An orally available small molecule BCL6 inhibitor effectively suppresses diffuse large B cell lymphoma cells growth in vitro and in vivo.

The transcription factor B cell lymphoma 6 (BCL6) is an oncogenic driver of diffuse large B cell lymphoma (DLBCL) and mediates lymphomagenesis through transcriptional repression of its target genes by recruiting corepressors to its N-terminal broad-complex/tramtrack/bric-a-brac (BTB) domain. Blocking the protein-protein interactions of BCL6 and its corepressors has been proposed as an effective approach for the treatment of DLBCL. However, BCL6 inhibitors with excellent drug-like properties are rare. Hence, the development of BCL6 inhibitors is worth pursuing. We screened our internal chemical library by luciferase reporter assay and Homogenous Time Resolved Fluorescence (HTRF) assay and a small molecule compound named WK500B was identified. WK500B engaged BCL6 inside cells, blocked BCL6 repression complexes, reactivated BCL6 target genes, killed DLBCL cells and caused apoptosis as well as cell cycle arrest. In animal models, WK500B inhibited germinal center (GC) formation and DLBCL tumour growth without toxic and side effects. Moreover, WK500B displayed strong efficacy and favourable pharmacokinetics and presented superior druggability. Therefore, WK500B is a promising candidate that could be developed as an effective orally available therapeutic agent for DLBCL.

Into Deep Water: Optimizing BCL6 Inhibitors by Growing into a Solvated Pocket.

We describe the optimization of modestly active starting points to potent inhibitors of BCL6 by growing into a subpocket, which was occupied by a network of five stably bound water molecules. Identifying potent inhibitors required not only forming new interactions in the subpocket but also perturbing the water network in a productive, potency-increasing fashion while controlling the physicochemical properties. We achieved this goal in a sequential manner by systematically probing the pocket and the water network, ultimately achieving a 100-fold improvement of activity. The most potent compounds displaced three of the five initial water molecules and formed hydrogen bonds with the remaining two. Compound 25 showed a promising profile for a lead compound with submicromolar inhibition of BCL6 in cells and satisfactory pharmacokinetic (PK) properties. Our work highlights the importance of finding productive ways to perturb existing water networks when growing into solvent-filled protein pockets.

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.

Progress toward B-Cell Lymphoma 6 BTB Domain Inhibitors for the Treatment of Diffuse Large B-Cell Lymphoma and Beyond.

B-cell lymphoma 6 (BCL6) is a master regulator of germinal center formation that produce antibody-secreting plasma cells and memory B-cells for sustained immune responses. The BTB domain of BCL6 (BCL6BTB) forms a homodimer that mediates transcriptional repression by recruiting its corepressor proteins to form a biologically functional transcriptional complex. The protein-protein interaction (PPI) between the BCL6BTB and its corepressors has emerged as a therapeutic target for the treatment of DLBCL and a number of other human cancers. This Perspective provides an overview of recent advances in the development of BCL6BTB inhibitors from reversible inhibitors, irreversible inhibitors, to BCL6 degraders. Inhibitor design and medicinal chemistry strategies for the development of novel compounds will be provided. The binding mode of new inhibitors to BCL6BTB are highlighted. Also, the in vitro and in vivo assays used for the evaluation of new compounds will be discussed.

Identification BCL6 and miR-30 family associating with Ibrutinib resistance in activated B-cell-like diffuse large B-cell lymphoma.

Ibrutinib has clear efficacy for activated B-cell-like diffuse large B cell lymphoma (ABC-DLBCL) in previous clinical researches. However, the resistance of Ibrutinib has limited its therapeutic benefit and the potential mechanism remains unclear. This study was aimed to identify potential candidate genes and miRNA targets to overcome Ibrutinib resistance in ABC-DLBCL. First, two expression profiles were downloaded from the GEO database, which used to identify the DEGs related to Ibrutinib resistance in ABC-DLBCL cell lines by GEO2R analysis separately. And the common DEGs were obtained though Venn diagram. Then Gene ontology (GO) and pathway enrichment analysis were conducted by DAVID database. From STRING database, BCL6, IL10, IL2RB, IRF4, CD80, PRDM1and GZMB were determined to be the hub genes by protein-protein interaction (PPI) network. Through miRNA-mRNA targeting network, we found that BCL6, IRF4, CD80, and PRDM1 were common target genes of miR-30 family. The cBioPortal database showed that BCL6 had the highest level of genetic alterations among DLBCL. In addition, another expression profile from GEO database showed that BCL6 was significantly high expression in no responsive patients after Ibrutinib treatment, and the receiver operating characteristic (ROC) curve which was used to evaluate the relationship between BCL6 expression and its effect was 0.67. MTT assay showed that treatment with FX1 (a BCL6 inhibitor) can enhance the sensitivity of Ibrutinib in C481S BTK HBL-1 cells. The results suggested that BCL6 and miR-30 family maybe associate with Ibrutinib resistance in ABC-DLBCL.

BCL6 maintains survival and self-renewal of primary human acute myeloid leukemia cells.

B-cell lymphoma 6 (BCL6) is a transcription repressor and proto-oncogene that plays a crucial role in the innate and adaptive immune system and lymphoid neoplasms. However, its role in myeloid malignancies remains unclear. Here, we explored the role of BCL6 in acute myeloid leukemia (AML). BCL6 was expressed at variable and often high levels in AML cell lines and primary AML samples. AMLs with higher levels of BCL6 were generally sensitive to treatment with BCL6 inhibitors, with the exception of those with monocytic differentiation. Gene expression profiling of AML cells treated with a BCL6 inhibitor revealed induction of BCL6-repressed target genes and transcriptional programs linked to DNA damage checkpoints and downregulation of stem cell genes. Ex vivo treatment of primary AML cells with BCL6 inhibitors induced apoptosis and decreased colony-forming capacity, which correlated with the levels of BCL6 expression. Importantly, inhibition or knockdown of BCL6 in primary AML cells resulted in a significant reduction of leukemia-initiating capacity in mice, suggesting ablation of leukemia repopulating cell functionality. In contrast, BCL6 knockout or inhibition did not suppress the function of normal hematopoietic stem cells. Treatment with cytarabine further induced BCL6 expression, and the levels of BCL6 induction were correlated with resistance to cytarabine. Treatment of AML patient-derived xenografts with BCL6 inhibitor plus cytarabine suggested enhanced antileukemia activity with this combination. Hence, pharmacologic inhibition of BCL6 might provide a novel therapeutic strategy for ablation of leukemia-repopulating cells and increased responsiveness to chemotherapy.

Inhibition of the BCL6/miR-31/PKD1 axis attenuates oxidative stress-induced neuronal damage.

Ischemic stroke (IS) is one of the most common cerebrovascular diseases worldwide. The aberrant expression of BCL6 has been previously implicated in the pathogenesis of IS. Meanwhile, miR-31 is known as a target of BCL6, and has also been suggested to diminish cell damage by suppressing the PKD1 expression. Expanding on this relationship, the current study set out to investigate whether BCL6 participates in ischemic stroke by targeting PKD1. Firstly, IS models were established in vitro and in vivo. TUNEL staining and MTT assay were performed to examine the apoptosis and cell survival. In addition, qRT-PCR and Western blot analysis were applied to examine the expression patterns of the BCL6/miR-31/PKD1 axis and its downstream pathway. Bioinformatics analysis was used to predict the target of miR-31. It was found that BCL6 over-expression promoted ODG-induced increase of apoptosis and decreased the cell survival and miR-31 expression levels, whereas the opposite effects were noted in vitro and in vivo models of IS that were treated with shBCL6. Furthermore, miR-31 down-regulation blocked the effect of BCL6 on ODG-induced cell injury. It was also verified that miR-31 directly-targets PKD1. Also, OGD induced the PKD1 expression and activation of the JAK2/STAT3 pathway, while down-regulation of PKD1 inhibited the OGD-induced cell injury and JAK2/STAT3 pathway activation. Lastly, down-regulation of BCL6 in brain brought about a significant reduction in the size of cerebral infarction and oxidative stress levels in IS mice. Collectively, our findings suggest that inhibition of BCL6 may attenuate oxidative stress-induced neuronal damage by targeting the miR-31/PKD1 axis.

TBL1XR1 Mutations Drive Extranodal Lymphoma by Inducing a Pro-tumorigenic Memory Fate.

The most aggressive B cell lymphomas frequently manifest extranodal distribution and carry somatic mutations in the poorly characterized gene TBL1XR1. Here, we show that TBL1XR1 mutations skew the humoral immune response toward generating abnormal immature memory B cells (MB), while impairing plasma cell differentiation. At the molecular level, TBL1XR1 mutants co-opt SMRT/HDAC3 repressor complexes toward binding the MB cell transcription factor (TF) BACH2 at the expense of the germinal center (GC) TF BCL6, leading to pre-memory transcriptional reprogramming and cell-fate bias. Upon antigen recall, TBL1XR1 mutant MB cells fail to differentiate into plasma cells and instead preferentially reenter new GC reactions, providing evidence for a cyclic reentry lymphomagenesis mechanism. Ultimately, TBL1XR1 alterations lead to a striking extranodal immunoblastic lymphoma phenotype that mimics the human disease. Both human and murine lymphomas feature expanded MB-like cell populations, consistent with a MB-cell origin and delineating an unforeseen pathway for malignant transformation of the immune system.

BTB/POZ zinc finger protein ZBTB16 inhibits breast cancer proliferation and metastasis through upregulating ZBTB28 and antagonizing BCL6/ZBTB27.

BACKGROUND: Breast cancer remains in urgent need of reliable diagnostic and prognostic markers. Zinc finger and BTB/POZ domain-containing family proteins (ZBTBs) are important transcription factors functioning as oncogenes or tumor suppressors. The role and regulation of ZBTB16 in breast cancer remain to be established. METHODS: Reverse-transcription PCR and methylation-specific PCR were applied to detect expression and methylation of ZBTB16 in breast cancer cell lines and tissues. The effects of ZBTB16 in breast cancer cells were examined via cell viability, CCK8, Transwell, colony formation, and flow cytometric assays. Xenografts and immunohistochemistry analyses were conducted to determine the effects of ZBTB16 on tumorigenesis in vivo. The specific mechanisms of ZBTB16 were further investigated using Western blot, qRT-PCR, luciferase assay, and co-IP. RESULTS: ZBTB16 was frequently downregulated in breast cancer cell lines in correlation with its promoter CpG methylation status. Restoration of ZBTB16 expression led to induction of G2/M phase arrest and apoptosis, inhibition of migration and invasion, reversal of EMT, and suppression of cell proliferation, both in vitro and in vivo. Furthermore, ectopically expressed ZBTB16 formed heterodimers with ZBTB28 or BCL6/ZBTB27 and exerted tumor suppressor effects through upregulation of ZBTB28 and antagonistic activity on BCL6. CONCLUSIONS: Low expression of ZBTB16 is associated with its promoter hypermethylation and restoration of ZBTB16 inhibits tumorigenesis. ZBTB16 functions as a tumor suppressor through upregulating ZBTB28 and antagonizing BCL6. Our findings also support the possibility of ZBTB16 being a prognostic biomarker for breast cancer.

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.

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.

Perspectives on chemotherapy for the management of double-hit lymphoma.

INTRODUCTION: Double-hit (DHL) and triple-hit lymphomas (THL) have long been among the most clinically aggressive molecular subtypes of diffuse large B-cell lymphomas. In the 2016 revised WHO classification, they represent a new entity called high-grade B-cell lymphoma with rearrangements of MYC and BCL2 and/or BCL6. Unlike most B-cell lymphomas, they have poor response to standard R-CHOP therapy, tend to quickly develop resistance to cytotoxic chemotherapies, and are associated with higher central nervous system (CNS) infiltration. This can lead to increased risk of relapse and worse prognosis. DHL/THL represent a subset of lymphomas with unmet medical need. AREA COVERED: The authors present the available data for the current treatment regimens including intensive chemotherapy regimens, hematopoietic stem-cell transplantation (HSCT), and CNS prophylaxis. They also discuss treatment for relapsed disease including targeted therapies. EXPERT OPINION: There is currently no accepted standard of care for DHL/THL. For frontline therapy, we recommend enrollment in a well-designed clinical trial if possible, otherwise DA-EPOCH-R with CNS prophylaxis is a commonly used first-line therapy. The authors recommend close surveillance for patients achieving complete response, but for those who fail to achieve a complete response, then clinical trials, more aggressive salvage chemotherapy regimens, or cellular therapies are usually considered.

Synthesis and Biological Evaluation of B-Cell Lymphoma 6 Inhibitors of N-Phenyl-4-pyrimidinamine Derivatives Bearing Potent Activities against Tumor Growth.

The transcriptional repressor B-cell lymphoma 6 (BCL6) is frequently misregulated in diffuse large B-cell lymphoma (DLBCL) and has emerged as an attractive drug target for the treatments of lymphoma. In this article, a series of N-phenyl-4-pyrimidinamine derivatives were designed and synthesized as potent BCL6 inhibitors by optimizing hit compound N4-(3-chloro-4-methoxyphenyl)-N2-isobutyl-5-fluoro-2,4-pyrimidinediamine on the basis of the structure-activity relationship. Among them, compound 14j displayed the most potent activities, which significantly blocked the interaction of BCL6 with its corepressors, reactivated BCL6 target genes in a dose-dependent manner, and had better effects compared with the two positive controls. Further studies indicated that a low dose of 14j could effectively inhibit germinal center formation. More importantly, 14j not only showed potent inhibition of DLBCL cell proliferation in vitro but also strongly suppressed the growth of DLBCL in vivo.

BCL6 BTB-specific inhibition via FX1 treatment reduces Tfh cells and reverses lymphoid follicle hyperplasia in Indian rhesus macaque (Macaca mulatta).

BACKGROUND: The BTB domain of B-cell lymphoma 6 (BCL6) protein was identified as a therapeutic target for B-cell lymphoma. This study compared the pharmacokinetics (PK) of the BCL6 BTB inhibitor (FX1) between mice and macaques, as well as evaluating its lymphoid suppressive effect in uninfected macaques with lymphoid hyperplasia. MATERIALS AND METHODS: Eight uninfected adult Indian rhesus macaques (Macaca mulatta) were used in the study, four animals carrying lymphoid tissue hyperplasia. Plasma FX1 levels were measured by HPLC-MS/MS. Lymph node biopsies were used for H&E and immunohistochemistry staining, as well as mononuclear cell isolation for flow cytometry analysis. RESULTS: Inhibition of the BCL6 BTB domain with FX1 led to a reduction in the frequency of GC, Tfh CD4+ , and Tfh precursor cells, as well as resolving lymphoid hyperplasia, in rhesus macaques. CONCLUSIONS: B-cell lymphoma 6 inhibition may represent a novel strategy to reduce hyperplastic lymphoid B-cell follicles and decrease Tfh cells.

Suppression of BCL6 function by HDAC inhibitor mediated acetylation and chromatin modification enhances BET inhibitor effects in B-cell lymphoma cells.

Multiple genetic aberrations in the regulation of BCL6, including in acetyltransferase genes, occur in clinically aggressive B-cell lymphomas and lead to higher expression levels and activity of this transcriptional repressor. BCL6 is, therefore, an attractive target for therapy in aggressive lymphomas. In this study romidepsin, a potent histone deacetylase inhibitor (HDACi), induced apoptosis and cell cycle arrest in Burkitt and diffuse large B-cell lymphoma cell lines, which are model cells for studying the mechanism of action of BCL6. Romidepsin caused BCL6 acetylation at early timepoints inhibiting its function, while at later timepoints BCL6 expression was reduced and target gene expression increased due to chromatin modification. MYC contributes to poor prognosis in aggressive lymphoma. MYC function is reduced by inhibition of chromatin readers of the bromodomain and extra-terminal repeat (BET) family, which includes BRD4. The novel combination of romidepsin and JQ1, a BRD4 inhibitor was investigated and showed synergy. Collectively we suggest that the combination of HDACi and BRD4i should be pursued in further pre-clinical testing.

BCL6 inhibitor FX1 attenuates inflammatory responses in murine sepsis through strengthening BCL6 binding affinity to downstream target gene promoters.

BACKGROUND: Sepsis occurs when an infection triggers deranged inflammatory responses. There exists no efficacious treatment for this condition. The transcriptional repressor B-cell Lymphoma 6 (BCL6) is known to act as an inhibitor of macrophage-mediated inflammatory responses. FX1, a novel specific BCL6 BTB inhibitor, is able to attenuate activity of B cell-like diffuse large B cell lymphoma (ABC-DLBCL). Nevertheless, the effect of FX1 in inflammatory responses and sepsis remains unknown. OBJECTIVES: Here, we explored the effect and potential mechanisms of FX1 on the regulation of LPS-induced inflammatory responses in murine sepsis. METHOD: Mice models of LPS-induced sepsis were monitored for survival rate following FX1 administration. ELISA was used to assess how FX1 administration affected pro-inflammatory cytokines present in macrophages exposed to LPS and in the serum of mice sepsis models. Flow cytometric analysis, Western blot and qRT-PCR were performed to evaluate differences in macrophages immune responses after FX1 pre-treatment. Finally, the affinity of BCL6 binding to downstream target genes was checked by ChIP. RESULTS: The survival rate of mice models of LPS-induced sepsis was improved in following FX1 administration. FX1 decreased the production of inflammatory cytokines, attenuated macrophage infiltration activities and reduced monocytes chemotaxis activities, all of which suggest that FX1 exert anti-inflammatory effects. Mechanistically, FX1 may enhance the affinity of BCL6 binding to downstream target pro-inflammatory genes. CONCLUSIONS: These findings illustrated the anti-inflammatory properties and potential mechanisms of FX1 in sepsis caused by LPS. FX1 could potentially become a new immunosuppressive and anti-inflammatory drug candidate in sepsis therapy.

Bcl6 knockdown aggravates hypoxia injury in cardiomyocytes via the P38 pathway.

B-cell lymphoma 6 (Bcl6) functions as a sequence-specific transcriptional repressor and negative regulator of many signaling proteins. The effects of Bcl6 on cardiomyocyte injury are not clear. This study was designed to determine whether Bcl6 affects hypoxia-induced cardiomyocyte injury and, if so, to identify the underlying mechanism. To meet this aim, cardiomyocytes were exposed to hypoxia and Bcl6 siRNA was used to silence Bcl6 in cardiomyocytes. Bcl6 knockdown under physiological conditions caused increased oxidative stress, apoptosis, and expression of pro-inflammatory cytokines. Increased inflammatory response, oxidative stress, and apoptosis were observed after cells were exposed to hypoxia for 24 h. Bcl6 knockdown aggravated cardiomyocyte injury when exposed to hypoxia. Bcl6 knockdown increased P38 activation without affecting JNK and ERK phosphorylation levels. Treatment with a P38 inhibitor reversed the Bcl6 silencing-induced deteriorating phenotype, as evidenced by reduced inflammatory response, improved oxidative stress response, and increased cell viability. The results indicate that Bcl6 knockdown causes cardiomyocyte injury at baseline conditions and aggravates cardiomyocyte hypoxia injury via activating the P38 pathway.

Development of a Novel B-Cell Lymphoma 6 (BCL6) PROTAC To Provide Insight into Small Molecule Targeting of BCL6.

B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.

Identification of Thiourea-Based Inhibitors of the B-Cell Lymphoma 6 BTB Domain via NMR-Based Fragment Screening and Computer-Aided Drug Design.

Protein-protein interactions (PPI) between the transcriptional repressor B-cell lymphoma 6 (BCL6) BTB domain (BCL6BTB) and its corepressors have emerged as a promising target for anticancer therapeutics. However, identification of potent, drug-like inhibitors of BCL6BTB has remained challenging. Using NMR-based screening of a library of fragment-like small molecules, we have identified a thiourea compound (7CC5) that binds to BCL6BTB. From this hit, the application of computer-aided drug design (CADD), medicinal chemistry, NMR spectroscopy, and X-ray crystallography has yielded an inhibitor, 15f, that demonstrated over 100-fold improved potency for BCL6BTB. This gain in potency was achieved by a unique binding mode that mimics the binding mode of the corepressor SMRT in the aromatic and the HDCH sites. The structure-activity relationship based on these new inhibitors will have a significant impact on the rational design of novel BCL6 inhibitors, facilitating the identification of therapeutics for the treatment of BCL6-dependent tumors.

Activation and Induction of Antigen-Specific T Follicular Helper Cells Play a Critical Role in Live-Attenuated Influenza Vaccine-Induced Human Mucosal Anti-influenza Antibody Response.

There is increasing interest recently in developing intranasal vaccines against respiratory tract infections. The antibody response is critical for vaccine-induced protection, and T follicular helper cells (TFH) are considered important for mediating the antibody response. Most data supporting the role for TFH in the antibody response are from animal studies, and direct evidence from humans is limited, apart from the presence of TFH-like cells in blood. We studied the activation and induction of TFH and their role in the anti-influenza antibody response induced by a live-attenuated influenza vaccine (LAIV) in human nasopharynx-associated lymphoid tissue (NALT). TFH activation in adenotonsillar tissues was analyzed by flow cytometry, and anti-hemagglutinin (anti-HA) antibodies were examined following LAIV stimulation of tonsillar mononuclear cells (MNC). Induction of antigen-specific TFH by LAIV was studied by flow cytometry analysis of induced TFH and CD154 expression. LAIV induced TFH proliferation, which correlated with anti-HA antibody production, and TFH were shown to be critical for the antibody response. Induction of TFH from naive T cells by LAIV was shown in newly induced TFH expressing BCL6 and CD21, followed by the detection of anti-HA antibodies. Antigen specificity of LAIV-induced TFH was demonstrated by expression of the antigen-specific T cell activation marker CD154 upon challenge by H1N1 virus antigen or HA. LAIV-induced TFH differentiation was inhibited by BCL6, interleukin-21 (IL-21), ICOS, and CD40 signaling blocking, and that diminished anti-HA antibody production. In conclusion, we demonstrated the induction by LAIV of antigen-specific TFH in human NALT that provide critical support for the anti-influenza antibody response. Promoting antigen-specific TFH in NALT by use of intranasal vaccines may provide an effective vaccination strategy against respiratory infections in humans.IMPORTANCE Airway infections, such as influenza, are common in humans. Intranasal vaccination has been considered a biologically relevant and effective way of immunization against airway infection. The vaccine-induced antibody response is crucial for protection against infection. Recent data from animal studies suggest that one type of T cells, TFH, are important for the antibody response. However, data on whether TFH-mediated help for antibody production operates in humans are limited due to the lack of access to human immune tissue containing TFH In this study, we demonstrate the induction of TFH in human immune tissue, providing critical support for the anti-influenza antibody response, by use of an intranasal influenza vaccine. Our findings provide direct evidence that TFH play a critical role in vaccine-induced immunity in humans and suggest a novel strategy for promoting such cells by use of intranasal vaccines against respiratory infections.