BCL6 Function Research Articles

Integrative Analysis of Ikaros-Dependent Changes of Transcriptional Regulation and Tyrosine Phosphorylation Events in Ph+ ALL

AbstractAbstract 528 Background:Tyrosine kinase-driven B-cell lineage acute lymphoblastic leukemia such as Ph+ ALL, is associated with particularly poor clinical outcome and defined by deletions of the IKZF1 gene encoding the Ikaros transcription factor in >80% of the cases. While the role of Ikaros in lymphoid differentiation has been extensively studied, it remains elusive how Ikaros functions as tumor suppressor in Ph+ALL. Here we demonstrate that Ikaros orchestrates a set of gene expression changes that affects leukemia cell proliferation and survival at multiple levels. Results:A comprehensive gene expression analysis revealed that reconstitution of Ikaros function in patient-derived Ph+ ALL cells mimics gene expression changes induced by Pax5 (a B cell specific transcription factor), μ-chain and BLNK (pre-B cell receptor components), and treatment with Imatinib (a BCR-ABL1 kinase inhibitor), while it reverses Myc- and Stat5a/b-dependent survival signals. In combining seven experimental perturbations (reconstitution of Ikzf1, Pax5, μ-chain, Blnk, treatment with Imatinib or deletion of Stat5a/bfl/fl and Mycfl/fl), we identified a commonly regulated gene set, including known tumor suppressors SPIB, BTG1, and BTG2 and highly enriched for Myc-related gene signatures. These findings suggest that reconstitution of PAX5 and IKZF1 transcription factors converges with pre-B cell receptor-mediated transcriptional changes. We next connected our analysis at the transcriptional level with Ikaros-dependent changes at the level of tyrosine phosphorylation events: The BCR-ABL1 kinase in Ph+ ALL drives proliferation and survival through phosphorylation of Stat5, AKT, ERK, SRC and multiple other signaling intermediates. A global mass spectrometry analysis of phosphotyrosine proteins in patient-derived Ph+ ALL cells showed that reconstitution of Ikaros leads to global loss of phospho-tyrosine, in a comparable manner as treatment with TKI. The vast majority of Ikaros-mediated changes resulted in loss of tyrosine phosphorylation such as the SRC tyrosine kinase LCK (Y504 and Y192), the proximal BCR-ABL1 adapter molecule NEDD9 (Y166, Y240, Y344 and Y578) and ABL1 itself (Y257). For a small number of molecules, Ikaros-reconstitution resulted in increased tyrosine phosphorylation including the phosphatases Shp2 (Ptpn11Y584) and Ship2 (Inppl1Y1161). A focus phospho-tyrosine analysis of individual signaling molecules analysis revealed that Stat5, SRC, AKT and ERK are all affected by Ikaros-induced de-phosphorylation. These results suggest that that Ikaros induces dephosphorylation of multiple signaling intermediates in BCR-ABL1 signaling. To identify Ikaros-mediated gene expression changes that were directly induced by Ikaros promoter binding, we matched Ikaros-dependent gene expression changes with Ikaros ChIP-seq data. This analysis confirmed enriched binding of Ikaros on Btg1, Myc and Smyd2 promoters. Among Ikaros target genes, we identified a set of genes that predicts clinical outcome for adult and pediatric patients with ALL. According to this predictor, IkarosUP (FBXO33, HBP1, HIVEP1, DTX1, KIF13B, RHOQ) and IkarosDOWN (PFKP, ASNS, ETV6, PSTPIP2, SMYD2, CDKL5), patients with B-ALL were segregated into two groups with significantly different outcomes, with a 5-year RFS of 76% vs 29% (COG P9906 trial, n=110; p=0.006) and a 5-year OS of 39% vs 0% (ECOG E2993 trial, n=25; p=0.007). Further biochemistry and mouse experiments showed that both the ability of Ikaros to dephosphorylate Stat5 and to function as tumor suppressor requires cooperation with the pre-B cell receptor linker molecule BLNK but not the proximal effector kinase Syk. Conclusions:Importantly, Ikaros binds to and transcriptionally activates CDKN2A (Arf) and TP53 loci and thereby opposes the function of BCL6, which acts as transcriptional repressor at these loci. Comparing the gene expression microarray and ChIP-seq or ChIP-on-Chip data of BCL6 and Ikaros, we identified a set of target genes that is regulated by both BCL6 and Ikaros and found that for the vast majority of Ikaros/BCL6 target genes, Ikaros-reconstitution recapitulated a BCL6−/− situation, i.e. genes that are induced by Ikaros are repressed by BCL6. These findings are of potential clinical relevance since pharmacological inhibition of BCL6 restores sensitivity of IKZF1-mutant Ph+ ALL cells to conventional tyrosine kinase inhibitors. Disclosures:Druker:ARIAD: OHSU receives clinical trial funding. Dr. Druker is currently principal investigator or co-investigator on Novartis, Bristol-Myers Squibb, and ARIAD clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and da Other; Bristol-Myers Squibb: OHSU receives clinical trial funding, OHSU receives clinical trial funding Other; Novartis: OHSU receives clinical trial funding, OHSU receives clinical trial funding Other; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Scientific Founder; OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and man, Scientific Founder; OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and man Other.

BCL6 Interacting Corepressor (BCOR) Functions As Lineage-Specific Tumor Suppressor in B Lymphoid and Myeloid Leukemia

Abstract 1301 Background:BCL6-interacting corepressor (BCOR) has been first characterized as a BCL6-binding protein and co-repressor of BCL6 along with NCOR and SMRT. The main function of BCOR is to potentiate the transcriptional repression by BCL6. BCOR is also capable of binding with different histone deacetylases(HDACs), demethylase and H2A ubiquitin ligase which suggests chromatin modification as a mechanism of BCL6-independent transcriptional repression by BCOR. Using next generation sequencing, it has been shown that BCOR is recurrently mutated in myelodysplastic syndrome (MDS; Tiacci et al. Blood 2012) and acute myeloid leukemia (AML; Yoshida et al. Nature 2011). BCOR mutations in AML are typically associated with lack or low expression of a truncated BCOR protein which suggest a tumor-suppressor role for BCOR in MDS and AML. Recently we have shown that BCOR's main ligand, BCL6 promotes drug-resistance and survival in B cell lineage Ph+ALL and CML (Duy et al., Nature 2011). Considering the interactive function of BCL6 and BCOR we hypothesized that BCOR, although a tumor suppressor in MDS and AML, acts in concert with BCL6 to promote survival and drug-resistance in Ph+ ALL and CML. Results:To study the role of BCOR in a genetic experiment, we developed a CML and Ph+ ALL leukemia model based on bone marrow progenitor cells from male mice carring a loxP-flanked allele of Bcor (Bcorfl/Y) and mice in which BCOR overexpression can be induced by excision of a loxP-flanked Stop-cassette (BcorLSL). In Bcorfl/Ymice, exon 3 of Bcor is flanked by loxP sites and in BcorLSL mice an extra allele of Bcor has been inserted into Rosa26 locus with a stop codon flanked by loxP sites upstream of the start codon. On the basis of this model, bone marrow hematopoietic stem and progenitor cells from Bcorfl/Y and BcorLSL mice were transformed with BCR-ABL1 under B lymphoid (IL7; Ph+ ALL) and stem/progenitor cell (IL3, IL6, SCF; CML) conditions. Cre-mediated deletion of loxP sequences causes the deletion of Bcor in Bcorfl/Y cells and the over-expression of Bcor in BcorR26cells. Transduction of the ALL and CML cells with tamoxifen (4-OHT)-inducible Cre-ERT2 or ERT2 empty vectors were used to inducibly activate Cre in Ph+ ALL- and CML-like leukemia cells. Inducible deletion of Bcor in B cell lineage (CD19+ B220+) Ph+ ALL results in massive cell death. Surprisingly, deletion of Bcor had no significant effect on CML-like (LSK and myeloid phenotype) cells. Gain-of-function studies with the BcorLSL system revealed that inducible overexpression of BCOR significantly increases the ability of Ph+ ALL-like leukemia cells to form colonies in semi-solid methylcellulose culture, whereas inducible activation of BCOR in BcorLSL CML-like cells had the opposite effect. Comparing the basal level of Bcor between Ph+ ALL and CML shows that there is significantly higher amount of Bcor in Ph+ ALL as compared to CML. In vivo injection and Cre-mediated deletion of BCOR in Bcorfl/YPh+ ALL shows that the Bcor is required to initiate B cell lineage Ph+ ALL in vivo and cause fatal disease (p=0.001). Five out of 7 mice, injected with Bcorfl/-Ph+ ALL survive with no sign of tumor growth until the end of experiment (240 days after injection) while all mice injected with wild type cells died within the first 15 days. In addition, we tested the impact of Bcor deletion and Bcor overexpression on TKI-sensitivity of Ph+ ALL and CML-like leukemia cells. While Bcor-deletion dramatically sensitized Ph+ALL cells to TKI-treatment (e.g. Imatinib), no effects on TKI-sensitivity were observed in CML-like leukemia. Conclusion:Our genetic studies demonstrate an unexpected lineage-specific effect of BCOR function in B cell lineage Ph+ ALL and myeloid lineage CML. While both leukemia types are induced by BCR-ABL1, BCOR function was critical for the survival and leukemia-initiation of Ph+ ALL, but not CML. The findings in B cell lineage Ph+ ALL are consistent with our hypothesis that BCOR mediates TKI-resistance and survival in Ph+ ALL in concert with BCL6. Our findings in CML support a role of BCOR as tumor suppressor in myeloid leukemia as was observed for MDS and AML. In myeloid leukemias, BCOR activity may predominantly involve HDACs and, hence, BCL6-independent pathways of transcriptional repression. Disclosures:No relevant conflicts of interest to declare.

Roles of BCL6 in normal and transformed germinal center B cells

BCL6 is a transcriptional repressor required in mature B cells during the germinal center (GC) reaction. Multiple mechanisms act coordinately to timely modulate BCL6 expression at transcriptional and post-transcriptional levels. BCL6 prevents premature activation and differentiation of GC B cells and provides an environment tolerant of the DNA breaks associated with immunoglobulin gene remodeling mechanisms involved in the production of high-affinity antibodies of different isotypes. The critical functions exerted by BCL6 during normal B-cell development can be hijacked by the malignant transformation process. Indeed, BCL6 is targeted by genetic aberrations and acts as an oncogene in GC-derived lymphomas. The aberrations affecting BCL6 interfere with the multiple levels of regulation that grant a fine tuning of BCL6 expression and activity in physiologic conditions. This review summarizes the current knowledge on BCL6 function and its role in lymphomagenesis.

BCL6-Mediated Repression of p53 Is Critical for Leukemia Stem Cell Survival in Chronic Myeloid Leukemia

Abstract 446 Background:Chronic myeloid leukemia (CML) is induced by the oncogenic BCR-ABL1 tyrosine kinase and can be effectively treated for many years with tyrosine kinase inhibitors (TKI). However, unless CML patients take TKI-treatment life-long, leukemia will eventually recur, which is attributed to the failure of TKI-treatment to eradicate leukemia-initiating cells (LIC; Corbin et al., J Clin Invest 2011). Persistence of LIC in CML can result in acquisition of secondary events eventually leading to TKI-resistant blast crisis, which is fatal within months. Recent work demonstrated that FoxO factors are critical for maintenance of CML-initiating cells (Naka et al., Nature 2010), however the mechanism of FoxO-dependent leukemia-initiation remained elusive. Results:Here we identified the BCL6 protooncogene as a critical effector downstream of FoxO in self-renewal signaling of CML-initiating cells. ChIP-seq analysis demonstrated that BCL6 directly binds to and represses Arf and p53 promoters in human CML cells. Genetic deletion of the BCL6 gene in a mouse model of CML results in progressive depletion of Lin- Sca-1+ c-Kit+ LIC. BCL6-deficient LIC exhibit excessively high expression levels of Arf and p53 and propensity to cellular senescence and apoptosis. As a consequence, BCL-deficient CML cells lack the ability to form colonies and to initiate leukemia in transplant recipient animals. To investigate whether these effects are indeed owing to the role of BCL6 as repressor of Arf/p53, we induced activation of a dominant-negative BCL6-mutant in p53+/+ and p53−/− CML cells. While dominant-negative BCL6 compromised colony formation and self-renewal in p53+/+ CML cells, BCL6 inhibition only had minor effect on p53−/− CML cells. We conclude that BCL6 enables survival of LIC in CML mainly through transcriptional repression of p53.To test potential clinical relevance of these findings, we used a recently developed retro-inverso BCL6 peptide inhibitor (RI-BPI, Cerchietti et al., 2009), which inhibits BCL6 function as transcriptional repressor. RI-BPI is currently under clinical trial for the treatment of BCL6-dependent diffuse large B cell lymphoma (Dr. Ari Melnick, LLS TAP Program). Importantly, peptide inhibition of BCL6 in human CML cells compromises colony formation and leukemia-initiation in transplant recipients and selectively eradicates CD34+ CD38− LIC in patient-derived CML samples. Conclusions:These findings identify pharmacological inhibition of BCL6 as a novel strategy to eradicate LIC in CML. Clinical validation of this concept could limit the duration of TKI-treatment in CML patients, which is currently life-long, and substantially decrease the risk of blast crisis transformation. Based on these findings, we propose a dual targeting strategy, in which (1) tyrosine kinase inhibitors (e.g. Imatinib) to target the transient amplifying pool of CML cells are coupled with (2) BCL6 inhibition that will target quiescent LIC. Disclosures:Hochhaus:Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Ariad: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Shah:Bristol-Myers Squibb: Consultancy, Research Funding; Novartis: Consultancy; Ariad: Consultancy, Research Funding. Druker:Novartis: ; Bristol-Myers Squibb: ; ARIAD Pharmaceuticals: ; OHSU patent #843: Mutated ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership.

Transcriptional Repressor BCL6 Controls Th17 Responses by Controlling Gene Expression in Both T Cells and Macrophages

The transcriptional repressor protein BCL6 regulates T cell differentiation by repressing Th2 responses and promoting follicular Th cell responses. However, little is known about the role of BCL6 in Th17 responses. We found that memory T cells from BCL6-deficient mice had increased IL-17 production. Additionally, BCL6 expression is upregulated in CD4 T cells cultured under Th17 conditions. T cells from BCL6-deficient mice showed defective Th17 differentiation and enhanced IL-4 production in vitro; however, normal Th17 differentiation was obtained with BCL6-deficient T cells under culture conditions when highly pure naive CD4 T cells were used, when IL-4 production was inhibited, or when TGF-beta levels were increased. Retrovirus-mediated expression of BCL6 in CD4 T cells repressed IL-4 and augmented basal IL-17 mRNA expression. These data support the idea that BCL6 promotes Th17 differentiation through suppression of Th2 differentiation. BCL6-deficient T cells transplanted into Rag1(-/-) mice produced wild-type levels of IL-17, indicating that, in vivo, BCL6-deficient T cells develop relatively normal Th17 responses. Macrophages from BCL6-deficient mice showed strikingly increased expression of the Th17-promoting cytokines IL-6, IL-23, and TGF-beta, and conditioned media from BCL6-deficient macrophages promoted augmented IL-17 expression by T cells. We propose that the increased Th17 activity in BCL6-deficient mice is due, in part, to BCL6-deficient macrophages promoting increased Th17 differentiation in vivo. T cells may require BCL6 for optimal Th17 differentiation; however, BCL6 function in macrophages critically regulates Th17 differentiation in vivo. We hypothesize that increased Th17 differentiation aggravates the severe Th2-type inflammatory disease in BCL6-deficient mice.

Abstract B236: A small molecule disrupts the BCL6 transcriptional repressor complex and kills lymphoma cells in vitro and in vivo

Abstract The BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B-cell lymphoma (DLBCL) and is also implicated in solid tumors. BCL6 depletion by siRNA or BCL6 blockade using peptide inhibitors kills DLBCL cells, demonstrating that lymphoma cells are addicted to BCL6. Based on these data we hypothesized that BCL6 could serve as an excellent therapeutic target in lymphomas and other cancers. From the mechanistic standpoint BCL6 mediates transcriptional repression through recruitment of the SMRT, N-CoR and BCOR corepressors to a lateral groove motif on its BTB domain. Our crystallographic, biochemical, and functional studies suggested that a particular section of the BTB lateral groove was amenable to development of small molecule inhibitors. Since these residues are unique to BCL6 and no other BTB proteins we predicted that molecules designed to bind to this region would be specific. To identify such molecules we performed a computer-aided drug design (CADD) screen of over 1,000,000 compounds. After two rounds of docking with increasing stringency, target flexibility simulation and chemical diversity clustering, we generated a priority list of compounds likely to bind the BCL6 BTB lateral groove. A number of these compounds indeed blocked the BCL6 - corepressor interaction and inhibited the transcriptional repressor function of BCL6. Analogs of the 10 leading compounds were subjected to biological testing. One of these families, the 79 series, was selected for further study because this group contained the largest number of active compounds. X-ray crystallography of the most active 79 family compound, called 79-6, confirmed binding to the critical BTB lateral groove region. 79-6 penetrated and accumulated in intact lymphoma cells as determined by HPLC-MS. 79-6 blocked the repressor activity of BCL6 but not other BTB proteins, blocked the recruitment of corepressors to endogenous BCL6 target genes as shown by ChIP, and induced the re-expression of BCL6 target genes in BCL6-positive (but not BCL6 negative) lymphoma cells as shown by q-PCR. Most importantly, 79-6 killed BCL6-positive lymphoma cells by apoptosis induction but had no effect on BCL6-negative lymphoma cells. Extensive toxicological studies performed in C57/BL mice revealed no toxic effects (by complete serum chemistry, CBC, histopathological examination of every organ including bone marrow, etc). Pharmacokinetic analysis (by HPLC-MS/MS) in SCID mice showed that 79-6 penetrates and accumulates in tumors after intraperitoneal administration. 79-6 potently inhibited the growth of established human BCL6-positive xenografts (Ly7 and SUDHL6) but not of BCL6-negative tumors (Toledo), again without toxicity to other organs. The compound also killed primary BCL6-positive DLBCL explants from human patients. 79-6 is currently being optimized for used in humans. These results demonstrate that therapeutic chemical manipulation of transcriptional repression via modulation of protein-protein interactions is a viable option for development of potent and non-toxic anti-cancer drugs. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B236.

BCL6-Dependent Negative Regulation of Cell Cycle Checkpoint Regulators Enables Drug-Resistance in Ph+ Acute Lymphoblastic Leukemia.

Abstract 765BCR-ABL1 tyrosine kinase inhibitors (TKI) are widely used for the treatment of patients with Ph+ ALL and CML. To elucidate mechanisms of TKI-resistance in Ph+ ALL, we studied gene expression changes of a set of 11 primary cases of Ph+ ALL in response to TKI-treatment (16 hours; 2 μmol/l Imatinib). Sorting genes based on the ratio of gene expression values in the presence vs the absence of TKI-treatment, the BCL6 gene consistently ranked #1 in this analysis. As confirmed by quantitative RT-PCR and Western blotting, BCL6 is upregulated by 60- to 90-fold in response to TKI-treatment of Ph+ ALL cells. The BCL6 transcription factor functions as a protooncogene in germinal center (GC)-derived B cell lymphomas. We focused our analysis on BCL6, because it functions a transcriptional repressor of p53 and other cell cycle check point regulators and thereby protects GC-derived B cell lymphoma cells from apoptosis. Of note, BCL6 protein levels in TKI-treated Ph+ ALL cells were as high as in GC-derived B cell lymphoma. We hypothesize that dramatic upregulation of the BCL6 gene in response to TKI-treatment represents a defense mechanism of Ph+ ALL to evade cell death induced by activation of p53 and cell cycle checkpoint regulators. We tested the role of BCL6 in Ph+ ALL in a genetic loss-of-function experiment: To this end, B cell precursors from bone marrow of BCL6+/+ and BCL6−/− mice were transformed with BCR-ABL1. Upon treatment with 1 mmol/l Imatinib for three days, viability of BCL6−/− BCR-ABL1 ALL cells (0.4% ± 0.2%) was lower by two log orders compared to BCL6+/+ leukemia cells (46.0% ± 8.2%; p=0.001). Consistent with our hypothesis that p53 and other cell cycle checkpoint regulators are transcriptional targets of BCL6 in Ph+ ALL, we observed in a comprehensive gene expression analysis that mRNA levels of p53, p21, Arf and p27 were significantly higher in BCL6−/− compared to BCL6+/+ and BCR-ABL1 ALL cells (confirmed by quantitative RT-PCR). To identify target genes of the BCL6 transcriptional repressor, we performed a ChIP-chip analysis for BCL6 both in Ph+ ALL and GC-derived B cell lymphoma. Among the 1,235 target genes of BCL6 in Ph+ ALL, 736 were shared targets with GC-derived B cell lymphoma and 499 were exclusive for Ph+ ALL. These findings suggest that the function of BCL6 in Ph+ ALL does not replicate all aspects of its function in GC-B cell lymphoma. Single locus ChIP analysis confirmed strong recruitment of BCL6 to the promoter regions of the p53, p21 (CDKN1A), Arf (CDKN2A) and p27 (CDKN1B) genes in human Ph+ ALL cells after TKI-treatment. In a genetic loss-of-function experiment, we tested whether transcriptional suppression of p53, p21, Arf and p27 represents a major function of BCL6-upregulation in response to TKI-treatment in Ph+ ALL. To this end, we measured sensitivity of wildtype and Arf−/−, p53−/−, p21−/− and p27−/− BCR-ABL1 ALL cells to either Imatinib alone or to a combination of Imatinib and a novel BCL6 peptide inhibitor (RI-BPI). TKI-treatment alone had approximately the same effect in wildtype BCR-ABL1 ALL cells compared to Arf−/−, p53−/−, p21−/− and p27−/− counterparts. However, when TKI-treatment was combined with BCL6 inhibition (5 μmol/l RI-BPI), the viability of Arf−/−, p53−/−, p21−/− and to lesser degree, p27−/−BCR-ABL1 ALL cells was significantly higher than survival of the respective wildtype control leukemia. These findings confirm that transcriptional suppression of Arf, p53 and p21 (to lesser extent p27) represents an important characteristic of BCL6-mediated drug-resistance in response to TKI-treatment.We next tested the potential therapeutic usefulness of concomitant TKI-treatment and BCL6 peptide inhibition: To this end, 2 × 106 BCR-ABL1 ALL cells were luciferase-labeled and injected into sublethally irradiated NOD/SCID mice. Mice were treated six times between days 7 and 21 after leukemia cell injection with either the BCR-ABL1 kinase inhibitor Nilotinib (75 mg/kg) alone or a combination of Nilotinib and RI-BPI (20 mg/kg). Addition of RI-BPI resulted in significantly prolonged median survival (29 days) compared to Nilotinib alone (22 days; 12 mice per group; p=0.004). We conclude that BCL6-mediated transcriptional repression of p53 and other cell cycle checkpoint regulators represents a novel and critical mechanism of drug-resistance in Ph+ ALL. Combination of TKI-treatment with BCL6 peptide inhibition represents a new promising approach to target drug-resistance in Ph+ ALL. Disclosures:No relevant conflicts of interest to declare.

BCL6 Is Critical for the Development of a Diverse Primary B Cell Repertoire.

Abstract 91Through DNA strand breaks resulting from somatic hypermutation and class-switch recombination, germinal center (GC) B cells are exposed to a high level of DNA damage stress. At the GC stage of development, B cells are protected against apoptosis by specific expression of BCL6, which functions as transcriptional repressor of genes in the DNA damage response pathway. In the absence of BCL6, GC formation is abrogated. During normal B cell development, BCL6 expression was only found in GCs, where the secondary B cell repertoire is shaped. Extensive DNA damage, however, also occurs during the development of the primary B cell repertoire in the bone marrow. B cell precursors in the bone marrow sustain DNA damage during V(D)J recombination at immunoglobulin heavy and light chain loci. It is currently unclear, through which mechanisms early B cell precursors are protected against extensive DNA damage stress caused by V(D)J recombination.Here we report that BCL6 plays a critical role during early B cell development by protecting pre-B cells from DNA damage-induced apoptosis during V(D)J recombination. At the transition from IL7-dependent to IL7-independent stages of B cell development, BCL6 is activated, reaches similar expression levels as in GC B cells. Compared to IL7-dependent pro-B cells and large cycling pre-B cells, BCL6 mRNA and protein levels in IL7-independent small resting pre-B cells were increased by 60- to 90-fold, respectively. We identified STAT5 as a critical negative regulator of BCL6 downstream of IL7 receptor signaling in pre-B cells. Expression of a constitutively active STAT5 mutant prevented BCL6 upregulation in differentiating pre-B cells at the transition from IL7-dependent to IL7-independent stages of B cell development.BCL6 function was then tested in bone marrow precursor cells from BCL6−/− and BCL6+/+ mice: Comparing the gene expression pattern of BCL6−/− and BCL6+/+ pre-B cells, we found that BCL6 is required for transcriptional repression of the ARF (Cdkn2a), p21 (Cdkn1a), Gadd45a and p53 genes, which all contribute to cellular senescence and cell cycle arrest. In agreement with gene expression analyses, ChIP-chip and single-locus q-ChIP studies identified ARF (Cdkn2a), p21 (Cdkn1a), Gadd45a and p53 as transcriptional targets of BCL6 in pre-B cells. BCL6-dependent transcriptional repression of these genes in pre-B cells is critical because BCL6+/+ but not BCL6−/− pre-B cells were capable to proliferate in vitro and to form pre-B cell colonies in semisolid agar. Of note, peptide-inhibition of BCL6 suppressed growth and colony formation in ARF+/+ but not ARF−/− pre-B cells, suggesting that ARF-deficiency rescues lack of BCL6 function. We conclude that BCL6-mediated transcriptional repression of ARF is critical for pre-B cell self-renewal.To determine whether BCL6 function is also required for normal early B cell differentiation in vivo, we performed a comprehensive analysis of B cell differentiation stages in bone marrow from BCL6−/− and BCL6+/+ mice. In agreement with previous studies, the overall number of B cell precursors in the bone marrow was only slightly reduced and pro-B cell and large pre-B cell populations were normal. However, the pools of small-resting pre-B cells and new emigrant B cells were reduced in BCL6−/− mice by 3- and 7-fold, respectively. While the overall numbers of mature B cells in BCL6−/− mice were normal, we found that their clonal repertoire was extremely restricted. Using spectratype analysis, we found a broad polyclonal primary B cell repertoire in BCL6+/+ mice, whereas the B cell repertoire in their BCL6−/− counterparts was strictly oligoclonal. We conclude that pre-B cell self-renewal and polyclonal B cell production critically depends on BCL6. While the self-renewal defect of BCL6-deficient pre-B cells can be numerically compensated by increased proliferation at later stages of development, the diversity of the B cell repertoire in BCL6−/− mice is permanently restricted. We conclude that BCL6 is required for pre-B cell self-renewal and the formation of a normal polyclonal B cell repertoire. Disclosures:No relevant conflicts of interest to declare.

BCL6 Is Required for Leukemia-Initiation and Self-Renewal Signaling in Chronic Myeloid Leukemia.

Abstract 2167Poster Board II-144The transcriptional repressor BCL6 was discovered as a potent protooncogene in diffuse large B-cell lymphoma (DLBCL) and is also expressed in normal germinal center (GC) B cells. BCL6 protects DLBCL and GC B cells against DNA damage-induced apoptosis by suppressing target genes including p53, p21, ATR and p27. In preliminary experiments for this study, we found that treatment of BCR-ABL1-driven Ph+ ALL cells with Imatinib results in a 60- to 90-fold up-regulation of BCL6, reaching protein levels as high as in DLBCL and GC B cells. Since the oncogenic BCR-ABL1 kinase also drives leukemic transformation in CML cells, we studied regulation of BCL6 in human CML cells upon TKI-treatment. Like in Ph+ ALL, TKI-treatment resulted in rapid BCL6 mRNA and protein upregulation in CML cells, albeit to lesser degree than in Ph+ ALL and DLBCL. We hypothesized that upregulation of BCL6 in CML cells in response to TKI-treatment serves the same function as in DLBCL and protects from p53-mediated apoptosis. Through transcriptional repression of p53, p21, ATR and p27 as in DLBCL, upregulation of BCL6 in response to TKI-treatment would represent a defense mechanism of CML cells to evade TKI-induced cell death.We tested the role of BCL6 in CML in a genetic loss-of-function experiment: To this end, myeloid progenitor cells from bone marrow of BCL6+/+ and BCL6-/- mice were transformed with BCR-ABL1. Compared to their BCL6+/+ counterparts, BCL6-/- mouse CML cells were highly sensitive to Imatinib-treatment. In a complementary approach, we treated human CML cells with either Imatinib alone or with a novel BCL6 peptide inhibitor (Retro-inverso BCL6-peptide inhibitor, RI-BPI). Consistent with findings in BCL6-/- mouse CML cells, also human CML cells were significantly sensitized to Imatinib by BCL6 inhibition (using 5 mmol/l RI-BPI). Of note, the BCL6 inhibitory peptide RI-BPI alone induced no significant toxicity in human CML cells but synergized with Imatinib-treatment.Since RI-BPI alone had no cytotoxic effect on CML cells, we tested whether loss of BCL6 function affects self-renewal capacity of CML cells. This possibility was explored in two complementary colony formation assays: In one set of experiments, we tested the ability of BCL6+/+ and BCL6-/- mouse CML cells to form colonies in semisolid methylcellulose agar. The comparison between BCL6+/+ (94 ± 11 colonies) and BCL6-/- (<1 ± 1 colonies; p<0.004) mouse CML cells revealed that BCL6 represents an absolute requirement for self-renewal signaling in CML cells. In line with these results, we observed that BCL6-/- mouse CML cells undergo replicative senescence and cell cycle arrest under cell culture conditions after ∼2 months, whereas leukemic growth proceeds undiminished in BCL6+/+ CML cells. These results were confirmed studying BCL6 function in human CML cells using RI-BPI. Despite lack of toxicity, 5 mmol/l RI-BPI drastically reduced the number of colonies that were formed by human CML cells (980 ± 79 vs 262 ± 174; p= 0.001). Based on these findings we conclude that inhibition of BCL6 using a novel peptide inhibitor effectively disrupts self-renewal signaling and may be developed as a novel therapeutic approach for leukemia stem cell eradication in CML. Disclosures:No relevant conflicts of interest to declare.

BCL6 suppression of BCL2 via Miz1 and its disruption in diffuse large B cell lymphoma

The BCL6 proto-oncogene encodes a transcriptional repressor that is required for germinal center (GC) formation and whose deregulation by genomic lesions is implicated in the pathogenesis of GC-derived diffuse large B cell lymphoma (DLBCL) and, less frequently, follicular lymphoma (FL). The biological function of BCL6 is only partially understood because no more than a few genes have been functionally characterized as direct targets of BCL6 transrepression activity. Here we report that the anti-apoptotic proto-oncogene BCL2 is a direct target of BCL6 in GC B cells. BCL6 binds to the BCL2 promoter region by interacting with the transcriptional activator Miz1 and suppresses Miz1-induced activation of BCL2 expression. BCL6-mediated suppression of BCL2 is lost in FL and DLBCL, where the 2 proteins are pathologically coexpressed, because of BCL2 chromosomal translocations and other mechanisms, including Miz1 deregulation and somatic mutations in the BCL2 promoter region. These results identify an important function for BCL6 in facilitating apoptosis of GC B cells via suppression of BCL2, and suggest that blocking this pathway is critical for lymphomagenesis.

BCL6 cooperates with CD40 stimulation and loss of p53 function to rapidly transform primary B cells

The BCL6 transcriptional repressor protein has been shown to promote B-cell lymphoma in transgenic mouse models. The mechanism by which BCL6 transforms primary B cells is unclear, although repression of the p53 tumor suppressor is thought to play a role. Here, we showed that BCL6 has critical oncogene functions that are independent of p53 repression. We found that BCL6 cooperates with constitutive CD40 signaling to rapidly transform p53-deficient primary mouse B cells in vitro. Constitutive CD40 signaling alone does not transform p53-deficient B cells, indicating that BCL6 acts specifically as an immortalizing oncogene in this system. The BCL6 transformed B cells are polyclonal and form polyclonal tumors. At the initiation of the cultures, BCL6 does not significantly alter cell cycle progression, but it does promote increased cell survival. Early cultures of BCL6-expressing B cells exhibited marked repression of ATR and p27kip1 but not other BCL6 target genes, suggesting that the ATR and p27kip1 genes have key early roles in mediating BCL6 transformation function. BCL6-transformed cell lines exhibited further decreases of ATR and p27kip1 expression plus strong decreases in Blimp1 and PDCD2 expression. Our study provides important clues about the critical target genes used by BCL6 to transform primary B cells and indicates that the CD40 signaling pathway can collaborate with BCL6 in the transformation of primary B cells. Thus, our study demonstrates a rapid in vitro system to analyze the transformation function of BCL6.

S2032 Pancreatic Tumor Cells Exhibit An Aberrantly Organized Golgi Apparatus That Is Regulated By SRC Kinase and the Large GTPase Dynamin 2

BCL6 and nuclear translocation of HB-EGF-CTF in gastric cancers. Method: We examined BCL6 and cyclinD2 expression level under the 12-0-tetradecanoylphorbor-13-acetate (TPA) stimulation. KB-R7785 was used to suppress HB-EGF-CTF nuclear translocation. We also investigated the interaction and translocation of BCL6 with HB-EGF-CTF by using immunofluorescence microscopy and immunoprecipitation. Furthermore, we immunohistochemically studied 100 surgical specimens of advanced gastric cancers to analyze the expression of HB-EGF, BCL6, and cyclin D2. Results: TPA treatment resulted in BCL6 degradation and cyclin D2 up-regulation. This phenomenon was inhibited by the suppression of the nuclear translocation of HB-EGF-CTF. The HB-EGF-CTF nuclear translocation lead to the interaction of BCL6 with HB-EGF-CTF and the nuclear export of BCL6, and the ubiquitin/proteasome pathway mediated the BCL6 degradation. siRNA targeting BCL6 revealed that the upregulation of cyclin D2 depends on BCL6 degradation. BCL6 interacts with nuclear translocated HB-EGF-CTF and the nuclear export and degradation of BCL6 (caused by this interaction and the ubiquitin/proteasome pathway) induces cyclin D2 up-regulation. In human gastric cancer tissues, BCL6 expression is associated with differentiated gastric cancers. The inverse correlation between BCL6 and cyclin D2 was also found in HB-EGF-positive human gastric cancers. BCL6 degradation caused by the HB-EGF-CTF also induced cyclin D2 expression In Vivo (human cancer tissues). Conclusion: Inhibition of HB-EGF-CTF nuclear translocation and the resulting maintenance of BCL6 function are important for the regulation of gastric cancer progression.

S2034 CEA Increases Metastatic Potential of CRC Cells By Targeting Multiple Signaling Pathways

BCL6 and nuclear translocation of HB-EGF-CTF in gastric cancers. Method: We examined BCL6 and cyclinD2 expression level under the 12-0-tetradecanoylphorbor-13-acetate (TPA) stimulation. KB-R7785 was used to suppress HB-EGF-CTF nuclear translocation. We also investigated the interaction and translocation of BCL6 with HB-EGF-CTF by using immunofluorescence microscopy and immunoprecipitation. Furthermore, we immunohistochemically studied 100 surgical specimens of advanced gastric cancers to analyze the expression of HB-EGF, BCL6, and cyclin D2. Results: TPA treatment resulted in BCL6 degradation and cyclin D2 up-regulation. This phenomenon was inhibited by the suppression of the nuclear translocation of HB-EGF-CTF. The HB-EGF-CTF nuclear translocation lead to the interaction of BCL6 with HB-EGF-CTF and the nuclear export of BCL6, and the ubiquitin/proteasome pathway mediated the BCL6 degradation. siRNA targeting BCL6 revealed that the upregulation of cyclin D2 depends on BCL6 degradation. BCL6 interacts with nuclear translocated HB-EGF-CTF and the nuclear export and degradation of BCL6 (caused by this interaction and the ubiquitin/proteasome pathway) induces cyclin D2 up-regulation. In human gastric cancer tissues, BCL6 expression is associated with differentiated gastric cancers. The inverse correlation between BCL6 and cyclin D2 was also found in HB-EGF-positive human gastric cancers. BCL6 degradation caused by the HB-EGF-CTF also induced cyclin D2 expression In Vivo (human cancer tissues). Conclusion: Inhibition of HB-EGF-CTF nuclear translocation and the resulting maintenance of BCL6 function are important for the regulation of gastric cancer progression.

BCL6 Is Up-regulated in Th17 cells and Is Required to Repress GATA3 Activity During Th17 Differentiation (47.27)

Abstract One important goal for immunology is a complete understanding of the regulatory mechanisms controlling the differentiation of CD4 T helper cells. The BCL6 gene, originally identified as an oncogene for B cell lymphoma, encodes a transcriptional repressor protein. We have shown previously that BCL6 is a potent inhibitor of Th2 cell differentiation, and BCL6-deficient mice develop greatly exaggerated Th2 responses and Th2-type inflammation. We have recently found that BCL6-deficient T cells are severely impaired in their ability to undergo Th17 differentiation, indicating that BCL6 function is required for normal Th17 differentiation. The cytokine IL-6 can promote Th17 differentiation, but the Th2 cytokine IL-4 strongly blocks Th17 differentiation by promoting Th2 differentiation. We have found that BCL6 is necessary to repress GATA3-mediated Th2 differentiation induced by IL-6 during Th17 differentiation. Further, we have found that BCL6 is significantly up-regulated in T cells stimulated under Th17 conditions, indicating a unique requirement for BCL6 in Th17 differentiation. These data show a novel, critical role for BCL6 in promoting Th17 responses.

BCL6 degradation caused by the interaction with the C-terminus of pro-HB-EGF induces cyclin D2 expression in gastric cancers

BCL6 is a transcriptional repressor that has important functions in lymphocyte differentiation and lymphomagenesis, but there have been no reports of BCL6 expression in gastric cancers. In the present study, we investigated the BCL6 function in gastric cancers. Treatment with TPA resulted in BCL6 degradation and cyclin D2 upregulation. This phenomenon was inhibited by the suppression of the nuclear translocation of HB-EGF-CTF (C-terminal fragment of pro-HB-EGF). The HB-EGF-CTF nuclear translocation leads to the interaction of BCL6 with HB-EGF-CTF and the nuclear export of BCL6, and after that BCL6 degradation was mediated by ubiquitin/proteasome pathway. Real-time RT–PCR and siRNA targeting BCL6 revealed that BCL6 suppresses cyclin D2 expression. Our data indicate that BCL6 interacts with nuclear-translocated HB-EGF-CTF and that the nuclear export and degradation of BCL6 induces cyclin D2 upregulation. We performed immunohistochemical analyses of BCL6, HB-EGF and cyclin D2 in human gastric cancers. The inverse correlation between BCL6 and cyclin D2 was also found in HB-EGF-positive human gastric cancers. BCL6 degradation caused by the HB-EGF-CTF also might induce cyclin D2 expression in human gastric cancers. Inhibition of HB-EGF-CTF nuclear translocation and maintenance of BCL6 function are important for the regulation of gastric cancer progression.

Integrated Biochemical and Computational Approach Identifies BCL6 Direct Target Genes Controlling Multiple Pathways in Normal Germinal-Center B Cells

BCL6 is a transcriptional repressor required by mature B cells for germinal center (GC) formation, whose deregulated expression, through mutations and translocations, is implicated in lymphomagenesis. The normal function of BCL6 is only partially known since a limited number of direct target genes have been identified by the analysis of cell lines derived from GC-derived lymphomas. However, the complete set of targets functionally relevant for the role of BCL6 in normal GC B cell physiology has not been completely uncovered and the possibility that BCL6 function may be altered in transformed cells has not been excluded. To address these issues, we used an integrated biochemical/computational/functional approach including:identification of BCL6-bound promoters by genome-wide chromatin immunoprecipitation (ChIP-chip) in normal human GC B cells;a computational algorythm inferring transcriptional relationships starting from gene expression data (ARACNe, Basso et al. Nature Genetics 2005);validation of physiologic relevance of the candidate target genes by selection of those either not expressed in normal GC B cells or downregulated compared to pre- and post- GC B cells.This approach identified a set of 1,207 genes which were then subjected to pathway analysis using several databases. The results showed that BCL6 regulates important signaling pathways via direct transcriptional repression of multiple genes acting at different levels from the cell surface (receptors), through signaling molecules to the nucleus. These pathways include:apoptosis, by impairing the expression of both pro- and anti-apoptotic molecules including several TNF-type receptors, signaling molecules (e.g. TRADD, A20, XIAP, TOSO) and effectors (e.g. CASP8);JAK-STAT signaling, through the repression of multiple interleukin and interferon receptors and the transcription factors STAT1, STAT3 and STAT5A;B cell receptor signaling, via repression of signaling molecules (e.g. lyn, vav), several MAP kinases, and transcription factors (e.g. JUN and NF-kB components);Toll-like receptor signaling, by regulating receptors (e.g. TLR1, 7 and 9);DNA damage sensing and response (e.g. TP53BP1, ATM).Overall, these results provide a comprehensive picture of BCL6 function suggesting that one role of BCL6 is to prevent GC centroblasts from receiving activation and differentiation signals before completion of the phase of proliferative expansion and somatic hypermutation leading to their selection based on antibody affinity maturation.

BCL6-Mediated Survival Signaling Promotes Drug-Resistance in BCRABL1- Driven Acute Lymphoblastic Leukemia

During B cell development, the transcriptional repressor BCL6 is almost exclusively expressed in germinal center B cells and promotes germinal center B cell survival by suppression of Arf and p53 signaling. Compared to germinal center B cells, BCL6 is expressed at extremely low levels in pro- and pre-B cells. Also in acute lymphoblastic leukemia (ALL) derived from BCR-ABL1-transformed pre-B cells, BCL6 expression is hardly detectable at both the mRNA and protein levels. Upon BCR-ABL1 kinase inhibition by Imatinib-treatment, however, BCL6 expression levels increased by 30 to 70- fold at the mRNA and protein levels, respectively. To test whether upregulation of BCL6 in response to Imatinib-treatment has a function we transformed bone marrow pre-B cells from Bcl6−/− mice and wildtype littermates with a retrovirus encoding BCR-ABL1. After two weeks, both Bcl6−/− and wildtype bone marrow gave rise to BCR-ABL1-driven ALL. Compared to BCR-ABL1-induced ALL from wildtype bone marrow, Bcl6−/− ALL cells grow at a significantly lower proliferation rate, which suggests that even low levels of BCL6 expression in wildtype leukemia cells has some biological significance. We next treated Bcl6−/− and wildtype BCR-ABL1 ALL cells with low dose (1 μmol/l) Imatinib. About 50% of wildtype BCR-ABL1 ALL cells were still viable after four days in culture. In contrast, induction of apoptosis in Bcl6−/−BCR-ABL1 ALL cells was greatly accelerated and no viable cells could be detected after four days of Imatinib-treatment. In addition, Bcl6-deficiency significantly delayed leukemia cell engraftment in a transplantation experiment: Upon tail vein injection of 3 × 106 firefly luciferase-labeled Bcl6−/− or wildtype BCR-ABL1 ALL cells in each ten sublethally irradiated NOD/SCID mice, engraftment of Bcl6−/−BCR-ABL1 ALL cells was delayed by approximately one week as compared to wildtype ALL cells by luciferase bioimaging. Whereas most recipient mice that were injected with wildtype BCR-ABL1 ALL cells died within three weeks despite treatment with 100 mg/kg/day Imatinib i.p., all mice injected with Bcl6−/−BCR-ABL1 ALL cells are currently still alive. We conclude that BCL6 decreases sensitivity of BCR-ABL1 ALL cells to BCR-ABL1 kinase inhibition by Imatinib both in vitro and in vivo. We next investigated whether a peptidomimetic inhibitor of BCL6 (RI-BPI, Cerchietti et al., 2008) was active to sensitize human BCR-ABL1 ALL cells to Imatinib-treatment. At 20 μmol/l but not 5 μmol/l, the RI-BPI induced apoptosis in untreated human BCR-ABL1 ALL cells, so we decided to study 5 μmol/l RI-BPI in combination with Imatinib: Whereas about 30% of human BCR-ABL1 ALL cells survived treatment with 10 μmol/l Imatinib for four days, viability of human BCR-ABL1 ALL cells was decreased to less than 5% if Imatinib was combined with 5 μmol/l RI-BPI. To mechanistically address the function of BCL6 in Imatinib-treated BCR-ABL1 ALL cells, we investigated whether the cytotoxic effect of the RI-BPI requires Arf and p53. Previous work had shown that BCL6 negatively regulates Arf and p53-dependent apoptosis in germinal center B cells. To this end, we transduced bone marrow from Arf−/−, Tp53−/− and wildtype mice with BCR-ABL1. After establishment of growth factor-independent ALL, leukemia cells were treated with Imatinib and with or without RI-BPI at various concentrations. Arf−/−, Tp53−/− and wildtype BCR-ABL1 ALL cells showed a similar degree of sensitivity to treatment with Imatinib alone. A combination of Imatinib and R-BPI at 20 μmol/l was toxic for all BCR-ABL1 ALL cells, regardless of genotype. In contrast, Imatinib in combination with RI-BPI at 5 μmol/l induced apoptosis selectively in wildtype but not in Arf−/− and Tp53−/− BCR-ABL1 ALL cells. We conclude that BCL6 mediates survival signaling in Imatinib-treated BCR-ABL1 ALL by suppression of Arf/p53 signaling. Inhibition of BCL6 using a peptidomimetic inhibitor represents a novel treatment concept to prevent drug-resistance in BCR-ABL1-driven ALL.

Direct Transcriptional Repression of BCL2 by BCL6 in Germinal Centre B Cells and Its Disruption in B Cell Lymphomas with BCL2 Locus Alterations

The human proto-oncogene BCL6 encodes a BTB/POZ-zinc finger transcriptional repressor that is required for germinal centre (GC) development and is expressed in the majority of normal GC B cells and in the majority of B cell lymphoma (B-NHL), including follicular lymphoma (FL) and a subset of diffuse large B cell lymphomas (DLBCLs). Deregulation of BCL6, by chromosomal translocation or somatic hypermutation, is implicated in the pathogenesis of B-NHL. The precise function of BCL6 in GC development and lymphomagenesis is still unclear also due to the very few direct BCL6 target genes that have been identified. In order to identify physiologically relevant BCL6 direct target genes, we used a novel approach combining high-throughput biochemical (ChIP-on-chip), bioinformatics (ARACNe) (Basso et al., Nature Genetics, 2005) and gene expression profile analysis (Klein et al., Proc. Natl. Acad. Sci., 2004) of normal GC B cells. The results (see abstract by Saito et al.) have identified a core of bona fide target genes whose promoter is bound by BCL6, whose transcription is dynamically linked to BCL6, and whose expression is down-regulated in GC B cells. Among the genes meeting these stringent criteria, we found BCL2, encoding the anti-apoptotic molecule whose expression is deregulated by chromosomal translocations or gene amplification in the majority of FL and in a subset of DLBCL. Further investigations showed that BCL6 represses BCL2 transcription by binding specific DNA sites within the BCL2 promoter region. Suppression of BCL6 expression via specific siRNAs leads to increased levels of BCL2, and, conversely, constitutive BCL6 expression prevents B cell receptor (BCR)-induced upregulation of BCL2 in B cells. Consistent with a physiological role for BCL6- mediated BCL2 suppression, immunohistochemical analysis shows that BCL2 expression is absent in GC B cells where BCL6 is highly expressed. Notably, the comparative analysis of BCL6 and BCL2 expression in B-NHL cell lines showed a conserved inverse correlation between BCL6 and BCL2 levels in cell lines carrying a normal BCL2 locus. However, cell lines carrying chromosomal translocations or amplifications affecting the BCL2 gene displayed the pathologic co-expression of both proteins, suggesting that the alterations affecting the BCL2 locus prevent BCL6-mediated suppression. These results indicate that one critical role of BCL6 is the modulation of antiapoptotic function in GC B cells via BCL2 transcriptional repression, suggesting a mechanism by which B cells may die in the GC if not rescued by BCR and CD40 engagement, both of which downregulate BCL6 while inducing BCL2 expression. Alterations of the BCL2 locus may contribute to lymphomagenesis by making the gene resistant to suppression by BCL6, and therefore allowing B cells to avoid apoptosis in the absence of antigen- and T-cell-mediated rescue.

Structure of a BCOR Corepressor Peptide in Complex with the BCL6 BTB Domain Dimer

The transcriptional corepressors BCOR, SMRT, and NCoR are known to bind competitively to the BCL6 BTB domain despite the fact that BCOR has no detectable sequence similarity to the other two corepressors. We have identified a 17 residue motif from BCOR that binds directly to the BCL6 BTB domain and determined the crystal structure of the complex to a resolution of 2.6 A. Remarkably, the BCOR BCL6 binding domain (BCOR(BBD)) peptide binds in the same BCL6 binding site as the SMRT(BBD) peptide despite the lack of any significant sequence similarity between the two peptides. Mutations of critical BCOR(BBD) residues cause the disruption of the BCL6 corepression activities of BCOR, and a BCOR(BBD) peptide blocks BCL6-mediated transcriptional repression and kills lymphoma cells.

The Balance between Myc and Bcl6 Determines Self-Renewal or Differentiation of Pre-B Cells.

Myc and Bcl6 represent classical proto-oncogenes in B-cell malignancies, mainly through translocation into the immunoglobulin (Ig) heavy chain locus in Burkitt's (MYC) and diffuse large B cell lymphoma (BCL6). While BCL6 was previously established as a factor regulating differentiation of germinal center B cells, the function of MYC and BCL6 in early B-cell development was not previously studied. Investigating requirements for the differentiation of pre-B cells into immature B-cells, we found that both withdrawal of IL7 from murine pre-B-cell cultures and inhibition of BCR-ABL1 in BCR-ABL1-transformed pre-B-cells terminates self-renewal and initiates differentiation into Ig light chain-expressing immature B-cells. Interestingly, IL7 and BCR-ABL1 are exchangeable at this checkpoint: Both IL7 and BCR-ABL1 promote self-renewal and prevent differentiation of pre-B-cells. While inhibition of BCR-ABL1 usually induces apoptosis and partial differentiation, both effects were entirely suppressed by IL7. These findings indicate that IL7 may confer resistance to BCR-ABL1 inhibitors in patients with BCR-ABL1-transformed acute lymphoblastic leukemia. Likewise, inhibition of either IL7 or BCR-ABL1 signaling resulted in complete silencing of Myc expression and strong de novo expression of Bcl6. Because expression of Myc and Bcl6 are mutually exclusive at the pre-B to immature B-cell checkpoint, we tested whether the two proto-oncogenes have distinct functions at this transition. Interestingly, forced expression of Myc rendered BCR-ABL1-transformed pre-B-cells resistant to induction of differentiation upon inhibition of BCR-ABL1. Besides downregulation of Myc, also de novo expression of Bcl6 is critical for the pre-B to immature B-cell differentiation: shmiR-mediated silencing of Bcl6 suppressed B-cell differentiation even if Myc was downregulated. However, forced expression of Bcl6 alone only modestly induced differentiation of pre-B cells if Myc was not downregulated. To test the interplay between Myc and Bcl6 at the pre-B to immature B cell transition more systematically, we analyzed bone marrow pre-B cells from Mycfl/fl mice. Mycfl/fl pre-B cells that also carry MxCre deleted the Myc locus on both alleles upon stimulation with IFNß. As controls, Mycfl/fl pre-B cells without MxCre were used. Pre-B cells were also transduced with a retroviral vector encoding Bcl6/GFP or GFP alone. Upon Myc deletion, more than 80 precent of the Bcl6/GFP transduced pre-B cells underwent differention as compared to 25 percent GFP-transduced pre-B cells. In the absence of Myc deletion, about 15 percent of Bcl6/GFP-transduced pre-B cells initiated differentiation as compared to 5 percent of GFP-transduced pre-B cells. These findings establish that Myc and Bcl6 have critical and antagonistic functions in early B cell development and that both downregulation of Myc together with upregulation Bcl6 are required to initiate differentiation of pre-B cells. The MYC/BCL6 balance may also be a target of leukemic transformation of human pre-B cells: The ratio of MYC/BCL6 mRNA levels in normal human pro- and pre-B cells at 0.52 is dramatically increased in various subtypes of acute lymphoblastic leukemia (6.4 for BCR-ABL1-, 2.6 for E2A-PBX1-, 14.4 for MLL-AF4- and 3.3 for TEL-AML1-transformed acute lymphoblastic leukemia).