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The B cell-specific transcription factor BACH2 is required for affinity maturation of mature B cells. Here, we show that Bach2-mediated activation of p53 is required for stringent elimination of pre-B cells that failed to productively rearrange immunoglobulin VH-DJH gene segments. Upon productive VH-DJH gene rearrangement, pre-B cell receptor signaling ends negative selection through BCL6-mediated repression of p53. In patients with pre-B ALL, BACH2-mediated checkpoint control is frequently compromised. Low levels of BACH2 expression represent a strong independent predictor of poor clinical outcome. Bach2+/+ pre-B cells resist leukemic transformation by Myc through Bach2-dependent upregulation of p53, and fail to initiate fatal leukemia in transplant recipient mice. ChIP-seq and gene expression analyses reveal that BACH2 competes with BCL6 for promoter binding and reverses BCL6-mediated repression of p53 and other checkpoint control genes. These findings identify Bach2 as a critical mediator negative selection at the pre-B cell receptor checkpoint and a safeguard against leukemogenesis.
In mice, bone marrow progenitor cells produce approximately 10 million pre-B cells daily3. Newly formed pre-B cells, however, are destined to die unless they productively rearrange VH-DJH gene segments and are rescued by pre-B cell receptor signals into the long-lived peripheral B cell pool4-5. We recently identified the transcriptional repressor BCL6 as critical survival factor that rescues pre-B cells that productively rearranged VH-DJH gene segments and emerged from the pre-B cell receptor checkpoint6-7. However, the mechanisms leading to clearance of other pre-B cells that failed to productively rearrange VH-DJH gene segments and thus lack pre-B cell receptor expression are poorly understood.
To identify factors that mediate negative selection at the pre-B cell receptor checkpoint in humans, we studied gene expression changes during human B cell development at the pro-B to pre-B cell transition8. We identified 18 genes with specific upregulation at the pre-B cell receptor checkpoint including components of the pre-B cell receptor itself (IGLL1, VPREB1), effectors of VH-DJH recombination (RAG1, RAG2, DNTT), and mediators of survival signaling (Supplementary Fig. 1). We next studied gene expression changes at the pre-B cell receptor checkpoint under two experimental conditions, namely (i) initiation of V(D)J recombination upon inducible activation of Pax59 and, (ii) inducible expression of a productively rearranged VH-DJH encoding a functional immunoglobulin μ heavy chain (μHC), which initiates pre-B cell receptor signaling7 (Fig. 1a). We reason that pre-B cell receptor checkpoint control should occur between these two events. As predicted, Pax5-mediated initiation of V(D)J recombination is reflected by strong upregulation of Rag1, Rag2 and caused upregulation of Arf/p53 and Bach2. By contrast, inducible activation of μHC expression induced expression of Bcl6, a transcriptional repressor of Arf/p53 (Figs. 1a-c). Bcl6-mediated repression of Arf/p53 is needed to rescue μHC+ pre-B cells once they have passed the pre-B cell receptor checkpoint6-7. Consistent with findings in human B cell differentiation (Supplementary Fig. 1), we found high levels of Bach2 prior to (Pro-B and pre-BI cells) and high levels of Bcl6 subsequent to (pre-BII cells) passage of the pre-B cell receptor checkpoint in mice (Fig. 1d). These findings imply that Bach2 potentially contributes to negative selection of pre-B cells that failed to productively rearrange VH-DJH gene segments at the pre-B cell receptor checkpoint. To test this hypothesis, we assayed pre-B cell receptor checkpoint control in pro-B and pre-BI cells from Bach2–/– mice2 and wildtype controls under experimental conditions. Activation of GFP-tagged Pax5 induces accumulation of Arf/p53 and results in rapid elimination of Bach2+/+ but not Bach2–/– pro-B and pre-BI cells (Figs. 1e-g).
Previous studies by our group identified Bcl6 as a strong transcriptional repressor of Arf/p53 in normal pre-B cells6 and pre-B ALL10. Of note, ChIP-seq analysis and QChIP validation revealed that both BCL6 and BACH2 bind to overlapping promoter regions of CDKN2A (Arf) and TP53 (p53) and other checkpoint regulators (CDKN1A (p21), CDKN1B (p27), GADD45A, GADD45B) (Fig. 2a and Supplementary Figs. 2 and 3). We therefore, tested the hypothesis that BACH2 and BCL6 compete for binding to promoter regions of checkpoint regulator genes, and that the ratio between the two determines negative (Bach2>Bcl6) and positive (Bach2<Bcl6) selection events at the pre-B cell receptor checkpoint (Fig. 2b). Binding of either BCL6 or BACH2 to CDKN2A and TP53 promoters affects gene expression in opposite directions: mRNA and protein levels of Arf and p53 are significantly reduced in the absence of Bach2 but strongly increased in the absence of Bcl6 (Figs. 2d, 2f and Supplementary Fig. 6) or upon inducible overexpression of Bach2 (Fig. 2c and Supplementary Figure 4). Likewise, mRNA levels of the p53-dependent tumor suppressor Btg2 were reduced by >20-fold in the absence of Bach2 but increased by 3-fold in the absence of Bcl6 (Fig. 2f and Supplementary Fig. 4). To test whether Bach2 negatively regulates the ability of Bcl6 to bind to Cdkn2a (Arf) and Tp53 (p53) promoters, we performed Bcl6-ChIP experiments with Bcl6–/– (negative control), Bach2+/+ and Bach2–/– cells. Binding of Bcl6 to Cdkn2a (Arf) and Tp53 (p53) was significantly increased in Bach2–/– compared to Bach2+/+ cells (Fig. 2e and Supplementary Fig. 5). Gene expression analysis for a subset of common Bach2- and Bcl6-target genes revealed that Bcl6 and Bach2 affect gene expression levels of checkpoint regulators including Cdkn2a (Arf), Tp53, Gadd45b, Btg1 and Btg2, in opposite directions (Figs. 2d, 2f and Supplementary Fig. 6). These findings collectively indicate that the balance between Bcl6 and Bach2 determines repression or transcriptional activation of Cdkn2a (Arf), Tp53 (p53) and related checkpoint molecules.
We measured functional consequences of Bach2-deficiency at the pre-B cell receptor checkpoint in Bach2–/– and wildtype mice. Using a classical pre-B cell differentiation model based on tyrosine kinase inhibition (Imatinib; IM) of BCR-ABL1-transformed pre-B cells12-13, we studied gene expression changes upon differentiation of Bach2–/– and Bach2+/+ pre-BI cells (Fig. 3a). In this analysis, Bach2-deficiency was associated with increased expression of the early progenitor antigen Ly6f (Sca-1) and reduced expression of the pre-B cell antigen Il2ra (CD25; Figs. 3a-b). Importantly, mRNA levels of Rag1 and Rag2, critical effectors of V(D)J recombination, were reduced by ~20-fold. Likewise, Imatinib (IM)-induced differentiation induced strong upregulation of Rag1/Rag2 expression in Bach2+/+ pre-BI cells as previously described6,12-13, but failed to upregulate Rag1/Rag2 beyond baseline levels in Bach2–/–cells (Fig. 3c). Inducible overexpression of Bach2 was sufficient to drive Rag1/Rag2 upregulation (Fig. 3d) and single-locus ChIP revealed that BACH2 binds RAG1 and RAG2 promoters, which was enhanced by IM-treatment (Fig. 3e).
To test if defective expression of Rag1/Rag2 results in impaired V(D)J recombination activity, we transduced Bach2–/– and Bach2+/+ pre-BI cells with a V(D)J recombination RSS substrate. Consistent with the massively increased Rag1/Rag2 expression, IM-treatment resulted in a 6-fold increase of baseline recombination of the RSS substrate in Bach2+/+ pre-B cells. By contrast, IM-induced V(D)J recombination activity was reduced by 20-fold in Bach2–/– pre-BI cells as compared to wildtype counterparts (Fig. 3f and Supplementary Fig.7).
We next studied the composition of B cell progenitor populations in bone marrows and spleens of Bach2+/+ and Bach2–/– mice. Total numbers of CD19+ B220+ B cells were only slightly reduced in the bone marrow and spleen of Bach2–/– mice (Fig. 3g and Supplementary Fig. 8). While pre-B cells from Bach2+/+ bone marrow were stringently selected for productive in-frame VH-DJH gene rearrangements, pre-B cells from Bach2–/– mice lacked selection for VH-DJH junctions with coding capacity (Fig. 3g and Supplementary Fig. 8). Based on random V(D)J recombination, only one of three pre-B cell clones achieve in-frame rearrangements and coding capacity for a functional pre-B cell receptor. Effective negative pre-B cell selection causes clearance of cells with non-functional out-of-frame VH-DJH rearrangements and, hence, results in a length distribution of VH-DJH junctions with size peaks that are spaced by three nucleotides14. Fragment length analysis revealed a peak size distribution of VH-DJH junctions that were selected for multiples of 3 bp among Bach2+/+ as compared to random distribution in Bach2–/– counterparts (Fig. 3g and Supplementary Fig. 8). Sequence analysis of VH-DJH junctions confirmed that out-of-frame VH-DJH rearrangements were stringently cleared from the repertoire in Bach2+/+ pre-B cells as opposed to accumulation of a large fraction of clones with non-functional rearrangements among Bach2–/– pre-B cells (Fig. 3g and Supplementary Fig. 8).
To test if activation of Bach2 downstream of Pax5 is sufficient to clear the pre-B cell repertoire from non-functional VH-DJH rearrangements, we developed a vector system for inducible expression of Bach2. IL7-dependent Bach2+/+ and Bach2–/– pre-B cells were transduced with tamoxifen (4-OHT)-inducible Bach2-ERT2 and ERT2 empty vector controls. Cells were treated with 4-OHT for 24 hours and sequence analysis of VH-DJH junctions was performed (Fig. 3h and Supplementary Figs. 9 and 10). While inducible overexpression of Bach2 had no significant effect on the repertoire composition of Bach2+/+ pre-B cells, inducible Bach2-reconstitution in Bach2–/– pre-B cells resulted in rapid elimination of clones carrying non-functional VH-DJH rearrangements (gray shaded in Supplementary Fig. 10). We conclude that Bach2 is required and sufficient for stringent clearance of pre-B cells that failed to productively rearrange VH-DJH segments at the pre-B cell receptor checkpoint.
In a high throughput screen for retroviral integrations in mouse pre-B cell leukemia (RTCGD), we found Bach2 introns 1-3 as common integration sites (Supplementary Fig. 11), raising the possibility that Bach2 functions as tumor suppressor in human pre-B ALL.
We therefore, tested whether Bach2 affects survival and proliferation of human pre-B ALL cells. To this end, we transduced xenografts from clinically-derived Ph+ ALL cells with vectors for either Bach2 or empty vector controls (EV). Primary Ph+ ALL cells carrying GFP-tagged Bach2 were rapidly depleted (Figs 4a-b and Supplementary Fig. 12). While Ph+ ALL cells expressed p53 protein in all cases studied, in 6 of 10 cases Ph+ ALL cells had low expression of ARF (Fig. 4c). Despite low ARF expression, the Ph+ ALL cells underwent cell death upon Bach2 overexpression (Fig. 4b). While Bach2 is required for upregulation of both ARF and TP53, these findings indicate BACH2 induces cell death in Ph+ ALL cells independently of ARF.
To test the requirements of Arf/p53 downstream of Bach2 in a genetic experiment, Cdkn2a-/- and Tp53-/- pre-B ALL cells were transduced with GFP-tagged Bach2 or empty vector controls (Figs. 4d-e). While Bach2 caused rapid elimination of Cdkn2a-/- and wildtype leukemia cells, the effect of Bach2 on Tp53-/- leukemia cells was delayed and substantially diminished (Fig. 4e and Supplementary Fig. 13). We conclude that p53 is an important effector of Bach2-mediated cell death, whereas Arf is dispensable. Our Arf/p53 Western blot analysis (Fig. 4c) agrees with previous findings that defects of p53 are rare in Ph+ ALL, whereas deletions of ARF are frequent (52%)15.
To identify potential defects in Bach2 function in human leukemia, leukemic blasts from 83 cases of Ph+ ALL (ECOG 2993) and pre-B cells from 12 normal bone marrow samples were analyzed with the HELP assay for promoter methylation (Log HpaII/MspI)16 of the BACH2 gene. The BACH2 promoter was more hypermethylated in Ph+ ALL cells as compared to normal human bone marrow pre-B cells (n=83; P=0.0026). In addition, sequence analysis of the BACH2 coding region of primary Ph+ ALL samples revealed BACH2 BTB domain missense mutations in 2 of 10 cases studied (Supplementary Fig. 14). Given that p53 is intact in most Ph+ ALLs, we conclude that inactivation of Bach2 upstream of p53 represents an important and non-redundant lesion.
Our earlier experiments showed that forced expression of Pax5 induces upregulation of Bach2 at the pre-B cell receptor checkpoint (Fig. 1a-b). Recent studies demonstrated that deletions and rearrangements of the PAX5 gene resulting in dominant-negative fusion molecules occur in ~20% of cases of pre-B ALL15,19. We therefore tested whether loss of PAX5 function results in transcriptional inactivation of BACH2 in human pre-B ALL cells. While overexpression of wildtype PAX5 increases transcriptional activity of the BACH2 promoter as measured by a luciferase reporter construct, overexpression of the dominant-negative PAX5-ETV6 fusion suppressed BACH2 promoter activity (Fig. 4f). Likewise, mRNA levels of BACH2 were increased by expression of wildtype PAX5 but decreased by dominant-negative PAX5-C20orf112 and PAX5-ETV6 fusion genes19 (Figs. 4g-i). Next, we studied gene expression values for three BACH2 probe sets in 160 cases of childhood pre-B ALL. Compared to 54 cases carrying either PAX5 deletions or somatic mutations of PAX5, BACH2 mRNA levels were significantly higher in 106 wildtype PAX5 pre-B ALL cases (Fig. 4i). We conclude that BACH2 links PAX5 to activation of p53 and, hence, represents an important tumor suppressor in human pre-B ALL.
BACH2 levels in ALLs carrying E2A-PBX1 and TEL-AML1 gene rearrangements were similar to those in normal pre-B cells from healthy bone marrow donors. However, Bach2 mRNA levels were significantly lower in MLL-AF4- and Ph+ ALL subtypes (Supplementary Fig. 14). We also studied BACH2 mRNA levels in matched sample pairs from 49 patients that had a relapse of leukemia after initial successful treatment. Comparing matched sample pairs from the time of diagnosis and subsequent relapse of leukemia, BACH2 mRNA levels were substantially reduced in relapse samples (Fig. 5a and Supplementary Fig. 15). In addition, BACH2 expression levels at the time of diagnosis predicted detection of minimal residual disease (MRD) in a bone marrow biopsy taken on day 29 of treatment (Fig. 5b). Importantly, loss of BACH2 expression strongly correlated with poor overall outcome among 207 patients with childhood ALL (P9906 Children's Oncology Group). Patients with higher than median expression levels of BACH2 had a relapse-free survival (RFS) of 77% whereas those with lower than median BACH2 expression levels had a RFS of 47% (Supplementary Fig. 16b).
To study whether Bach2 mRNA levels represent an independent predictor of clinical outcome, we performed multivariate analyses using three established predictors of poor clinical outcome as reference, namely MRD, high white blood cell counts (WBC>100,000/μl) and deletion of the IKZF1 tumor suppressor17. Studying high risk groups of patients on the basis of these three factors, we found that lower than median expression levels of BACH2 defined subgroups of patients with even worse clinical outcome. Lower than median expression levels of BACH2 at diagnosis predicted inferior clinical outcome for patients with positive MRD on day 29, patients with WBC>100,000/μl (Figs. 5c-e and Supplementary Fig. 16), and for patients with IKZF1-deletion (Supplementary Fig. 17).
Our data in Figs. 1 and and22 demonstrated that both Bach2 and Bcl6 bind Cdkn2a and Tp53 promoters but inversely regulate protein levels of Arf and p53 mRNA. Interestingly, the antagonistic function of BACH2 relative to BCL6 in the regulation of ARF/P53 and multiple other checkpoint genes is mirrored by their opposite association with clinical outcome. While higher than median mRNA levels of BACH2 at the time of diagnosis predict favorable clinical outcome, the opposite is true for ALL patients with BCL6 mRNA at higher than median levels (COG P9906). Studying BACH2 and BCL6 mRNA levels in a bivariate analysis identified subgroups of patients with particularly favorable (BACH2High-BCL6Low) and particularly poor (BACH2Low-BCL6High) outcome based on the ratio of BACH2:BCL6 mRNA levels (Fig. 5f).
BACH2 is located at chromosome 6q15 and deletions of 6q encompassing 6q15 were found in 32% of cases of B cell lineage ALL18. We therefore studied B cell lineage ALL cell lines (n=9) and primary cases (n=4) with 6q deletions by high-resolution SNP analysis and found that BACH2 lies within a region of minimal deletion (RMD) on 6q (Fig. 5g). Studying bone marrow samples from primary ALL cases at the time of (i) diagnosis, (ii) clinical remission and (iii) relapse of leukemia, we found that deletion of the BACH2 locus was acquired at the time of leukemia relapse in 3 of 4 cases studied and was pre-existing in the 4th case (Figs. 5g-j).
Ph+ ALL cells critically depend on tyrosine kinase signaling from (i) the BCR-ABL1 oncogene, (ii) downstream phosphorylation of Stat5 and (iii) transcriptional activation of Myc20. Interestingly, a global analysis of Bach2-dependent gene expression changes revealed that these changes significantly overlap with gene expression changes caused by (i) BCR-ABL1 kinase inhibition, inducible deletion of (ii) Stat5a/b and (iii) Myc (Supplementary Fig. 18). These findings suggest that Bach2-mediated gene expression changes oppose a transcriptional program of survival (Stat5) and proliferation (Myc) downstream of BCRABL1 in Ph+ ALL cells.
Consistent with this scenario, BCR-ABL1-transformed Bach2–/– pre-B ALL cells are significantly less sensitive to BCR-ABL1 tyrosine kinase inhibition (TKI; IM) than their wildtype counterparts (Figs. 6a-b). In the absence of Bach2, IM-induced apoptosis was reduced by 3-fold (Fig. 6a) and the fraction of cells that remained in cell cycle despite IM-treatment was increased by 2-fold (Fig. 6b). While BCR-ABL1-transformed Bach2+/+ cells are able to form colonies only after a lag phase of ~10 days, colony formation of Bach2–/– pre-B ALL cells was significantly accelerated (Fig. 6c).
Overexpression of the Myc proto-oncogene frequently leads to increased proliferation and malignant transformation. However, Myc-driven proliferation can only be increased within certain limits, and Myc alone is not sufficient to cause malignant growth21-22. A number of failsafe mechanisms restrain Myc-induced proliferation and tumorigenesis23. These failsafe mechanisms are critical to terminate pre-malignant clones and, hence, represent a powerful safeguard against malignant transformation. Studying progressive transformation of pre-B cells in BCR-ABL1-tg mice in vivo, we found that mRNA levels of Bach2 and Myc are inversely regulated. In wildtype and young (<30 days, non-leukemic) BCR-ABL1-tg mice, mRNA levels of Bach2 are high whereas mRNA levels of Myc are low. Between ages of 30 and 120 days, BCR-ABL1-tg mice undergo progressive leukemic transformation24. Fully transformed leukemia cells express high levels of Myc and low levels of Bach2, whereas treatment of mice with TKI (Nilotinib) restored normal expression levels for Bach2 (high) and Myc (low; Fig. 6d). We conclude that expression of Bach2 and Myc are mutually exclusive during oncogenic transformation of pre-B cells.
We next tested the hypothesis that Bach2 negatively regulates the susceptibility of pre-B cells to Myc-mediated transformation. In normal pre-B cells, excessively high levels of Myc result in activation of Arf/p53 and oncogene-induced senescence21-23. Retroviral overexpression of Myc was inefficient, induced cell death and caused significant toxicity in Bach2+/+ pre-B cells but not in Bach2–/– pre-B cells (Fig. 6e-f and Supplementary Figs 19 and 20). These findings indicate that Bach2 strongly reduces susceptibility of pre-B cells to malignant transformation by Myc.
Cell cycle analysis revealed that overexpression of Myc in Bach2+/+ pre-B cells increased the fraction of cells in S-phase to a maximum threshold of about 50%. However, Myc overexpression in Bach2–/– pre-B cells caused an increase of proliferation beyond this limit with about 70% of pre-B cells in S-phase (Supplementary Fig. 19b). Consistent with these findings, overexpression of Myc in Bach2+/+ pre-B cells resulted in activation of both Arf and p53. By contrast, p53 levels were very low in Bach2–/– pre-B cells and not increased by Myc-overexpression (Supplementary Fig. 19b). We conclude that in the absence of Bach2, p53-dependent failsafe mechanisms are not activated, obviating the requirement of additional genetic lesions for leukemic transformation.
To confirm a critical role of Bach2 in Myc-induced failsafe control in an in vivo setting, IL7-dependent Bach2+/+ and Bach2–/– pre-B cells were transduced with retroviral Myc-GFP. Myc-GFP+ pre-B cells (Fig. 6e) were then injected into sublethally irradiated NSG recipient mice. While Bach2–/– MycGFP pre-B cells readily initiated fatal leukemia that killed the NSG recipient mice within 25 days, all NSG mice receiving Bach2+/+ MycGFP pre-B cells were still healthy at 70 days after injection (Fig. 6g). After 70 days, the mice in the surviving Bach2+/+ MycGFP cohort were sacrificed and analyzed together with mice receiving Bach2–/– MycGFP pre-B cells. In contrast to mice injected with Bach2–/– MycGFP pre-B cells, no CD19+ GFP+ cells (i.e. leukemia) were found in mice receiving Bach2+/+ MycGFP pre-B cells (Fig. 6h and Supplementary Fig. 21). Minimal residual disease (MRD) PCR for GFP revealed small numbers of persisting leukemia cells in most of the surviving mice at 70 days after injection. However, MRD levels were 4-5 log10 orders lower in the wildtype group as compared to mice injected with Bach2–/– MycGFP pre-B cells (Fig. 6i).
This study identifies Bach2-mediated activation of p53 as a critical mediator of pre-B cell receptor checkpoint control. During normal B cell development, Bach2 determines stringency of negative selection of pre-B cell clones that failed to productively rearrange productively rearrange VH-DJH gene segments. In addition, Bach2-mediated activation of p53 represents a critical failsafe mechanism to terminate pre-malignant pre-B cell clones before they can complete malignant transformation. Thereby, Bach2 limits pre-B cell proliferation and oncogene signaling through activation of p53. Both in normal pre-B cells and in pre-B ALL, Bach2-mediated activation of p53 is opposed by Bcl6, a potent transcriptional repressor of p53. While inactivation of TP53 in ALL is rare, BACH2 is frequently inactivated in established pre-B ALL clones or at the time of leukemia relapse. Consistent with their opposing role in TP53-regulation and checkpoint control, the ratio of BACH2 (favorable) and BCL6 (poor) expression levels also represents a strong predictor of clinical outcome in patients with ALL (Fig. 5f). While BACH2 function cannot be reinstated in patients with ALL carrying BACH2 deletions, we propose that pharmacological inhibition of BCL6 (e.g. with RI-BPI) will restore the balance of BACH2/BCL6-mediated checkpoint control and relieve BCL6-mediated transcriptional repression of p53 (Supplementary Fig. 22).
Methods and any associated references are available in the online version of this paper. Sequence data are available from EMBL/GenBank under accession numbers provided in Supplementary Table 7. Microarray data are available from GEO under accession numbers provided in Supplementary Table 8.
We would like to thank Hilda Ye (Albert Einstein College of Medicine, Bronx, NY) for sharing BCL6-/- mice and wildtype controls with us, Lothar Hennighausen (NIDDK, Bethesda, MD) for Stat5fl/fl mice. Samples used in this research include those obtained from the Newcastle Haematology Biobank-http://www.ncl.ac.uk/nbb/collections/nhb/index.htm). This work is supported by grants from the NIH/NCI through R01CA137060, R01CA139032, R01CA157644, R01CA169458 and (to M.M.), Translational Research Program grants from the Leukemia and Lymphoma Society (grants 6132-09 and 6097-10), a Leukemia and Lymphoma Society SCOR (grant 7005-11, PI Brian J Druker), the William Laurence and Blanche Hughes Foundation and a Stand Up To Cancer-American Association for Cancer Research Innovative Research Grant (IRG00909, to M.M.), the California Institute for Regenerative Medicine (CIRM; TR2-01816 to MM) and Leukaemia and Lymphoma Research (VR and AGH). A.M. and M.M. are Scholars of the Leukemia and Lymphoma Society. VR is a Leukaemia and Lymphoma Research Bennett Fellow.
The authors have no conflicting financial interests.