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.⁹ (B) BCR-ABL1 pre-B ALL cells were transduced with 4-hydroxy-tamoxifen (4-OHT)–inducible vectors for BCL6-ER^T2 and ER^T2 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).

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 mice² 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 Pten^fl/fl mice (supplemental Table 4) were transduced with 4-OHT–inducible Cre-ER^T2 or an ER^T2 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).