B-cell Lymphoma 9 Research Articles

BCL9 is a Risk Factor of Neck Lymph Nodes Metastasis and Correlated with Immune Cell Infiltration in Papillary Thyroid Carcinoma.

B-cell lymphoma 9 (BCL9), a key transcription co-activator of the Wnt pathway, contributed to tumor progression and metastasis in various tumors, whereas, the role of BCL9 in papillary thyroid cancer (PTC) has not been investigated. We acquired PTC gene expression data from The Cancer Genome Atlas (TCGA) database. Fifty-nine PTC tissues were applied to validate the clinical significance of BCL9. Cell experiments were applied to investigate the role of BCL9. Bioinformatics analysis was employed to investigate the biological functions of BCL9. We found that BCL9 was higher expressed (P < 0.05) and an independent risk factor for lymph node metastasis (OR = 3.770, P = 0.025), as well as associated with poorer progression-free survival (PFS) (P = 0.049) in PTC. BCL9 knockdown inhibited proliferation and invasion of PTC cells. BCL9 was positively associated with the key genes of Wnt/β-catenin and MAPK pathway by co-expression analysis. GO, KEGG and GSEA analysis showed BCL9 might participated in PPAR, cAMP, and focal adhesion pathway. CIBERSORT analysis found BCL9 was negatively associated with CD8+ T cells and NK cell infiltration and positively with PD-L1 expression. Therefore, BCL9 was associated with lymph node metastasis and shorter PFS of PTC, due to promotion of PTC cell proliferation and invasion, activation of Wnt/β-catenin and MAPK pathway, inhibition of CD8+ T and NK cell infiltration, and promotion of PD-L1 expression.

Abstract LB165: Preclinical evaluation of carnosic acid (CA) and rosemary extract standardized to 40% carnosic acid (RE40CA) for the prevention of invasive breast cancer

Abstract Background: There is an unmet need for the development of safer and more effective therapies for the prevention of human invasive breast cancer, for which ductal carcinoma in situ (DCIS) is a precursor. Currently, anti-hormonal therapies are the only option in individuals previously diagnosed with hormone receptor positive breast cancer. However, these agents are associated with many side effects. Furthermore, there are currently no preventive options for hormone receptor negative breast cancers. To study DCIS progression, we developed a model referred to as MIND (Mouse-INtraDuctal) in which patient-derived DCIS cells and cell lines are injected intraductally and studied as they progress to invasive ductal carcinoma (IDC). Previously we provided strong evidence for the role of B Cell Lymphoma-9 (BCL9) as a potential driver of DCIS malignancy. We chose to target BCL9 using RE40CA and its active ingredient CA previously identified in a screen for inhibitors of β-catenin binding to BCL9. Experimental Strategy: CA and RE40CA were administered to DCIS.COM MIND animal models for four weeks by oral gavage or mixed in a powder diet formulation. Drug plasma levels were evaluated by LC-MS. Four weeks following drug administration, mammary glands were excised and evaluated for efficacy and expression of biomarkers of aggressive DCIS, nuclear β-catenin and BCL9. For efficacy studies, mammary glands containing DCIS like lesions were processed and subjected to immunofluorescence staining using anti-human CK-5, anti-SMA, Dapi, β-catenin and BCL9. Extent of DCIS in vivo growth, number of invasive lesions, tumor number and tumor volume were evaluated for each mammary gland. Results: Efficacy studies showed a significant reduction in the number of invasive lesions by administration of CA by oral gavage (CA OG:10mg/kg), CA at 200mg/kg diet (CA200), CA400 and RE40CA 500. A significant reduction in extent of DCIS in vivo growth was achieved by CA OG and RE40CA 500. A significant reduction in the number of tumors was achieved by CA OG, RE40CA OG (25mg/kg equivalent to CA 10mg/kg), RE40CA 100, RE40CA 500 and RE40CA 1000. A significant reduction in tumor volume was achieved by CA OG, RE40CA OG, RE40CA 100, RE40CA 500 and RE40CA 1000. Linear regression modelling was used to compare statistical differences among the groups and differences that reached p-value &amp;lt;0.05 were considered significant. Administration of CA or RE40CA by oral gavage was also associated with a significant loss of nuclear BCL9/β-catenin expression in DCIS epithelial cells. Dietary administration of CA and RE40CA as OG or mixed in powder diet showed an increasing trend in plasma concentrations as the dose was increased. Evaluation of HE stained sections of animal organs by histopathology showed no evidence in any tissue of necrosis, degeneration, inflammation or neoplasia. Additionally, no consistent lesions attributable to treatment were observed in any of the animals or groups. Conclusion: Administration of CA and RE40CA are safe and effective in prevention of DCIS malignancy. Future studies are aimed at testing the efficacy of CA/RE40CA using DCIS cell lines representing other DCIS subtypes (ER+/PR+ and HER2 overexpressing) as well as patient derived DCIS MIND animal models. Supported by NCI PREVENT Cancer Preclinical Drug Development Program, 75N91019D00016/75N91020F00001. Citation Format: Yan Hong, Aditi Rastogi, Aryamitra Banerjee, Brandon L. Plattner, Matthew Lindeblad, Alexander V. Lyubimov, Timothy Fields, Seema A. Khan, Altaf Mohammed, Jeremy J. Johnson, Fariba Behbod. Preclinical evaluation of carnosic acid (CA) and rosemary extract standardized to 40% carnosic acid (RE40CA) for the prevention of invasive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB165.

Endometrial epithelial cells-derived exosomes deliver microRNA-30c to block the BCL9/Wnt/CD44 signaling and inhibit cell invasion and migration in ovarian endometriosis

Endometriosis (EMs) is a benign gynecological disorder showing some tumor-like migratory and invasive phenotypes. This study intended to investigate the role of microRNA-30c (miR-30c) in EMs, which is involved with B-cell lymphoma 9 (BCL9), an activator of the Wnt/β-catenin signaling pathway. EMs specimens were clinically collected for determination of miR-30c and BCL9 expression. Exosomes were isolated from endometrial epithelial cells (EECs), and the uptake of exosomes by ectopic EECs (ecto-EECs) was characterized using fluorescence staining and confocal microscopy. The binding of miR-30c to BCL9 was validated by dual-luciferase reporter assay. Artificial modulation (up- and down-regulation) of the miR-30c/BCL9/Wnt/CD44 regulatory cascade was performed to evaluate its effect on ecto-EEC invasion and migration, as detected by Transwell and wound healing assays. A mouse model of EMs was further established for in vivo substantiation. Reduced miR-30c expression and elevated BCL9 expression was revealed in EMs ectopic tissues and ecto-EECs. Normal EECs-derived exosomes delivered miR-30c to ecto-EECs to suppress their invasive and migratory potentials. Then, miR-30c was observed to inhibit biological behaviors of ecto-EECs by targeting BCL9, and the miR-30c-induced inhibitory effect was reversed by BCL9 overexpression. Further, miR-30c diminished the invasion and migration of ecto-EECs by blocking the BCL9/Wnt/CD44 axis. Moreover, miR-30c-loaded exosomes attenuated the metastasis of ecto-EEC ectopic nodules. miR-30c delivered by EECs-derived exosomes repressed BCL9 expression to block the Wnt/β-catenin signaling pathway, thus attenuating the tumor-like behaviors of ecto-EECs in EMs.

Response to vaccination against SARS‐CoV‐2 in 168 patients with Waldenström macroglobulinaemia: A French Innovative Leukaemia Organization study

Response to vaccination against <scp>SARS‐CoV</scp>‐2 in 168 patients with Waldenström macroglobulinaemia: A French Innovative Leukaemia Organization study

BCL9 regulates CD226 and CD96 checkpoints in CD8+ T cells to improve PD-1 response in cancer

To date, the overall response rate of PD-1 blockade remains unsatisfactory, partially due to limited understanding of tumor immune microenvironment (TIME). B-cell lymphoma 9 (BCL9), a key transcription co-activator of the Wnt pathway, is highly expressed in cancers. By genetic depletion and pharmacological inhibition of BCL9 in tumors, we found that BCL9 suppression reduced tumor growth, promoted CD8+ T cell tumor infiltration, and enhanced response to anti-PD-1 treatment in mouse colon cancer models. To determine the underlying mechanism of BCL9’s role in TIME regulation, single-cell RNA-seq was applied to reveal cellular landscape and transcription differences in the tumor immune microenvironment upon BCL9 inhibition. CD155-CD226 and CD155-CD96 checkpoints play key roles in cancer cell/CD8+ T cell interaction. BCL9 suppression induces phosphorylation of VAV1 in CD8+ T cells and increases GLI1 and PATCH expression to promote CD155 expression in cancer cells. In The Cancer Genome Atlas database analysis, we found that BCL9 expression is positively associated with CD155 and negatively associated with CD226 expression. BCL9 is also linked to adenomatous polyposis coli (APC) mutation involved in patient survival following anti-PD-1 treatment. This study points to cellular diversity within the tumor immune microenvironment affected by BCL9 inhibition and provides new insights into the role of BCL9 in regulating CD226 and CD96 checkpoints

BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins

Colorectal cancer (CRC) is the third most commonly diagnosed cancer, which despite recent advances in treatment, remains incurable due to molecular heterogeneity of tumor cells. The B-cell lymphoma 9 (BCL9) oncogene functions as a transcriptional co-activator of the Wnt/β-catenin pathway, which plays critical roles in CRC pathogenesis. Here we have identified a β-catenin-independent function of BCL9 in a poor-prognosis subtype of CRC tumors characterized by expression of stromal and neural associated genes. In response to spontaneous calcium transients or cellular stress, BCL9 is recruited adjacent to the interchromosomal regions, where it stabilizes the mRNA of calcium signaling and neural associated genes by interacting with paraspeckle proteins. BCL9 subsequently promotes tumor progression and remodeling of the tumor microenvironment (TME) by sustaining the calcium transients and neurotransmitter-dependent communication among CRC cells. These data provide additional insights into the role of BCL9 in tumor pathogenesis and point towards additional avenues for therapeutic intervention.

Low BCL9 expression inhibited ovarian epithelial malignant tumor progression by decreasing proliferation, migration, and increasing apoptosis to cancer cells

BackgroundAbnormal activation of the classic Wnt signaling pathway is closely related to the occurrence of epithelial cancers. B-cell lymphoma 9 (BCL9), a transcription factor, is a novel oncogene discovered in the classic Wnt pathway and promotes the occurrence and development of various tumors. Ovarian cancer is the gynecological malignant tumor with the highest mortality because it is difficult to diagnose early, and easy to relapse and metastasis. The expression and role of BCL9 in epithelial ovarian cancer (EOC) have not been studied. Thus, in this research, we aimed to investigate the expression and clinical significance of BCL9 in EOC tissues and its effect on the malignant biological behavior of human ovarian cancer cells.MethodsWe detect the expression of BCL9 in ovarian epithelial tumor tissues and normal ovarian tissues using immunohistochemistry and analyzed the relationship between it and clinicopathological parameters and patient prognosis. The expression of proteins was detected by Western blot. The MTT assay, flow cytometry, the scratch assay, and the transwell assay were used to detect cell proliferation, apoptosis, migration, and invasion, respectively. A total of 374 ovarian cancer tissue samples were collected using TCGA database. A gene set enrichment analysis of BCL9 was performed.ResultsBCL9 was overexpressed in EOC tissues. The level of BCL9 expression was correlated with the 5-year progression-free survival rate and overall survival rate in ovarian cancer patients and independently predicted the risk of ovarian cancer recurrence. Low BCL9 expression inhibited proliferation, invasion and migration of EOC cells, decreased MMP2 and MMP9 expression of ES-2 cell line, increased the BAX/BCL2 ratio and promoted apoptosis of EOC cells.ConclusionBCL9 is overexpressed in epithelial ovarian tumors, resulting in a poor prognosis for ovarian cancer patients. Low BCL9 expression can promote ovarian cancer cell apoptosis, inhibit proliferation and migration. BCL9 promotes the development of ovarian cancer.

Inhibition of β-catenin/B cell lymphoma 9 protein−protein interaction using α-helix–mimicking sulfono-γ-AApeptide inhibitors

The rational design of α-helix-mimicking peptidomimetics provides a streamlined approach to discover potent inhibitors for protein-protein interactions (PPIs). However, designing cell-penetrating long peptidomimetic scaffolds equipped with various functional groups necessary for interacting with large protein-binding interfaces remains challenging. This is particularly true for targeting β-catenin/BCL9 PPIs. Here we designed a series of unprecedented helical sulfono-γ-AApeptides that mimic the binding mode of the α-helical HD2 domain of B Cell Lymphoma 9 (BCL9). Our studies show that sulfono-γ-AApeptides can structurally and functionally mimic the α-helical domain of BCL9 and selectively disrupt β-catenin/BCL9 PPIs with even higher potency. More intriguingly, these sulfono-γ-AApeptides can enter cancer cells, bind with β-catenin and disrupt β-catenin/BCL9 PPIs, and exhibit excellent cellular activity, which is much more potent than the BCL9 peptide. Furthermore, our enzymatic stability studies demonstrate the remarkable stability of the helical sulfono-γ-AApeptides, with no degradation in the presence of pronase for 24 h, augmenting their biological potential. This work represents not only an example of helical sulfono-γ-AApeptides that mimic α-helix and disrupt protein-protein interactions, but also an excellent example of potent, selective, and cell-permeable unnatural foldameric peptidomimetics that disrupt the β-catenin/BCL9 PPI. The design of helical sulfono-γ-AApeptides may lead to a new strategy to modulate a myriad of protein-protein interactions.

MiR-30c exerts tumor suppressive functions in colorectal carcinoma by directly targeting BCL9.

Colorectal carcinoma (CRC) remains a leading health threat worldwide due to its high mortality. MicroRNA (miR-30c) is an important tumor suppressor in various cancers. B cell lymphoma 9 (BCL9) is one of the candidate genes for cancers. The synergistic effects of miR-30c and BCL9 in CRC progression remain to be carefully elucidated. Fifty pairs of CRC samples and matched adjacent non-tumor tissues were collected from Yantai Yuhuangding Hospital between 2015 and 2017. MiR-30c and BCL9 expression levels were measured by quantitative Real-time polymerase chain reaction (qRT-PCR) in CRC tissues and cell lines. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the influence of miR-30c on the proliferation ability of CRC cells. Target Scan was used to predict the potential target of miR-30c. Then, luciferase assay was performed to confirm the prediction. In addition, we also investigated the biological influence of BCL9 on miR-30c-mediated functions in CRC. We found that miR-30c was significantly decreased in CRC tissues and cell lines while the BCL9 expression level was prominently increased in CRC tissues and cells. Additionally, the miR-30c expression was negatively correlated with BCL9 expressions in CRC tissues. Furthermore, the findings of this study also showed that BCL9 was a direct target of miR-30c in CRC and miR-30c could inhibit the CRC proliferation by binding to its 3'-UTR. This study showed that miR-30c overexpression inhibited CRC proliferation via the regulation of BCL9, suggesting that miR-30c may be a new molecular therapeutic target for CRC.

Mechanism of Low Expression of miR-30a-5p on Epithelial-Mesenchymal Transition and Metastasis in Ovarian Cancer.

Metastasis of ovarian cancer is regulated by microRNAs. This study focused on the effects of miR-30a-5p on ovarian cancer migration and invasion. Our results showed that the miR-30a-5p and mucin type O-glycan biosynthesis are closely related to ovarian cancer, and that miR-30a-5p was downregulated in ovarian cancer cells. miR-30a-5p overexpression reduced cell viability and inhibited migration and invasion in HO-8910 and HO-8910PM cells. S phase kinase-associated protein 2 (SKP2), B cell lymphoma 9 (BCL9), and NOTHC1 are direct target genes of miR-30a-5p. MTDH, SKP2, BCL9, and NOTCH1 genes were overexpressed in ovarian cancer cells, and they are direct target genes of miR-30a-5p. miR-30a-5p overexpression inhibited epithelial-mesenchymal transition (EMT) process, while upregulation of SKP2, BCL9, and NOTCH1 gene expression levels reduced the inhibition of EMT process by miR-30a-5p. miR-30a-5p was lowly expressed in ovarian cancer, and such a phenomenon is related to ovarian cancer metastasis. miR-30a-5p might inhibit the migration and invasion of ovarian cancer cells by downregulating the expression of SKP2, BCL9, and NOTCH1 genes.

SOX7 Suppresses Wnt Signaling by Disrupting β-Catenin/BCL9 Interaction.

The Wnt signaling is involved in angiogenesis and tumor development. β-catenin is the core component of the Wnt pathway, which mediates oncogenic transcription and regulated by a series of proteins. Sex-determining region Y-box 7 (SOX7) is a member of high-mobility-group transcription factor family, which inhibits oncogenic Wnt signaling in lots of tumor cells with unknown mechanism. By coimmunoprecipitation (co-IP) and super Topflash reporter assay, SOX7 can bind β-catenin and inhibit β-catenin/T cell factor (TCF)-mediated transcription. Meanwhile, B cell lymphoma 9 (BCL9) drives Wnt signaling path through direct binding-mediated β-catenin. Finally, we found that SOX7 inhibits oncogenic β-catenin-mediated transcription by disrupting the β-catenin/BCL9 interaction. Mechanistically, SOX7 compete with BCL9 to bind β-catenin. Our results show SOX7 inhibited Wnt signaling as suppressor and could be an important target for anticancer therapy.

Abstract B01: B cell lymphoma 9 mediates a cross talk between the canonical Wnt and EGFR signaling in breast cancer

Abstract Human ductal carcinoma in situ (DCIS) are the most common type of non-invasive breast cancers. The five-year survival rate for women diagnosed with non-invasive DCIS is 98% while the five-year survival plummets to 83-27% for breast cancers that have become invasive and have spread to distant sites [also referred to as invasive ductal carcinoma (IDC)]. To study DCIS pathobiology and factors that promote their transition to IDC, we have developed a novel in vivo DCIS model, MIND (mouse-intraductal), that involves intraductal injection of epithelial cells derived from primary human DCIS biopsy and surgical samples thus mimicking the entire process of DCIS to IDC transition. As a complementary approach, we have utilized human DCIS/IDC tandem lesions, which are patient DCIS that show a transition to IDC within the same breast. Analysis of RNA and protein at distinct stages of in situ to IDC using both models showed B cell lymphoma-9 (BCL9) up-regulation to be associated with DCIS transition to IDC. BCL9 is a recently identified co-activator of Wnt-stimulated beta-catenin-mediated transcription. Our studies showed that in vivo silencing of BCL9 led to inhibition of DCIS invasion and reversal of EMT. We have also demonstrated a direct binding interaction between BCL9 and beta-catenin and showed suppression of beta-catenin-mediated transcription by BCL9 knockdown. Analysis of patient DCIS samples revealed a significant correlation between high nuclear BCL9 expression and pathologic characteristics associated with DCIS recurrence: Estrogen receptor (ER) negative and Ki67. Furthermore, analysis of the TCGA data showed BCL9 gene to be upregulated in 26% of breast cancers. This is a significant gene alteration when compared to HER2 (ERBB2) gene (19%) and estrogen receptor (ESR1) gene (8%) alterations in breast cancers. Interestingly, a significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification suggesting that BCL9 may predispose to the development of basal breast cancers. We have performed an RPPA analysis on our DCIS cell lines KD BCL9 vs. control. This analysis indicated that BCL9 KD showed down-regulation in a number of genes in the EGFR signaling pathway including p-EGFR, p-HER2, p-STAT3, and p-Src. Conclusion: BCL9 is a molecular driver of DCIS invasive progression. The molecular mechanism for BCL9's role in breast cancer progression is through the enhancement in the canonical Wnt and EGFR signaling. Citation Format: Hanan Elsarraj, Hong Yan, Jennifer Knapp, Anna Tsimelzon, Shixia Huang, Andrew Godwin, Sue Hilsenbeck, Dean Edwards, Fariba Behbod. B cell lymphoma 9 mediates a cross talk between the canonical Wnt and EGFR signaling in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B01.

BCL9, a coactivator for Wnt/β-catenin transcription, is targeted by miR-30c and is associated with prostate cancer progression.

B-cell lymphoma 9 (BCL9), a component of aberrantly activated Wnt signaling, is an important contributing factor to tumor progression. Our previous data indicated that downregulation of the tumor suppressor microRNA-30c (miR-30c) was a frequent pathogenetic event in prostate cancer (PCa). However, a functional link between miR-30c and BCL9/Wnt signaling, and their clinical and pathological significance in PCa, have not been well established. The present study demonstrated that miR-30c serves as a key negative regulator targeting BCL9 transcription in PCa cells. Ectopic expression of miR-30c was associated with reduced expression of Wnt pathway downstream targets, including c-Myc, cluster of differentiation 44 and sex determining region Y-box 9 in DU145 human PCa cells. Examination of clinical prostate specimens revealed higher levels of BCL9 expression in PCa compared with that in benign prostate tissues. After substantiating this finding by patient sample analysis, BCL9 expression or activity was observed to be closely correlated with PCa biochemical recurrence (BCR) and disease progression, whereas it was inversely associated with miR-30c. Furthermore, overexpression of BCL9 in PCa acted cooperatively with miR-30c low expression to predict earlier BCR in PCa. These findings indicate that inhibition of BCL9/Wnt signaling by miR-30c is important in the progression of PCa. Furthermore, the combined analysis of miR-30c and BCL9 may be valuable tool for prediction of BCR in PCa patients following radical prostatectomy.

Expression profiling of in vivo ductal carcinoma in situ progression models identified B cell lymphoma-9 as a molecular driver of breast cancer invasion.

IntroductionThere are an estimated 60,000 new cases of ductal carcinoma in situ (DCIS) each year. A lack of understanding in DCIS pathobiology has led to overtreatment of more than half of patients. We profiled the temporal molecular changes during DCIS transition to invasive ductal carcinoma (IDC) using in vivo DCIS progression models. These studies identified B cell lymphoma-9 (BCL9) as a potential molecular driver of early invasion. BCL9 is a newly found co-activator of Wnt-stimulated β-catenin-mediated transcription. BCL9 has been shown to promote progression of multiple myeloma and colon carcinoma. However BCL9 role in breast cancer had not been previously recognized.MethodsMicroarray and RNA sequencing were utilized to characterize the sequential changes in mRNA expression during DCIS invasive transition. BCL9-shRNA knockdown was performed to assess the role of BCL9 in in vivo invasion, epithelial-mesenchymal transition (EMT) and canonical Wnt-signaling. Immunofluorescence of 28 patient samples was used to assess a correlation between the expression of BCL9 and biomarkers of high risk DCIS. The cancer genome atlas data were analyzed to assess the status of BCL9 gene alterations in breast cancers.ResultsAnalysis of BCL9, by RNA and protein showed BCL9 up-regulation to be associated with DCIS transition to IDC. Analysis of patient DCIS revealed a significant correlation between high nuclear BCL9 and pathologic characteristics associated with DCIS recurrence: Estrogen receptor (ER) and progesterone receptor (PR) negative, high nuclear grade, and high human epidermal growth factor receptor2 (HER2). In vivo silencing of BCL9 resulted in the inhibition of DCIS invasion and reversal of EMT. Analysis of the TCGA data showed BCL9 to be altered in 26 % of breast cancers. This is a significant alteration when compared to HER2 (ERBB2) gene (19 %) and estrogen receptor (ESR1) gene (8 %). A significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification.ConclusionBCL9 is a molecular driver of DCIS invasive progression and may predispose to the development of basal like invasive breast cancers. As such, BCL9 has the potential to serve as a biomarker of high risk DCIS and as a therapeutic target for prevention of IDC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0630-z) contains supplementary material, which is available to authorized users.

Association of BCL9 expression with prostate cancer clinicopathological features and survival

B-cell lymphoma 9 (BCL9) as a co-activator for β-catenin-mediated transcription is highly expressed in tumors. The aim of our study is to investigate the expression of BCL9, and its clinicopathological significance in prostate cancer (PCa). Immunohistochemistry was conducted to analyze the expression of BCL9 in 98 PCa samples and 20 benign prostate hyperplasia (BPH) samples. The associations of BCL9 expression with clinicopathological features and prognosis in PCa patients were analyzed with various statistical methods, such as chi-square test, Kaplan-Meier method and log-rank test. The positive rate of BCL9 was 53.1% (52/98) in prostate cancer group, and 25.0% (5/20) in benign group (P=0.022). In addition, BCL9 expression in PCa tissues was significantly associated with Gleason score (P=0.016), and biochemical recurrence (P=0.020). Moreover, Kaplan-Meier survival analysis showed that the higher expression of BCL9 was correlated with shorter biochemical recurrence-free survival (P=0.037). However, BCL9 was not an independent prognostic predictor for biochemical recurrence-free survival in patients with PCa after the multivariate analysis was conducted (Hazard ratio =1.73, P=0.384). Our study demonstrates the up-regulation of BCL9 is associated with PCa's early diagnosis and malignant degree.

Targeted disruption of the BCL9/β-catenin complex inhibits oncogenic Wnt signaling.

Deregulated Wnt/β-catenin signaling underlies the pathogenesis of a broad range of human cancers, yet the development of targeted therapies to disrupt the resulting aberrant transcription has proved difficult because the pathway comprises large protein interaction surfaces and regulates many homeostatic functions. Therefore, we have directed our efforts toward blocking the interaction of β-catenin with B cell lymphoma 9 (BCL9), a co-activator for β-catenin-mediated transcription that is highly expressed in tumors but not in the cells of origin. BCL9 drives β-catenin signaling through direct binding mediated by its α-helical homology domain 2. We developed a stabilized α helix of BCL9 (SAH-BCL9), which we show targets β-catenin, dissociates native β-catenin/BCL9 complexes, selectively suppresses Wnt transcription, and exhibits mechanism-based antitumor effects. SAH-BCL9 also suppresses tumor growth, angiogenesis, invasion, and metastasis in mouse xenograft models of Colo320 colorectal carcinoma and INA-6 multiple myeloma. By inhibiting the BCL9-β-catenin interaction and selectively suppressing oncogenic Wnt transcription, SAH-BCL9 may serve as a prototype therapeutic agent for cancers driven by deregulated Wnt signaling.

Rapid Infusion Rituximab in Maintenance Therapy: Is It Feasible?

Abstract 4973 Background/Rationale:Rituximab is an anti-CD-20 monoclonal antibody used in the management of lymphoproliferative disorders. Rituximab is indicated in maintenance therapy for follicular cell lymphoma and can be administered once every 2 months, once every 3 months, or weekly for 4 weeks every 6 months. The use of maintenance rituximab has improved progression free survival and overall survival in low grade follicular lymphomas. Although rapid rituximab infusions have been studied extensively, there is little data on the use of rapid infusions during maintenance therapy for low grade lymphomas. Herein we report our experience with rapid infusion rituximab in patients receiving maintenance therapy. Methods:All patients who received rapid infusion rituximab as maintenance therapy for low grade lymphoma between December 2007 and June 2011 were included. Patients were considered eligible for rapid infusions if they tolerated previous rituximab infusions and presented with a peripheral lymphocyte count of less than 4.8 k/μl. Rapid rituximab infusions were administered over 90 minutes (150 mL/hr for 30 minutes and then increased to 275 mL/hr until completion). Patients were monitored for signs and symptoms of reactions. In case of an infusion reaction supportive care medications were readily available. Demographic, laboratory and clinical data were collected. Adverse events were assessed through vital signs, symptoms of infusion related reaction and supportive care measures administered and graded according to the Common Terminology Criteria for Adverse Events Version 4. The primary and secondary objectives of this retrospective study were to evaluate the incidence of grade 3/4 and all grade infusion reactions with rapid rituximab infusions during maintenance therapy, respectively. Maintenance schedules were also compared with regards to incidence of infusion reactions. Results:A total of 105 patients received 629 rapid rituximab infusions. Patient demographics, laboratory, and clinical data are summarized in Table 1. All patients were eligible for rapid infusion rituximab according to institutional criteria. Two grade 2 infusion reactions and 4 grade 3 reactions were reported (1 patient experienced a grade 2 and 3 reaction); however none of these patients required hospitalization. All 5 patients received pharmacological and/or supportive care to relieve the symptoms associated with the reaction. Of these 5 patients, 3 patients went on to receive rapid infusions of rituximab; 2 patients were switched to standard infusion per physician request. The sample size was too small to determine if a correlation existed between infusion related reactions and the schedule of maintenance rituximab.Table 1Patient Demographicsn=105Male, n (%)55 (52.4)Median BSA (range), m21.97 (1.36-2.84)Diagnosisn (%)Follicular Lymphoma65 (62)Marginal Zone Lymphoma10 (9.5)Large B-cell lymphoma9 (8.6)Mantle Cell Lymphoma8 (7.6)CLL/SLL5 (4.8)Mucosa Associated Lymphoid Tissue3 (2.8)Other5 (4.8)Baseline Laboratory Parametersk/μL (range)Median lymphocyte count1.1 (0.04-8.21)edian WBC5.14 (1.34-13.07)RituximabMedian Dose, mg750 (510-1090)Median No. Doses5 (1-12)Mean Infusion Time, min90 (85-97) Conclusion:The rapid infusion of rituximab in patients receiving maintenance therapy is well tolerated with minimal incidence of infusion-related reactions. Although the sample size was insufficient to assess differences in adverse effects according to the maintenance schedule, the low overall incidence of adverse effects suggests that rapid rituximab infusions are well tolerated regardless of maintenance schedule. Our study concludes rapid infusion rituximab is a safe and feasible option for maintenance therapy. Disclosures:Ho:Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Maintenance Rituximab Therapy in Non-Hodgkin's Lymphoma. Cultrera:Genentech: Speakers Bureau. Sotomayor:Genentech: Membership on an entity's Board of Directors or advisory committees. Wetzstein:Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees.

A co‐dependent requirement of xBcl9 and Pygopus for embryonic body axis development in Xenopus

The Wnt/beta-catenin transcriptional activation complex requires the adapter protein Pygopus (Pygo), which links the basal transcription machinery to beta-catenin, by its association with legless (Lgs)/ B-cell lymphoma-9 (Bcl9). Pygo was shown to be required for development in vertebrates, but the role of Lgs/Bcl9 is unknown. We identified an amphibian orthologue of Lgs/Bcl9, XBcl9, which interacted biochemically with Xbeta-catenin and XPygo2. The body axis promoting ability of Xbeta-catenin was diminished when residues required for its interaction with XBcl9 were mutated. In blastula embryos, XBcl9 was transiently preferentially expressed in nuclei of dorsoanterior cells and ectopically expressed XBcl9 required XPygo2 to localize to nuclei. Furthermore, while neither XBcl9 nor XPygo2 alone affected development when ectopically expressed, both were required to induce supernumerary axis and dorsal gene activation. Like XPygo2, depletion of maternal XBcl9 alone caused dorsal defects. These results indicated an essential role of the Pygo-Bcl9 duet in vertebrate body axis formation.

Analysis of the Interaction of BCL9 with β-Catenin and Development of Fluorescence Polarization and Surface Plasmon Resonance Binding Assays for this Interaction

The transcriptional activator beta-catenin is the primary mediator of the canonical Wnt signaling pathway and is frequently upregulated in many types of human cancer. Recent studies have suggested that the interaction of beta-catenin and its cofactor, B-cell lymphoma 9 (BCL9), is crucial for its transcriptional activity. Targeting this interaction using small molecules will improve our understanding of the beta-catenin/Wnt signaling pathway and may lead to the development of a new class of anticancer drugs. In this study, we developed a fluorescence polarization (FP)-based BCL9 binding assay. Using our initial FP assay, we performed extensive mutational analysis on four critical hydrophobic residues in the BCL9 peptide and determined the precise region in BCL9 responsible for binding to beta-catenin. These results led to further optimization of our FP assay, making it amenable for high-throughput screening (HTS). We also developed and validated a complementary surface plasmon resonance (SPR)-based binding assay and showed that our synthetic BCL9-based peptides are capable of fully inhibiting the binding of beta-catenin to wild-type BCL9 protein. Our studies provide not only further insight into the interaction between BCL9 and beta-catenin but also quantitative and reliable biochemical binding assays for the discovery of potent and specific small-molecule inhibitors of this interaction.