BACH2 mediates negative selection and p53-dependent tumor suppression at the pre-B cell receptor checkpoint

Abstract

The B cell-specific transcription factor BACH2 is required for affinity maturation of 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. After productive VH-DJH gene rearrangement, pre-B cell receptor signaling ends BACH2-mediated negative selection through B cell lymphoma 6 (BCL6)-mediated repression of p53. In patients with pre-B acute lymphoblastic leukemia, the BACH2-mediated checkpoint control is compromised by deletions, rare somatic mutations and loss of its upstream activator, PAX5. Low levels of BACH2 expression in these patients represent a strong independent predictor of poor clinical outcome. In this study, we demonstrate that Bach2(+/+) pre-B cells resist leukemic transformation by Myc through Bach2-dependent upregulation of p53 and do not initiate fatal leukemia in transplant-recipient mice. Chromatin immunoprecipitation sequencing and gene expression analyses carried out by us revealed that BACH2 competes with BCL6 for promoter binding and reverses BCL6-mediated repression of p53 and other cell cycle checkpoint-control genes. These findings identify BACH2 as a crucial mediator of negative selection at the pre-B cell receptor checkpoint and a safeguard against leukemogenesis.

Figure 1. Bach2 and Bcl6 maintain balance between negative selection and survival of early B cells at the pre-B cell receptor checkpoint

(a) Gene expression changes at the pre-B cell receptor checkpoint were studied under two experimental conditions, namely (i) initiation of V(D)J recombination upon inducible activation of Pax5 compared to empty vector control (EV) and (ii) inducible expression of a functional immunoglobulin μ heavy chain (μHC). (b) Quantitative RT-PCR and (c) Western blot depicting regulation of Bach2 and Bcl6 by Pax5 and μHC-induction, respectively. (d) Quantitative RT-PCR for Bach2 and Bcl6 in multi-lineage progenitor cells (MPP), Pro-B, Pre-BI and pre-BII cells sorted from bone marrow of C57BL/6 mice. (e-f) GFP-tagged Pax5 or empty vector controls (EV) were transduced in transformed pro-B and pre-BI cells from Bach2^-/- and wildtype mice and GFP⁺ cells were monitored (day 1 and day 10) by flow cytometry. (g) Bach2^+/+ and Bach2^-/- pro-B and pre-BI cells transduced with Pax5-GFP or EV were sorted and studied for protein expression of Arf and p53 by Western blotting, using β-actin as loading control.

Figure 2. Bach2-dependent activation of Arf/p53 is reversed by Bcl6 upon expression of a functional μ-heavy chain

(a) ChIP-seq analysis was performed in a lymphoma cell line using antibodies against BACH2 (red) and BCL6 (green) and peaks with significant enrichment relative to background (input; black) are annotated by ChIP-seeqer with bold underlines. Overlapping peaks between BACH2 and BCL6 binding are indicated by shades. (b) The proposed scenario for BACH2-BCL6 interactions at the pre-B cell receptor checkpoint. (c) Effects of presence or absence of Bach2, presence or absence of Bcl6 and inducible overexpression of Bach2 (tamoxifen-inducible Bach2-ER^T2 vector) on mRNA levels of Arf and p53 measured by qRT-PCR. (d) Effects of presence or absence of Bach2 and presence or absence of Bcl6 on protein levels of Arf and p53 measured by Western blot using β-actin as loading control. (e) To directly test the hypothesis that Bach2 negatively regulates the ability of Bcl6 to bind to Cdkn2a (Arf) and Tp53 (p53) promoters, Bcl6-ChIP experiments with Bcl6^-/- (negative control), Bach2^+/+ and Bach2^-/- cells were performed. (f) A systematic ChIP-seq analysis revealed that 134 of the 541 BCL6- and 565 Bach2-target genes are shared (not shown). Gene expression analysis (Affymetrix GeneChip) for a subset of common Bach2- and Bcl6-target genes showing that Bcl6 and Bach2 affect gene expression levels of checkpoint regulators like Cdkn2a (Arf), Tp53, Gadd45b, Btg1 and Btg2, in opposite directions.

Figure 3. Bach2 mediates V(D)J recombination and μ-heavy chain checkpoint control during early B cell development

(a) Using a classical pre-B cell differentiation model based on tyrosine kinase inhibition (Imatinib; IM) of BCR-ABL1-transformed pre-B cells^,, gene expression changes upon inducible differentiation of Bach2^-/- and Bach2^+/+ pre-BI cells were measured by microarray analysis. (b) Increased expression of the early progenitor antigen Ly6f (Sca-1) and reduced expression of the pre-B cell antigen Il2rα (CD25) in Bach2-deficient Pre-BI cells was validated by flow cytometry. (c) Reduced mRNA levels of Rag1 and Rag2 in Bach2-deficient Pre-BI ALL (BCR-ABL1) cells in the presence and absence of IM were validated by qRTPCR. (d) The effect of inducible overexpression of Bach2 on Rag1 and Rag2 mRNA levels was measured by qRT-PCR. (e) Single-locus ChIP analysis using IgG and BACH2-specific antibodies depicting the binding of BACH2 to Rag1 and Rag2 promoters, which is further enhanced by IM-treatment. (f) To test functional consequences of defective expression of Rag1/Rag2, Bach2^+/+ and Bach2^-/- pre-BI cells were transduced with a puromycin-selectable V(D)J recombination substrate carrying an inverted GFP flanked by recombination signal sequences (RSS). Recombination activity of the RSS substrate in Bach2^+/+ and Bach2^-/- pre-BI cells was measured by flow cytometry (inversion of the GFP cassette in the correct orientation; percentages of GFP⁺ cells are given) in the presence and absence of IM treatment. (g) Comparison of the composition of B cell progenitor populations in the bone marrow of Bach2^+/+ and Bach2^-/- mice by flow cytometry. Fragment length analysis revealed a size peak distribution of V_H-DJ_H junctions that were indeed selected for multiples of 3 bp among Bach2^+/+ compared to random distribution in Bach2^-/- pre-B cells. (h-i) IL7-dependent Bach2^+/+ and Bach2^-/- pre-B cells were transduced with tamoxifen (4-OHT)-inducible Bach2-ER^T2 and ER^T2 empty vector controls. Cells were treated with 4-OHT for 24 hours and sequence analysis of V_H-DJ_H junctions was performed. (i) Amino acid sequences of V_H-DJ_H junctions with non-functional V_H-DJ_H rearrangements shaded in gray.

Figure 4. BACH2 mediates PAX5-dependent tumor suppression in pre-B ALL through activation of p53

(a) Patient-derived Ph⁺ ALL cells were propagated on OP9 stroma and transduced with vectors for Bach2^GFP or GFP empty vectors (EV; a), and (b) GFP⁺ cells were monitored by flow cytometry. (c) Ph⁺ ALL cases were assayed for ARF and TP53 protein expression by Western blot using β-actin as loading control. (d-e) Flow cytometry for GFP in Cdkn2a^-/- and Tp53^-/- pre-B ALL cells (BCR-ABL1) transduced with Bach2^GFP or EV^GFP. (f) Transcriptional regulation of BACH2 by PAX5 assayed by the overexpression of dominant-negative PAX5-ETV6 fusion in 293T cells and Nalm6 pre-B ALL cells transduced with a BACH2 promoter luciferase reporter construct. (g-h) mRNA levels of BACH2 were increased by expression of wildtype PAX5 but decreased by dominant-negative PAX5-C20orf112 and PAX5-ETV6 fusion genes. (i) Gene expression values for three BACH2 probe sets in 160 cases of childhood pre-B ALL, 54 of which carry PAX5 deletions or somatic mutations (221234_s_at, 227173_s_at, 215907_at; COG P9906).

Figure 5. BACH2 is an independent predictor of poor clinical outcome in ALL patients

(a) Comparison of BACH2 mRNA levels in matched sample pairs from 49 patients at the time of diagnosis and relapse of leukemia (pair-wise t-test). (b) Comparison of BACH2 mRNA levels in MRD^- and MRD⁺ patients (MRD⁺; n=67; COG P9906 trial). Multivariate analyses for BACH2 mRNA levels as independent predictors of clinical outcome performed using established predictors of poor clinical outcome, namely, (c) detection of MRD and (d-e) high white blood cell counts (WBC>100,000/μl). (f) Studying BACH2 and BCL6 mRNA levels in a bivariate analysis identified subgroups of patients with particularly favorable (BACH2^High-BCL6^Low) and particularly poor (BACH2^Low-BCL6^High) outcome. P values are from logrank test. (g) High resolution SNP analysis identified BACH2 as a region of minimum deletion (RMD) in ALL cell lines (n=9) and primary cases (n=4) with 6q deletions. Longitudinal bone marrow samples at the time of diagnosis, remission and relapse of ALL were studied by (h) genomic PCR using the Taqman probe Hs01527432_cn, (i) qRT-PCR using the Taqman probe Hs00222364_m1 and (j) Western blot to assay allelic status and expression of BACH2.

Figure 6. Bach2 prevents leukemic transformation by Myc

(a) Induction of cell death (Annexin V/7AAD staining) and (b) cell cycle arrest (BrdU staining; G0/1, S and G2/M phase distribution) in BCR-ABL1 transformed Bach2^+/+ and Bach2^-/- pre-B ALL cells before and after imatinib treatment. (c) Comparison of colony forming abilities of Bach2^+/+ and Bach2^-/- pre-B ALL cells plated in methylcellulose on day 7. Bach2^+/+ pre-B ALL cells were able to form colonies after a lag phase of ~10 days (not shown). (d) To study progressive leukemic transformation in vivo, bone marrow pre-B cells were isolated from wildtype mice and BCR-ABL1-tg mice of various ages reflecting different stages of transformation. mRNA levels of Bach2 and Myc were plotted against age of mice [days], TKI denotes in vivo treatment of leukemic mice with the tyrosine kinase inhibitor Nilotinib. (e) Bach2^+/+ and Bach2^-/- IL-7-dependent pre-B cells were retrovirally transduced with Myc^GFP or EV^GFP and transduction efficiency was monitored by flow cytometry on days 0 and 3. (f) Plating efficiency of Myc^GFP-transduced Bach2^+/+ and Bach2^-/- IL-7-dependent pre-B cells in methylcellulose was monitored by fluorescence microscopy. (g) Sorted Myc^GFP-transduced Bach2^+/+ and Bach2^-/- IL-7-dependent pre-B cells were also tested for their capacity to initiate fatal leukemia in NOD/SCID recipient mice and Kaplan-Meier analysis was performed to compare overall survival of transplant recipient mice in the two groups. (h) After 70 days, the mice in the surviving Bach2^+/+ Myc^GFP cohort were sacrificed and analyzed together with mice receiving Bach2^-/- Myc^GFP pre-B cells for CD19⁺ GFP⁺ cells in bone marrow and spleen (i.e. leukemia) by flow cytometry. (i) Minimal residual disease (MRD) qRT-PCR for leukemic cells infiltrating bone marrow or spleens was performed in triplicates for mice in each group using GFP-specific primers.