoprecipitation (ChIP) analysis detected region is associated with glucocorticoid receptor (GR) binding at the KLF13 promoter to trigger KLF13 expression BIM transcription and only in sensitive PDXs. Next, KLF13 bound to the MYB promoter, deactivating MYB ex- dexamethasone sensitivity in pression only in sensitive PDXs. Sustained MYB expression in resistant PDXs resulted in pediatric ALL cells in vivo. maintenance of BCL2 expression and inhibition of apoptosis. ChIP sequencing analysis revealed a novel GR binding site in a BIM intronic region (IGR) that was engaged only in dexamethasone-sensitive PDXs. The absence of GR binding at the BIM IGR was associated with BIM silencing and dexamethasone resistance. This study has identified novel mechanisms of opposing BCL2 and BIM gene regulation that control glucocorticoid-induced apoptosis in pediatric ALL cells in vivo. (Blood. 2015;125(2):273-283) Introduction

Glucocorticoids, ie, dexamethasone and prednisolone, are commonly can lead to gene transactivation by binding to multiple DNA loci used to treat lymphoid malignancies including pediatric acute lympho- including proximal promoter regions and/or distal sites.9-12 The GR blastic leukemia (ALL).1,2 The current standard for combination can interact with histone acetyltransferases, such as cAMP-response chemotherapy adopted by the Berlin-Frankfurt-Munster (BFM) group element binding protein–binding protein (CBP) and P300, as well as involves an initial week of glucocorticoid monotherapy combined other coactivators, to induce histone acetylation.13-17 The GR can with a single intrathecal dose of methotrexate.3,4 Patients who respond also interact with the switch/sucrose nonfermentable chromatin- poorly to this initial week of treatment (prednisolone poor responders) remodeling complex and the boundary element/insulator-binding experience poorer outcome than prednisolone good responders, protein CTCF to alter chromatin structure.12,18 Histone acetylation despite their therapy being intensified due to increased risk of relapse.5 and chromatin remodeling can alter nucleosomal packaging to allow A greater understanding of the mechanisms associated with resistance increased access of transacting factors and components of the basal to glucocorticoids in pediatric ALL would facilitate the design of transcriptional machinery to the local DNA.19,20 In addition, the GR can treatment strategies to overcome resistance and improve outcome. also repress gene transcription via DNA-independent interactions with The glucocorticoid-induced apoptotic response is mediated through transcriptionfactors such as activator protein-1 and nuclear factor the glucocorticoid receptor (GR), a member of the nuclear receptor kB.21,22 GR-induced activation or repression of gene transcription family of ligand-dependent transcription factors.6,7 On ligand binding, controls apoptosis of normal and malignant lymphocytes. the GR dissociates from the large protein complex that maintains it in A critical role for BCL2 family proteins in glucocorticoid- an inactive conformation in the cytoplasm. On activation, the GR induced apoptosis of malignant lymphocytes has been identified translocates to the nucleus and binds as a dimer to activate target genes by our group and others.23-25 A proapoptotic member of the BCL2 via direct interaction with specific palindromic DNA sequences family, BIM, is upregulated on glucocorticoid stimulation, which known as glucocorticoid response elements (GREs).8-10 GR binding antagonizes antiapoptotic members such as BCL2, BCL-XL, and

Submitted May 23, 2014; accepted October 10, 2014. Prepublished online as The publication costs of this article were defrayed in part by page charge Blood First Edition paper, October 21, 2014; DOI 10.1182/blood-2014-05- payment. Therefore, and solely to indicate this fact, this article is hereby 576470. marked “advertisement” in accordance with 18 USC section 1734.

BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2 273 274 JING et al BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2

MCL-1.26,27 Disequilibrium of pro- and antiapoptotic proteins leads to Information’s Gene Expression Omnibus41 (accession no. GSE57795). Full the activation of BAX, disrupting mitochondrial transmembrane details are provided in the supplemental Methods. potential and promoting activation of caspases.27-29 However, it is still For ChIP and ChIP-seq experiments, spleen-harvested cells were fixed unclear which specific glucocorticoid-regulated genes are involved with 1% formaldehyde for 10 minutes at room temperature immediately after fi in transducing the apoptotic signal, upstream of BIM/BCL2 dynamic harvesting. Nuclei were extracted from xed cells by 10-minute incubation interactions. in lysis buffer (0.2% NP40 in 10 mM Tris buffer, pH 8.0) followed by centrifugation at 1250 g for 5 minutes at4°C.The nuclei samples were snap We have previously shown that the in vivo and in vitro dexametha- frozen and stored at 280°C for further use. sone responses of a panel of childhood ALL biopsies established as fl xenografts in NOD/SCID mice re ected the clinical outcome of the ChIP and ChIP-seq patients from whom they were derived.30-32 In contrast to findings 35,42-44 with in vitro-cultured cell lines, dexamethasone resistance in xeno- ChIP and ChIP-seq were carried out as previously described. Details of grafts was not attributed to a dysfunctional GR.25,33,34 We found transcriptional factor binding sites, primer design, and ChIP/ChIP-seq protocols are provided in the supplemental Methods. The ChIP-seq data were deposited in that there was a clear disparity between dexamethasone-sensitive National Center for Biotechnology Information’s Gene Expression Omnibus41 and -resistant xenografts in their ability to induce transcription of (accession no. GSE58266). BIM. Although BIM lacks GREs in its promoter, we observed that the epigenetic regulation of the BIM promoter, including histone Assessment of dexamethasone sensitivity acetylation and methylation status, were significantly different between sensitive and resistant xenografts.35,36 In this study, gene modula- In vitro dexamethasone sensitivity was assessed using the assessment of tion on in vivo dexamethasone treatment was assessed by micro- mitochondrial activity by Alamar blue assay. The in vivo dexamethasone sensitivity was determined by the leukemia growth delay (LGD) using array analysis, and whole-genome GR binding sites and histone established methods.31 Full details are provided in the supplemental acetylation status were detected using chromatin immunopreci- Methods. pitation (ChIP) and ChIP sequencing (ChIP-seq) analyses. The combination of these techniques provided an understanding of Reverse transcription-polymerase chain reaction and dexamethasone-induced signaling cascades in ALL cells in vivo. western blotting BCL2 We revealed a novel signaling pathway of KLF13-mediated 2 repression in sensitive xenografts and identified a novel GR binding Real-time quantitative reverse-transcription PCR (RT -PCR) and western blot analysis were carried out as previously described.35 Full details are provided in site in a distal region of the BIM locus that is likely to regulate BIM the supplemental Methods. expression. These findings provide novel links between the GR and pro/antiapoptotic proteins, which are likely to be important in Lentiviral gene knockdown the understanding of mechanisms of glucocorticoid resistance in lymphoid malignancies. KLF13 gene knockdown was performed using pLKO.1 lentiviral constructs (Sigma-Aldrich, St. Louis, MO) in Nalm6 cells. Full details are provided in the supplemental Methods.

GR-DNA binding enzyme-linked immunosorbent assay Materials and methods GR-DNA binding affinity in nuclear extracts was quantified by TransAM ALL xenograft model and primary patient samples Transcription Factor enzyme-linked immunosorbent assay (Active Motif, Carlsbad, CA), as previously described.25 Full details are provided in the The process by which continuous xenografts from childhood ALL biopsies supplemental Methods. have been established in immunodeficient NOD/SCID mice has been 35 previously described in detail. Full details are provided in the supplemental Luciferase reporter assay Methods, available on the Blood Web site. All investigations using human tissue were performed in accordance with the principles embodied in the Luciferase reporter assays were performed using pGL2B and pGL2P vectors declaration of Helsinki. All animal studies had previous approval from the from Promega (Madison, WI), and luminescence was detected using the Dual- Animal Care and Ethics Committee of the University of New South Wales Glo system from Promega. Full details are provided in the supplemental Methods. whereas experiments that used patient biopsy material were approved by the Human Research Ethics Committees of the South East Sydney and Illawara Statistics Area Health Service and the University of New South Wales. Microarray and in vivo ChIP studies were performed using spleen-harvested cells from 3 randomized ALL-engrafted mice at each condition of treatment.45 In vitro and in vivo dexamethasone treatment, sample In vitro gene expression and time course ChIP studies were performed with preparation, and analysis 3 independent experiments. Quantitative variables of normally distributed data ALL xenograft cells were inoculated by tail-vein injection into NOD/SCID mice, were compared by the Student t test, and non-normally distributed data were and engraftment was monitored weekly as previously described.25 For in vivo compared by the Mann-Whitney U test. All statistical tests were 2-sided, and , fi efficacy studies mice were randomized and treated with either dexamethasone P .05 was considered statistically signi cant. (15 mg/kg) or vehicle control by intraperitoneal injection when the %huCD451 cells in the peripheral blood reached 1% (see below and supplemental Methods). For cell and molecular biology experiments, mice were treated when there were 1 .70% %huCD45 cells in the peripheral blood and culled 8 hours thereafter. Results Cell suspensions of spleens were prepared and mononuclear cells were enriched to .97% human by density gradient centrifugation. Xenograft selection and dexamethasone response For gene expression and microarray studies, RNA was extracted immediately after harvesting. The microarray was performed using Illumina We previously established a panel of dexamethasone-sensitive and HumanWG-6 v3 Expression BeadChips and analyzed as previously -resistant ALL xenografts derived from primary biopsy specimens of described.37-40 The data were deposited in National Center for Biotechnology B-cell precursor ALL patients,25,30,31 as well as a panel of xenografts BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2 COORDINATED REGULATION OF BIM AND BCL2 BY THE GR 275

Table 1. Patient demographics and in vivo/in vitro responses to dexamethasone Age at diagnosis, CR1 duration Current In vivo LGD In vitro In vivo Xenograft (month)/sex Cytogenetics (months) clinical status In vitro IC50 (days) stratification stratification ALL-28 20/M Hyperdiploid 194 CR1 .10 mM ND Resistant ND ALL-50 131/M normal 30 DOD .10 mM 21.1 Resistant Resistant ALL-55 176/M t(9;22) 136 CR1 .10 mM 30.7 Resistant Resistant ALL-56 120/M t(9;22) 55 CR2 9.0 nM 4.4 Sensitive Resistant ALL-57 72/F t(1;19) 127 CR1 .10 mM 7.5 Resistant Resistant ALL-26 43/F t(12;21) 196 CR1 1.9 nM 70.0 Sensitive Sensitive ALL-51 19/M dic(7;9) 192 CR1 .10 mM 69.7 Resistant Sensitive ALL-52 138/M t(7;15) 180 CR1 142 nM 67.0 Sensitive Sensitive ALL-53 87/M t(12;21) 1102 CR1 1.0 nM ND Sensitive ND ALL-54 89/M normal 193 CR1 59 nM 46.3 Sensitive Sensitive

CR1, first complete remission; CR2, second complete remission; DOD, dead of disease; LGD, leukemia growth delay (days); ND, not determined; resistant, IC50 . 1 mM, LGD , 40 days; sensitive, IC50 , 1 mM, LGD . 40 days; 1, no event. derived from prednisolone poor responders and prednisolone good responses. Four pairs of xenografts closely matched in basal GEPs responders (Table 1).46 To select a molecularly matched pair of but differing in their responses to dexamethasone treatment were xenografts to study mechanisms associated with dexamethasone identified (ie, ALL-7/-25; ALL-52/-55; ALL-50/-54; and ALL-51/- resistance, we first performed microarray analysis of basal gene ex- 56). To avoid bias toward specific cytogenetic mutations, ALL-50 pression profiles (GEPs) of all xenografts. Unsupervised hierarchical and ALL-54 were chosen for further detailed study because they clustering of the xenografts and 3 key characteristics (in vitro and exhibited normal cytogenetics. in vivo dexamethasone sensitivity and cytogenetics) are shown in Figure 1B-D illustrates the in vivo and in vitro dexamethasone Figure 1A. As expected, some xenografts (eg, ALL-11, -26, and -53) sensitivities of ALL-50 and ALL-54. Kaplan-Meier survival plots of clustered according to dexamethasone sensitivity. However, in more mouse event-free survival (EFS) revealed a longer LGD of 46.3 days than half of the cases, basal GEPs did not predict dexamethasone in ALL-54 (Figure 1C) compared with 21.1 days for ALL-50 (Figure 1B).

Figure 1. Hierarchical clustering of xenografts and their dexamethasone sensitivities in vivo and in vitro. (A) Unsupervised cluster dendrogram of the 37 879 probes across 19 BCP-ALL xenografts. The 10 framed xenografts are resistant to dexamethasone, according to their LGD in vivo and IC50 in vitro. The 9 unframed xenografts are sensitive to dexamethasone. (B-C) Kaplan-Meier curves of EFS following treatment with dexamethasone 15 mg/kg intraperitoneally Monday throughout Friday for 4 weeks (solid line) or vehicle control (dashed line) for (B) ALL-50 and (C) ALL-54. LGD calculated as the difference between median EFS of treated and control groups. (D) ALL-50 and ALL-54 were treated with increasing concentrations of dexamethasone, and viability was assessed by Alamar Blue assay after 48-hour incubation. Values are expressed as a percentage of the untreated control. Data represent mean 6 standard error of the mean for n 5 3 experiments. (E-G) ALL-50 and ALL-54 cultured cells in vitro were exposed to dexamethasone (1 mmol/L) and were harvested at times up to 16 hours and processed for real-time RT-PCR quantitation. Data are expressed relative (fold) to solvent-treated controls at each time point. Each data point represents the mean 6 standard error of the mean from 3 independent experiments. Dex, dexamethasone; IC50, the dexamethasone half maximal inhibitory concentration; LGD, leukemia growth delay; ND, not determined. 276 JING et al BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2

Figure 2. Microarray analysis of in vivo dexamethasone-induced gene expression. ALL-50 and ALL-54 engrafted mice were treated with dexamethasone 15 mg/kg or vehicle control, and cells harvested after 8 hours. GEPs of spleen-harvested cells were evaluated using microarray. The data for each condition are presented with samples from 3 randomized ALL-engrafted mice. (A) Principal component analysis of global GEPs of the ALL-50 and ALL-54 samples before (circles) and after (squares) treatment with dexamethasone. (B) Heatmap of differentially expressed genes between ALL-50 and ALL-54 after dexamethasone treatment. A list of 457 genes was generated and ranked using Limma with a formula of (x54.Dex-x54.Con)/2-(x50.Dex-x50.Con)/2 (false discovery rate [FDR] ,0.05). (C) Nine genes related to either GR signaling or epigenetic modulation were selected from the Limma gene list. Relative gene expressions of the 9 selected genes and BIM (BCL2L11) in ALL-50 and ALL-54 during dexamethasone treatment were presented in a heatmap. (D) Heatmap of the dexamethasone-induced fold changes of 10 genes in a panel of 10 xenografts including 5 dexamethasone-resistant xenografts (ALL-28/-50/-55/-56/-57) and 5 dexamethasone-sensitive xenografts (ALL-26/-51/-52/-53/-54). Hierarchical clustering of the 10 genes was performed based on their fold changes on dexamethasone treatment. (E-F) Scatter plots of dexamethasone-induced fold changes of KLF13 and MYB expression in the 2 groups of dexamethasone-resistant and -sensitive xenografts. *P , .05 (Mann-Whitney U test). (G) Correlation curve of dexamethasone-induced fold changes of MYB and BCL2 expression levels in the 10 xenografts. (H) Correlation curve of dexamethasone-induced fold changes of BCL2 and BIM expression levels in the 10 xenografts. Con, vehicle control; Dex, dexamethasone.

The results of the cytotoxicity assays in vitro were consistent contrast, ALL-50, ALL-54, and Nalm6 cells only expressed the with the in vivo data and showed profound resistance in ALL-50 entire wild-type GR sequence. These results indicate that the (IC50 .10 mM) compared with ALL-54 (IC50 5 59 nM), a distinct responses of ALL-50 and -54 to dexamethasone were not difference of .200-fold (Figure 1D). Next, we detected that the due to a dysfunctional GR, in contrast to the observation in most GR in both xenografts translocated from the cytoplasm to nucleus glucocorticoid-resistant cell lines.25,33 (supplemental Figure 1) and bound consensus GRE oligos by GR- We then assessed the expression of candidate dexamethasone- DNA binding enzyme-linked immunosorbent assay (supplemental regulated genes following treatment of ex vivo-cultured ALL-50 Figure 2) following dexamethasone stimulation. The entire GR cDNA and -54 cells and observed downregulation of BCL2 and upregulation from ALL-50, ALL-54, Nalm6, CEM-WT, and CEM-MTXR3 cells of BIM in ALL-54 (sensitive) but markedly less so in ALL-50 was sequenced following RT-PCR ampliﬁcation. Consistent with our (resistant) (Figure 1E-F). In contrast, GILZ, a primary target of the previous results,47 CEM-WT cells were heterozygous for a wild-type GR and not directly involved in apoptosis, was upregulated in both codon TTA and a mutated codon TTT at 2259 bp from the translation xenografts on dexamethasone treatment (Figure 1G). These results start site, whereas CEM-MTXR3 cells exhibited loss of heterozygosity suggested that coordinated regulation of BIM and BCL2 controls and only expressed the mutated codon (supplemental Figure 3). In dexamethasone-induced apoptosis of ALL xenograft cells. BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2 COORDINATED REGULATION OF BIM AND BCL2 BY THE GR 277

Table 2. Comparison of gene regulation in 5 dexamethasone- sensitive and -resistant xenografts (Figure 2A). Limma analysis sensitive and 5 dexamethasone-resistant xenografts by Limma identified 457 genes that were significantly differentially regulated Gene LogFC (S vs R) Direction (S vs R) P value FDR by dexamethasone between ALL-50 and ALL-54 (FDR ,0.05; KLF13 0.7958 Up .01 0.02* Figure 2B). Among these genes, 9 were previously reported to have NCOA7 1.0064 Up .02 0.02* functional relevance to either GR signaling or epigenetic modulation MYB 20.8249 Down .02 0.02* (eg, histone acetylation by P300/CBP; a library of GR-, CBP-, and BCL2 20.6976 Down .09 0.08 P300-related genes is shown in supplemental Table 1). It was not TCF12 20.5206 Down .16 0.11 anticipated that BIM (BCL2L11) would be selected by Limma 2 NRIP1 0.7081 Down .18 0.11 analysis because its peak of induction was around 16 hours BCL2L11 0.1959 Up .28 0.14 (Figure 1F). CEBPB 0.4671 Up .44 0.19 KHDRBS3 20.1222 Down .80 0.29 Expression of the 9 Limma-selected genes plus BIM are presented KLF8 0.0742 Up .81 0.29 in heatmap format in Figure 2C. We next extended the study of these 10 genes to the panel of 10 xenografts including 5 each of dexamethasone- R, dexamethasone-resistant xenografts; S, dexamethasone-sensitive xenografts. sensitive and -resistant xenografts (Table 1). The genes down- or up- *Significantly different between sensitive and resistant xenografts. regulated in ALL-54 still clustered together in this extended xenograft Microarray analysis of in vivo dexamethasone-induced panel (Figure 2D). These results were further analyzed using Limma gene expression and the Mann-Whitney U test, where we found the changes in expression of KLF13, NCOA7,andMYB were significantly greater in the To further understand the basis for differential in vivo glucocorticoid group of 5 sensitive xenografts in comparison with the 5 resistant sensitivity of pediatric ALL xenografts, microarray analysis of gene xenografts (FDR ,0.05; Table 2; Figure 2E-F). Furthermore, the expression was carried out on ALL-50 and ALL-54 cells harvested differential regulation of MYB, a known activator of BCL2,48-50 was 8 hours after treatment. Principal component analysis demonstrated significantly correlated with BCL2 regulation on dexamethasone that the transcriptomes of the control and dexamethasone-treated treatment in the 10 xenografts (P , .05; Figure 2G). In addition, the ALL-50 and ALL-54 samples clustered into distinct groups, indicating downregulation of BCL2 was significantly correlated with BIM reproducible gene expression differences between dexamethasone- upregulation (P , .05; Figure 2H). These results again supported

Figure 3. ChIP-seq data of GR binding and histone acetylation. ALL-54 engrafted mice were treated with dexamethasone 15 mg/kg or vehicle control and cells were harvested after 8 hours. ChIP sequencing identified GR binding and histone acetylation on the (A) KLF13 locus, (B) MYB locus, (C) BCL2 locus, and (D) BIM locus as determined using the University of California Santa Cruz Genome Browser. The frames with solid lines refer to promoter regions and downstream regions near the transcription start site. The frames with dashed lines refer to the intronic/distal GR binding regions. Dex, dexamethasone; AcHistone, histone acetylation. 278 JING et al BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2

Figure 4. ChIP assay on KLF13 to BCL2 signaling pathway. ChIP was performed using spleen-harvested cells from ALL-engrafted mice treated with dexamethasone 15 mg/kg or vehicle control for 8 hours. The data for each point were generated with samples from 3 randomized ALL-engrafted mice. Time course ChIP study was performed with 3 independent experiments in vitro. (A) GR binding at the KLF13 locus in ALL-50 and ALL-54 following dexamethasone treatment in vivo and time course studies of GR binding at region 2 of the KLF13 locus in ALL-54 in vitro. Black bars refer to GR binding sites predicted in silico. (B) GR binding at the GILZ promoter in ALL-50 and ALL-54 following dexamethasone treatment in vivo. (C) KLF13 and SP1 binding at the MYB promoter in ALL-50 and ALL-54 following dexamethasone treatment in vivo. Dashed bars refer to GC-rich sequences; solid bars refer to CACCC box. (D) c-MYB/v-MYB binding at the BCL2 locus in ALL-50 and ALL-54 following dexamethasone treatment in vivo. Black bars refer to MYB binding sites predicted in silico. (E-F) SP1 binding at the (E) BCL2 and (F) BCL-XL promoter in ALL-50 and ALL-54 following dexamethasone treatment in vivo. TSS, transcription start site; Dex, dexamethasone. *P , .05. coordinated regulation of these genes in controlling the dexameth- always accompanied by an increase in histone acetylation on both asone responses of ALL cells. the promoter and IGRs. No GR binding sites were found on the MYB, BCL2,andNRIP1 loci (Figure 3B-C; supplemental Figure 4), but GR binding and histone acetylation in xenograft in vivo by a decrease in histone acetylation was observed on their promoters, ChIP-seq study consistent with downregulation of these genes. No GR binding sites were identiﬁed at the BIM promoter (Figure 3D), but interestingly, To identify genes directly targeted by the GR, we performed ChIP- a novel IGR was recognized, which was accompanied by increased seq with ALL-54 cells harvested from engrafted mice 8 hours after histone acetylation at the BIM promoter and IGR following dexameth- treatment with dexamethasone or vehicle control. We found GR asone treatment. The BIM IGR may be responsible for long-distance binding sites at the KLF13 promoter (Figure 3A), which indicated regulation of BIM transcription. that the GR directly triggered KLF13 transcription. We also detected intronic GR binding regions (IGRs) in the KLF13 locus, which could Mechanism of transcriptional regulation of BCL2 be distal regulation sites of the GR. Similar to KLF13, GR binding sites were also found on the promoters and IGRs of the NCOA7 The above ChIP-seq data were conﬁrmed and expanded to compare and GILZ loci (supplemental Figure 4). Furthermore, GR binding was mechanisms of gene regulation in a sensitive and a resistant ALL BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2 COORDINATED REGULATION OF BIM AND BCL2 BY THE GR 279

Figure 5. KLF13 knockdown in Nalm6 cells. Nalm6 cells were transduced with either scrambled shRNA or 2 distinct KLF13 shRNAs using lentivirus. (A) Time course of KLF13, c-MYB, BCL2, BIM, and Actin protein expression following 1 mM dexamethasone treatment in vitro. (B) Quantified expression of c-MYB normalized to actin and then to Scr 0-hour samples. N 5 3; *P , .05. (C) Quantified expression of BCL2 normalized to actin and then to Scr 0-hour samples. N 5 3; *P , .05. Dex, dexamethasone; Scr, scrambled shRNA; SH1, KLF13 shRNA 1; SH2, KLF13 shRNA 2.

xenograft. Because the ChIP-seq data indicated that BCL2 is not dexamethasone treatment, and this binding was virtually eradicated a direct target of the GR, we studied the possible roles of KLF13 following dexamethasone treatment in ALL-54 but not ALL-50 and MYB in BCL2 gene regulation. We designed primers to target (Figure 4D). Besides MYB, SP1 binding at region 4 of the BCL2 the in silico predicted GREs of the KLF13 locus as shown in promoter was also significantly decreased following dexamethasone Figure 4A. Consistent with the ChIP-seq data, GR binding at region treatment of ALL-54 but not ALL-50 (Figure 4E). The decrease in 2oftheKLF13 promoter was prominent in the dexamethasone- MYB and SP1 binding at the BCL2 promoter specificallyinALL-54is sensitive ALL-54 cells 8 hours after in vivo dexamethasone treat- consistent with inhibition of BCL2 transcription (Figure 1E). For these ment, but not in the dexamethasone-resistant ALL-50 cells. An in experiments, the BCL-XL promoter served as a control, where SP1 vitro time course ChIP study revealed a peak of GR binding at the binding was also detected but not modulated by dexamethasone KLF13 promoter in ALL-54 within 4 hours of dexamethasone treatment in ALL-54 (Figure 4F). treatment (Figure 4A). However, no GR binding was observed on To further verify the role of KLF13 in regulating BCL2,we the KLF13 promoter of ALL-50. In contrast, the GILZ promoter knocked down KLF13 expression in Nalm6 cells using a lentiviral served as a positive control, where GR binding was detected in both shRNA vector. As shown in Figure 5A, knocking down KLF13 with ALL-50 and ALL-54 following dexamethasone treatment in vivo 2 different shRNAs blocked KLF13 expression in Nalm6 cells after (Figure 4B). dexamethasone treatment compared with the scrambled shRNA KLF13 has been shown to deactivate gene transcription by control. A time course of protein expression revealed that c-MYB competing for DNA binding sites with SP1.51,52 Therefore, we (Figure 5A-B) and BCL2 (Figure 5A,C) levels were maintained at studied KLF13 and SP1 binding at the MYB and BCL2 promoters 18 and 24 hours, respectively, following dexamethasone treatment in following dexamethasone treatment. First, we designed primers to KLF13 knockdown samples compared with the significant decrease target CACCC boxes and GC-rich sequences on the MYB and BCL2 of c-MYB and BCL2 in the control samples. In contrast, BIM ex- promoters (Figure 4C,E). The structures of the SP1 and KLF13 pression was upregulated in all transduced lines (Figure 5A). proteins are similar, containing 3 highly homologous C-terminal zinc fi nger motifs, but SP1 preferentially binds GC-rich sites, whereas Mechanism of BIM transcriptional regulation by the GR KLF13 prefers the sequence CACCC.51 SP1 binding in cells exposed to vehicle control was observed inregions 3 and 4 of the MYB promoter Because it was previously unclear how BIM transcription was up- (Figure 4C). Importantly, on dexamethasone treatment, KLF13 bound regulated by glucocorticoids in lymphoid cells, we next studied GR to region 5 of the MYB promoter in ALL-54 but not ALL-50 cells and RNA Polymerase II (RNA Pol II) binding and histone acetylation (Figure 4C),which indicates that KLF13 deactivated the SP1-triggered at the BIM locus using conventional ChIP assays. As shown in MYB transcription in ALL-54. An in vitro time course study of Figure 6A, 9 pairs of primers were designed to cover up to 3 kb of ALL-54 revealed a clear temporal relationship between the levels BIM promoter and intron 1. First, we found a strong increase in of KLF13 and MYB proteins, with MYB protein downregulation histone acetylation and RNA Pol II binding at the BIM promoter and occurring after KLF13 upregulation (supplemental Figure 5). first intron in ALL-54 but not ALL-50 8 hours after dexamethasone Because MYB has been shown to suppress apoptosis by inducing treatment in vivo (Figure 6B-E), which was consistent with the BCL2 expression in multiple cell types,48-50 weassessed MYBbinding AcHistone ChIP-seq data shown in Figure 3D. This was further sites on the BCL2 locus following dexamethasone treatment and confirmed by a time course ChIP study in vitro with up to 16 hours included MYB binding sites that were predicted in silico (MBS1) of dexamethasone treatment, showing the dexamethasone-inducing as well as other published binding sites (MBS2-4).50 MYB was modulation of GR and RNA Pol II binding and histone acetylation associated with MBS1 and MBS4 at the BCL2 locus before on the BIM and GILZ promoters in the 2 xenografts (supplemental 280 JING et al BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2

Figure 6. Dexamethasone-induced modulation of the BIM locus. (A) Schematic demonstrating the primers used in the ChIP study covering the BIM promoter and first intron. (B-C) RNA Pol II binding and (D-E) histone acetylation at the BIM locus were determined in (B,D) ALL-50 and (C,E) ALL-54 following 8-hour treatment with dexamethasone or vehicle control in vivo. The data for each point were generated with samples from 3 randomized ALL-engrafted mice. (F) Schematic showing GR binding at the BIM IGR (intronic GR binding region) from the ChIP-seq data. Four pairs of primers were designed to cover 5 to 10 kb up- and downstream of the BIM IGR region. (G) GR binding at the BIM IGR in ALL-50 and ALL-54 following treatment with dexamethasone or vehicle control in vivo (n 5 3). (H) Time course study of GR binding at the BIM IGR in ALL-54 following dexamethasone treatment in vitro (n 5 3). (I) Regression curve of dexamethasone-induced fold changes of BIM expression vs enrichment of GR binding at the BIM IGR in 11 xenografts, P 5 .03. BIM expression was detected by RT-PCR using 3 independent experiments in vitro. (J) RNA Pol II binding at the BIM IGR as well as up- and downstream regions in ALL-54 following 8-hour treatment with dexamethasone or vehicle control in vivo (n 5 3). TSS, transcription start site; Dex, dexamethasone. *P , .05.

Figure 6). Overall, these data indicate that the BIM promoter was only treatment in vivo (Figure 6F-G). Time course ChIP studies revealed activated in the dexamethasone-sensitive xenograft ALL-54 both in a gradual increase in GR binding at the IGR in ALL-54 up to 8 hours vitro and in vivo. after dexamethasone treatment in vitro (Figure 6H). Extending this In the ChIP-seq study (Figure 3D), we identified a GR binding study to 11 xenografts, we found a significant correlation between site at the BIM IGR that we proposed to be a long-range regulatory GR-IGR binding and BIM induction in vitro following dexameth- domain. We next performed conventional ChIP and detected GR asone treatment (R2 5 0.41, P 5 .034; Figure 6I). Next, we identified binding at the IGR in ALL-54 but not ALL-50 following dexamethasone RNA Pol II binding at the IGR but not at 5 to 10 kb up- or downstream BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2 COORDINATED REGULATION OF BIM AND BCL2 BY THE GR 281

Figure 7. The BIM IGR functions as a dexamethasone- inducible enhancer of transcription. (A) The GR binding motif identified by GR ChIP-seq in in vivo dexamethasone- treated ALL-54 cells. (B) Two potential GREs at the BIM IGR were revealed by sequence analysis, both located within the first peak of the GR ChIP-seq signal. (C) Locations and sequences of the potential GREs, which were mutated as shown (mutated residues are under- lined). (D) The effects of single or dual GRE mutations on dexamethasone-induced reporter gene activity in Nalm6 cells. Reporter plasmid with firefly luciferase and control plasmid with renilla luciferase were cotransfected into Nalm6 cells. Firefly luminescence was determined after 16-hour treatment of dexamethasone and normalized to the renilla luminescence signal. The fold inductions were then calculated by normalizing to pGL2P control. N 5 5; *P , .05.

of the IGR (Figure 6J), indicating a role for the GR in recruiting RNA Many genes have been previously published for their roles in GR- Pol II to trigger gene transcription. induced apoptosis. However, in our microarray study, most of these genes were modulated similarly in ALL-50 and ALL-54 by BIM IGR enhances GRE-dependent gene transcription dexamethasone in vivo, including some well-recognized genes such as CEBPB, FOXO3, FOS, HSP90AA1, JUN, NCOA1/2, NFKB1, The GR binding motif (Figure 7A) was identified from the in vivo POU2F1/2, SMAD3,andSTAT5A.7-10,17,56,57 This disparity could be GR ChIP-seq data, similar to motifs previously identified on the due to differences between experimental model systems compared basis of in vitro models.53-55 Next, we found 2 potential GREs with our use of in vivo treated patient-derived xenografts, in which we containing the conserved GR binding motif at the first GR-binding observed a similar trend of activation of GR downstream signaling peak within the BIM IGR (Figure 7B). Conventional ChIP detected pathways in sensitive and resistant xenografts. no significant GR binding at the region of the 39 minor peak in To identify the genes that were differentially regulated between Figure 7B (data not shown). To study the function of the potential dexamethasone-sensitive and -resistant xenografts, we next performed GREs, we cloned the BIM IGRwithGREs1and2inwild-typeand a 1-step Limma analysis on the data from 4 conditions, ie, ALL-50 mutated sequences 1 kb downstream of the luciferase gene in the and ALL-54 with and without dexamethasone treatment. Among the pGL2P reporter vector (Figure 7C) and carried out reporter assays identified genes, KLF13, MYB, BCL2, and BIM were especially in- in the Nalm6 ALL cell line. Dexamethasone treatment signifi- teresting. By combining ChIP and ChIP-seq data, we demonstrated cantly augmented luciferase expression exclusively in the con- that GR binding at the KLF13 locus and BIM IGR in dexamethasone- struct containing the wild-type BIM IGR (pGL2PE) (Figure 7D). sensitive ALL-54 represented 2 distinct signaling pathways. The Mutating the individual GREs (pGL2PE1 and 2) or both (pGL2PE3) former induced KLF13 expression, which subsequently inhibited SP1- abolished dexamethasone-induced luciferase expression. These triggered MYB transcription and transcription of BCL2,adownstream results indicate that the BIM IGR functions as an enhancer in target of MYB. GR binding at the BIM IGR appears to act as an enhancer response to GR binding at its GREs. of BIM transcription, which may play a key role in glucocorticoid- induced apoptosis of ALL cells.23-27,35 In contrast to ALL-54, the lack of GR binding at the KLF13 and BIM loci in ALL-50 correlated with its resistance to dexamethasone. Discussion In an effort to further understand the underlying basis for defective GR binding at the BIM IGR in dexamethasone-resistant xenografts in The mechanism of dexamethasone-induced apoptosis in ALL cells has the context of wild-type GR expression, we analyzed the abundance of been studied using a variety of cell types in vitro and in vivo.7,25,32-36 In DNaseI hypersensitive sites (DHSs) at the BIM IGR in 78 normal and this study, we performed microarray, ChIP, and ChIP-seq assays using malignant human cell types from Encyclopedia of DNA Elements in vivo dexamethasone-treated human pediatric ALL xenografts. (ENCODE)/OpenChrom (Duke University)58 using the University 282 JING et al BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2 of California Santa Cruz Genome Browser.59 All 18 datasets of normal and malignant B and T lymphocytes showed high or medium Acknowledgments DHS intensity at the BIM IGR, whereas 56 of 60 other cell types had low/nil DHS intensity (supplemental Table 2 and Figure 7). These The authors thank Drs Chintanu Sarma and Santi Suryani for findings indicate that the BIM IGR exists in an open chromatin assistance in analyzing the microarray gene expression data. conformation exclusively in lymphoid cells, which may allow The authors acknowledge the ENCODE Consortium and the the GR and other transcription factors to bind and enhance BIM Duke, University of North Carolina-Chapel Hill, University of transcription, thereby contributing to the acute sensitivity of nor- Texas-Austin, and European Bioinformatics Institute ENCODE mal and malignant lymphocytes to glucocorticoid-induced apoptosis. groups for generating DNaseI HS datasets and Transcription Next, we analyzed potential transcription factor binding data generated Factor ChIP-seq (161 factors) datasets. The Children’s Cancer 58 from a large collection of ChIP-seq experiments by ENCODE. As Institute Australia is affiliated with University of New South shown in supplemental Figure 8, there is potential binding of 2 Wales Australia and The Sydney Children’s Hospitals Network. transcription factors at the first GR-binding peak in the BIM IGR in This research was funded by grants from the National Health and which the 2 GREs are located, whereas 9 transcription factors could Medical Research Council of Australia and the Cancer Council New bind at the second GR-binding peak. In particular, transcription South Wales. V.A.B. was supported by fellowships from the Leu- factors such as CTCF could contribute to modifying the DNA kaemia Foundation of Australia and the Steven Walter Foundation. 60 structure to bridge the BIM promoter and the regulatory domains. R.B.L. is supported by a fellowship from the National Health and Although GR binding at the BIM IGR may regulate gene transcrip- Medical Research Council of Australia. tion via long-range chromatin remodeling, the transcription factor(s) involved in activating the BIM promoter remain to be defined. We previously identified reduced Foxo3a binding at the BIM promoter in dexamethasone-resistant vs -sensitive ALL xenografts.35 In this study, Authorship we observed a decrease in phosphorylated-Foxo3a protein (inactive form) inthe cytoplasm of ALL-54 on dexamethasone treatment in vitro Contribution: D.J., V.A.B., D.B., J.A.I.T., J.E.P., and R.B.L. designed and a concomitant increase in Foxo3a (active form) in the nucleus, the study; D.J., V.A.B., D.B., J.A.I.T., N.A.Y., J.W.H.W., and K.K. neither of which were observed in ALL-50 (supplemental Figure 9). generated and analyzed the data; and D.J. and R.B.L. interpreted the These data support the published mechanism of Foxo3a in recruiting data and wrote the manuscript. histone acetyltransferases and triggering BIM transcription.16,61,62 Conflict-of-interest disclosure: The authors declare no competing In conclusion, in this study, we compared the signal pathways financial interests. involved in BIM/BCL2 gene regulation in dexamethasone-sensitive Correspondence: Richard B. Lock, Children’s Cancer Institute and -resistant ALL xenografts in vivo and identified novel mecha- Australia for Medical Research, Lowy Cancer Research Centre, nisms of opposing BIM and BCL2 gene regulation that control UNSW, PO Box 81, Randwick, NSW 2031, Australia; e-mail: rlock@ glucocorticoid-induced apoptosis in pediatric ALL cells in vivo. ccia.unsw.edu.au.

References

1. Inaba H, Pui CH. Glucocorticoid use in acute 7. Schaaf MJ, Cidlowski JA. Molecular mechanisms distinct coactivator complexes and promote lymphoblastic leukaemia. Lancet Oncol. 2010; of glucocorticoid action and resistance. J Steroid distinct patterns of local chromatin modification. 11(11):1096-1106. Biochem Mol Biol. 2002;83(1-5):37-48. Mol Cell Biol. 2003;23(11):3763-3773. 2. Pui CH, Evans WE. A 50-year journey to cure 8. Schoneveld OJ, Gaemers IC, Lamers WH. 16. Wang F, Marshall CB, Yamamoto K, et al. childhood acute lymphoblastic leukemia. Semin Mechanisms of glucocorticoid signalling. Biochim Structures of KIX domain of CBP in complex with Hematol. 2013;50(3):185-196. Biophys Acta. 2004;1680(2):114-128. two FOXO3a transactivation domains reveal 9. Wang JC, Derynck MK, Nonaka DF, promiscuity and plasticity in coactivator 3. Schrappe M, Reiter A, Zimmermann M, et al. Khodabakhsh DB, Haqq C, Yamamoto KR. recruitment. Proc Natl Acad Sci USA. 2012; Long-term results of four consecutive trials in Chromatin immunoprecipitation (ChIP) scanning 109(16):6078-6083. childhood ALL performed by the ALL-BFM study identifies primary glucocorticoid receptor target group from 1981 to 1995. Berlin-Frankfurt- 17. Lützner N, Kalbacher H, Krones-Herzig A, Rösl F. genes. Proc Natl Acad Sci USA. 2004;101(44): Münster. Leukemia. 2000;14(12):2205-2222. FOXO3 is a glucocorticoid receptor target and 15603-15608. regulates LKB1 and its own expression based on 4. Flohr T, Schrauder A, Cazzaniga G, et al; 10. John S, Sabo PJ, Johnson TA, et al. Interaction of cellular AMP levels via a positive autoregulatory International BFM Study Group (I-BFM-SG). the glucocorticoid receptor with the chromatin loop. PLoS ONE. 2012;7(7):e42166. Minimal residual disease-directed risk landscape. Mol Cell. 2008;29(5):611-624. 18. Johnson TA, Elbi C, Parekh BS, Hager GL, John stratification using real-time quantitative PCR S. Chromatin remodeling complexes interact analysis of immunoglobulin and T-cell 11. Hakim O, John S, Ling JQ, Biddie SC, Hoffman dynamically with a glucocorticoid receptor- receptor gene rearrangements in the AR, Hager GL. Glucocorticoid receptor activation regulated promoter. Mol Biol Cell. 2008;19(8): international multicenter trial AIEOP-BFM of the Ciz1-Lcn2 locus by long range interactions. 3308-3322. ALL 2000 for childhood acute J Biol Chem. 2009;284(10):6048-6052. lymphoblastic leukemia. Leukemia. 12. Paakinaho V, Makkonen H, Jääskeläinen T, 19. Kouzarides T. Chromatin modifications and their 2008;22(4):771-782. Palvimo JJ. Glucocorticoid receptor activates function. Cell. 2007;128(4):693-705. poised FKBP51 locus through long-distance 5. Möricke A, Reiter A, Zimmermann M, et al; 20. John S, Sabo PJ, Thurman RE, et al. Chromatin interactions. Mol Endocrinol. 2010;24(3):511-525. German-Austrian-Swiss ALL-BFM Study Group. accessibility pre-determines glucocorticoid Risk-adjusted therapy of acute lymphoblastic 13. Spencer TE, Jenster G, Burcin MM, et al. receptor binding patterns. Nat Genet. 2011;43(3): leukemia can decrease treatment burden and Steroid receptor coactivator-1 is a histone 264-268. improve survival: treatment results of 2169 acetyltransferase. Nature. 1997;389(6647): 21. Herrlich P. Cross-talk between glucocorticoid unselected pediatric and adolescent patients 194-198. receptor and AP-1. Oncogene. 2001;20(19): enrolled in the trial ALL-BFM 95. Blood. 2008; 14. Lambert JR, Nordeen SK. CBP recruitment and 2465-2475. 111(9):4477-4489. histone acetylation in differential gene induction 22. Schmidt S, Rainer J, Ploner C, Presul E, Riml S, 6. Greenstein S, Ghias K, Krett NL, Rosen ST. by glucocorticoids and progestins. Mol Kofler R. Glucocorticoid-induced apoptosis and Mechanisms of glucocorticoid-mediated apoptosis Endocrinol. 2003;17(6):1085-1094. glucocorticoid resistance: molecular mechanisms in hematological malignancies. Clin Cancer Res. 15. Li X, Wong J, Tsai SY, Tsai MJ, O’Malley BW. and clinical relevance. Cell Death Differ. 2004; 2002;8(6):1681-1694. Progesterone and glucocorticoid receptors recruit 11(Suppl 1):S45-S55. BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2 COORDINATED REGULATION OF BIM AND BCL2 BY THE GR 283

23. Abrams MT, Robertson NM, Yoon K, Wickstrom 36. Piazza R, Magistroni V, Mogavero A, et al. dependent regulation of bcl-2 promoter activity. E. Inhibition of glucocorticoid-induced apoptosis Epigenetic silencing of the proapoptotic gene BIM Proc Natl Acad Sci USA. 1997;94(7):3296-3301. by targeting the major splice variants of BIM in anaplastic large cell lymphoma through an mRNA with small interfering RNA and short MeCP2/SIN3a deacetylating complex. Neoplasia. 49. Lang G, Gombert WM, Gould HJ. A transcriptional hairpin RNA. J Biol Chem. 2004;279(53): 2013;15(5):511-522. regulatory element in the coding sequence of the 55809-55817. human Bcl-2 gene. Immunology. 2005;114(1): 37. Suryani S, Carol H, Chonghaile TN, et al. Cell and 25-36. 24. Erlacher M, Michalak EM, Kelly PN, et al. BH3- molecular determinants of in vivo efficacy of the only proteins Puma and Bim are rate-limiting for BH3 mimetic ABT-263 against pediatric acute 50. Drabsch Y, Robert RG, Gonda TJ. MYB gamma-radiation- and glucocorticoid-induced lymphoblastic leukemia xenografts. Clin Cancer suppresses differentiation and apoptosis of apoptosis of lymphoid cells in vivo. Blood. 2005; Res. 2014;20(17):4520-4531. human breast cancer cells. Breast Cancer Res. 2010;12(4):R55. 106(13):4131-4138. 38. Johnson WE, Li C, Rabinovic A. Adjusting batch 25. Bachmann PS, Gorman R, Papa RA, et al. effects in microarray expression data using 51. Kaczynski J, Zhang JS, Ellenrieder V, et al. The Divergent mechanisms of glucocorticoid empirical Bayes methods. Biostatistics. 2007;8(1): Sp1-like protein BTEB3 inhibits transcription via resistance in experimental models of pediatric 118-127. the basic transcription element box by interacting acute lymphoblastic leukemia. Cancer Res. 2007; 39. Diffner E, Beck D, Gudgin E, et al. Activity of with mSin3A and HDAC-1 co-repressors and 67(9):4482-4490. a heptad of transcription factors is associated with competing with Sp1. J Biol Chem. 2001;276(39): 26. Schmidt S, Rainer J, Riml S, et al. Identification of stem cell programs and clinical outcome in acute 36749-36756. glucocorticoid-response genes in children with myeloid leukemia. Blood. 2013;121(12): 52. Natesampillai S, Fernandez-Zapico ME, Urrutia acute lymphoblastic leukemia. Blood. 2006; 2289-2300. R, Veldhuis JD. A novel functional interaction 107(5):2061-2069. 40. Reich M, Liefeld T, Gould J, Lerner J, Tamayo P, between the Sp1-like protein KLF13 and SREBP- 27. Krishna S, Low IC, Pervaiz S. Regulation of Mesirov JP. GenePattern 2.0. Nat Genet. 2006; Sp1 activation complex underlies regulation of low mitochondrial metabolism: yet another facet in the 38(5):500-501. density lipoprotein receptor promoter function. J Biol Chem. 2006;281(6):3040-3047. biology of the oncoprotein Bcl-2. Biochem J. 41. Edgar R, Domrachev M, Lash AE. Gene 2011;435(3):545-551. Expression Omnibus: NCBI gene expression and 53. Watson LC, Kuchenbecker KM, Schiller BJ, Gross 28. Planey SL, Abrams MT, Robertson NM, Litwack hybridization array data repository. Nucleic Acids JD, Pufall MA, Yamamoto KR. The glucocorticoid G. Role of apical caspases and glucocorticoid- Res. 2002;30(1):207-210. receptor dimer interface allosterically transmits regulated genes in glucocorticoid-induced 42. Pimanda JE, Chan WY, Wilson NK, et al. sequence-specific DNA signals. Nat Struct Mol apoptosis of pre-B leukemic cells. Cancer Res. Endoglin expression in blood and endothelium is Biol. 2013;20(7):876-883. 2003;63(1):172-178. differentially regulated by modular assembly of 54. So AY, Chaivorapol C, Bolton EC, Li H, 29. Webb MS, Miller AL, Johnson BH, et al. Gene the Ets/Gata hemangioblast code. Blood. 2008; Yamamoto KR. Determinants of cell- and gene- networks in glucocorticoid-evoked apoptosis of 112(12):4512-4522. specific transcriptional regulation by the leukemic cells. J Steroid Biochem Mol Biol. 2003; 43. Beck D, Thoms JA, Perera D, et al. Genome-wide glucocorticoid receptor. PLoS Genet. 2007; 85(2-5):183-193. analysis of transcriptional regulators in human 3(6):e94. 30. Lock RB, Liem N, Farnsworth ML, et al. HSPCs reveals a densely interconnected network The nonobese diabetic/severe combined of coding and noncoding genes. Blood. 2013; 55. Bryne JC, Valen E, Tang MH, et al. JASPAR, the immunodeficient (NOD/SCID) mouse model of 122(14):e12-e22. open access database of transcription factor- binding profiles: new content and tools in the 2008 childhood acute lymphoblastic leukemia reveals 44. Chacon D, Beck D, Perera D, Wong JW, Pimanda intrinsic differences in biologic characteristics at update. Nucleic Acids Res. 2008;36(Database JE. BloodChIP: a database of comparative issue):D102-D106. diagnosis and relapse. Blood. 2002;99(11): genome-wide transcription factor binding profiles 4100-4108. in human blood cells. Nucleic Acids Res. 2014; 56. Chen DW, Saha V, Liu JZ, Schwartz JM, Krstic- 31. Liem NL, Papa RA, Milross CG, et al. 42(Database issue):D172-D177. Demonacos M. Erg and AP-1 as determinants of glucocorticoid response in acute lymphoblastic Characterization of childhood acute lymphoblastic 45. Bhadri VA, Cowley MJ, Kaplan W, Trahair TN, leukemia. Oncogene. 2013;32(25):3039-3048. leukemia xenograft models for the preclinical Lock RB. Evaluation of the NOD/SCID xenograft evaluation of new therapies. Blood. 2004;103(10): model for glucocorticoid-regulated gene 57. Heidari N, Miller AV, Hicks MA, Marking CB, 3905-3914. expression in childhood B-cell precursor acute Harada H. Glucocorticoid-mediated BIM induction 32. Bachmann PS, Gorman R, Mackenzie KL, Lutze- lymphoblastic leukemia. BMC Genomics. 2011; and apoptosis are regulated by Runx2 and c-Jun Mann L, Lock RB. Dexamethasone resistance in 12:565. in leukemia cells. Cell Death Dis. 2012;3:e349. B-cell precursor childhood acute lymphoblastic 46. Wong NC, Bhadri VA, Maksimovic J, et al. 58. ENCODE Project Consortium. An integrated leukemia occurs downstream of ligand-induced Stability of gene expression and epigenetic nuclear translocation of the glucocorticoid encyclopedia of DNA elements in the human profiles highlights the utility of patient-derived genome. Nature. 2012;489(7414):57-74. receptor. Blood. 2005;105(6):2519-2526. paediatric acute lymphoblastic leukaemia 33. Hillmann AG, Ramdas J, Multanen K, Norman xenografts for investigating molecular 59. Kent WJ, Sugnet CW, Furey TS, et al. The human MR, Harmon JM. Glucocorticoid receptor gene mechanisms of drug resistance. BMC Genomics. genome browser at UCSC. Genome Res. 2002; mutations in leukemic cells acquired in vitro and in 2014;15:416. 12(6):996-1006. vivo. Cancer Res. 2000;60(7):2056-2062. 47. Catts VS, Farnsworth ML, Haber M, Norris MD, 60. Ong CT, Corces VG. CTCF: an architectural 34. Tissing WJ, Meijerink JP, Brinkhof B, et al. Lutze-Mann LH, Lock RB. High level resistance to protein bridging genome topology and function. Glucocorticoid-induced glucocorticoid-receptor glucocorticoids, associated with a dysfunctional Nat Rev Genet. 2014;15(4):234-246. expression and promoter usage is not linked to glucocorticoid receptor, in childhood acute glucocorticoid resistance in childhood ALL. Blood. lymphoblastic leukemia cells selected for 61. Gilley J, Coffer PJ, Ham J. FOXO transcription 2006;108(3):1045-1049. methotrexate resistance. Leukemia. 2001;15(6): factors directly activate bim gene expression and 929-935. promote apoptosis in sympathetic neurons. J Cell 35. Bachmann PS, Piazza RG, Janes ME, et al. Biol. 2003;162(4):613-622. Epigenetic silencing of BIM in glucocorticoid poor- 48. Salomoni P, Perrotti D, Martinez R, Franceschi C, responsive pediatric acute lymphoblastic Calabretta B. Resistance to apoptosis in CTLL-2 62. van der Heide LP, Smidt MP. Regulation of FoxO leukemia, and its reversal by histone deacetylase cells constitutively expressing c-Myb is associated activity by CBP/p300-mediated acetylation. inhibition. Blood. 2010;116(16):3013-3022. with induction of BCL-2 expression and Myb- Trends Biochem Sci. 2005;30(2):81-86.