BCL6 BTB Research Articles

A platform to induce and mature biomolecular condensates using chemicals and light.

Biomolecular condensates are membraneless compartments that impart spatial and temporal organization to cells. Condensates can undergo maturation, transitioning from dynamic liquid-like states into solid-like states associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Huntington's disease. Despite their important roles, many aspects of condensate biology remain incompletely understood, requiring tools for acutely manipulating condensate-relevant processes within cells. Here we used the BCL6 BTB domain and its ligands BI-3802 and BI-3812 to create a chemical genetic platform, BTBolig, allowing inducible condensate formation and dissolution. We also developed optogenetic and chemical methods for controlled induction of condensate maturation, where we surprisingly observed recruitment of chaperones into the condensate core and formation of dynamic biphasic condensates. Our work provides insights into the interaction of condensates with proteostasis pathways and introduces a suite of chemical-genetic approaches to probe the role of biomolecular condensates in health and disease.

Design and Synthesis of BCL6 BTB Inhibitor OICR12694

Key words non-Hodgkin lymphoma - BCL6 - protein–protein interaction - structure-based drug design

Mechanisms of CP190 Interaction with Architectural Proteins in Drosophila Melanogaster.

Most of the known Drosophila architectural proteins interact with an important cofactor, CP190, that contains three domains (BTB, M, and D) that are involved in protein–protein interactions. The highly conserved N-terminal CP190 BTB domain forms a stable homodimer that interacts with unstructured regions in the three best-characterized architectural proteins: dCTCF, Su(Hw), and Pita. Here, we identified two new CP190 partners, CG4730 and CG31365, that interact with the BTB domain. The CP190 BTB resembles the previously characterized human BCL6 BTB domain, which uses its hydrophobic groove to specifically associate with unstructured regions of several transcriptional repressors. Using GST pull-down and yeast two-hybrid assays, we demonstrated that mutations in the hydrophobic groove strongly affect the affinity of CP190 BTB for the architectural proteins. In the yeast two-hybrid assay, we found that architectural proteins use various mechanisms to improve the efficiency of interaction with CP190. Pita and Su(Hw) have two unstructured regions that appear to simultaneously interact with hydrophobic grooves in the BTB dimer. In dCTCF and CG31365, two adjacent regions interact simultaneously with the hydrophobic groove of the BTB and the M domain of CP190. Finally, CG4730 interacts with the BTB, M, and D domains of CP190 simultaneously. These results suggest that architectural proteins use different mechanisms to increase the efficiency of interaction with CP190.

BCL6 BTB-specific inhibitor reversely represses T-cell activation, Tfh cells differentiation, and germinal center reaction in vivo.

Inhibition of the BCL6 BTB domain results in killing Diffuse Large B-cell Lymphoma (DLBL) cells, reducing the T-cell dependent germinal center (GC) reaction in mice, and reversing GC hyperplasia in nonhuman primates. The available BCL6 BTB-specific inhibitors are poorly water soluble, thus, limiting their absorption in vivo and our understanding of therapeutic strategy targeting GC. We synthesized a prodrug (AP-4-287) from a potent BCL6 BTB inhibitor (FX1) with improved aqueous solubility and pharmacokinetics (PK) in mice. We also evaluated its in vivo biological activity on humoral immune responses using the sheep red blood cells (SRBC)-vaccination mouse model. AP-4-287 had a significant higher aqueous solubility and was readily converted to FX1 in vivo after intraperitoneally (i.p.) administration, but a shorter half-life in vivo. Importantly, AP-4-287 treatment led to a reversible effect on (1) the reduction in the frequency of splenic Ki67+ CD4+ T cells, Tfh cells, and GC B cells; (2) lower GC formation following vaccination; and (3) a decrease in the titers of antigen-specific IgG and IgM antibodies. Our study advances the preclinical development of drug targeting BCL6 BTB domain for the treatment of diseases that are associated with abnormal BCL6 elevation.

Functionalization of the BCL6 BTB domain into a noncovalent crystallization chaperone.

The production of diffraction-quality protein crystals is challenging and often requires bespoke, time-consuming and expensive strategies. A system has been developed in which the BCL6 BTB domain acts as a crystallization chaperone and promiscuous assembly block that may form the basis for affinity-capture crystallography. The protein of interest is expressed with a C-terminal tag that interacts with the BTB domain, and co-crystallization leads to its incorporation within a BTB-domain lattice. This strategy was used to solve the structure of the SH3 domain of human nebulin, a structure previously solved by NMR, at 1.56 Å resolution. This approach is simple and effective, requiring only routine protein complexation and crystallization screening, and should be applicable to a range of proteins.

Abstract 1288: BCL6 drives an oncogenic transcriptional and epigenetic program in NSCLC whose inhibition results in antineoplastic activity in vivo

Abstract BCL6 is an oncogenic transcription factor that represses multiple target genes by recruiting three essential co-repressors (SMRT, NCOR, BCOR) via its BTB domain. These, in turn, serve as scaffold to form distinct complexes with various chromatin remodelers. We found that BCL6 is required to sustain oncogenic features in NSCLC such as cell proliferation and colony formation. Furthermore, we demonstrated that BCL6 allows NSCLC cells to survive several stress conditions including exposure to DNA damaging agents. Although these data demonstrated a critical role of BCL6 in NSCLC oncogenesis, the mechanism underlying its oncogenic activity remains unknown. To elucidate the role of BCL6 in NSCLC, we used the small molecule FX1 which specifically disrupts the interaction between the BTB domain and its essential corepressors (Kd 7+/- 3 uM). An in vitro screening demonstrated that FX1 impaired the survival of 6/15 (40%) BCL6-positive NSCLC cell lines that we considered as BCL6-dependent. Furthermore, FX1 (25mg/kg, every other day) significantly decreased the tumor growth of two BCL6-dependent human NSCLC xenograft models (p< 0.001) without toxicity to normal tissues. To gain insight into the transcriptional and epigenetic mechanisms that explain BCL6 oncogenic activity, we exposed two BCL6-dependent cell lines (H1155 and H2122) to FX1 for short-term treatment and performed parallel ATAC-sequencing (chromatin accessibility) and spike-in normalized RNA-sequencing (transcriptional changes) experiments. At the genome-wide level, we found that inhibition of BCL6 broadly affected the epigenome landscape of both cell lines. Notably, 34% (H1155) and 27% (H2122) of the regions for which chromatin accessibility was significantly altered mapped to promoters. Accordingly, we identified 4,825 shared genes that were significantly re-expressed and 1,425 shared genes that were significantly repressed in cells treated with FX1 vs. vehicle. Re-expressed genes were enriched for pathways regulating immune signaling (i.e. CD247, IRF7 and HLAB/C/F), cell-cell interaction (i.e. TNF, IL4R, IL2RB, CXCR4), extracellular matrix organization (i.e. COL1A2, COL2A1, COL5A1, BGN) and neuronal system differentiation (i.e. NEUROD1, SCG3, SCG5). Interestingly, repressed genes included oncogenic transcriptional factors among which MYC, NOTCH1 and NOTCH3. Accordingly, repressed genes were enriched for MYC target genes and genes regulating epithelial to mesenchymal transition (TGFB2, TGFB1, WNT5A, VIM) as well as extracellular matrix organization (COL4A5, COL4A5, COL5A2, MMP10). Overall these findings suggest that BCL6, via its BTB domain, transcriptionally and epigenetically regulates multiple oncogenic pathways in NSCLC and may shape the cellular and extracellular composition of the tumor microenvironment. Furthermore, they provide preliminary evidence of the feasibility of BCL6 BTB inhibition as therapeutic strategy for NSCLC. Citation Format: Rossella Marullo, Maria V. Revuelta, Yong Ai, Fengtian Xue, Leandro Cerchietti. BCL6 drives an oncogenic transcriptional and epigenetic program in NSCLC whose inhibition results in antineoplastic activity in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1288.

BCL6 Evolved to Enable Stress Tolerance in Vertebrates and Is Broadly Required by Cancer Cells to Adapt to Stress.

Several lines of evidence link the canonical oncogene BCL6 to stress response. Here we demonstrate that BCL6 evolved in vertebrates as a component of the HSF1-driven stress response, which has been co-opted by the immune system to support germinal center formation and may have been decisive in the convergent evolution of humoral immunity in jawless and jawed vertebrates. We find that the highly conserved BTB corepressor binding site of BCL6 mediates stress adaptation across vertebrates. We demonstrate that pan-cancer cells hijack this stress tolerance mechanism to aberrantly express BCL6. Targeting the BCL6 BTB domain in cancer cells induces apoptosis and increases susceptibility to repeated doses of cytotoxic therapy. The chemosensitization effect upon BCL6 BTB inhibition is dependent on the derepression of TOX, implicating modulation of DNA repair as a downstream mechanism. Collectively, these data suggest a form of adaptive nononcogene addiction rooted in the natural selection of BCL6 during vertebrate evolution. SIGNIFICANCE: We demonstrate that HSF1 drives BCL6 expression to enable stress tolerance in vertebrates. We identify an HSF1-BCL6-TOX stress axis that is required by cancer cells to tolerate exposure to cytotoxic agents and points toward BCL6-targeted therapy as a way to more effectively kill a wide variety of solid tumors.This article is highlighted in the In This Issue feature, p. 565.

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.

Cutting edge: Bcl6-interacting corepressor contributes to germinal center T follicular helper cell formation and B cell helper function.

CD4(+) germinal center (GC)-T follicular helper (Tfh) cells help B cells become long-lived plasma cells and memory cells. The transcriptional repressor Bcl6 plays a key role in GC-Tfh formation by inhibiting the expression of genes that promote differentiation into other lineages. We determined whether BCOR, a component of a Polycomb repressive complex that interacts with the Bcl6 BTB domain, influences GC-Tfh differentiation. T cell-targeted BCOR deficiency led to a substantial loss of peptide:MHC class II-specific GC-Tfh cells following Listeria monocytogenes infection and a 2-fold decrease following immunization with a peptide in CFA. The reduction in GC-Tfh cells was associated with diminished plasma cell and GC B cell formation. Thus, T cell-expressed BCOR is critical for optimal GC-Tfh cell differentiation and humoral immunity.

BCL6 Mediates a Stress Tolerance Phenotype through Its BTB Domain

Diffuse large B-cell lymphomas (DLBCLs) arise from germinal center (GC) B-cells. Normal GC B-cells clonally expand and undergo somatic hypermutation of their immunoglobulin loci to produce high-affinity antibodies. Induction of BCL6, a transcription factor that represses genes involved in DNA damage sensing and checkpoint activation, is essential for GC B-cells to tolerate replicative and genotoxic stress without inducing cell cycle arrest. We previously showed that BCL6 forms a complex with tumor enriched HSP90 (TE-HSP90) to repress target genes in DLBCL cells.Based on these facts, we hypothesized that BCL6 is a component of a conserved stress response program, required for GC formation and maintenance of established lymphoma cells. Along these lines, we report that treatment of mice with TE-HSP90-selective inhibitor PU-H71 completely abrogates GC formation after immunization by T-cell dependent antigen. Stress responses are coordinated by the transcription factor heat shock factor 1 (HSF1), which is activated by phosphorylation. Immunofluorescence of human tonsillar sections revealed HSF1-pSer326-positive cells within BCL6+ GC B-cells. We found that HSF1-/- mice manifest a 40% decrease in GC B-cells after immunization and significantly (p=0.0073) decreased titers of high-affinity immunoglobulin compared to WT mice, indicating defective affinity maturation. Mixed chimera experiments revealed that the GC defect is exclusively due to malfunction of GC B-cells and not other cell types. Because of this GC B-cell defect, we reasoned that HSF1 might induce BCL6 expression. We identified three conserved heat shock elements (HSEs) in the BCL6 promoter. Quantitative ChIP analysis demonstrated HSF1 binding to these HSEs in human GC B-cells. Heat shock induced the BCL6 promoter in reporter assays and resulted in an increase in BCL6 nascent transcripts and protein. However this induction did not occur in HSF1-/-B-cells or after HSF1 knockdown. These data suggest that BCL6 is a stress response gene downstream of HSF1.To determine whether BCL6 is involved in mediating a stress tolerant phenotype, we performed serial stress response assays (using heat shock) in B220+ splenocytes from BCL6+/+ or BCL6-/- mice. Whereas BCL6+/+ cells were able acquire stress tolerance if preconditioned with an initial heat shock, BCL6-/- splenocytes failed to adapt to stress and died. To understand the mechanistic basis of this finding, we generated knockin mice with point mutations that disrupt the repressor activity of the BCL6 BTB (BCL6BTB) or the BCL6 RD2 (BCL6RD2) domain. Interestingly while splenocytes from BCL6RD2 mutant mice displayed normal stress tolerance responses, BCL6BTB mutant B-cells were completely deficient similar to BCL6-/-B-cells. Likewise the BCL6 BTB domain inhibitor RI-BPI also abrogated the BCL6 stress tolerance function.DLBCLs are dependent on many of the same molecular mechanisms as normal GC B-cells (e.g. BCL6). Indeed the lentiviral transduction of HSF1 shRNAs in DLBCL cell lines reduced BCL6 protein levels by more than 50% resulting in a 70%-90% loss in viability. HSF1 is known to help tumor cells survive exposure to chemotherapy drugs, and the BCL6 BTB domain is required for stress tolerance. Thus we hypothesized that BCL6 BTB domain targeted therapy (RI-BPI) would synergistically kill DLBCL when combined with chemotherapy. We treated 7 DLBCL cell lines with RI-BPI in combination with doxorubicin, vincristine, dexamethasone, mechlorethamine (in place of cyclophosphamide), and their combination CHOP. Almost all combinations resulted in an additive or synergistic effect on DLBCL growth inhibition. Moreover the combination of RI-BPI and doxorubicin in a DBLCL xenograft model was more potent and significantly (p<0.001) better at reducing tumor growth than either drug alone. Using an ex vivo coculture system for primary human DLBCL specimens, the combination of RI-BPI and CHOP had at least an additive effect on growth inhibition in 80% of primary human DLBCL cells with the majority demonstrating a synergistic anti-lymphoma effect.Collectively we demonstrate that BCL6 is an HSF1-dependent stress tolerance factor and mediates this effect via its BTB domain. This phenomenon occurs during normal GC formation and in lymphoma cells. Thus targeting the BTB domain of BCL6 pharmacologically in combination with other chemotherapy is a viable strategy to eradicate lymphomas. DisclosuresNo relevant conflicts of interest to declare.

The Bcl6 RD2 Domain Is Essential For Pre-Germinal Center B Cell Development

The transcriptional repressor Bcl6 is a master regulator of the germinal center (GC) reaction through directing naïve B cells and CD4+ T cells to differentiate into GC B cells and follicular T helper (TFH) cells respectively. Bcl6 mediates its action largely by recruitment of co-repressors through its N-terminal BTB domain and its middle second repression domain (RD2). The BTB domain repression function is critical for GC B cell survival and proliferation, but not important for TFH cell differentiation. However, the in vivobiological function of RD2 remains unknown.To explore the specific role of RD2 transcriptional repression in the GC reaction, we generated a knockin mouse model in which the endogenous Bcl6 locus encodes a mutant form of the protein that specifically disrupts RD2 mediated transcriptional repression. RD2 mutant mice were developmentally indistinguishable from wild-type mice and displayed normal B cell development prior to the GC phase. However, these mice failed to accumulate GCs after immunization with sheep blood cells and exhibited remarkably impaired production of high-affinity antibodies 21 days after T-cell dependent antigen immunization, indicative of severe deficiency of the GC reaction. Mixed bone marrow transplantation experiments showed that RD2 loss of function led to complete loss of GC B cells and partial impairment of TFH cell differentiation in cell-intrinsic manner. Intravital imaging analysis indicated that RD2-deficent antigen-engaged B cells migrate normally to the inter-follicular zone of lymph nodes and interacted normally with cognate T helper cells. To further understand the nature of the functional defect of RD2 mutant B-cells, hen egg lysosome (HEL)-specific RD2-deficient GFP B cells and wild type RFP B cells (with the ratio 1:1) were transferred together with non-fluorescent ovalbumin (OVA)-specific T cells into SMARTA hosts, which were then immunized at the footpad with HEL-OVA two days later. On day 5 after immunization, draining popliteal lymph nodes were harvested and subjected for immunofluorescence histology analysis. At this time point, wild-type RFP B cells have started to cluster into tiny GC, whereas RD2-deficient GFP B cells did not form GCs. Moreover, wild-type B cells in the follicular interior were predominantly Bcl6hi, a characteristic of pre-GC B cells, suggesting that they could serve as a source of GC B cells. By contrast, RD2-deficient GFP B cells were primarily extra-follicular, and infrequently observed in the follicle interior. Most importantly, these cells were typically Bcl6lo, demonstrating that RD2 repression function is essential for pre-GC B cell differentiation.BCL6 knockout mice display a lethal inflammatory phenotype due to aberrant T-cell and macrophage activation. In striking contrast, RD2-deficient mice experienced normal healthy lives with no inflammation, and had nearly normal inflammation cytokine production in B cells and macrophages as well as differentiation of Th1,Th2 and Th17 subtypes. Hence the RD2 repression domain is specifically involved in humoral immunity but has minimal participation in the anti-inflammatory functions of BCL6. Instead we observed that the BCL6 zing finger domain plays the key role in anti-inflammatory functions in macrophages, and through ChIP-competition assays show that this is mediated by directly competing with STATs for binding to chemokine genes.These results highlight an essential role of RD2-mediated transcriptional repression in pre-GC B cell development specifically at the early B-cell activation phase. This is different than mice with BCL6 BTB mutations where early activation is normal and the defect occurs later on in the proliferative phase of GCs. The data suggest a surprising development and cellular context-specific biochemical functions of Bcl6 governing each distinct phase of the humoral immune response and inflammation. Disclosures:No relevant conflicts of interest to declare.

Abstract B14: A biochemical basis for toggling enhancers from the active to poised state in B-cells

Abstract The BCL6 transcriptional repressor is required for development of germinal center (GC) B-cells and diffuse large B-cell lymphomas (DLBCL). The BCL6 BTB domain corepressor interaction is an important mediator of BCL6 actions and a potential therapeutic target. BTB point mutations that disrupt corepressor recruitment inactivate BTB domain repressor function. A similar effect can be achieved using specific BCL6 BTB groove binding peptides or small molecules. Although BCL6 can recruit multiple corepressors, its transcriptional repression mechanism of action in normal and malignant B-cells is unknown. In this study we addressed the question of how BCL6 mediates transcriptional repression in normal and malignant B-cells through its association with SMRT or NCOR corepressor complexes. We combined extensive genomic binding (ChIP-seq) analysis of BCL6, its corepressors SMRT and NCOR and epigenetic chromatin patterns both in DLBCL and in primary human GC B-cells, with functional and biochemical experiments performed in vitro, in cell-based assays and in vivo to reveal the biological and transcriptional mechanism of BCL6. We find that in B-cells, virtually all SMRT/NCOR binding sites overlap with BCL6 binding sites indicating that in these cells BCL6 uniquely sequesters SMRT/NCOR complexes from other factors to mediate cell specific functions. Interestingly, these BCL6-SMRT/NCOR complexes preferentially localize at B cell enhancers. Moreover, BCL6-SMRT/NCOR complexes can inactivate these enhancers by deacetylating H3K27. The SMRT/NCOR-dependent H3K27 de-acetylation is mediated by HDAC3 and opposed by p300 histone acetyltransferase. Furthermore, we show that BCL6 BTB targeted inhibitors may reverse the enhancer silencing effect by disrupting the association of BCL6 with SMRT/NCOR complexes. Taken together these results point to an enhancer “toggling” mechanism that might provide the basis for B-cells to undergo rapid transcriptional changes in response to environmental cues. This mechanism might be particularly relevant to the shuttling of B-cells from the dark to the light zone during affinity maturation facilitating the rapid on/off gene expression changes necessary for this transition. For example genes associated with BCL6-SMRT/NCOR enhancers were highly downregulated in the dark zone compared to the light zone and they were also significantly enriched in genes upregulated by CD40 signaling which occurs in the light zone during B-cell activation by Tfh cells and disrupts the association of BCL6 and SMRT/NCOR. In summary we describe a novel mechanism of enhancer regulation. Reversible enhancer toggling may be critical for dynamic modulation of the BCL6 transcriptional program during the GC reaction as well for the therapeutic effects of BCL6 inhibitors. This mechanism is likely a general biological phenomenon that applies to enhancer regulation in a variety cell types and tumors. Citation Format: Katerina Chatzi, Yanwen Jiang, Chuanxin Huang, Paul Zumbo, Srividya Bhaskara, Alexander D. MacKerell, Jr., Fengtian Xue, Christopher E. Mason, Scott W. Hiebert, Leandro Cerchietti, Vivian J. Bardwell, Olivier Elemento, Melnick Ari. A biochemical basis for toggling enhancers from the active to poised state in B-cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B14.

Adaptor Protein Self-Assembly Drives the Control of a Cullin-RING Ubiquitin Ligase

The E3 ligases recruit substrate proteins for targeted ubiquitylation. Recent insights into the mechanisms of ubiquitylation demonstrate that E3 ligases can possess active regulatory properties beyond those of a simple assembly scaffold. Here, we describe the dimeric structure of the E3 ligase adaptor protein SPOP (speckle-type POZ protein) in complex with the N-terminal domain of Cul3 at 2.4Å resolution. We find that SPOP forms large oligomers that can form heteromeric species with the closely related paralog SPOPL. In combination, SPOP and SPOPL (SPOP-like) form a molecular rheostat that can fine-tune E3 ubiquitin ligase activity by affecting the oligomeric state of the E3 complex. We propose that adaptor protein self-assembly provides a graded level of regulation of the SPOP/Cul3 E3 ligase toward its multiple protein substrates.

A Small-Molecule Inhibitor of BCL6 Kills DLBCL Cells In Vitro and In Vivo

The BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B cell lymphoma (DLBCL). We combined computer-aided drug design with functional assays to identify low-molecular-weight compounds that bind to the corepressor binding groove of the BCL6 BTB domain. One such compound disrupted BCL6/corepressor complexes in vitro and in vivo, and was observed by X-ray crystallography and NMR to bind the critical site within the BTB groove. This compound could induce expression of BCL6 target genes and kill BCL6-positive DLBCL cell lines. In xenotransplantation experiments, the compound was nontoxic and potently suppressed DLBCL tumors in vivo. The compound also killed primary DLBCLs from human patients.

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 represses CHEK1 and suppresses DNA damage pathways in normal and malignant B-cells

BCL6 is a transcriptional repressor protein that is expressed in a developmentally regulated fashion during B-cell maturation. Specifically, BCL6 is required for formation of germinal centers in response to T-cell dependent antigen activation. Germinal center B-cells feature the ability to tolerate rapid proliferation and simultaneous genetic recombination. Genetic lesions that cause constitutive expression of BCL6 are commonly associated with diffuse large B-cell lymphomas (DLBCL). Recent studies show that BCL6 contributes to the germinal center phenotype by directly repressing genes involved in sensing or responding to DNA damage including ATR, TP53 and CDKN1A. The CHEK1 protein is activated through phosphorylation by the ATR kinase domain in response to DNA damage. Activated CHEK1 can phosphorylate and modulate the activity a number of proteins including p53, providing a link between ATR sensing of DNA damage and p53 checkpoint activity. Herein we show that BCL6 can directly bind to a DNA consensus element in the CHEK1 promoter and repress its expression in normal and malignant B-cells. DLBCL cells can be killed by a specific BCL6 peptide inhibitor (BPI) that interferes with corepressor binding to the BCL6 BTB domain. BPI could reactivate CHEK1 in DLBCL cells, suggesting that its induction might contribute to BPI anti-lymphoma effects. Therefore, BCL6 can suppress multiple genes involved in a common pathway sensing, transducing and responding to genotoxic stress.

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.

Design and Development of Small Molecules for Specific Targeted Therapy of Diffuse Large B-Cell Lymphoma.

BCL6 is the most commonly involved oncogene in diffuse large B-cell lymphoma (DLBCL). BCL6 mediates lymphomagenesis through direct transcriptional repression of target genes including p53 and ATR. BCL6 represses these genes by recruiting three corepressors: SMRT, N-CoR and BCoR. This interaction is mediated by an 18 amino acid corepressor sequence with a unique “lateral groove” motif located in the BCL6 BTB domain. We previously showed that a peptide mimic of the corepressor BCL6-binding sequence could inhibit BCL6 and kill DLBCL cells in vitro and in vivo, without toxicity to normal tissues. Although, transcription factors such as BCL6 that function through protein-protein interactions were traditionally regarded as “undruggable”, we hypothesized that a detailed structural and biochemical analysis might facilitate the design of small molecule inhibitors. Based on our crystal structure of the BCL6-corepressor complex, we used a computational strategy to screen one million small molecules for their ability to potentially dock to a specific lateral groove region. The chemistry of protein contacts in this region led us to predict that small molecules that could dock to this site would have the best chance of destabilizing the BCL6-corepressor complex. Among the top-scoring 100 molecules from this screen, we identified 10 compounds that could specifically inhibit the repressor activity of the BCL6 BTB domain in reporter assays and that displayed direct binding to purified BCL6 BTB domains. Using these leads as molecular scaffolds we generated small libraries of molecules derived from each parental compound. The most active of these families was called “the 57 series”. Series 57 compounds could all specifically block BCL6 repression in reporter assays, and disrupt corepressor/BCL6 complexes at low micromolar concentrations as shown in fluorescence polarization assays. X-ray crystallography of the most active member of the 57 family (called “57-6”) showed that the small molecule docked as predicted in the critical region of the lateral groove. Moreover, 57-6 induced an allosteric conformational change in the entire lateral groove that explains how these small molecules so effectively disrupt the BCL6/corepressor complex. 57-6 was also biologically active, since it could induce expression of BCL6 target genes including p53 and ATR in BCL6-positive DLBCL cells as shown by QPCR. 57-6 had no effect on negative control genes nor in BCL6-negative DLBCL cells. The mechanism of action was confirmed in ChIP assays showed that 57-6 abrogated BCL6 mediated corepressor recruitment to BCL6 target genes but had no effect on negative control genes. Most importantly, 57-6 specifically killed BCL6-positive DLBCL cells but had no effect on BCL6-negative DLBCL cells. A dose escalation experiment in mice revealed no toxic effects. In xenotransplantation experiments, 57-6 potently inhibited the growth of already established human DLBCL tumors in mice, again without toxicity to other organs. In summary, we used a rational approach to design specific and potent small molecule inhibitors of BCL6, which could serve as targeted agents for DLBCL in clinical trials. Our data show that transcription factors are druggable targets that can be harnessed to potentially improve cancer therapy.

Crystal structure of the BTB domain from the LRF/ZBTB7 transcriptional regulator

BTB-zinc finger (BTB-ZF) proteins are transcription regulators with roles in development, differentiation, and oncogenesis. In these proteins, the BTB domain (also known as the POZ domain) is a protein-protein interaction motif that contains a dimerization interface, a possible oligomerization surface, and surfaces for interactions with other factors, including nuclear co-repressors and histone deacetylases. The BTB-ZF protein LRF (also known as ZBTB7, FBI-1, OCZF, and Pokemon) is a master regulator of oncogenesis, and represses the transcription of a variety of important genes, including the ARF, c-fos, and c-myc oncogenes and extracellular matrix genes. We determined the crystal structure of the BTB domain from human LRF to 2.1 A and observed the canonical BTB homodimer fold. However, novel features are apparent on the surface of the homodimer, including differences in the lateral groove and charged pocket regions. The residues that line the lateral groove have little similarity with the equivalent residues from the BCL6 BTB domain, and we show that the 17-residue BCL6 Binding Domain (BBD) from the SMRT co-repressor does not bind to the LRF BTB domain.

Specific Peptide Disruption of the Bcl-6 Repression Complex Reveals Its Transcriptional Mechanism of Action in Normal and Malignant B-Cells and Is a Novel Therapeutic Approach for Diffuse Large B-Cell Lymphoma.

The Bcl-6 transcriptional repressor is required for the establishment of germinal centers during B-cell maturation. Regulatory elements of the BCL6 gene are frequently mutated in diffuse large B-cell lymphomas (DLBCL), leading to inappropriately timed expression of Bcl-6. However, it is unknown whether Bcl-6 is involved in lymphomagenesis. Within Bcl-6, the N-terminal BTB domain is involved in mediating transcriptional repression. We investigated the mechanism of action of the Bcl-6 BTB domain using X-ray crystallography and functional assays. We found that the SMRT, N-CoR and BCoR corepressors bind directly to Bcl-6 through a 16 residue “BBD motif”, which fits into a highly specific “lateral groove” surface feature of the Bcl-6 BTB domain. To determine the contribution of BTB lateral groove recruitment of corepressors to transcriptional and biological functions of Bcl-6, we engineered small Bcl-6 BTB peptide inhibitors (BPI). BPI penetrate cells and localize to the nuclei where they specifically bound to Bcl-6 and blocked its interaction and co-localization with co-repressors. This resulted in dose-dependent blockade of repression by Bcl-6 in reporter assays and reactivation of endogenous Bcl-6 target genes in Bcl-6 expressing B-cells measured by real-time PCR. Loss of repression was caused by disruption of the endogenous Bcl-6 transcriptional repression complex. We found that BPI specifically excluded corepressors from the promoters of endogenous Bcl-6 target genes, resulting in a switch from repressed to activated histone code settings with consequent gene reactivation. From the biological standpoint, BPI injection into mice reproduced the B-cell phenotype of Bcl-6 null animals, as germinal center formation in response to T-cell dependent antigens was abrogated. To address the question of whether Bcl-6 is required to maintain the malignant phenotype of DLBCL, a panel of Bcl-6 positive and negative DLBCL cells were exposed to BPI. BPI induced growth suppression, cell cycle arrest and apoptosis in Bcl-6 positive but not negative DLBCL cells. Expression array analysis showed immediate upregulation of checkpoint genes involved in proliferation and apoptosis, allowing us to identify novel direct Bcl-6 target genes. In contrast, BPI did not upregulate differentiation related genes and did not induce differentiation in lymphoma cells, indicating that the Bcl-6 BTB domain lateral groove controls proliferation and survival pathways but not differentiation. Finally, BPI completely suppressed growth of Bcl-6 positive human DLBCL xenotransplants without any toxicity to other organs. In summary, specific disruption of the Bcl-6 BTB domain repression mechanism allowed us to reveal the contribution of specific corepressor recruitment to the activities of Bcl-6 in B-cells and to demonstrate that Bcl-6 is in fact an oncogene, required to maintain the malignant phenotype of Bcl-6 positive DLBCL. Our pre-clinical studies indicate that Bcl-6 is a bona fide therapeutic target and that BPI is a specific, potent and non-toxic anti-lymphoma therapeutic agent.