Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis
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ABSTRACT: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphomas worldwide and is characterized by a high diversity of genetic and molecular alterations. Chromosomal translocations and mutations leading to deregulated expression of the transcriptional repressor BCL6 occur in a significant fraction of DLBCL patients. An oncogenic role of BCL6 in the initiation of DLBCL has been shown as the constitutive expression of BCL6 in mice recapitulates the pathogenesis of human DLBCL. However, the role of BCL6 in tumor maintenance remains poorly investigated due to the absence of suitable genetic models and limitations of pharmacological inhibitors. Here, we have utilized tetracycline-inducible CRISPR/Cas9 mutagenesis to study the consequences of BCL6 deletion in established DLBCL models in culture and in vivo. We show that BCL6 knock-out in SU-DHL-4 cells in vitro results in an anti-proliferative response 4-7 days after Cas9 induction that was characterized by cell cycle (G1) arrest. Conditional BCL6 deletion in established DLBCL tumors in vivo induced a significant tumor growth inhibition with initial tumor stasis followed by slow tumor growth kinetics. Our findings support a role of BCL6 in the maintenance of lymphoma growth and showcase the utility of inducible CRISPR/Cas9 systems for probing oncogene addiction.
Project description:We studied transcriptional changes by Affymetrix human microarrays in DLBCL cell lines as a result of treatment with GSK126, a potent, highly-selective, SAM-competitive, small molecule inhibitor of EZH2 In eukaryotes, epigenetic post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and is responsible for repressing target gene expression through methylation of histone H3 on lysine 27 (H3K27). Over-expression of EZH2 is implicated in tumorigenesis and correlates with poor prognosis in multiple tumor types. Recent reports have identified somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). The Y641 residue is the most frequently mutated residue, with 22% of GCB (Germinal Cell B-cell) DLBCL and FL harboring mutations at this site. These lymphomas exhibit increased H3K27 tri-methylation (H3K27me3) due to altered substrate preferences of the mutant enzymes. However, it is unknown whether direct inhibition of EZH2 methyltransferase activity alone will be effective in treating lymphomas carrying activating EZH2 mutations. Herein, we demonstrate that GSK126, a potent, highly-selective, SAM-competitive, small molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and dramatically inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma. 10 DLBCL cell lines (7 mutant and 3 wild type EZH2), that were differentially sensitive to GSK126 in proliferation assays, were treated for 72 hours, in duplicate (n=2), with either DMSO (vehicle) or 500nM of GSK126, a potent selective EZH2 inhibitor. EZH2 mutant cell lines are Pfeiffer, KARPAS-422, WSU-DLCL2, SU-DHL-10, SU-DHL-6, DB and SU-DHL-4. EZH2 wildtype cell lines are HT, OCI-LY-19 and Toledo.
Project description:MYC translocations are the biologic hallmark of Burkitt lymphomas but also occur in other mature B-cell lymphomas. If accompanied by chromosomal breaks targeting the BCL2 and/or BCL6 oncogenes, these MYC translocation-positive (MYC+) lymphomas are called double-hit lymphomas (DHLs); otherwise, the term single-hit lymphoma (SHL) is applied. In order to characterize the biologic features of these MYC+ lymphomas other than Burkitt lymphomas, we explored, after exclusion of molecular Burkitt lymphoma (mBL) as defined by gene expression profiling (GEP), the molecular, pathological and clinical aspects of 80 MYC translocation (MYC+) lymphomas (31 SHL, 26 BCL2+/MYC+, 14 BCL6+/MYC+, 6 BCL2+/BCL6+/MYC+ and 3 MYC+ lymphomas with unknown BCL6 status). Comparison of SHL and DHL revealed no difference in frequency of MYC partner (IG/non-IG), genomic complexity or MYC expression and no differences in GEP. DHL showed a more frequent GCB-like GEP and higher IGH and MYC mutation rates. GEP revealed 130 differentially expressed genes between BCL6+/MYC+ and BCL2+/MYC+ DHL. BCL2+/MYC+ DHL showed a more frequent GCB-like GEP. Analysis of all lymphomas according to MYC partner (IG/non-IG) revealed no substantial differences. In contrast to mBL and lymphomas without MYC break, SHL and DHL patients had similar poor outcome. Our data suggest that after excluding mBL, MYC+ lymphomas could be biologically widely lumped without further need for subclassification. 32 diffuse large B-cell lymphoma samples were hybridized to HG-U133A Affymetrix GeneChips. In addition, this study contains 30 already published samples, which contribute to GSE4475 (Hummel et al. 2006 (PMID 16760442)), as well as 18 already published samples from GSE22470 (Salaverria et al. 2011 (PMID 21487109)). No re-normalisation of the published samples was performed. The complete dataset representing: (1) the 32 diffuse large B-cell lymphoma Samples, (2) the 30 Samples from GSE4475 and (3) the 18 Samples from GSE22470, is linked below as a supplementary file
Project description:CC-122 is a next-generation cereblon E3 ligase modulating agent that has demonstrated promising clinical efficacy in relapsed or refractory diffuse large B‐cell lymphoma (R/R DLBCL) patients. Mechanistically, CC-122 induces the degradation of IKZF1/3, leading to T cell activation and robust cell-autonomous killing in DLBCL. Here, we report a genome-wide CRISPR/Cas9 positive selection screen for CC-122 in a DLBCL cell line SU-DHL-4 with follow-up mechanistic characterization in 6 DLBCL cell lines to identify genes regulating the response to CC-122. Top-ranked CC-122 resistance genes encode not only well-defined members or regulators of the CUL4-DDB1-RBX1-CRBN E3 ubiquitin ligase complex, but also key components of several signaling and transcriptional networks that have not previously been shown to modulate the response to other cereblon modulators. Ablation of CYLD, NFKBIA, TRAF2, or TRAF3 induces hyper-activation of the canonical and/or non-canonical NF-κB pathways and subsequently diminishes CC-122-induced apoptosis in 5 out of 6 DLBCL cell lines. Depletion of KCTD5, the substrate receptor of the CUL3-RBX1-KCTD5 ubiquitin ligase complex, promotes the stabilization of its cognate substrate, GNG5, resulting in CC-122 resistance in HT, SU-DHL-4, and WSU-DLCL2. Furthermore, knockout of AMBRA1 renders resistance to CC-122 in SU-DHL-4 and U-2932, whereas knockout of RFX7 leads to resistance specifically in SU-DHL-4. The ubiquitous and cell line-specific mechanisms of CC-122 resistance in DLBCL cell lines revealed in this work pinpoint genetic alternations that are potentially associated with clinical resistance in patients, and facilitate the development of biomarker strategies for patient stratification, which may improve clinical outcome of CC-122 for R/R DLBCL.
Project description:MYC translocations are the biologic hallmark of Burkitt lymphomas but also occur in other mature B-cell lymphomas. If accompanied by chromosomal breaks targeting the BCL2 and/or BCL6 oncogenes, these MYC translocation-positive (MYC+) lymphomas are called double-hit lymphomas (DHLs); otherwise, the term single-hit lymphoma (SHL) is applied. In order to characterize the biologic features of these MYC+ lymphomas other than Burkitt lymphomas, we explored, after exclusion of molecular Burkitt lymphoma (mBL) as defined by gene expression profiling (GEP), the molecular, pathological and clinical aspects of 80 MYC translocation (MYC+) lymphomas (31 SHL, 26 BCL2+/MYC+, 14 BCL6+/MYC+, 6 BCL2+/BCL6+/MYC+ and 3 MYC+ lymphomas with unknown BCL6 status). Comparison of SHL and DHL revealed no difference in frequency of MYC partner (IG/non-IG), genomic complexity or MYC expression and no differences in GEP. DHL showed a more frequent GCB-like GEP and higher IGH and MYC mutation rates. GEP revealed 130 differentially expressed genes between BCL6+/MYC+ and BCL2+/MYC+ DHL. BCL2+/MYC+ DHL showed a more frequent GCB-like GEP. Analysis of all lymphomas according to MYC partner (IG/non-IG) revealed no substantial differences. In contrast to mBL and lymphomas without MYC break, SHL and DHL patients had similar poor outcome. Our data suggest that after excluding mBL, MYC+ lymphomas could be biologically widely lumped without further need for subclassification.
Project description:EZH2 mediates the humoral immune response and drives lymphomagenesis through de novo formation of bivalent chromatin domains and critical germinal center (GC) B cell promoters. We show that such formation is dependent on the presense of BCL6 and the presence of non-canonical PRC1-BCOR complex. We observe that BCL6 and EZH2 cooperate to accelerate diffuse large B cell lymphoma (DLBCL) development and combinatorial targeting of these repressors results in enhanced anti-lymphoma activity in vitro, in vivo, and in primary human DLBCLs. DLBCL cell lines treated with BCL6 inhibitor 79-6.1085
Project description:BCL6 is a key oncogene in lymphoma pathogenesis. The expression of BCL6 in lymphoid cells can be deregulated by several mechanisms, including chromosomal translocations, somatic mutations in the promoter regulatory regions or reduced proteasome-mediated degradation. FBXO11 was recently identified as a major ubiquitin ligase involved in the degradation of BCL6 and is frequently inactivated in diffuse large B-cell lymphoma (DLBCL). In this work, we found that FBXO11 is frequently mutated in Burkitt lymphoma (BL) but rarely mutated in other BCL6-positive lymphomas, such as follicular lymphoma (FL). All mutations tested impaired FBXO11 mediated BCL6 degradation and FBXO11 knock-out completely stabilized BCL6 levels in human BL cell lines. Conditional deletion of one copy or both copies of FBXO11 in c-Myc-driven B cell lymphoma in mice accelerated lymphomagenesis, genetically confirming that FBXO11 is a haplo-insufficient oncosuppressor in lymphoma. In both FBOX11 WT and deficient BL mouse and human cell lines, blockade of BCL6 via a specific degrader or BCL6 inhibitors, impaired lymphoma growth in vitro and in vivo, an effect further enhanced by co-inhibition of c-Myc activity. Overall these findings not only establish FBXO11 as one of the most frequently mutated genes in BL, but also elucidate its biological functions in lymphomagenesis and thereby identify BCL6 as a specific therapeutic target in BL.
Project description:The BCL6 transcriptional repressor is a critical oncogene in diffuse large B-cell lymphomas (DLBCL). The specific BCL6 inhibitor RI-BPI potently kills DLBCL cells. We find that RI-BPI induces a particular gene expression signature in DLBCL. In order to identify classes of drugs that might synergize with RIBPI we examined the connectivity of this signature and found a strong association with HDAC and Hsp90 inhibitors. This was explained by the discovery that BCL6 directly represses the p300 lysine acetyltransferase and its co-factor BAT3. RI-BPI induced expression of p300 and BAT3, and p300 acetyltransferase activity, resulting in acetylation of p300 targets like p53 and Hsp90. As a consequence, RI-BPI could attenuate Hsp90 chaperone function, similar to the effect of Hsp90 and HDAC inhibitors. Induction of p300 and BAT3 was required for the anti-lymphoma effects of RI-BPI since specific blockade of either protein rescued DLBCL cells from the BCL6 inhibitor. RI-BPI synergistically killed DLBCL cells in combination with HDAC inhibitors (SAHA, TSA and VPA) and Hsp90 inhibitors (17-DMAG and PUH71). The combination of RI-BPI and SAHA, or RI-BPI and PU-H71 potently suppressed or even eradicated human DLBCL in mice. BCL6 repression of EP300 thus provides a basis for rational targeted combinatorial therapy for patients with DLBCL. Direct comparison of gene expression levels in DLBCL cell lines after 24hs of treatment with either a Bcl6 inhibitor peptide or control peptide
Project description:The multifunctional protein lipopolysaccharide-induced TNFalpha factor (LITAF) induces the secretion of inflammatory cytokines in monocytes and regulates protein degradation in neural cells. In B-cell lymphomas, LITAF is frequently inactivated by epigenetic mechanisms, but beyond these data little is known about its regulation and function. Immunohistochemical and gene expression profiling analyses of normal and malignant B-cells revealed that LITAF and BCL6 exhibited opposite expression patterns. Accordingly, chromatin immunoprecipitation and luciferase experiments showed that LITAF is transcriptionally repressed by BCL6 in germinal center (GC) lymphocytes and in B-cell lymphoma cells. Gain- and-loss-of-function assays demonstrated that LITAF does not exert any of its previous roles. Conversely, LITAF co-localized with autophagosomes in B-cells whereby activated autophagic responses, which were abrogated upon LITAF silencing. Therefore, BCL6-mediated transcriptional repression of LITAF may contribute to an appropriate GC reaction by suppressing autophagy in GC lymphocytes, whereas constitutive repression of autophagic responses may promote B-cell lymphoma development. Analysis of global gene expression in the diffuse large B-cell lymphoma (DLBCL) cell lines KARPAS-231 and VAL treated with a specific siRNA for LITAF or a scrambled control, and analysis of global gene expression in the DLBCL cell lines RL and SC-1 stably transfected with a tetracycline-inducible LITAF expression vector in the presence or absence of 4 mg/ml doxycycline in the culture medium. The over-expression experiments were performed in duplicate and the silencing experiments in triplicate, resulting in a total of 20 microarrays.
Project description:MicroRNAs (miRNAs) post-transcriptionally regulate gene expression by inhibiting protein synthesis of target messenger RNAs (mRNAs). MicroRNA-142 (miR-142), which has tumor-suppressive properties, was functionally deleted by CRISPR/Cas9 knockout in cell lines derived from diffuse large B-cell lymphoma (DLBCL), a highly aggressive tumor that represents about 30% of non-Hodgkin lymphoma worldwide. Mutations in miR-142 affect about 20% of all cases of DLBCL. By proteome analyses, the miR-142 knockout resulted in a consistent up-regulation of 52 but also down-regulation of 41 proteins in the GC-DLBCL lines BJAB and SUDHL4. Various mitochondrial ribosomal proteins were up-regulated in line with their pro-tumorigenic properties, while proteins necessary for MHC-I presentation were down-regulated in accordance with the finding that miR-142 knockout mice have a defective immune response. Of the deregulated proteins/genes, CFL2, CLIC4, STAU1, and TWF1 are known targets of miR-142, and we could additionally confirm AKT1S1, CCNB1, LIMA1, and TFRC as new targets of miR-142-3p or -5p. We further show that seed-sequence mutations of miR-142 can be used to confirm potential targets and that miRNA knockout cell lines might thus be used to identify novel targets of miRNAs. Due to the complex contribution of miRNAs within cellular regulatory networks, in particular when a miRNA highly present in the RISC complex is deleted and can be replaced by other endogenous miRNAs, primary effects on gene expression may be covered by secondary layers of regulation