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. The online version of this article contains a data supplement. There is an Inside Blood Commentary on this article in this issue. © 2015 by The American Society of Hematology 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