BCL6 Transcriptional Repressor Research Articles

Transcriptional signatures of fentanyl use in the mouse ventral tegmental area.

Synthetic opioids such as fentanyl contribute to the vast majority of opioid-related overdose deaths, but fentanyl use remains broadly understudied. Like other substances with misuse potential, opioids cause lasting molecular adaptations to brain reward circuits, including neurons in the ventral tegmental area (VTA). The VTA contains numerous cell types that play diverse roles in opioid use and relapse; however, it is unknown how fentanyl experience alters the transcriptional landscape in specific subtypes. Here, we performed single nuclei RNA sequencing to study transcriptional programs in fentanyl-experienced mice. Male and female C57/BL6 mice self-administered intravenous fentanyl (1.5μg/kg/infusion) or saline for 10days. After 24 h abstinence, VTA nuclei were isolated and prepared for sequencing on the 10× platform. We identified different patterns of gene expression across cell types. In dopamine neurons, we found enrichment of genes involved in growth hormone signalling. In dopamine-glutamate-GABA combinatorial neurons, and some GABA neurons, we found enrichment of genes involved in Pi3k-Akt signalling. In glutamate neurons, we found enrichment of genes involved in cholinergic signalling. We identified transcriptional regulators for the differentially expressed genes in each neuron cluster, including downregulated transcriptional repressor Bcl6, and upregulated transcription factor Tcf4. We also compared the fentanyl-induced gene expression changes identified in mouse VTA with a published rat dataset in bulk VTA, and found overlap in genes related to GABAergic signalling and extracellular matrix interaction. Together, we provide a comprehensive picture of how fentanyl self-administration alters the transcriptional landscape of the mouse VTA that serves as the foundation for future mechanistic studies.

Genomic deletion of Bcl6 differentially affects conventional dendritic cell subsets and compromises Tfh/Tfr/Th17 cell responses.

Conventional dendritic cells (cDC) play key roles in immune induction, but what drives their heterogeneity and functional specialization is still ill-defined. Here we show that cDC-specific deletion of the transcriptional repressor Bcl6 in mice alters the phenotype and transcriptome of cDC1 and cDC2, while their lineage identity is preserved. Bcl6-deficient cDC1 are diminished in the periphery but maintain their ability to cross-present antigen to CD8+ T cells, confirming general maintenance of this subset. Surprisingly, the absence of Bcl6 in cDC causes a complete loss of Notch2-dependent cDC2 in the spleen and intestinal lamina propria. DC-targeted Bcl6-deficient mice induced fewer T follicular helper cells despite a profound impact on T follicular regulatory cells in response to immunization and mounted diminished Th17 immunity to Citrobacter rodentium in the colon. Our findings establish Bcl6 as an essential transcription factor for subsets of cDC and add to our understanding of the transcriptional landscape underlying cDC heterogeneity.

Abstract 5777: Rewiring EWS/FLI1 with transcriptional chemical inducers of proximity (TCIPs)

Abstract The purpose of this study was to determine if EWS/FLI1 (E/F), the oncogenic fusion protein that drives Ewing Sarcoma (ES), can be rewired to enhance expression of BCL6-controlled genes using Transcriptional Chemical Inducers of Proximity (TCIPs), a novel bivalent small molecule platform. TCIP molecules recruit a transcriptional regulator, such as BRD4, to the transcriptional repressor BCL6. Upon binding, a ternary complex is formed on chromatin at BCL6-bound loci, causing transcriptional upregulation of BCL6 target genes. Since E/F does not have a bona fide ligand, we have used an exogenously expressed N terminal FKBPF36V-EWS/FLI1 (N-FK-E/F) fusion system to study how modulation of E/F by TCIPs impacts the transcriptome of ES cells. To eliminate background signal from endogenous E/F, we used CRISPR/Cas9 to knockout (KO) endogenous E/F. Our exogenously expressed N-FK-E/F has mutated PAM motifs and is resistant to KO. In our proof-of-concept study we used TCIP1 consisting of ortho-AP1867 (oAP), a synthetic ligand for FKBPF36V, linked to the BCL6 inhibitor BI3812 (BI) to rewire N-FK-E/F. We have also tested a negative control TCIP (TCIP NEG) that links oAP to a BI derivative that abolishes BCL6 binding. TCIP NEG has similar physiochemical properties to our active TCIP1; however, it cannot bind BCL6, serving as a negative control for ternary complex formation. TCIP1 rapidly and dose-dependently increases the RNA and protein expression of several BCL6 target genes, like p21, in multiple ES models that stably express N-FK-E/F. TCIP NEG does not increase the expression of BCL6 target genes in ES cells. TCIP1 induces ternary complex formation in ES cell lysates, and ternary complex formation is necessary for activity in ES cells. TCIP1 is specific and does not increase BCL6 target gene levels to the same extent in ES cells expressing a FKBP-GFP construct or in parental ES cells lacking N-FK-E/F. RNA sequencing of EWS502 N-FK-E/F expressing cells shows that TCIP1 treatment induces a larger increase in expression of BCL6 target gene RNA than BCL6 inhibition. Moreover, CUT&RUN experiments in EWS502 N-FK-E/F expressing cells suggest TCIP1 leads to an accumulation of N-FK-E/F at the promoters of BCL6 target genes. Motif analysis showed that TCIP1 increases the likelihood of N-FK-E/F binding to BCL6 consensus sequences as wells as increases the likelihood of BCL6 binding to E/F recognition sequences. In future studies we will conduct RNA sequencing, CUT&RUN, and other ‘omics’ approaches (e.g. DisP-seq) to compare TCIP1 to TCIP NEG in multiple cell lines at multiple concentrations and time points. Our data suggests that N-FK-E/F can be rewired to induce the expression of BCL6 target genes in ES. Since ES is driven solely by E/F, TCIPs may be promising next generation therapeutics. Our study provides a proof-of-concept that will be the foundation for the development of future TCIP molecules that recruit endogenous E/F once suitable ligands are developed. Citation Format: Michael J. Bond, Ryan P. Golden, Roman C. Sarott, Giulia DiGiovanni, Basel A. Karim, Briana Howard, Kenneth Ross, Nathanael S. Gray, Kimberly Stegmaier. Rewiring EWS/FLI1 with transcriptional chemical inducers of proximity (TCIPs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5777.

Pannexin-3 stabilizes the transcription factor Bcl6 in a channel-independent manner to protect against vascular oxidative stress.

Targeted degradation regulates the activity of the transcriptional repressor Bcl6 and its ability to suppress oxidative stress and inflammation. Here, we report that abundance of endothelial Bcl6 is determined by its interaction with Golgi-localized pannexin 3 (Panx3) and that Bcl6 transcriptional activity protects against vascular oxidative stress. Consistent with data from obese, hypertensive humans, mice with an endothelial cell-specific deficiency in Panx3 had spontaneous systemic hypertension without obvious changes in channel function, as assessed by Ca2+ handling, ATP amounts, or Golgi luminal pH. Panx3 bound to Bcl6, and its absence reduced Bcl6 protein abundance, suggesting that the interaction with Panx3 stabilized Bcl6 by preventing its degradation. Panx3 deficiency was associated with increased expression of the gene encoding the H2O2-producing enzyme Nox4, which is normally repressed by Bcl6, resulting in H2O2-induced oxidative damage in the vasculature. Catalase rescued impaired vasodilation in mice lacking endothelial Panx3. Administration of a newly developed peptide to inhibit the Panx3-Bcl6 interaction recapitulated the increase in Nox4 expression and in blood pressure seen in mice with endothelial Panx3 deficiency. Panx3-Bcl6-Nox4 dysregulation occurred in obesity-related hypertension, but not when hypertension was induced in the absence of obesity. Our findings provide insight into a channel-independent role of Panx3 wherein its interaction with Bcl6 determines vascular oxidative state, particularly under the adverse conditions of obesity.

The Tfh/Tfr Pathway Is Pivotal in FVIII Inhibitor Development in Mice

The development of neutralizing anti-FVIII antibodies (inhibitors) is one major complication of FVIII protein replacement therapy in hemophilia A patients. Our previous study demonstrated that the induction of activated follicular helper T (Tfh) cells in FVIII null mice is critical for FVIII inhibitor development. Follicular regulatory T (Tfr) cells are a specialized subset of Foxp3 + T cells identified in the germinal center (GC) that can modulate Tfh cell activation of B cells and antibody development. However, the role of Tfr cells in controlling anti-FVIII inhibitor development in vivo is still unclear. Here, we explored the activation and induction of Tfr cells and their functions in anti-FVIII inhibitor development in FVIII-immunized mice. We first characterized the features of activated Tfr cells in the spleen of immunized FVIII null mice by flow cytometry analysis. Activated Tfr cells were defined as CD4 +Foxp3 +CD44 +CD62L -CXCR5 +PD-1 +. Tfr cells express a high level of transcription factor BCL6 and costimulatory receptor ICOS. Activated Tfr cells also express a high level of costimulatory receptor CTLA4, which is similar to non-Tfr effector Treg cells, but CD25 expression is low on Tfr cells. FVIII immunization increases the frequency and the absolute number of activated Tfr cells in the spleen of mice that produce inhibitors. The percentage of activated CXCR5 hiPD-1 hi Tfr cells within the CD4 population significantly increased in inhibitor-producing mice compared to saline-treated controls and non-inhibitor-producing mice (0.88 ± 0.2 %, 0.33 ± 0.16 %, and 0.36 ± 0.2%, respectively). The Tfh/Tfr ratio also significantly increased in FVIII inhibitor-producing mice, suggesting a preferential expansion of Tfh cells relative to Tfr cells in inhibitor-producing mice. Interestingly, similar to Tfh cells as we reported before, the emergence of Tfr cells correlated with titers of anti-FVIII inhibitors in FVIII immunized mice. This indicates that a functional relationship exists between Tfr cell activation and FVIII inhibitor production after FVIII immunization. Using Tfr deficient Foxp3-Cre Bcl6 fl/fl (BCL6FC) mice, we determined the role of Tfr cells in inhibitor generation in vivo. Loss of Tfr cells led to significantly decreased FVIII inhibitor levels, with FVIII inhibitor titers in BCL6FC mice markedly lower than in wild-type (WT) mice upon recombinant human full-length FVIII (rhFVIII, Kogenate 200 U/kg/week IV x4) immunization (24 ± 16 and 131 ± 114 BU/ml, respectively). However, the total anti-FVIII IgG titers were similar between the BCL6FC and WT groups, suggesting that Tfr cells help generate neutralizing anti-FVIII antibodies. FVIII immunization could still efficiently induce Tfh cell activation in BCL6FC mice. However, both GC Tfh cells and GC B cells were reduced in FVIII immunized BCL6FC mice, suggesting that Tfr cells promote FVIII-specific GC responses. To further investigate the regulatory function of Tfr cells on FVIII immune responses, mice with phosphatase Pten deficiency in Treg cells (Foxp3-Cre Pten fl/fl [PtenFC]), a model with augmented Treg and Tfr cells, were immunized with rhFVIII and anti-FVIII antibody titers were measured at 1 week after the 4 th weekly immunization. Of note, FVIII inhibitor titers (8.06 ± 8.6 BU/ml) were significantly lower than those in WT controls under the same immunization protocol. There were significant positive correlations between the inhibitor titers and total anti-FVIII IgG in the WT and PtenFC groups but not in the BCL6FC group after rhFVIII immunization. To confirm the important role of the Tfh/Tfr pathway in FVIII inhibitor development, we used CD4-Cre Bcl6 fl/fl (CD4-Bcl6-cKO) mice, a model with Bcl6 deficiency in all CD4 + T cells. As expected, none of the CD4-Bcl6-cKO mice developed detectable levels of FVIII inhibitors, demonstrating that the transcription repressor BCL6 is pivotal for FVIII inhibitor development. In conclusion, we found that FVIII immunization induces Tfr cell activation and expansion. Tfr cells have a dual function in regulating FVIII immune responses in mice, and the Tfh/Tfr pathway is essential for FVIII inhibitor development in hemophilia A mice.

Optical tweezers experiments reveal anticancer drug BI-3802 binds DNA in the micromolar range.

B-cell lymphoma is a common form of cancer associated with the lymphatic system. A recently developed anticancer drug BI-3802 is known to target B-cell lymphoma by binding to transcription repressor BCL6 in order to kill cancerous cells. Many small molecule drugs like BI-3802 that are dissolved in DMSO have been a challenge to study with optical tweezers since DMSO changes the refractive index of the medium. Here we have carefully conducted control experiments to estimate the concentration limits of DMSO to study these drugs. Staying within these limits (1:400 volume ratio of DMSO to Buffer), we use optical tweezers to trap a single DNA molecule to explore the binding of BI-3802 with DNA. Once we trap a single DNA molecule, we stretch the DNA and measure the tension in the DNA as a function of extension. During this process, the DNA undergoes a phase transition from double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA) at a certain force which is commonly referred to as the overstretching transition. Based on its structure we believed that this small molecule drug would bind to DNA through intercalation. However, our results suggest that the force where the phase transition from dsDNA to ssDNA occurs is decreased as the concentration of BI-3802 is increased. This indicates that this drug destabilizes the naturally occurring double-stranded form of DNA, which is the opposite effect of intercalating drugs. These effects were observed only at micromolar concentrations of BI-3802, whereas it is used in nanomolar concentrations in targeting BCL6. Even though this is weak DNA binding, the outcome of these experiments may help us better understand BI-3802 interactions at the molecular level and help develop new therapeutics.

291 (PB071) - Improved binding affinity and pharmacokinetics enables sustained degradation of BCL6 in vivo

Background: The transcriptional repressor BCL6 is an oncogenic driver often found to be deregulated in lymphoid malignancies. We report the optimisation of our previously reported benzimidazolone molecular glue type degrader CCT369260 to CCT373566, a highly potent probe suitable for sustained depletion of BCL6 in vivo.

Improved Binding Affinity and Pharmacokinetics Enable Sustained Degradation of BCL6 In Vivo.

The transcriptional repressor BCL6 is an oncogenic driver found to be deregulated in lymphoid malignancies. Herein, we report the optimization of our previously reported benzimidazolone molecular glue-type degrader CCT369260 to CCT373566, a highly potent probe suitable for sustained depletion of BCL6 in vivo. We observed a sharp degradation SAR, where subtle structural changes conveyed the ability to induce degradation of BCL6. CCT373566 showed modest in vivo efficacy in a lymphoma xenograft mouse model following oral dosing.

The Cdkn2a gene product p19ARF (alternative reading frame) is a critical regulator of IFNβ-mediated Lyme arthritis

Abstract Lyme disease is caused by infection with the tick-borne spirochete Borrelia burgdorferi, with a spectrum of clinical disorders. Infected C3H mice develop severe arthritis while B6 mice display mild arthritis, allowing analysis of host genetic contribution to disease. B6 mice introgressed with the C3H allele of Bbaa1 on Chr4, B6.C3-Bbaa1, display increased severity of arthritis, and heightened expression of Type I IFN. IFNβ, was identified as the key effector for arthritis severity, acting through the muscle regulatory protein myostatin. To identify regulators of IFNβ in the Bbaa1 locus, candidate genes were subjected to siRNA silencing in macrophages. The Cdkn2a gene encoded protein p19ARF was identified as the modulator of IFNβ expression. B6 Arf−/− mice reconstituted with cells expressing the C3H allele of p19ARF developed severe arthritis, whereas mice reconstituted with cells expressing the B6 allele developed mild disease. IFNβ induction by p19ARF is inducible by other pathogens and a variety of PAMPs. P19ARF regulation of IFNβ involved the tumor suppressor p53 and transcription repressor BCL6 in myeloid cells. Indeed, targeted blocking of BCL6 enhanced IFNβ activation in the joint tissue of B6 mice and resulted in increased severity of Lyme arthritis. Similar responses in B6 Rag1−/− mice, indicate dependence on myeloid cells expression of BCL6. Thus, we have identified novel involvement of p19ARF in modulating IFNβ expression in Lyme arthritis development. Supported by R01 AR043521

The Cdkn2a gene product p19 alternative reading frame (p19ARF) is a critical regulator of IFNβ-mediated Lyme arthritis.

Type I interferon (IFN) has been identified in patients with Lyme disease, and its abundant expression in joint tissues of C3H mice precedes development of Lyme arthritis. Forward genetics using C3H mice with severe Lyme arthritis and C57BL/6 (B6) mice with mild Lyme arthritis identified the Borrelia burgdorferi arthritis-associated locus 1 (Bbaa1) on chromosome 4 (Chr4) as a regulator of B. burgdorferi-induced IFNβ expression and Lyme arthritis severity. B6 mice introgressed with the C3H allele for Bbaa1 (B6.C3-Bbaa1 mice) displayed increased severity of arthritis, which is initiated by myeloid lineage cells in joints. Using advanced congenic lines, the physical size of the Bbaa1 interval has been reduced to 2 Mbp, allowing for identification of potential genetic regulators. Small interfering RNA (siRNA)-mediated silencing identified Cdkn2a as the gene responsible for Bbaa1 allele-regulated induction of IFNβ and IFN-stimulated genes (ISGs) in bone marrow-derived macrophages (BMDMs). The Cdkn2a-encoded p19 alternative reading frame (p19ARF) protein regulates IFNβ induction in BMDMs as shown by siRNA silencing and overexpression of ARF. In vivo studies demonstrated that p19ARF contributes to joint-specific induction of IFNβ and arthritis severity in B. burgdorferi-infected mice. p19ARF regulates B. burgdorferi-induced IFNβ in BMDMs by stabilizing the tumor suppressor p53 and sequestering the transcriptional repressor BCL6. Our findings link p19ARF regulation of p53 and BCL6 to the severity of IFNβ-induced Lyme arthritis in vivo and indicate potential novel roles for p19ARF, p53, and BCL6 in Lyme disease and other IFN hyperproduction syndromes.

Exploring the binding of anticancer drug BI-3802 to DNA using optical tweezers

BI-3802 is a relatively new anticancer drug known to bind to the transcription repressor BCL6 and potently kill the most common form of cancer cells, B-cell lymphoma, found in the lymphatic system. Based on its structure we believe this small molecule drug can bind to DNA through intercalation. We use Optical tweezers to trap a single DNA molecule to explore the binding of this BI-3802 with DNA. Once we trap a single DNA molecule, we stretch the DNA and measure the tension in the DNA as a function of extension.

Making memories with Bcl-6.

For over a decade, mutual antagonism between the transcriptional repressors Bcl-6 and Blimp-1 has been appreciated as a key mechanistic determinant of lymphoid differentiation programs. Now, in this issue of JEM, Ciucci et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20202343) demonstrate that this relationship is "central" to the generation of T cell memory.

Dysregulation of Epigenetic Landscape Uncovered the Mechanisms Underlying the Relapse of Pediatric Acute Lymphoblastic Leukemia with NSD2 Mutation

Background: Relapse from acute lymphoblastic leukemia (ALL) is one of the most common causes of pediatric cancer-related death. Early relapse of ALL is associated with recurrent mutations of histone methyltransferase NSD2 (nuclear receptor binding SET domain protein 2), which is specific for H3K36me2, suggesting a link to therapy resistance or other mechanisms underlying relapse such as central neural system (CNS) infiltration. NSD2 p.E1099K affects gene expression through disturbing the balance of H3K36me2/H3K27me3. Using CRISPR/Cas9-edited isogenic ALL cell lines and PDX cells, we found that NSD2 p.E1099K drives oncogenic programming, CNS infiltration and glucocorticoid (GC) resistance. However, the molecular mechanisms underlying the relapse of this subtype of ALL are still under investigation.Aim: To elucidate the epigenetic landscape underlying the mechanism of the relapse of pediatric ALL with NSD2 mutation.Methods: We performed in vivo experiments to observe tumor burden, leukemia cell infiltration and survival of the NOD/SCID mice injected with a NSD2 p.E1099K mutation knock-out SEM cell line and knock-in CEM cell line. We determined transcriptome (RNA-Seq), chromatin accessibility (ATAC-Seq) in isogenic RCH-ACV, SEM, RPMI-8402 and CEM cell lines, transcription factor binding and histone modification (ChIP-Seq) and 3D organization (Hi-C) in RCH-ACV cells. Finally, we integrated analysis of RNA-Seq, ATAC-Seq, ChIP-Seq and Hi-C to comprehensively disclose the epigenetic landscape in ALL with NSD2 mutation.Results: NOD/SCID mice xenografted with NSD2 mutant cells developed high tumor burden and infiltration to spleen, liver and brain while the mice injected with WT cells accumulated significant less tumor cells and had extended survival. RNA-Seq analysis showed that reversion of NSD2 mutation to WT caused more genes downregulated while insertion of NSD2 mutation to WT cells led to more genes upregulated (Mutant vs WT: RCH-ACV 838 vs 494, SEM 1567 vs 1158, RPMI-8402 1922 vs 1745, CEM 1809 vs 1031). 50 upregulated genes and 3 downregulated genes were in common in B-ALL and T-ALL with NSD2 mutation. Most of the upregulated genes correlated with neural development and adhesion which might contribute to CNS infiltration (e.g., NCAM1 and NEO1). A few genes were associated with GC resistance such as decreased NR3C1 and increased NR3C2. Accordingly, ATAC-Seq showed that NSD2 mutant cells had more open chromatin peaks than those of WT (RCH-ACV 4853 vs 3212, SEM 10052 vs 7595, RPMI-8402 20392 vs 12133, CEM 10155 vs 6437). ChIP-Seq revealed general large gains of H3K36me2 in intergenic regions in NSD2 mutant cells. Importantly, genes upregulated with NSD2 mutation (e.g., NCAM1 and NEO1) lost H3K27me3 at promoters but gained H3K36me2 at promoters and whole gene bodies, accompanied with increased H3K27ac at enhancers. Conversely, a small subset of genes gained H3K27me3 and lost H3K36me2 in their promoters. Concentrated H3K36me2 in gene bodies diffused and broadened was less prominent and H3K27me3 accumulation became dominant. This for example was associated with repression of NR3C1 to drive GC resistance of NSD2 mutant cells. Genes upregulated in NSD2 mutant cells were enriched for binding sites for lymphoid transcriptional activators such as EBF1 and IRF2. The promoters of the downregulated genes had motifs for transcription factors poorly expressed in lymphoid cells and were enriched for binding sites for the BCL6 transcriptional repressor. Hi-C analysis revealed 430 topologically associated domains (TADs) with increased loop interactions while 136 TADs with decreased interactions were in NSD2 mutant cells compared to WT cells. Overall, 491 regions switched from compartment A to B and 444 regions switched from B to A in NSD2 mutant cells compared to WT cells. Compartment switching from inactive B to active A correlated with upregulated gene expression levels while the reverse was true for switching from A to B. Increased intra-TAD interactions were linked to upregulated genes while decreased intra-TAD interactions were linked to downregulated genes.Conclusions: The NSD2 mutation led to increased tumor burden, CNS infiltration and glucocorticoid resistance due to dysregulation of epigenetic patterns and 3D chromatin architecture, indicating mechanisms underlying the relapse of pediatric ALL and potential therapeutic targets in ALL with NSD2 mutation. DisclosuresLicht: Epizyme: Research Funding.

HOX gene regulation in glioma stem cells, mediators of resistance in breast cancer, and paradoxical therapeutic interventions

Therapy resistance remains a major challenge during the treatment of patients with advanced cancer. Due to the complex nature of the cellular and molecular mechanisms underlying therapy resistance, research efforts to tackle this challenge have focused on diverse aspects: from cancer stem cell (CSC) biology, therapy resistance-specific signatures to the development of new combinatorial therapeutic approaches. Among other articles, the August issue of Molecular Oncology includes several studies that directly or indirectly explore therapy resistance in patients with advanced cancer. In some cancers, therapeutic resistance has been linked to the presence of CSCs. In glioma, for example, glioma stem cells (GSCs) represent a tumor cell subpopulation underlying tumor relapse. Understanding the molecular mechanisms behind GSC gene expression may give new insights on tumorigenesis and tumor relapse. In their recent study, Le Boiteux et al. [[1]] analyzed six patient-derived GSC lines, as well as 70 primary adult diffuse glioma samples to map the mechanisms leading to deregulation of the HOX gene clusters, which have been previously implicated in the oncogenic potential of GSCs. The authors identified widespread reactivation of HOX genes in adult IDH wild-type (IDHwt) diffuse glioma samples and GSCs. HOX gene deregulation was associated with self- and cross-regulatory interactions between HOX transcription factors, but not with genomic rearrangements. Integrative analysis of high-throughput DNA methylation analysis, ChIPseq, and RNAseq data demonstrated that the absence of hypermethylation on canonical and alternative bivalent CpG islands in promoters, as well as loss of the H3K27me3 epigenetic mark, underlies aberrant gene expression of HOX clusters in IDHwt gliomas. Besides providing a comprehensive epigenetic analysis of HOX gene regulation in glioma, the study by Le Boiteux et al. highlights the complexity of HOX gene expression via alternative promoters in cancer. In a different setting, Mohammad Sultan and colleagues employed an in vivo genome-wide shRNA screen to identify potential mediators or biomarkers of paclitaxel resistance in patients with advanced breast cancer [[2]]. The transcriptional repressor BCL6 was identified among the top paclitaxel resistance hits, and BCL6 overexpression was detected in tumors of chemotherapy-resistant patients. To further test the role of BCL6 in paclitaxel resistance, the authors performed BCL6 knockdown and BCL6 inhibitor experiments both in vitro and in vivo, in a mouse model of triple-negative breast cancer. BCL6 targeting in preclinical models enhanced the efficacy of paclitaxel treatment. Thus, BCL6 may serve as a biomarker for drug resistance and may potentially be targeted for the treatment of patients with breast cancer that have developed paclitaxel resistance. In addition to chemotherapy, targeted therapies designed to block mitogenic signaling pathways are often used for the treatment of patients with cancer. However, drug resistance to these therapeutic agents represents a major challenge for the treatment of advanced cancer. In their perspective, Matheus Henrique Dias and Rene Bernards explore the prospects of a ‘paradoxical intervention’ strategy for the treatment of therapy-resistant cancers [[3]]. Such a strategy would involve overactivation, rather than inhibition, of mitotic signaling in cancer cells. In their view, such an approach would result in an overload of the stress response pathways and disruption of cancer cell homeostasis and viability. The authors review related data from several independent experimental models, discuss future perspectives, and explore the potential drawbacks of such a paradoxical intervention strategy for the treatment of patients with cancer.

Distinct roles of androgen receptor, estrogen receptor alpha, and BCL6 in the establishment of sex-biased DNA methylation in mouse liver

Sexual dimorphism in gene regulation, including DNA methylation, is the main driver of sexual dimorphism in phenotypes. However, the questions of how and when sex shapes DNA methylation remain unresolved. Recently, using mice with different combinations of genetic and phenotypic sex, we identified sex-associated differentially methylated regions (sDMRs) that depended on the sex phenotype. Focusing on a panel of validated sex-phenotype dependent male- and female-biased sDMRs, we tested the developmental dynamics of sex bias in liver methylation and the impacts of mutations in the androgen receptor, estrogen receptor alpha, or the transcriptional repressor Bcl6 gene. True hermaphrodites that carry both unilateral ovaries and contralateral testes were also tested. Our data show that sex bias in methylation either coincides with or follows sex bias in the expression of sDMR-proximal genes, suggesting that sex bias in gene expression may be required for demethylation at certain sDMRs. Global ablation of AR, ESR1, or a liver-specific loss of BCL6, all alter sDMR methylation, whereas presence of both an ovary and a testis delays the establishment of male-type methylation levels in hermaphrodites. Moreover, the Bcl6-LKO shows dissociation between expression and methylation, suggesting a distinct role of BCL6 in demethylation of intragenic sDMRs.

Bcl6-Independent In Vivo Development of Functional Type 1 Classical Dendritic Cells Supporting Tumor Rejection.

The transcriptional repressor Bcl6 has been reported as required for development of a subset of classical dendritic cell (cDCs) called cDC1, which is responsible for cross-presentation. However, mechanisms and in vivo functional analysis have been lacking. We generated a system for conditional deletion of Bcl6 in mouse cDCs. We confirmed the reported in vitro requirement for Bcl6 in cDC1 development and the general role for Bcl6 in cDC development in competitive settings. However, deletion of Bcl6 did not abrogate the in vivo development of cDC1. Instead, Bcl6 deficiency caused only a selective reduction in CD8α expression by cDC1 without affecting XCR1 or CD24 expression. Normal cDC1 development was confirmed in Bcl6cKO mice by development of XCR1+ Zbtb46-GFP+ cDC1 by rejection of syngeneic tumors and by priming of tumor-specific CD8 T cells. In summary, Bcl6 regulates a subset of cDC1-specific markers and is required in vitro but not in vivo for cDC1 development.

BCL6 Enables RAS-Driven Pre-B Cell Transformation in Acute Lymphoblastic Leukemia

Background and Hypothesis: The transcriptional repressor and proto-oncogene BCL6 was previously identified to be a factor that bypasses oncogene-induced senescence (OIS) caused by activation of oncogenic RAS (Shvarts A. et al. 2002). Recently, we discovered a novel BCL6-dependent mechanism of drug resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL; Duy et al., 2011). Given that activating genetic lesions in the RAS pathway are found in ~50% of high-risk childhood pre-B ALL at diagnosis (Zhang et al., 2012), we tested the concept that BCL6 may facilitate RAS-mediated transformation of pre-B cells by suppressing the OIS-program.Results: To examine the consequences of acute activation of RAS-ERK signaling on Bcl6 expression, we engineered murine pre-B cells to carry a doxycycline (Dox)-inducible TetOn- NRASG12D vector system. Following induction with Dox, strong upregulation of Bcl6 expression was observed at both the mRNA and protein levels, which were abrogated upon treatment with a MEK inhibitor (PD325901). These findings suggest that increased Bcl6 levels were a consequence of ERK activation. Similarly, inducible expression of BRAF V600E mutant resulted in elevated levels of Bcl6 in parallel with increased phosphorylation of ERK1/2- T202/Y204. Furthermore, studying a diagnostic (KRAS wild-type) and a relapsed (KRASG12V) sample from one pre-B ALL patient revealed hyperactivation of ERK in association with increased BCL6 levels in KRASG12V relapsed ALL cells.Previously, we showed that BCL6 expression is repressed by STAT5 in pre-B ALL (Duy C. et al. 2011). Interestingly, while overexpression of oncogenic NRASG12D resulted in hyperactivation of ERK in pre-B cells, phosphorylation of STAT5-Y694 was drastically inhibited. Reverse Phase Protein Array (RPPA) measurements revealed that phosphorylation levels of MEK-S217/221 and ERK1/2-T202/Y204 were inversely correlated with phosphorylation of STAT5-Y694 in newly diagnosed cases of adult ALL (MDACC, 1983-2007). In addition, genetic lesions causing activation of the RAS-ERK and STAT5 pathways were found to be mutually exclusive in the majority of high-risk childhood pre-B ALL cases studied (COG P9906). Mechanistically, expression of oncogenic NRASG12D resulted in activation of PTPN6 (SHP1), an inhibitory protein tyrosine phosphatase. Loss of Ptpn6 function resulted in elevated levels of phospho-STAT5-Y694 and suppressed imatinib-mediated induction of Bcl6 expression in BCR-ABL1 -driven pre-B ALL. Taken together, our findings suggest that oncogenic RAS induces BCL6 expression through PTPN6-mediated dephosphorylation of STAT5.Our observations that RAS-ERK signaling induces BCL6 expression prompted us to examine the role of BCL6 in RAS-mediated malignant transformation of pre-B cells. Activation of oncogenic RAS increased expression of cell cycle checkpoint regulators CDKN1A (p21) and CDKN1B (p27). BCL6 has previously been shown to mediate transcriptional repression of CDKN1A and CDKN1B (Duy C. et al. 2011). Notably, ChIP-seq analysis of patient-derived KRASG12V pre-B ALL cells revealed binding of BCL6 across the loci of cell cycle checkpoint regulators CDKN1A, CDKN1B, CDKN2D and TP53BP1, as well as pro-apoptotic molecules BIM and BMF . Consistent with these findings, Cre-mediated deletion of Bcl6 in pre-B cells expressing NRASG12D led to depletion of cells in competitive-growth assays and impaired the ability to form colonies in semi-solid methylcellulose. Moreover, expression of NRASG12Din Bcl6+/+ pre-B cells resulted in transformation and fatal leukemia in transplant recipient mice. In contrast, Bcl6-/- pre-B cells transduced with NRASG12D failed to initiate fatal disease in transplant recipients.Conclusions: Our findings suggest that activation of oncogenic RAS enables the switch from STAT5-driven signaling to activation of BCL6, and that RAS-induced dephosphorylation of STAT5 is mediated by PTPN6. Furthermore, we demonstrated that BCL6 is required for RAS -mediated transformation of pre-B cells, suggesting that inhibition of BCL6 may provide a new therapeutic avenue. DisclosuresHurtz:Incyte: Research Funding.

Reciprocal Regulation of BCL6 and MLL Expression As a Vulnerability in MLL -Rearranged Acute Lymphoblastic Leukemia

Background: Chromosomal translocations involving the Mixed Lineage Leukemia gene MLL account for ~70% of infant acute lymphoblastic leukemias (ALLs; Ayton and Cleary, 2001) and are associated with dismal prognosis. These findings underscore the importance of understanding the molecular mechanisms underlying disease progression of MLL-rearranged (MLLr) leukemia for the development of novel therapeutics. Recently, an integrative epigenomic analysis has identified the transcriptional repressor and proto-oncogene BCL6 as a transcriptional target in MLL-AF4 -driven pre-B ALL (Geng et al., 2012). BCL6 functions as a mediator of drug resistance to tyrosine kinase inhibitors in Ph+ ALL (Duy et al., 2011). Here, we tested the hypothesis that BCL6 is required for MLLr leukemia and represents a therapeutic target in MLLr pre-B ALL.Results: Western blot analysis of a panel of patient-derived pre-B ALL cells (MLLr : n= 7; ETV6-RUNX1 : n= 2; BCR-ABL1 : n= 1; not otherwise specified: n= 4) and normal CD19+ B cells (n= 3) revealed that BCL6 levels were elevated in MLL r ALL samples. Furthermore, immunohistochemistry staining in bone marrow biopsies from ALL patients (n= 72) showed that BCL6 levels were upregulated in MLLr cases. Expression of MLL-ENL or MLL-AF4 fusion protein increased Bcl6 protein levels (~10-fold) in murine pre-B cells. Moreover, ChIP-seq analysis of human MLLr pre-B ALL cell lines using antibodies against MLL, AF4 and ENL demonstrated direct binding to the BCL6 promoter. Studying methylation status revealed that the BCL6 promoter was hypomethylated in patients with MLLr pre-B ALL compared to patients with other subtypes of pre-B ALL (including BCR-ABL1, ETV6-RUNX 1 and hyperdiploid) as well as normal pre-B cells from healthy donors (St. Jude, n= 132, Figueroa, 2014). Consistent with these observations, multivariate analysis of high risk pre-B ALL patients showed that high BCL6 levels in combination with MLL rearrangements were associated with the worst clinical outcome (COG P9906). Collectively, our findings demonstrated that BCL6 levels are elevated in MLLr ALL, and that expression levels of MLL and BCL6 can serve as predictors for risk stratification in ALL.Interestingly, expression of wild-type MLL was positively correlated with that of BCL6 in both pediatric (COG P9906) and adult (ECOG E2993) pre-B ALL. Furthermore, multivariate analysis comparing MLLHighBCL6High patients with MLLLowBCL6Low patients showed that high expression levels of both genes correlated with poor clinical outcome (COG P9906). These findings prompted us to study whether MLL regulates BCL6 expression, and vice versa . Cre-mediated deletion of Mll abrogated imatinib-mediated induction of Bcl6 in BCR-ABL1 -driven pre-B ALL, indicating that Mll is required for upregulation of Bcl6 expression. While genetic inactivation of Bcl6 as well as treatment with the BCL6-specific retro-inverso peptide-inhibitor (RI-BPI) decreased Mll expression, doxycycline-induced expression of Bcl6 upregulated Mll expression. Combining chromatin immunoprecipitation and DNA microarray (ChIP-on-chip), we found that BCL6 was recruited to the MLL promoter in pre-B ALL. Taken together, these findings support our proposed scenario of reciprocal regulation of MLL and BCL6 expression.Notably, ChIP-seq analyses of MLLr pre-B ALL cells showed that there are 268 common gene targets of both MLL and BCL6. Among the common targets include cell cycle checkpoint regulators (CDKN1B, ETS2, BMI1, RB1), apoptosis facilitator BIM, and tumor suppressor TET2, indicating that BCL6 is a key regulator of the transcriptional program in MLLr pre-B ALL. In support of an essential role of BCL6 in MLLr ALL, expression of a dominant negative BCL6 mutant in patient-derived MLLr pre-B ALL cells induced rapid depletion from cell culture in competitive-growth assays. Furthermore, RI-BPI treatment resulted in cell cycle arrest, impaired colony forming ability and inhibited leukemia initiation in transplant recipient mice. Importantly, RI-BPI in combination with standard chemotherapy Vincristine exerted more potent effects on killing MLLr pre-B ALL cells than either agent alone.Conclusions: Our findings suggest that aberrant BCL6 expression levels are important for disease progression, and that pharmacological inhibition of BCL6 augments responsiveness to standard chemotherapy in the treatment of MLLr pre-B ALL. DisclosuresHurtz:Incyte: Research Funding. Armstrong:Cyteir: Consultancy; Imago: Consultancy; C4 Therapeutics: Consultancy; Epizyme: Consultancy.

Achieving In Vivo Target Depletion through the Discovery and Optimization of Benzimidazolone BCL6 Degraders.

Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein–protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.

Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis.

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.