Pre-B cell receptor-mediated activation of BCL6 induces pre-B cell quiescence through transcriptional repression of MYC - PubMed (original) (raw)

Pre-B cell receptor-mediated activation of BCL6 induces pre-B cell quiescence through transcriptional repression of MYC

Rahul Nahar et al. Blood. 2011.

Abstract

Initial cell surface expression of the pre-B cell receptor induces proliferation. After 2 to 5 divisions, however, large pre-BII (Fraction C') cells exit cell cycle to become resting, small pre-BII cells (Fraction D). The mechanism by which pre-BII cells exit cell cycle, however, is currently unclear. The checkpoint at the Fraction C'-D transition is critical for immunoglobulin light chain gene recombination and to prevent malignant transformation into acute lymphoblastic leukemia. Here we demonstrate that inducible activation of pre-B cell receptor signaling induces cell-cycle exit through up-regulation of the transcriptional repressor BCL6. Inducible activation of BCL6 downstream of the pre-B cell receptor results in transcriptional repression of MYC and CCND2. Hence, pre-B cell receptor-mediated activation of BCL6 limits pre-B cell proliferation and induces cellular quiescence at the small pre-BII (Fraction D) stage.

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Figures

Figure 1

Figure 1

Pre-BCR signaling and Ikaros up-regulate BCL6 in normal and leukemic pre-B cells. _Ighm_−/− pro-B cells and _Blnk_−/− pre-BII cells were transformed with BCR-ABL1 and pre-BCR signaling was reconstituted by retroviral expression of BLNK-GFP, CD8/μ-chain or GFP and CD8 empty vectors. BCL6 mRNA levels were measured by quantitative RT-PCR in (A; n = 2) and protein levels were determined by Western blot in (B; n = 2) using β-actin as loading control. IL-7–dependent pre-B cells from Rag2−/− tTA/μ-chain-transgenic mice were cultured in the presence of tetracycline, removal of which induced activation of transgenic μ-chain expression under endogenous transcriptional control elements. Inducible differentiation was verified by flow cytometry (supplemental Figure 2) and BCL6 and MYC protein levels were measured by Western blot (C; n = 2). Normal IL-7–dependent pre-B cells were transduced with a constitutively active (CA) mutant of FoxO1 or an empty vector (transduction efficiency shown in supplemental Figure 1). After inducible activation with 4-hydroxy-tamoxifen (4-OHT), cells were subjected to Western blot analysis for BCL6 and β-actin as loading control (D; n = 2). Likewise, normal IL-7–dependent pre-B cells from Ptenfl/fl mice (supplemental Table 4) were transduced with 4-OHT–inducible Cre-ERT2 or an ERT2 empty vector control and subjected to antibiotic selection. Cre-mediated deletion of Pten and BCL6-up-regulation on IL-7 withdrawal was studied by Western blot using β-actin as loading control (E; n = 2). Pre-B ALL cells from Bcl6+/+ bone marrow were transduced with retroviral expression vectors for BCL6-GFP or a GFP empty vector control. The relative change of the percentage of GFP+ cells over time was measured by flow cytometry (F; n = 2). The cell-cycle profile of transduced cells was measured by BrdU incorporation and flow cytometry (G; n = 2) with statistical analysis (H). Pre-B ALL cells transduced with BCL6-GFP or GFP alone were sorted for GFP and transplanted into sublethally irradiated NOD/SCID mice via tail vein injection (3 mice per group). Spleens of recipient mice and their weight (right) are shown (I).

Figure 2

Figure 2

Pre-B cell receptor–mediated up-regulation of BCL6 inhibits proliferation by transcriptional repression of Myc. Pre-B ALL cells from Bcl6+/+ and _Bcl6_−/− bone marrow were transduced with retroviral expression vectors for CD8/μ-chain or CD8 alone. The relative change of the percentage of CD8+ cells over time was measured by flow cytometry (A; n = 3). To identify BCL6-target genes, ChIP-on-chip analysis was performed for 3 human BCR-ABL1 pre-B ALL cell lines (BV173, Tom1, Nalm1) and 2 human diffuse large B-cell lymphoma cell lines (OCI-Ly1 and OCI-Ly7) as positive controls. High expression levels of BCL6 were induced by treatment of BCR-ABL1 pre-B ALL cell lines with 10μM STI571 for 24 hours (STI571). Recruitment to CCND2 and MYC (see supplemental Figure 3) promoters are shown and to the HPRT promoter as a negative control. The ChIP-on-chip data for Myc in Nalm1 cells is also published in Duy et al. (B) BCR-ABL1 pre-B ALL cells were transduced with 4-hydroxy-tamoxifen (4-OHT)–inducible vectors for BCL6-ERT2 and ERT2 empty vector control. Myc mRNA (C; n = 2) and protein (D; n = 2) levels on 4-OHT induction were measured by quantitative RT-PCR and Western blot, respectively. Induction of BCL6 also resulted in up-regulation of Cdkn1b (p27) protein levels (D). Overexpression of Myc was sufficient to partially rescue BLNK/BCL6-mediated inhibition of proliferation (E): _Blnk_−/− BCR-ABL1 pre-B ALL cells were transduced with Myc-Puro or a Puro-empty vector control and subjected to antibiotic selection. Subsequently, pre-BCR signaling was reconstituted by retroviral expression of BLNK-GFP or a GFP empty vector control and proliferation of the Myc-Puro versus Puro-transduced cells was measured as relative change of the percentage of BLNK-GFP+ or GFP+ cells (E; n = 2).

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References

    1. Decker DJ, Boyle NE, Koziol JA, Klinman NR. The expression of the Ig H chain repertoire in developing bone marrow B lineage cells. J Immunol. 1991;146(1):350–361. - PubMed
    1. Hess J, Werner A, Wirth T, Melchers F, Jäck HM, Winkler TH. Induction of pre-B cell proliferation after de novo synthesis of the pre-B cell receptor. Proc Natl Acad Sci U S A. 2001;98(4):1745–1750. - PMC - PubMed
    1. Kersseboom R, Middendorp S, Dingjan GM, et al. Bruton's tyrosine kinase cooperates with the B cell linker protein SLP-65 as a tumor suppressor in Pre-B cells. J Exp Med. 2003;198(1):91–98. - PMC - PubMed
    1. Jumaa H, Bossaller L, Portugal K, et al. Deficiency of the adaptor SLP-65 in pre-B-cell acute lymphoblastic leukaemia. Nature. 2003;423(6938):452–456. - PubMed
    1. Flemming A, Brummer T, Reth M, Jumaa H. The adaptor protein SLP-65 acts as a tumor suppressor that limits pre-B cell expansion. Nat Immunol. 2003;4(1):38–43. - PubMed

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