B cell–intrinsic TBK1 is essential for germinal center formation during infection and vaccination in mice

The germinal center (GC) is a site where somatic hypermutation and clonal selection are coupled for antibody affinity maturation against infections. However, how GCs are formed and regulated is incompletely understood. Here, we identified an unexpected role of Tank-binding kinase-1 (TBK1) as a crucial B cell–intrinsic factor for GC formation. Using immunization and malaria infection models, we show that TBK1-deficient B cells failed to form GC despite normal Tfh cell differentiation, although some malaria-infected B cell–specific TBK1-deficient mice could survive by GC-independent mechanisms. Mechanistically, TBK1 phosphorylation elevates in B cells during GC differentiation and regulates the balance of IRF4/BCL6 expression by limiting CD40 and BCR activation through noncanonical NF-κB and AKTT308 signaling. In the absence of TBK1, CD40 and BCR signaling synergistically enhanced IRF4 expression in Pre-GC, leading to BCL6 suppression, and therefore failed to form GCs. As a result, memory B cells generated from TBK1-deficient B cells fail to confer sterile immunity upon reinfection, suggesting that TBK1 determines B cell fate to promote long-lasting humoral immunity.

The Oncoprotein BCL6 Enables Cancer Cells to Evade Genotoxic Stress

Genotoxic agents remain the mainstay of cancer treatment. Unfortunately, the clinical benefits are often countered by a rapid tumor adaptive response. Here, we report that oncoprotein B cell lymphoma 6 (BCL6) is a core component that confers tumor adaptive resistance to genotoxic stress. Multiple genotoxic agents promoted BCL6 transactivation, which was positively correlated with a weakened therapeutic efficacy and a worse clinical outcome. Mechanistically, we discovered that treatment with the genotoxic agent etoposide led to the transcriptional reprogramming of multiple pro-inflammatory cytokines, among which the interferon-α and interferon-γ responses were substantially enriched in resistant cells. Our results further revealed that the activation of interferon/signal transducer and activator of transcription 1 axis directly upregulated BCL6 expression. The increased expression of BCL6 further repressed the tumor suppressor PTEN and consequently enabled resistant cancer cell survival. Accordingly, the targeted inhibition of BCL6 remarkably enhanced etoposide-triggered DNA damage and apoptosis both in vitro and in vivo. Our findings highlight the importance of BCL6 signaling in conquering tumor tolerance to genotoxic stress, further establishing a rationale for a combined approach with genotoxic agents and BCL6-targeted therapy.

UHRF1 downregulation promotes T follicular helper cell differentiation by increasing BCL6 expression in SLE

Background Transcription factor B cell lymphoma 6 (BCL6) is a master regulator of T follicular helper (Tfh) cells, which play a crucial role in the pathogenesis of systemic lupus erythematosus (SLE). However, the mechanisms by which BCL6 expression is regulated are poorly understood. Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an important epigenetic factor that regulates DNA methylation and histone modifications. In the present study, we assessed whether UHRF1 can regulate BCL6 expression and influence the differentiation and proliferation of Tfh cells. Results Compared to healthy controls, the mean fluorescence intensity of UHRF1 (UHRF1-MFI) in Tfh cells from SLE patients was significantly downregulated, whereas that of BCL6 (BCL6-MFI) was significantly upregulated. In vitro, UHRF1 knockdown led to BCL6 overexpression and promoted Tfh cell differentiation. In contrast, UHRF1 overexpression led to BCL6 downregulation and decreased Tfh cell differentiation. In vivo, conditional UHRF1 gene knockout (UHRF1-cKO) in mouse T cells revealed that UHRF1 depletion can enhance the proportion of Tfh cells and induce an augmented GC reaction in mice treated with NP-keyhole limpet hemocyanin (NP-KLH). Mechanistically, UHRF1 downregulation can decrease DNA methylation and H3K27 trimethylation (H3K27me3) levels in the BCL6 promoter region of Tfh cells. Conclusions Our results demonstrated that UHRF1 downregulation leads to increased BCL6 expression by decreasing DNA methylation and H3K27me3 levels, promoting Tfh cell differentiation in vitro and in vivo. This finding reveals the role of UHRF1 in regulating Tfh cell differentiation and provides a potential target for SLE therapy.

IRF4 overexpression promotes the transdifferentiation of tregs into macrophage‐like cells to inhibit the development of colon cancer

Background Interferon regulatory factor 4 (IRF4) is a transcription factor from the IRF factor family that exerts regulatory functions in the immune system and oncogenesis. However, the biological role of IRF4 in colon cancer is still unclear. The aim of this study is to investigate whether IRF4 participates in the immune response in colon cancer. Methods We compared the expression of IRF4, the number of regulatory T cells (Tregs) and macrophages in the colon cancer tissues and paracancerous colon tissues from colon cancer patients. Colon cancer mouse model was established by inoculation with colon cancer cells (SW480) as a xenograft tumor, and we observed tumor growth of colon cancer. Furthermore, the mechanism of action of IRF4 in transdifferentiation of Tregs into macrophage-like cells and the effect of IRF4 on colon cancer cells were investigated in vitro. Results IRF4 was severely down-regulated in the colon cancer tissues. Colon cancer tissues exhibited an increase in the number of regulatory T cells (Tregs) and macrophages. Furthermore, IRF4 overexpression repressed proliferation, migration and invasion of colon cancer cells (SW480 and HT116 cells). Moreover, IRF4 up-regulation ameliorated tumor growth of colon cancer by promoting the transdifferentiation of Tregs into macrophage-like cells through inhibition of BCL6 expression. Exosomes derived from colon cancer cells repressed IRF4 expression in Tregs by transmitting miR-27a-3p, miR-30a-5p and miR-320c. Conclusions IRF4 overexpression promoted the transdifferentiation of Tregs into macrophage-like cells to inhibit the occurrence and development of colon cancer. Thus, IRF4 may be a potential target for colon cancer treatment.

LncRNA PCED1B-AS1 is Overexpressed in Hepatocellular Carcinoma and Regulates miR-10a/BCL6 axis to Promote Cell Proliferation

Background: PCED1B-AS1 has been characterized as an oncogene in glioma, while its role in hepatocellular carcinoma (HCC) is unknown. This study was performed to analyze the involvement of PCED1B-AS1.Methods: Expression of PCED1B-AS1 in paired HCC and non-tumor tissues from 62 HCC patients was determined by RT-qPCR. The correlation between PCED1B-AS1 and miR-10a or BCL6 was analyzed by linear regression. The 62 HCC patients were followed up for 5 years to analyze the prognostic value of PCED1B-AS1 for HCC. The interactions among PCED1B-AS1, miR-10a and BCL6 were analyzed by overexpression experiments. Cell proliferation was analyzed by CCK-8 assay.Results: PCED1B-AS1 was upregulated in HCC and predicted poor survival. Across HCC tissues, PCED1B-AS1 was inversely correlated with miR-10a, and positively correlated with BCL6 mRNA. In HCC cells, PCED1B-AS1 overexpression mediated the downregulation of miR-10a and upregulation of BCL6. Moreover, PCED1B-AS1 overexpression reduced the inhibitory effects of miR-10a overexpression on BCL6 expression and cell proliferation. Conclusions: PCED1B-AS1 is overexpressed in HCC and regulates miR-10a/BCL6 axis to promote cell proliferation.

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

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 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 inhibitor 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 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 HSCs. Treatment with cytarabine (AraC) further induced BCL6 expression, and the levels of BCL6 induction were correlated with resistance to AraC. Treatment of AML patient derived xenografts (PDX) with BCL6 inhibitor plus Ara-C suggested enhanced anti-leukemia activity by this combination. Hence pharmacological inhibition of BCL6 might provide a novel therapeutic strategy for ablation of leukemia-repopulating cells and increased responsiveness to chemotherapy.