Transcriptional Suppression of BACH2 by the Bcr-Abl Oncoprotein Is Mediated by PAX5

ORIGINAL ARTICLE Transcriptional suppression of BACH2 by the Bcr-Abl oncoprotein is mediated by PAX5

DA Casolari1,4, M Makri2,4, C Yoshida2, A Muto3, K Igarashi3 and JV Melo1,2

Bach2 is a lymphoid-specific transcription factor with a prominent role in B-cell development and apoptosis-induction in response to oxidative stress. We previously showed that Bach2 is downregulated in chronic myeloid leukaemia (CML), and here we demonstrate the mechanism by which Bcr-Abl mediates this phenomenon. We have cloned a 3.9 Kb genomic DNA fragment upstream of the transcription initiation site, and delineated the core and proximal BACH2 promoter regions. Transient BCR-ABL expression led to significant reduction in BACH2 promoter activity and this effect was dependent on the kinase function of the oncoprotein. Sequential deletions disclosed several regulatory elements within the promoter region, as well as within BACH2 exonic sequences. Analysis of these elements and transient transfection assays led to the identification of the Pax5 transcription factor as a potent trans-activator of BACH2, whose effect is predominantly mediated through occupation of a binding site on the BACH2 promoter, as demonstrated by both in vitro and in vivo experiments. Overall, our data show that Pax5 functions as an intermediate effector in the Bcr-Abl-mediated transcriptional repression of BACH2. The current results, combined with previous reports, establish Pax5 and Bach2 as transcriptional targets of Bcr-Abl, whose downregulation may contribute to lymphoid blast crisis of CML.

Leukemia (2013) 27, 409–415; doi:10.1038/leu.2012.220 Keywords: Bach2; chronic myeloid leukaemia; Bcr-Abl; Pax5

INTRODUCTION recombination and somatic hypermutation of Ig genes, with 14 Chronic myeloid leukaemia (CML) is consistently associated with effects on AID, Blimp-1 and XBP-1 gene expression. We have the t(9;22)(q34;q11) chromosomal translocation, which generates previously described that Bcr-Abl not only downregulates BACH2 10 a BCR-ABL fusion gene on the Philadelphia (Ph) chromosome, expression, but also inhibits its nuclear translocation through encoding the constitutively active tyrosine kinase p210Bcr-Abl.1 serine phosphorylation via the PI-3 K/S6K pathway, suppressing, as 15 CML classically evolves in three clinical stages: chronic phase (CP), a consequence, Bach2 pro-apoptotic function. Those findings accelerated phase and blast crisis (BC). Progress to the advanced are consistent with a Bach2 role as tumour suppressor gene. stages is characterised by an accumulation of immature blasts Pax5 (also known as BSAP) is a prominent B-lymphoid in the bone marrow and increased genomic instability.2,3 The transcription factor. Like Bach2, its expression pattern is differ- transformed clone in BC is, in the majority of cases, of myeloid or, entiation stage-dependent, strictly regulated and reminiscent of less frequently, of B-lymphoid cell lineage.4 Despite increasing its role in B-lymphoid lineage commitment, where it fulfills a dual knowledge about a variety of additional genetic lesions in function by repressing alternative lineage genes while simulta- BC of CML, it is still unclear how these lesions originate and neously activating B-cell-specific genes (reviewed in Cobaleda 16 which factors determine transformation of myeloid or lymphoid et al. ). In addition, Pax5 influences processes taking place at nature. Inhibition of apoptosis, a prominent feature of Bcr-Abl late stages of B-cell differentiation, such as the control of B-cell 17 18 oncogenicity,5 could facilitate the accumulation of mutations and proliferation, IgH class switch and suppression of plasma cell 19 DNA mis-repair6,7 which, in normal cells, would result in cell differentiation. death. In addition, deregulated expression of key B-lymphoid In this study, we demonstrate that BACH2 is transcriptionally development genes could contribute to the emergence of a repressed by Bcr-Abl, and identify Pax5 as a potent trans-activator transformed clone in lymphoid BC. of BACH2, whose effect is mediated through direct binding to a The association between BACH2 and CML was uncovered when site on the BACH2 promoter. Furthermore, we show that the genes exemplified by BACH2, cyclin D2 and BCL-6 were identified expression profile of Pax5 in primary cells and CML cell lines as being transcriptionally regulated by Bcr-Abl.8–10 The lymphoid- parallels that of BACH2, supporting the possibility of an association specific11,12 transcription factor Bach2 functions as a homo- between disrupted expression of both the genes and CML disease or a heterodimer partner of the small Maf proteins (MafK, MafF, development. MafG).11 It acts predominantly as a transcription repressor,11 controlling processes such as the induction of apoptosis in 13 MATERIALS AND METHODS response to oxidative stress. In B-lymphoid development, Standard methods for cell culture, retroviral infection, rapid amplification Bach2 negatively regulates the immunoglobulin heavy chain of cDNA ends, real-time quantitative PCR and western blot analysis are (IgH) gene 30- enhancer12 and is instrumental for class switch described in the Supplementary Information.

1Department of Haematology, Centre for Cancer Biology, SA Pathology University of Adelaide, Adelaide, South Australia, Australia; 2Department of Haematology, Imperial College London, London, UK and 3Department of Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan. Correspondence: Professor JV Melo, Department of Haematology, Centre for Cancer Biology, SA Pathology University of Adelaide, Frome Road, Adelaide, South Australia 5000, Australia. E-mail: [email protected] 4These authors contributed equally to this work. Received 15 February 2012; revised 24 July 2012; accepted 27 July 2012; accepted article preview online 3 August 2012; advance online publication, 24 August 2012 Bach2 transcriptional suppression by Bcr-Abl DA Casolari et al 410 Luciferase reporter constructs RESULTS The luciferase reporters pGL3/BACH2P3912, pGL3/BACH2P3003, pGL3/ We first confirmed that Bach2 levels are downregulated by Bcr-Abl BACH2P1605, pGL3/BACH2P725, pGL3/BACH2P184, pGL3/BACH2P126 by ectopically expressing the fusion oncogene in the Ph-negative and pGL3/BACH2P11 þ , were constructed through PCR amplification of 0 lymphoid cell line 697, and demonstrated that either degradation the corresponding BACH2 5 - regions to the end of exon 1 from normal of Bcr-Abl or inhibition of its tyrosine kinase activity are able to leukocyte DNA, followed by restriction enzyme digestion and ligation reverse the oncoprotein effect on Bach2 expression (Supple- into the pGL3-basic vector. For the pGL3/BACH2P72 þ construct two complementary oligonucleotides were designed spanning the sequence mentary Figure 2). from 72 nucleotides downstream of the transcription initiation site (TIS) of BACH2, to the end of exon 1 (Supplementary Figure 1) and ligated into Cloning and characterisation of the BACH2 promoter pGL3-basic. The remaining reporter constructs, namely pGL3/BACH2P649, Next, the BACH2 TIS was identified by 50-rapid amplification pGL3/BACH2P640, pGL3/BACH2P568, pGL3/BACH2P521 and pGL3/BACH- 2P261, were generated using the TaKaRa deletion kit for kilo-sequencing of cDNA ends PCR as a G nucleotide on exon 1, located 601 bp (TaKaRa, Kyoto, Japan). All constructs were checked for mutations by upstream the ATG codon on exon 6 (Supplementary Figure 1). The sequencing. BACH2 proximal promoter was cloned and characterised by engineering several DNA constructs encompassing sequences 50 of BACH2 exon 1, and cloning them into pGL3. The full range of Luciferase assays reporter constructs was transiently transfected into 697 cells and The cell line 697 was transfected using FUGENE6 (Roche, Penzberg, the highest increase in luciferase activity was observed with the Germany), following the manufacturer’s instructions. BV173 cells were electroporated as described previously.20 Luciferase activity was measured pGL3/BACH2P725 reporter construct (Figure 1), suggesting that using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, maximum BACH2 promoter activity is retained within 725 USA). All results were normalised against the luciferase activity of the nucleotides upstream of the TIS. A series of progressive deletions control pEF-sea pansy plasmid. Nonparametric Mann–Whitney test was within this region led to gradual and significant (po0.05) decrease used for statistical analysis. in reporter activity, and highlighted the regulatory importance of two regions of the BACH2 promoter, À 725/ À 568 and À 126/ Electrophoretic mobility shift assay þ 72 (Figure 1). Nuclear proteins were prepared as described previously.21 The double- stranded probe, corresponding to a specific sequence on the BACH2 Repressive effect of Bcr-Abl on BACH2 promoter activity 32 promoter (Supplementary Figure 1) was 50- labelled with g- P. Each 20 ml Co-transfection of BCR-ABL with the pGL3/BACH2P725 promoter of binding reaction contained 3 mg of nuclear protein extract, 2 mg poly reporter in 697 cells led to B60% downregulation in BACH2 (deoxyinosinic-deoxycytidylic) (GE, Fairfield, CT, USA), 15 000–25 000 promoter activity (Figure 2a). This effect was dependent on the counts per minute of purified labelled probe and 5 ml of 70% glycerol, in 1 Â binding buffer.22 Probe binding was detected by autoradiography on kinase activity of the oncoprotein, as treatment for 24 h with Kodak-X-OMAT AR (Sigma, St Louis, MO, USA). imatinib (IM) led to complete reconstitution of promoter activity in a dose-dependent manner (Figure 2a). The dependence of the Bcr-Abl-mediated effect on its kinase activity was also demon- Chromatin immunoprecipitation (ChIP) strated by co-transfection of pGL3/BACH2P725 with a kinase dead Chromatin fixation, purification and immunoprecipitation were done as BCR-ABL construct, which produced no significant effect on the previously described.23 Immunoprecipitation was performed using 2–5 mg of antibody or normal rabbit or goat (Sigma) serum. The precipitated DNA reporter activity (Figure 2a). was used as template for PCR with primers spanning the regions of interest Similar results were obtained by transfecting the CML cell line on the BACH2, CD19 (positive control) and KRAS (negative control) BV173 with the pGL3/BACH2P725 construct and treating it with IM promoters. Input DNA, fixed and sonicated but not subjected to for 24 h. The basal promoter activity was increased upon exposure immunoprecipitation, was used as positive control. to IM in a dose-dependent manner (Figure 2b). Overall, these

LUC -3912 LUC -3003 * LUC -1605- LUC -725 * LUC -649 LUC * -640 LUC * -568 * LUC -521 LUC -261 LUC -184 LUC

-126 LUC * 11+ LUC * 72+ LUC 0 20 4060 80 100 120 140 160 Relative Luciferase activity Figure 1. BACH2 promoter activity in a pre-B-cell leukaemia line. Luciferase activity was measured 48 h post transfection of 697 cells with reporter constructs containing varying lengths of the BACH2 promoter. The TIS is depicted by a vertical line. Results are expressed as the mean±s.d. of three experiments. *Po0.05.

Leukemia (2013) 409 – 415 & 2013 Macmillan Publishers Limited Bach2 transcriptional suppression by Bcr-Abl DA Casolari et al 411 250 Control LUC 200 Pax5

150 -725 12 3 LUC

100

-521 2 3 LUC % Luciferase activity Luciferase % 50

0 pGL3-Basic + + ++------261261 3 LUC pcDNA3.1 -+- - - +- - - - - P725 - - - - + + + + + + + BCR-ABL - - + - - - - + - + + BCR-ABL KD - - - + - - - - + - - 72+ LUC IM (M) 000 0 0 0 5 0 0 1 5 025050 100 150 200 Relative luciferase activity 60

50 B NE C N-19 C-20 Actin 40 1 2 3 4 5 6 30

activity 20

Relative luciferase Relative 10 SS 0 pGL3-Basic + - - - Pax5 P725 - + + + IM (M) 010 5

LUC -725 LUC -649 Free LUC probe

-640 LUC Input Precipitated -568 1/10 1/100 Chromatin LUC --- + -Pax5 (C-20) - - + - IgG -126 LUC Site III 11+ LUC Control Site I Bcr-Abl 72+ LUC Figure 3. (a) Ectopic PAX5 expression induces BACH2 promoter 0 5 10 15 20 25 30 activity. Luciferase activity was measured 24 h post transfection of Relative Luciferase activity 697 cells with the pSG5/Pax5 plasmid and reporter constructs containing varying lengths of the BACH2 promoter. Results are Figure 2. (a) Bcr-Abl-mediated repression of BACH2 promoter expressed as the mean±s.d. of three experiments. (b) Electro- activity. Luciferase activity was measured 24 h post transfection of phoretic mobility shift assay and supershift assay demonstrating 697 cells with BCR-ABL or BCR-ABL-KD expression plasmids and the in vitro binding of Pax5 on the BACH2 promoter. Nuclear extracts BACH2P725 reporter construct. one or 5 mM IM were added from 697 cells were incubated with a probe speciﬁc for the immediately after transfection. Results are expressed as the Pax5-binding site III ( þ 15/ þ 42) on the BACH2 promoter. Lane 1: no mean±s.d. of three experiments. (b) IM effect on BACH2 promoter nuclear extract; lane 2: with nuclear extract; lane 3: competition with activity. Luciferase activity was measured 48 h post electroporation 100-fold excess unlabelled oligo; lane 4: supershift with the N-19 of BV173 cells with BACH2P725 reporter plasmid or empty control. Pax-5 antibody; lane 5: supershift with the C-20 Pax-5 antibody; lane IM treatment was initiated 24 h after electroporation. Results are 6: supershift using the actin antibody (negative control). The Pax5 expressed as the mean±s.d. of three experiments. (c) Bcr-Abl effect and supershifted (SS) bands and the free probe are indicated by on different regions of the BACH2 promoter. Luciferase activity was arrows. Results are representative of three experiments. (c) ChIP measured 24 h post transfection of 697 cells with a BCR-ABL assay demonstrating in vivo binding of Pax5 on the BACH2 promoter. expression plasmid and reporter constructs containing varying Protein-DNA complexes from 697 cells were co-immunoprecipitated lengths of the BACH2 promoter. Results are expressed as the with a Pax5 antibody (C-20) or normal goat serum (negative control). mean±s.d. of three experiments. The precipitated DNA was used for PCR of Pax5-binding site III (-126/exon 1) and I (-759/-483) of the BACH2 promoter. Results are representative of two experiments. experiments demonstrate that BACH2 is transcriptionally regulated by Bcr-Abl and that this regulation depends on the kinase activity of the oncoprotein. constructs maintained a 2–3-fold repression of promoter activity We next analysed the regions in the BACH2 promoter necessary (Figure 2c). The suppression was eliminated only when BCR-ABL for Bcr-Abl control by co-transfecting 697 cells with BCR-ABL and was co-expressed with pGL3/BACH2P72 þ (Figure 2c). These reporter constructs containing different lengths of the BACH2 results strongly indicate that transcription factors binding in promoter. Co-expressing BCR-ABL with the majority of these the region between þ 11 and þ 72 nucleotides downstream of

& 2013 Macmillan Publishers Limited Leukemia (2013) 409 – 415 Bach2 transcriptional suppression by Bcr-Abl DA Casolari et al 412 the BACH2 TIS might be important for Bcr-Abl-mediated suppres- the region encompassing site I ( À 577/ À 550) occurred (lane 4, sion of BACH2 transcription. Figure 3c). This experiment demonstrates in vivo binding of Pax5 to the BACH2 promoter at position þ 15/ þ 42. Bcr-Abl does not influence histone acetylation/methylation on the BACH2 promoter Pax5 mediates BACH2 repression in CML cells As histone modifications on gene promoters affect the gene We noticed that the Pax5-binding site III ( þ 15/ þ 42) was within a expression,24 we analysed the Bcr-Abl influence on histone region whose deletion reversed the suppressive effect of Bcr-Abl acetylation or methylation patterns on two regions of the BACH2 on BACH2 promoter activity (Figure 2c). We therefore asked promoter that prevailed for their regulatory importance, À 759/ whether Bcr-Abl can suppress Pax5 expression in a manner similar À 483 and À 126/exon 1 (Figure 1). ChIP assays performed with to that seen for Bach2, and thus mediate BACH2 promoter BV173 cells suggest that, in the presence of Bcr-Abl, the BACH2 repression through Pax5 downregulation. promoter is in an open-chromatin conformation (Supplementary Indeed, ectopic expression of BCR-ABL in 697 cells resulted in a Figure 3). Moreover, IM treatment appears to have no effect on twofold reduction of PAX5 mRNA levels, and treatment with IM histone modification on the BACH2 promoter. completely rescued its expression (Figure 4a), demonstrating that the Bcr-Abl kinase activity is responsible for the downregulation of BACH2 is a transcriptional target of Pax5 PAX5. In addition, treatment of the lymphoid CML cell lines BV173 and TOM1 with IM induced an increase in BACH2 and PAX5 The previous results suggest that regulation of the BACH2 expression (Figure 4b), whereas there was no noticeable increase promoter is predominantly dependent on transcription factor in either mRNA when 697 (BCR-ABL-negative) and K562 (myeloid binding. Therefore, we used MatInspector (http://www.genomatix. CML) cells were treated with IM. As expected, Pax5 and Bach2 de/cgi-bin/matinspector_prof/mat_fam.pl) for prediction of tran- protein expression was also induced after IM treatment of BV173 scription factor binding sites contained within À 725 bp from the and TOM1 cells in a time-dependent manner (Figure 4c). These TIS of BACH2. A candidate transcription factor thus identified was results demonstrate that upon inhibition of Bcr-Abl by IM, the B-cell differentiation factor Pax5, previously shown as being 25 evidenced by inhibition of phosphorylation of the Bcr-Abl specific able to induce Bach2 expression in mouse pro-B cells. Based on target CRKL, Pax5 expression is increased in parallel with the conserved expression pattern between Bach2 and Pax5 upregulation of Bach2 levels. proteins, as well as the functional implication of both in B-cell Moreover, analysis of mononuclear cells from patients with development, we investigated the possibility that Pax5 regulates B-lymphoid BC of CML, although showing higher BACH2 and PAX5 BACH2 transcription in human cells. expression than CP and myeloid BC of CML (as expected, as The MatInspector analysis identified three potential Pax5- myeloid cells don’t express either of these genes), showed binding sites within -725 nucleotides from the BACH2 TIS significantly lower levels of BACH2 (Po0.0028) and PAX5 (Supplementary Figures 4 and 5). Therefore, we co-transfected (Po0.0031) expression than the lineage correspondent cells from 697 cells with the pSG5/Pax5 and the pGL3/BACH2P725 constructs Ph-negative B-progenitor acute lymphoblastic leukaemia (B-ALL) and found that, indeed, ectopic PAX5 expression led to a 2.7-fold patients (Figure 4d). increase in BACH2 promoter activity (Figure 3a). Finally, we observed, by ChIP assay, low binding of Pax5 to the In order to identify which of the three sites is responsible for the BACH2 promoter in BV173 cells and enhanced binding after the Pax5 effect we co-transfected 697 cells with a PAX5-expressing inhibition of Bcr-Abl by IM in a dose-dependent manner plasmid and reporter constructs devoid of regions containing (Figure 4e). The same IM dose-dependent increase in relative Pax5-binding sites I ( À 559/ À 532), I and II ( À 458/ À 431) and I, II enrichment was observed for the Pax5 regulated gene CD19. This and III ( þ 15/ þ 42), respectively. Whereas Pax5 increased reporter result suggests that inhibition of Pax5 expression by Bcr-Abl activity in the absence of sites I and II, no induction in reporter results in its decreased binding to the BACH2 promoter and, activity was observed in the absence of site III (Figure 3a). These therefore, reduced BACH2 expression. experiments indicate that Pax5-binding site III ( þ 15/ þ 42) is essential for Pax5 regulation of BACH2 expression, and that sites I and II are dispensable for the Pax5 effect. DISCUSSION Transformation by the Bcr-Abl oncoprotein is mediated through Pax5 binds to the BACH2 promoter both in vitro and in vivo activation of various signalling pathways, ultimately leading to the Binding of Pax5 to site III ( þ 15/ þ 42) on the BACH2 promoter was regulation of genes conferring the malignant phenotype of CML investigated by electrophoretic mobility shift assay. When 697 and Ph þ ALL.1 Here, we demonstrate that BACH2 is an indirect nuclear extracts were incubated with a radiolabelled probe transcriptional target of Bcr-Abl, and propose that the repressive specific for the Pax5-binding site III ( þ 15/ þ 42), a single band effect on BACH2 expression is actively maintained, at least partly, was present (lane 2; Figure 3b), suggesting Pax5 binding. The via suppression of Pax5 expression. specificity of this binding was demonstrated by the absence of the The cloning of the BACH2 promoter and data from BCR-ABL- band from lanes 1 (no nuclear extract) and 3 (competition with overexpression experiments demonstrate that BACH2’s decreased 100-fold excess unlabelled oligo). Incubation with the C-20 expression is due to a Bcr-Abl-dependent effect on promoter antibody, which recognises both Pax5a and Pax5b isoforms,22 activity, which is reversed by induced degradation of Bcr-Abl or led to supershift of the specific band (lane 5), confirming Pax5 as inhibition of its kinase activity. A similar type of regulation was the binding protein in the complex. Incubation of nuclear extracts described for the pro-apoptotic factor Bim, where IM treatment with the N-19, specific for the Pax5a splice variant, (lane 4) or actin induced accumulation of a trans-activator, FOXO3a, and its (lane 6) antibodies, failed to supershift the band, indicating it is a subsequent binding to the BIM promoter.26 Subsequently, the Pax5b-specific complex. These results suggest that at least one of IM-mediated transcriptional and posttranslational activation of the two Pax5 isoforms (Pax5b) binds in vitro to the Pax5-binding Bim was suggested to have a major role in IM-induced apoptosis site III ( þ 15/ þ 42) on the BACH2 promoter. of BCR-ABL-transformed cells,27 a phenomenon which is likely to To demonstrate in vivo binding of Pax5 we performed a ChIP assay also apply to the pro-apoptotic function of Bach2.13 Our data do on 697 cells. The Pax5 antibody (C-20) successfully immunoprecipi- not exclude the possible existence of additional mechanisms of tated the BACH2 promoter region containing Pax5-binding site III Bcr-Abl-mediated regulation of BACH2 expression, such as effects ( þ 15/ þ 42) (lane 4, Figure 3c); however, no immunoprecipitation of on enhancer elements, or on BACH2 mRNA stability, processing or

Leukemia (2013) 409 – 415 & 2013 Macmillan Publishers Limited Bach2 transcriptional suppression by Bcr-Abl DA Casolari et al 413 nucleocytoplasmic shuttling.28–31 In fact, we have previously promoter. A similar model of regulation in the context of CML was shown that even the little amount of Bach2 produced in proposed for cyclin D2. In the presence of Bcr-Abl, Stat5 binds to Bcr-Abl-positive cells is constitutively serine-phosphorylated.15 the promoter and activates transcription of cyclin D2, while upon This prevents the translocation of Bach2 to the nucleus for IM treatment Stat5 is replaced by the Bcl-6 transcription trans-repression of its anti-apoptotic target genes, such as HO-1 repressor.20 We thus hypothesised that, in a reverse fashion, Bcr- which, as a consequence, is upregulated in CML.15 Taken together, Abl may suppress BACH2 transcription by inhibiting the activity of these data suggest that Bach2 is a crucial pro-apoptotic protein, BACH2 trans-activators, consequently allowing for putative which needs to be suppressed by all means by Bcr-Abl in order to repressive elements to suppress BACH2 expression (Figure 5). ensure the latter’s oncogenic activity. Our experiments revealed that Pax5 binds to BACH2 exon 1 and We also investigated the possibility of Bcr-Abl-dependent induces BACH2 expression on human Bcr-Abl-positive and epigenetic changes within the BACH2 promoter. Our ChIP data negative pro-B-cell lines. The presence of transcription factor revealed, however, that the pattern of histone modiﬁcations on binding sites within untranslated exonic sequences is not rare; an the BACH2 promoter in Bcr-Abl-positive cells is reminiscent of an example is the PAX5 gene itself, which contains an important accessible chromatin conformation, even though the BACH2 gene repressive element within the 50-untranslated region of its expression is repressed. Therefore, it is more likely that Bcr-Abl alternative exon 1A.32 Several lines of evidence support our maintains low levels of BACH2 expression through the regulation ﬁnding of BACH2 as a transcriptional target of Pax5 in human pro- of transcription factor expression and/or binding onto the BACH2 B cells. Firstly, Pax5 was found to bind to the BACH2 promoter and enhancer and to induce BACH2 expression in mouse pro-B 25,33 Control cells. Secondly, both Bach2 and Pax5 are expressed from 12 3.5 IM 1 M pro-B to mature B-cell stages. In addition, Pax5 is known to 3.0 confer transcriptional activation/repression through directly 2.5 binding to the promoter or enhancer regions of several genes 2.0 associated with B-cell development,25 like BACH2 and IgH. 1.5 1.0 Furthermore, our result showing that Pax5 induces BACH2

PAX5/GAPDH 0.5 expression without changing chromatin conformation in Bcr-Abl- 0 positive cells (Supplementary Figure 3) is also supported by a 697/MIGR1 697/MIGP210 recent study demonstrating that BACH2 presented an active þ / þ 4.0 histone mark in PAX5 murine pro-B cells, and deletion of Pax5 did not alter this chromatin status.33 Although our data do not 3.5 PAX5/GAPDH address speciﬁcally the issue of regulation of Bach2 transcription 3.0 BACH2/GAPDH during B-cell differentiation, taken together with the evidence 2.5 from the literature,12,25,33 they could suggest a model in which 2.0 during early stages of B-cell development Pax5 induces BACH2 1.5 transcription (Figure 5). 1.0 Several studies have associated deregulated PAX5 expression 0.5 with human cancer.34–38 Chromosomal translocations have Fold change in relative ratio 0 BV173BV173 TOM1TOM1 697 697 K562 K562 implicated PAX5 as an oncogene in a subset of B-ALL and non- 16 +IM +IM +IM +IM Hodgkin lymphomas (reviewed in Cobaleda et al. ), with a subsequent demonstration that PAX5 fusions function as 697 BV173 TOM1 dominant-negative suppressors of Pax5.39 Moreover, 0 0 18 24 0 18 24 (h in 1M IM) somatic mutations on PAX5 (including deletions, ampliﬁcations, Bach2

Pax5 Actin Figure 4. (a) Reduction in PAX5 mRNA upon ectopic BCR-ABL p-Crkl Crkl expression. The 697 cells transduced with BCR-ABL were treated with 1 mM IM for 12 h and PAX5 mRNA levels were assessed by 7.001.28 1.57 4.42 0.51 0.55 p-CRKL/CRKL quantitative RT-PCR (Q-RT-PCR). Results are expressed as the mean±s.d. of four replicates. (b) Changes in BACH2 and PAX5 ** ** 100 102 mRNA levels after IM treatment. BV173, TOM1, 697 and K562 cells 101 were treated for 24 h with 1 mM IM. PAX5, BACH2 mRNA levels relative 100 to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were 10 10-1 assessed by Q-RT-PCR. Changes after IM treatment are presented 10-2 relative to the respective untreated cells (normalised as 1). (c) -3 1 10 Changes in Pax5 and Bach2 protein levels after IM treatment. -4

PAX5/GAPDH 10

BACH2/GAPDH Immunoblotting for Pax5, Bach2, Actin and CRKL was performed 10-5 with BV173, TOM1 and 697 protein extracts, after 0, 18 and 24 h 0.1 10-6 N CP MBC LBC Ph-ALL N CP MBC LBC Ph-ALL treatment with 1 mM IM. Results are representative of three experiments. (d) BACH2 and PAX5 mRNA levels in normal and CML cells. BACH2, PAX5 and GAPDH mRNA expression in peripheral blood 14 Control mononuclear cells of normal individuals (n ¼ 9), CP CML (n ¼ 13), myeloid BC (MBC) CML (n 8), B-lymphoid BC (LBC) CML (n 6) and 12 IM 1M ¼ ¼ Ph-negative B-ALL (Ph-ALL) patients (n ¼ 2) were analysed by Q-RT- IM 5M 10 PCR. **Po0.01. (e) Bcr-Abl inhibition results in increased Pax5 8 binding to the BACH2 promoter. BV173 cells treated with 1 or 5 mM 6 IM for 24 h were subjected to ChIP using a Pax5 antibody (C-20). The precipitated DNA was used for Q-PCR of the BACH2 promoter 4 of Pax5 binding (region À 126/exon 1) and the promoters of CD19 (positive control) Relative enrichment 2 and KRAS (negative control). Relative enrichment was normalised 0 against the input and KRAS enrichment. Results are expressed as the KRAS CD19 BACH2 mean±s.d. of two experiments.

& 2013 Macmillan Publishers Limited Leukemia (2013) 409 – 415 Bach2 transcriptional suppression by Bcr-Abl DA Casolari et al 414

Ph- Ph+

? Bcr-Abl

Methyl transferase Activator Inhibitor

Met Met PAX5 Repressor Met BACH2 PAX5 Repressor BACH2

apoptosis apoptosis IgH IgH Figure 5. A hypothetical model for trans-regulation of BACH2 transcription by the Pax5 transcription factor. In normal (Ph-) cells and at early stages of B-lymphoid differentiation, Pax5 activates BACH2 transcription. As a result, Bach2 represses the IgH 3’ enhancer and cells undergoing unproductive IgH transcription/rearrangement are eliminated by apoptosis. In CML (Ph þ ) cells, Bcr-Abl suppresses Pax5 expression, through still unknown mechanisms, which results in repression of BACH2 transcription. This will ultimately result in a block in apoptosis and in deregulated IgH transcription and rearrangement.

mutations and translocations) are detected in over 30% childhood frequency of IgH gene rearrangements,14 which may also correlate B-ALL38 and adult Ph þ ALL.37 Overall, these mutations result in with an increase in pathogenic translocations.47,48 CML cells reduced expression of Pax5, which consequently alters expression carrying such translocations would avoid elimination due to an of its target genes,40 suggesting that PAX5 deletion could inherent resistance to apoptosis,1 mediated in part by the contribute to leukemogenesis.38 suppression in Bach2 expression,13 and eventually be Here, we demonstrate that Pax5 expression is repressed in responsible for progression to BC (Figure 5). It is possible that Bcr-Abl-positive cells, and this repression is reversed by IM the degree of Pax5 and Bach2 suppression by BCR-ABL correlates treatment. More importantly, the parallel expression profiles of with the risk of lymphoid BC transformation, so that serial PAX5 and BACH2 in primary B-lymphoid BC of CML as compared measurements of their levels of expression during CP may provide with Ph-negative B-ALL cells indicate that their pattern of prognostic information. This is currently being addressed in a regulation is not an artefact of transfection systems or immortal large prospective clinical trial. cell lines. In agreement with our findings, microarray screens Our results identify BACH2 and PAX5 as Bcr-Abl targets. Ultimately, detected downregulation of both genes in Ph-positive B-ALL a detailed knowledge of the role of Bach2 in the pathogenesis and patients compared with Ph À negative B-ALL.41,42 Similarly, PAX5 evolution of Ph-positive leukaemias may lead to therapeutic expression was reported to be reduced in cells from lymphoid BC strategies involving genetic/chemical manipulation of this transcrip- of CML compared with germinal centre normal counterpart B tion factor and/or its targets. The clinical relevance of such biological cells,43 corroborating our results on the effect of Bcr-Abl on PAX5 knowledge is exemplified by the finding that anticancer drugs that expression in lymphoid cells. In addition, they reported a higher produce reactive oxygen species, such as etoposide, doxorubicin PAX5 expression in lymphoid BC than in CP of CML, as we and cytarabine, and which facilitate nuclear accumulation and observed here (Figure 4d); however, such relative increase is only activation of Bach2, can act synergistically with IM treatment apparent when comparing PAX5 expression in the lymphoid BC through manipulating the pro-apoptotic function of Bach2.49 cells with the myeloid CP cells as it reflects the natural silencing of the B-cell-specific gene in the myeloid lineage. Intriguingly, a study employing tissue microarrays and immunohistochemical CONFLICT OF INTEREST analysis revealed increased PAX5 expression in 72% of early pre-B- The authors declare no conflict of interest. ALL, 100% of CML in lymphoid BC, 50% of AML and 50% of mixed B-cell ALL and AML;44 however, the number of cases investigated was small. We postulate that the Bcr-Abl-dependent suppression of BACH2 ACKNOWLEDGEMENTS expression is partly mediated through repression of Pax5 We thank Dr Andreas Reimold and Dr Brian Druker for donation of plasmids, expression via a currently unknown mechanism. As a result, CML Dr Elizabeth Buchdunger for donation of imatinib, Drs Hiromu Sato, Gina Mollica and cells with decreased PAX5 and BACH2 expression may still be Helen Ambrose (Imperial College London) for their advice on electrophoretic mobility 45 shift assay experiments, and Drs Eleanor Need and Grant Buchanan (Freemasons capable of committing to the B-lymphoid lineage, but may be Foundation Centre for Mens Health, University of Adelaide) for advice on ChIP impaired at late, antigen-driven stages of B-cell development, and experiments. This work was sponsored by the Leukaemia & Lymphoma Research at increased risk of premature and/or deregulated IgH Fund, UK, the LEUKA Trust, UK, the NIHR Biomedical Research Centre Funding 12,46 transcription. This may, in turn, lead to an increase in the Scheme, UK and CAPES, Ministry of Education, Brazil.

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