Silencing Of BCL6 Research Articles

Immediate Adaptation Analysis Implicates BCL6 as an EGFR-TKI Combination Therapy Target in NSCLC

Drug resistance is a major obstacle to curative cancer therapies, and increased understanding of the molecular events contributing to resistance would enable better prediction of therapy response, as well as contribute to new targets for combination therapy. Here we have analyzed the early molecular response to epidermal growth factor receptor (EGFR) inhibition using RNA sequencing data covering 13,486 genes and mass spectrometry data covering 10,138 proteins. This analysis revealed a massive response to EGFR inhibition already within the first 24 h, including significant regulation of hundreds of genes known to control downstream signaling, such as transcription factors, kinases, phosphatases and ubiquitin E3-ligases. Importantly, this response included upregulation of key genes in multiple oncogenic signaling pathways that promote proliferation and survival, such as ERBB3, FGFR2, JAK3, and BCL6, indicating an early adaptive response to EGFR inhibition. Using a library of more than 500 approved and experimental compounds in a combination therapy screen, we could show that several kinase inhibitors with targets including JAK3 and FGFR2 increased the response to EGFR inhibitors. Further, we investigated the functional impact of BCL6 upregulation in response to EGFR inhibition using siRNA-based silencing of BCL6. Proteomics profiling revealed that BCL6 inhibited transcription of multiple target genes including p53, resulting in reduced apoptosis which implicates BCL6 upregulation as a new EGFR inhibitor treatment escape mechanism. Finally, we demonstrate that combined treatment targeting both EGFR and BCL6 act synergistically in killing lung cancer cells. In conclusion, or data indicates that multiple different adaptive mechanisms may act in concert to blunt the cellular impact of EGFR inhibition, and we suggest BCL6 as a potential target for EGFR inhibitor-based combination therapy.

Ubiquitin-specific protease 14 regulates ovarian cancer cisplatin-resistance by stabilizing BCL6 oncoprotein

Ubiquitin-specific protease 14 (USP14) is one of the three proteasome-associated deubiquitinating enzymes and implicated in the progression of various cancers. However, the role of USP14 in ovarian cancer remains unknown. By using an unbiased qRT-PCR screen, here we show that USP14 is considerably increased in cisplatin-resistant ovarian cancer cells. Overexpression of USP14 confers resistance to cisplatin-sensitive ovarian cancer cells. Genetic or pharmacological inhibition of USP14 is able to reverse cisplatin-resistance of ovarian cancer cells, which was accompanied by decreased protein expression of BCL6. Besides, BCL6 protein level was also increased in cisplatin-resistant ovarian cancer cells and silencing of BCL6 in these cells restored their sensitivity to cisplatin. At the molecular lever, we found that USP14 interacted with BCL6 and prevented it from proteasomal-dependent degradation. Thus, our results provide a rationale to target USP14-BCL6 axis in ovarian cancer that may be therapeutically beneficial.

Bcl6 gene-silencing facilitates PMA-induced megakaryocyte differentiation in K562 cells.

Targeted therapy via imatinib appears to be a promising approach for chronic myeloid leukemia (CML) therapy. However, refractory and resistance to imatinib therapy has encouraged many investigators to get involved in development of new therapeutic agents such as Phorbol 12-myrestrat 13-acetate (PMA) for patients with CML. In that line, we attempted to investigate the chemosensitizing effect of PMA on the imatinib-resistant cells. Based on our western blot analyses, resistant K562 cells (K562R) showed high levels of FoxO3a and Bcl6 expressions which were not modulated by imatinib treatment. However, upon PMA treatment, the levels of both FoxO3a and Bcl6 were up-regulated among both the sensitive and the resistant cells and this treatment was associated with initiation of megakaryocytic differentiation of the cells. SiRNA-silencing of FoxO3a led to augmentation of megakaryocytic differentiation of the cells. Similarly, siRNA gene silencing of Bcl6 enhanced the differentiation and induced cell apoptosis among both types of cells. Regarding these results, it might be concluded that Bcl6 knockdown combined with PMA therapy could present a new therapeutical strategy for refractory CML patients to imatinib.

BCL6 promotes glioma and serves as a therapeutic target

ZBTB transcription factors orchestrate gene transcription during tissue development. However, their roles in glioblastoma (GBM) remain unexplored. Here, through a functional screening of ZBTB genes, we identify that BCL6 is required for GBM cell viability and that BCL6 overexpression is associated with worse prognosis. In a somatic transgenic mouse model, depletion of Bcl6 inhibits the progression of KrasG12V-driven high-grade glioma. Transcriptome analysis demonstrates the involvement of BCL6 in tumor protein p53 (TP53), erythroblastic leukemia viral oncogene homolog (ErbB), and MAPK signaling pathways. Indeed, BCL6 represses the expression of wild-type p53 and its target genes in GBM cells. Knockdown of BCL6 augments the activation of TP53 pathway in response to radiation. Importantly, we discover that receptor tyrosine kinase AXL is a transcriptional target of BCL6 in GBM and mediates partially the regulatory effects of BCL6 on both MEK-ERK (mitogen-activated protein/extracellular signal-regulated kinase kinase-extracellular signal-regulated kinase) and S6K-RPS6 (ribosomal protein S6 kinase-ribosomal protein S6) axes. Similar to BCL6 silencing, depletion of AXL profoundly attenuates GBM proliferation both in vitro and in vivo. Moreover, targeted inhibition of BCL6/nuclear receptor corepressor 1 (NCoR) complex by peptidomimetic inhibitor not only significantly decreases AXL expression and the activity of MEK-ERK and S6K-RPS6 cascades but also displays a potent antiproliferative effect against GBM cells. Together, these findings uncover a glioma-promoting role of BCL6 and provide the rationale of targeting BCL6 as a potential therapeutic approach.

Abstract 1271: The transcription factor BCL6 is a rational target in non-small cell lung cancer (NSCLC)

Abstract BCL6 is a transcription factor that promotes lymphomagenesis by repressing target genes involved in DNA damage sensing and response. In NSCLC patients, BCL6 is amplified in 40% of Squamous Cell Carcinomas (197/501, TCGA) and in 2.2% of Adenocarcinomas (5/230, TCGA), suggesting a potential oncogenic role for BCL6 in lung cancer. To determine whether BCL6 plays a role in sustaining NSCLC, we first measured the expression of BCL6 in a panel of 15 NSCLC cell lines and found BCL6 expression in 15/15 cell lines. Furthermore, in 7/15 cells lines (46%) we found BCL6 gene amplification. To gain insight into BCL6 function, we engineered two NSCLC cell lines with BCL6 amplification (H1299 and H838) to stably express BCL6-shRNA. BCL6 silencing resulted in 3-5 fold mRNA increase of key DNA damage response genes such as ATR, Chek1, p21 and p53. BCL6-mediated repression of DNA damage response genes has functional effects as BCL6 silencing resulted into i) G1 cell-cycle arrest as determine by flow cytometry analysis, ii) 1.5-2 fold increase in cell doubling time, iii) 40-60% reduction in colony formation. These results suggest that a major role of BCL6 in lung cancer could be to enable proliferation under genotoxic stress, a function that BCL6 exerts physiologically in B-cells during antibody generation. Under these premises, BCL6 inhibition should preferentially affect the proliferation of cells with elevated genomic instability. Thus, we measured the amount of chromosomal aberration harbored by the proliferating fractions of BCL6-shRNA cells compared to the isogenic parental cells using micronuclei assay. BCL6 silencing resulted in 30-40% reduction of micronuclei amount in the proliferating fraction of cells, supporting our hypothesis. Moreover, exposing NSCLC cells to DNA damaging agents leads to 2-3 fold increase in BCL6 expression regardless the presence of BCL6 amplification. In lymphomas, BCL6 represses genes by recruiting co-repressors to form “repressosome” complexes in gene promoters and enhancers. In particular, repression of DNA damage response genes is achieved by the recruitment of three co-repressors (SMRT, N-CoR, and BCOR) to the BTB-domain of BCL6. To determine whether the BTB-domain of BCL6 is mediating the repression of DNA damage genes in NSCLC, we employed FX1085, a small molecule that specifically interacts with the BCL6 BTB domain and prevents the formation of the repressosome complex. Exposure to FX1085 resulted in 3-10 fold increase in ATR, Chek1, p21 and p53 mRNA levels and prevented the proliferation of NSCLC cells with elevated genomic instability. Moreover, FX1085 affected the survival of 6/15 NSCLC cell lines (40%) at GI50 doses comparable to those effective in lymphoma cells, indicating that BCL6 maybe a suitable therapeutic target in NSCLC. Overall, our data indicate a role for BCL6 in sustaining NSCLC genomic instability and suggest that this protein may be a potential therapeutic target in this disease. Citation Format: Rossella Marullo, Haelee Ahn, Mariano Cardenas, Ari Melnick, Fengtian Xue, Leandro Cerchietti. The transcription factor BCL6 is a rational target in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1271.

LITAF, a BCL6 target gene, regulates autophagy in mature B‐cell lymphomas

We have previously reported that LITAF is silenced by promoter hypermethylation in germinal centre-derived B-cell lymphomas, but beyond these data the regulation and function of lipopolysaccharide-induced tumour necrosis factor (TNF) factor (LITAF) in B cells are unknown. Gene expression and immunohistochemical studies revealed that LITAF and BCL6 show opposite expression in tonsil B-cell subpopulations and B-cell lymphomas, suggesting that BCL6 may regulate LITAF expression. Accordingly, BCL6 silencing increased LITAF expression, and chromatin immunoprecipitation and luciferase reporter assays demonstrated a direct transcriptional repression of LITAF by BCL6. Gain- and loss-of-function experiments in different B-cell lymphoma cell lines revealed that, in contrast to its function in monocytes, LITAF does not induce lipopolysaccharide-mediated TNF secretion in B cells. However, gene expression microarrays defined a LITAF-related transcriptional signature containing genes regulating autophagy, including MAP1LC3B (LC3B). In addition, immunofluorescence analysis co-localized LITAF with autophagosomes, further suggesting a possible role in autophagy modulation. Accordingly, ectopic LITAF expression in B-cell lymphoma cells enhanced autophagy responses to starvation, which were impaired upon LITAF silencing. Our results indicate that the BCL6-mediated transcriptional repression of LITAF may inhibit autophagy in B cells during the germinal centre reaction, and suggest that the constitutive repression of autophagy responses in BCL6-driven lymphomas may contribute to lymphomagenesis.

MicroRNA-155 promotes atherosclerosis by repressing Bcl6 in macrophages

Macrophages in atherosclerotic plaques drive inflammatory responses, degrade lipoproteins, and phagocytose dead cells. MicroRNAs (miRs) control the differentiation and activity of macrophages by regulating the signaling of key transcription factors. However, the functional role of macrophage-related miRs in the immune response during atherogenesis is unknown. Here, we report that miR-155 is specifically expressed in atherosclerotic plaques and proinflammatory macrophages, where it was induced by treatment with mildly oxidized LDL (moxLDL) and IFN-γ. Leukocyte-specific Mir155 deficiency reduced plaque size and number of lesional macrophages after partial carotid ligation in atherosclerotic (Apoe-/-) mice. In macrophages stimulated with moxLDL/IFN-γ in vitro, and in lesional macrophages, loss of Mir155 reduced the expression of the chemokine CCL2, which promotes the recruitment of monocytes to atherosclerotic plaques. Additionally, we found that miR-155 directly repressed expression of BCL6, a transcription factor that attenuates proinflammatory NF-κB signaling. Silencing of Bcl6 in mice harboring Mir155-/- macrophages enhanced plaque formation and CCL2 expression. Taken together, these data demonstrated that miR-155 plays a key role in atherogenic programming of macrophages to sustain and enhance vascular inflammation.

LITAF, a BCL6 Target Gene, Regulates Autophagia in B Cells and Is Essential for T-Cell Dependent Humoral Responses

Abstract 1391LITAF was discovered as a p53-induced transcript that promoted TNFa secretion in monocytes in response to LPS. We previously reported that LITAF is inactivated by deletion or promoter hypermethylation in germinal center-derived B-cell lymphomas. However, the function of LITAF in B lymphocytes is unknown. Using gene expression analysis of isolated B-cell subpopulation and immunohistochemical studies of tonsil lymphoid follicles we found that LITAF is expressed in naïve B lymphocytes and is repressed within the germinal centers (GCs). Thus, LITAF showed an opposite expression to BCL6, an essential regulator of GC development and function. Likewise, expression of LITAF and BCL6 were inversely correlated in cell lines and biopsies from patients with B-cell lymphoma, further suggesting a link between LITAF and BCL6. ChIP-on-chip and ChIP-sequencing analyses of B cells coupled with luciferase reporter assays revealed that BCL6 repressed LITAF expression by binding to its promoter. Accordingly, BCL6 silencing with siRNAs or after exposure to a BCL6-inhibitor peptide increased LITAF expression, indicating that LITAF is transcriptionally repressed by BCL6 in GC B lymphocytes and in B-cell lymphoma cells. To initially elucidate the function of LITAF in B cells, gain-and-loss of function experiments were performed in different cellular models. LITAF expression was not related to TNFa secretion after LPS exposure, nor modulated cell proliferation or apoptosis in B cells. However, sustained expression of LITAF in B-cell lymphoma cells increased cell size, lysosome content and mitochondrial mass. Gene expression microarray studies defined a LITAF-related transcriptional signature containing genes involved in the regulation of endomembranes, vesicle trafficking and protein transport. Accordingly, immunofluorescence analysis co-localized LITAF with lysosomes and with autophagosomes expressing LC3, the mammalian homolog of yeast autophagy-related protein (Atg8), as well as with the lysosomal sorting-associated proteins NEDD4 and TSG101, both in normal CD19+ B lymphocytes and in B-cell lymphoma cells. In addition, LITAF expression induced autophagic activity in B cells, shown by an increase in the FL1/FL3 ratio after acridine orange staining and by converting LC3-I to LC3-II, which were more evident upon cell starvation. Together, these data suggest that LITAF may play a role in the processing of proteins in autophagosomes through regulating autophagy. To investigate LITAF function in vivo, we generated mice with targeted deletion of the Litaf gene in B lymphocytes by using the Cre-loxP system. Litaf -mb1-Cre (Litaf−/− ) mice developed healthy and showed normal distribution of hematopoietic cell subpopulations. However, Litaf−/− mice were unable to develop full T-cell dependent immune responses, presenting PNA-stained, Litaf-negative GCs that were absent or had marked reduction in size and number. Accordingly, reduced amounts of IgM, IgG1 and IgG3 antibodies as a consequence of abnormal class switch recombination (CSR) were detected in immunized mice. However, in experiments testing CSR in vitro, in which B cells are artificially activated in the absence of T cells, the amounts of IgM/IgG1/IgG3 did not differ between knock-out and control groups. Similarly, mouse immunization with a T-cell independent antigen did not induce differences in immunoglobulin production. Further studies of GCs in T-cell immunized Litaf−/− mice using an antibody for the Class II-associated invariant chain peptide (CLIP) revealed that the atrophic GCs in Litaf−/− mice showed strong CLIP expression in comparison to wild-type littermates. In normal immune responses, CLIP peptides bind to MHC class II molecules in endolysosomes, until they are displaced by the antigen, then releasing CLIP and allowing MHC II-antigen complexes to be transported to the cell membrane for T-cell presentation. The failure to develop appropriate immune responses together with the accumulation of CLIP peptides in Litaf -deficient mice indicate that Litaf is essential for adequate T-cell dependent immune responses in GC B lymphocytes, possibly through facilitating the presentation of the antigens to MHC II molecules in the endolysosomes. Once this process is assembled and the T-cell activated B lymphocytes enter the GCs, BCL6 represses LITAF to prevent additional interactions between B and T cells during BCR editing. Disclosures:No relevant conflicts of interest to declare.

BCL6 represses NFκB activity in diffuse large B‐cell lymphomas

BCL6 is a transcriptional repressor whose deregulated expression plays a key role in diffuse large B-cell lymphomas (DLBCLs). BCL6 expression characterizes one of the two main subtypes (GC type) of DLBCL, while the other (ABC type) is recognized by increased NFkappaB activation. The mechanistic basis of this distinction remains unclear and the BCL6 targets have been only partially explored. Here we describe how NFkappaB activity is increased after BCL6 silencing by shRNA in DLBCL cells, leading us to propose that BCL6 represses NFkappaB activity. We also demonstrate that this repression is brought about by a mechanism involving protein-protein interaction between BCL6 and NFkappaB members, both in vitro and in vivo. Analysis of a series of DLBCLs shows a negative correlation between the expression of NFkappaB target genes and BCL6. This combined approach using silenced cells and a series of human DLBCL samples leads us to a better understanding of the role of BCL6 as an NFkappaB regulator in B-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).