Journal: Clinical and Translational Medicine

Article Title: IRF4 contributes to chemoresistance in IGH::BCL2‐positive diffuse large B‐cell lymphomas by mediating BCL2‐induced SOX9 expression

doi: 10.1002/ctm2.70336

Figure Lengend Snippet: Silencing BCL2 suppresses sex‐determining region Y (SRY)‐box 9 protein (SOX9) expression in BCL2 overexpressed germinal centre B‐cell‐like (GCB) diffuse large B‐cell lymphoma (DLBCL) subsets. (A and B) Immunoblotting and real‐time polymerase chain reaction (PCR) assay of BCL2 and SOX9 protein and mRNA levels in a panel of DLBCL cell lines (Karpas‐422, DB, SUDHL2 and SUDHL8), DB cells were transduced either with lentiviral encoding scramble control, shBCL2#1 or shBCL2#2 plasmids for 72 h, respectively, prior to subject flow cytometry sorting of Green fluorescent protein (GFP)‐positive cells to generate stable transfectants. (C and D) Immunoblotting or real‐time PCR assays of protein or mRNA level of BCL2 and SOX9. Two‐way analysis of variance (ANOVA) was used to compare scramble Ctrl to shSOX9#1 and shSOX9#2. (E and F) Immunoblotting assay of BCL2 and SOX9 protein levels in Karpas‐422 and DB cells were either treated with indicated doses of ABT‐737 or ABT‐199 for 48 h. (G and H) Real‐time PCR of BCL2 and SOX9 mRNA levels in Karpas‐422 and DB cells were either treated with 2.5 µM ABT‐737 or ABT‐199 for 48 h. Two‐way ANOVA was performed to compare vehicle Ctrl to treated. (I) BCL2 and SOX9 immunostainings were denoted as (+), (++) and (+++) to indicate the expression levels of BCL2 and SOX9. Negative control was included to eliminate the false‐positive staining. Positivity of BCL2 or SOX9 immunostaining was measured using ImageJ software, the simple linear regression statistical analysis was carried out to determine the association between SOX9 and BCL2 positives ( n = 6, 200× magnification). β‐Tubulin or GAPDH was included as indications of equal loading. Protein levels were quantified (normalised to housekeeping genes) using ImageJ software and graph was generated using GraphPad version 9.0. All experiments were repeated three times, and graph with error bars show the data represent the mean ± standard deviation (SD) from technical triplicates ( **** p < .005).

Article Snippet: SUDHL2 (CRL‐2956), SUDHL6 (CRL‐2959), SUDHL8 (CRL‐2961) and DB (CRL‐2289) cells were obtained from the American Type Culture Collection.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Control, Flow Cytometry, Negative Control, Staining, Immunostaining, Software, Generated, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: IRF4 contributes to chemoresistance in IGH::BCL2‐positive diffuse large B‐cell lymphomas by mediating BCL2‐induced SOX9 expression

doi: 10.1002/ctm2.70336

Figure Lengend Snippet: In vivo targeting IRF4 impedes lymphomagenesis and resensitising diffuse large B‐cell lymphoma (DLBCL) to chemotherapy. SUDHL8 and SUDHL2 cells were incubated either with vehicle control, 1 or 5 µM Ctrl ASO, hIRF4 ASO#1, hIRF4 ASO#2 or hIRF4 ASO#3 for 72 h, respectively, followed by (A) real‐time polymerase chain reaction (PCR) analysis of IRF4 mRNA level, (B) flow cytometry assay to determine cell viability, (C) expressions of IRF4 target genes, (D) MFI of IRF4 or (E) cell cycle distribution. Ten million of IRF4 deficient OCI‐LY3 cells were subcutaneously injected into each nude mouse ( n = 5). Three weeks post‐transplantation, mice were sacrificed and (F) tumour volume, (G) tumour cell proliferation and (H) apoptosis were measured. SUDHL8 (solid lines) and SUDHL2 (dash lines) DLBCL xenografts were administrated either with Ctrl ASO (25 mg/kg) or hIRF4 ASO (25 mg/kg) five times per week for 3 weeks, mice were sacrificed prior to tumourigenesis indexes measurement as demonstrated in (I) tumour volume and (J) IRF4 mRNA level. SUDHL8 (solid lines) and SUDHL2 (dash lines) DLBCL xenografts were administrated either with Ctrl ASO (25 mg/kg) or hIRF4 ASO (25 mg/kg) five times per week for 3 weeks or DOX (2.5 mg/kg) every 2 days for four times alone, or combination. Phosphate‐buffered saline (PBS) control was also included as a vehicle control. Three weeks post‐drug administration, mice were sacrificed prior to subject to tumourigenesis indexes measurements. (K) Tumour volume and (L) cell viability was assessed. All experiments were repeated three times, and graph with error bars show data represent the mean ± standard deviation (SD) from technical triplicates ( * p < .01; ** p < .05; *** p < .001; **** p < .005).

Article Snippet: SUDHL2 (CRL‐2956), SUDHL6 (CRL‐2959), SUDHL8 (CRL‐2961) and DB (CRL‐2289) cells were obtained from the American Type Culture Collection.

Techniques: In Vivo, Incubation, Control, Real-time Polymerase Chain Reaction, Flow Cytometry, Injection, Transplantation Assay, Saline, Standard Deviation

Journal: Scientific Reports

Article Title: Coordinated loss of microRNA group causes defenseless signaling in malignant lymphoma

doi: 10.1038/srep17868

Figure Lengend Snippet: ( a ) Cell survival of lymphoma cell lines depends on BCR signaling. SUDHL4, SUDHL8, and BCBL1 cells were treated with SYK inhibitor R406 (1 μM) or IKKβ inhibitor BMS345541 (5 μM) for 3 days. The apoptotic cells were detected by Annexin V staining. ( b ) RISC-capture assays in B cell lymphoma cell lines. Two independent anti-Ago2 antibodies were used for RISC immunoprecipitation. Immunoprecipitated levels of BCR factor mRNAs are plotted. Representatives of two independent experiments are shown. ( c ) Quantification of RISC-associated mRNA in normal resting B cells (n = 3) and DLBCL samples (n = 3) assessed by RISC-capture assay. mRNA inputs were comparable among all tested samples (data not shown). ( d ) BCR target gene upregulation in frozen DLBCL samples used in ex vivo experiments (RISC-capture assay and Polysome analysis). Expression levels of BCR signaling-inducible genes relative to normal resting B cells are shown. ( e ) Scatter plot showing relationship between 3′UTR length and RISC incorporation efficiency in BCR factor mRNAs. Average values of the RISC-incorporated mRNA levels from DLBCL samples (n = 3) and normal B cells (n = 3) are plotted.

Article Snippet: DLBCL cell lines SUDHL4, SUDHL6, and SUDHL8 were purchased from DSMZ.

Techniques: Staining, Immunoprecipitation, Ex Vivo, Expressing

Journal: Scientific Reports

Article Title: Coordinated loss of microRNA group causes defenseless signaling in malignant lymphoma

doi: 10.1038/srep17868

Figure Lengend Snippet: ( a ) Hierarchical clustering of miRNA expression in AIDS-associated DLBCL (n = 8), non-AIDS DLBCL (n = 5), tonsil (n = 8), and normal CD19 + B cell (n = 3). ( b ) Lymphoma cell lines were transduced with lentivirus vectors carrying pri-miRNA (or pre-miRNA, data not shown), followed by long-term culture in complete medium. Results as percentage of each Venus-positive population transduced with shRNA series are plotted (n = 3). ( c ) miRNA expression affects proliferation of human CD19 + B cells induced by anti-IgM (5 μg/ml). miRNA expression was monitored by Venus expression. Representative results of proliferation time-course experiments (n = 3) are shown. ( d ) miRNA quantification in DLBCL cohort (total, n = 83; GC, n = 31;, non-GC, n = 34; AIDS, n = 20; non-AIDS, n = 63, EBV + , n = 30; EBV−, n = 48) and tonsil (n = 10). CD19 + B cells (n = 3) were used for normal control samples. ( e ) RISC-associated miRNA levels in normal B cells (n = 3, mean values), LCL (n = 3, mean values), primary DLBCL samples (n = 4), and three DLBCL cell lines evaluated by RISC-capture assay. ( f ) Transduction of miRNA lentivirus could express functional miRNA in lymphoma cells. Graphs show the levels of total miRNA (left) and RISC-incorporated miRNA (right) in SUDHL8 with miRNA lentivirus (n = 3, average values). ( g ) Overall survival probabilities of DLBCL cases with high miRNA (dashed line) and low miRNA (solid line).

Article Snippet: DLBCL cell lines SUDHL4, SUDHL6, and SUDHL8 were purchased from DSMZ.

Techniques: Expressing, Transduction, shRNA, Control, Functional Assay

Journal: Scientific Reports

Article Title: Coordinated loss of microRNA group causes defenseless signaling in malignant lymphoma

doi: 10.1038/srep17868

Figure Lengend Snippet: ( a ) Scatter plot showing RISC-associated mRNA levels of BCR signaling factors (SUDHL8/miRNA vs. SUDHL8/Empty). Average values of three independent experiments are shown. ( b ) Schematic of the miRNA target sites in 3′UTR of the BCR signaling factors based on predictions by computational algorithms and results of RISC-capture screening, 3′UTR reporter assay, qRT-PCR, and western blotting. ( c ) Radar charts showing association between miRNA and targets in BJAB cells. Solid lines indicate mRNA level affected by miRNA expression (n = 3); filled lines indicate 3′UTR luciferase value (n = 3~4). ( d ) BCR factors expression in miRNA-expressing SUDHL8 analyzed by western blotting and flow cytometry (for CD79B). NIK was visualized by MG132 pretreatment. ( e ) Pull-down assay with biotinylated miRNA (Bi-miRNAs). Bi-miRNAs or mock were transfected into BJAB cells. The biotinylated miRNA and targeted mRNA complex were pull-downed by streptavidin beads. The incorporated miRNA (right graph) and mRNA of BCR factors (left graph) were quantified by qRT-PCR (n = 3, mean values). ( f ) mRNA quantification of sucrose-gradient fractions in miRNA-expressing SUDHL8. The fractionated mRNA levels (miRNA expression vs. Empty) are plotted. Representatives of two independent experiments are shown. ( g ) Polysome analysis of DLBCL samples (n = 3, solid lines) and B cell (dashed line). The mRNA levels of BCR factors in each fraction were quantified by qRT-PCR and calculated as relative to the lightest fraction (no.1). Line colors indicate different individuals. ( h ) Polysome analysis in shAgo2 or shCtrl-expressing LCL. BCR mRNA levels in each fraction were quantified. Representative of two independent experiments is shown.

Article Snippet: DLBCL cell lines SUDHL4, SUDHL6, and SUDHL8 were purchased from DSMZ.

Techniques: Reporter Assay, Quantitative RT-PCR, Western Blot, Expressing, Luciferase, Flow Cytometry, Pull Down Assay, Transfection

Journal: Scientific Reports

Article Title: Coordinated loss of microRNA group causes defenseless signaling in malignant lymphoma

doi: 10.1038/srep17868

Figure Lengend Snippet: ( a ) BJAB cells were transduced with miRNA or shSYK-encoding lentivirus, followed by stimulation with anti-IgM at indicated concentrations for 2 days. CD25 expression level (mean fluorescence intensity, MFI) was analyzed by flow cytometry. ( b ) miR 3 suppresses NF-κB activation in B cell. BJAB cells with miRNA or shRNA targeting BCR factor were treated with indicated B cell stimulants for 24 hours. Relative NF-κB activity was analyzed by 6 × κB luciferase assay (n = 3). ( c ) Depletion of BCR signaling factors suppresses B cell lymphoma cell proliferation. SUDHL8 cells were treated with shRNA-expressing lentivirus, followed by long-term culture in complete medium. Results as percentage of each Venus-positive population transduced with shRNA series are plotted. Similar result was observed in SUDHL4 cells (data not shown). We confirmed that the knockdown efficiencies were comparable among the shRNAs (mean fold change vs. shCtrl is 0.255 ± 0.09 in 293T cells). ( d ) Results of qRT-PCR in DLBCL cohort (total, n = 68; GC, n = 31; non-GC, n = 35) shown as value relative to normal B cells. ( e ) Western blots showing total and phosphorylated Akt and PIK3CD (p110δ) levels in SUDHL8/miRNA cells. ( f ) Western blots showing cellular levels of RasGRP3, Ras-GTP (active form of Ras), total Ras, phosphorylated and total Erk1/2 and Akt in SUDHL8/shRasGRP1, /shRasGRP3, /miR-200c, and /miR-203 cells.

Article Snippet: DLBCL cell lines SUDHL4, SUDHL6, and SUDHL8 were purchased from DSMZ.

Techniques: Transduction, Expressing, Fluorescence, Flow Cytometry, Activation Assay, shRNA, Activity Assay, Luciferase, Knockdown, Quantitative RT-PCR, Western Blot

Journal: Clinical Cancer Research

Article Title: Interactome of Aiolos/Ikaros Reveals Combination Rationale of Cereblon Modulators with HDAC Inhibitors in DLBCL

doi: 10.1158/1078-0432.CCR-21-3347

Figure Lengend Snippet: Sensitivity to lenalidomide and avadomide is associated with baseline chromatin state at Aiolos and Ikaros–regulated genes. A, mRNA from indicated DLBCL cells were purified following treatment with DMSO, lenalidomide, or avadomide (1 μmol/L) for 18 hours and gene expression via RNA-seq was performed. Gene set variation analysis (GSVA) Hallmark Interferon-α response genes enrichment plot is shown for a category identified as positively enriched with treatment. Values are scaled as Z-scores. Negative and positive values do not have a specific meaning, but they are rather part of the same continuous scale. B, Comparison of chromatin states (poised: H3K27ac, H3K4me3; repressive: H3K27me3, H3K9me3) for promoters of Interferon-α response genes in indicated DLBCL cells. C, Scatterplot demonstrating correlation between average log2 fold change in gene expression and log2 fold change in H3K27ac, H3K4me3 abundance at promoters of Interferon-α response genes after 18 hours treatment with avadomide in indicated DLBCL cells. D, ChIP-seq peaks demonstrating direct binding of Aiolos and Ikaros at representative promoters of Interferon-α response genes in TMD8 DLBCL cells.

Article Snippet: DLBCL (TMD8, U2932, WSU-DLCL2, SUDHL-4, OCI-LY18, RL, WILL1, Pfeiffer, SUDHL-8, Riva) were obtained from ATCC and DSMZ, authenticated through use of AmpFLSTR (ThermoFisher) and cultured in RPMI1640 containing 10% FBS, 1% Penicillin/Streptomycin, and 1 mmol/L sodium pyruvate.

Techniques: Purification, Gene Expression, RNA Sequencing, Comparison, ChIP-sequencing, Binding Assay

Journal: Clinical Cancer Research

Article Title: Interactome of Aiolos/Ikaros Reveals Combination Rationale of Cereblon Modulators with HDAC Inhibitors in DLBCL

doi: 10.1158/1078-0432.CCR-21-3347

Figure Lengend Snippet: Targeting NuRD complex through targeting Aiolos/Ikaros and HDAC1/2 induces synergistic antiproliferative effect in DLBCL cells. A, TMD8 and SUDHL-4 DLBCL cells were treated with DMSO, avadomide (0.1–1 μmol/L), CC-241 (0.1–1 μmol/L), or a combination of both for 18 hours and gene expression was assayed for IFIT3, DDX58, and β-actin by qPCR. B, WSU-DLCL2 and SUDHL-4 GCB-DLBCL cells treated with DMSO, avadomide (0.25–1 μmol/L), CC-241 (0.5–1 μmol/L), or a combination of both for 3 days. Cell lysates were separated by SDS-PAGE and levels of Aiolos, IFIT3, DDX58, IRF7, and Tubulin were assessed. C, Top, indicated DLBCL cell lines were treated with DMSO, avadomide, and CC-241 (0.01–10 μmol/L) for 5 days. Proliferation for all cell lines was determined using the H-thymidine incorporation method. Results of three independent experiments are shown (mean ± SEM). Bottom, TMD8, WSU-DLCL2, and SUDHL-4 (left to right) were treated with DMSO, CC-241 (0.01–10,000 nmol/L), avadomide (1 μmol/L), or a combination of citarinostat (CC-241) with avadomide for 5 days. Proliferation for all cell lines was determined using the H-thymidine incorporation method. Results of three independent experiments are shown (mean ± SEM).

Article Snippet: DLBCL (TMD8, U2932, WSU-DLCL2, SUDHL-4, OCI-LY18, RL, WILL1, Pfeiffer, SUDHL-8, Riva) were obtained from ATCC and DSMZ, authenticated through use of AmpFLSTR (ThermoFisher) and cultured in RPMI1640 containing 10% FBS, 1% Penicillin/Streptomycin, and 1 mmol/L sodium pyruvate.

Techniques: Gene Expression, SDS Page

Journal: Cell reports

Article Title: Targeting Excessive EZH1 and EZH2 Activities for Abnormal Histone Methylation and Transcription Network in Malignant Lymphomas.

doi: 10.1016/j.celrep.2019.10.083

Figure Lengend Snippet: Figure 5. EZH1- and EZH2-Dependent H3K27me3 in DLBCL (A) EZH1 and EZH2 levels in primary frozen DLBCL (GC, n = 31; non-GC, n = 35; EBV[], n = 45; EBV[+], n = 23) and CD19+ normal B cells (n = 7). *p < 0.01 and **p < 107. (B) EZH2 promoter activity in BJAB cells treated with anti-IgM. n = 4, mean ± SD, *p < 0.05. (C) EZH2 promoter activity in BCR pathway-activating cells. n = 4, mean ± SD, *p < 0.05. (D) SUDHL8 (non-GC type, EZH2WT/WT, BCL6/CD10) and WSU-DLCL2 (GC type, EZH2WT/Y641F, BCL6+/CD10+) were treated with shEZH1 or shEZH2, and results as Venus-competitive assay (representative of n = 2) are shown. Functional PRC2 was detected by SUZ12 co-IP. (E) SUDHL8 and WSU-DLCL2 with shCtrl or shEZH1 (#1, #2) were cultured for 14 days in the presence of several dose GSK126. n = 3, mean ± SD. (F) DLBCL cell lines were cultured for 14 days in the presence of several dose EZH inhibitors. n = 3, mean ± SD.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER HCT116 ATCC Cat# CCL-247 EOL-1 ECACC Cat# EC94042252-F0 SUDHL4 DSMZ Cat# ACC 495 SUDHL6 DSMZ Cat# ACC 572 SUDHL8 DSMZ Cat# ACC 573 WSU-DLCL2 DSMZ Cat# ACC 575 Toledo ATCC Cat# CRL-2631 Pfeiffer ATCC Cat# CRL-2632 B95.8 EBV-transformed B lymphoblastoid cell lines (LCLs) Horie R. N/A 293T Watanabe T. N/A 293FT Yamochi T. N/A Experimental Models: Organisms/Strains NOD.Cg-PrkdcscidIl2rgtm1Sug/Jic (NOG) female mice In-Vivo Science Inc N/A Non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) female mice Charles River Strain code 394 Oligonucleotides See Table S1 N/A N/A Recombinant DNA CS-RfA-EVBsd RIKEN Cat# RDB06090 pENTR4-H1 RIKEN Cat# RDB04395 pCAG-HIVgp RIKEN Cat# RDB04394 pCMV-VSV-G-RSV-Rev RIKEN Cat# RDB04393 pSINsi-U6 TAKARA Cat# 3661 pGP (gag-pol coding) TAKARA Cat# 6161 pE-ampho (env coding) TAKARA Cat# 6161 pRx-puro Watanabe T. N/A Software and Algorithms FlowJo FlowJo LLC 9.9.5 R The R Foundation 3.2.3 DAVID Bioinformatics Resources https://david.ncifcrf.gov/ 6.8 GeneSpring Agilent Technologies 12.5 GraphPad Prism GraphPad Software 4.03 Scaffold 4 Proteome Software Inc 4.8.6 Other ATL gene expression data (Expression array) Yamagishi et al., 2012 GEO: GSE33615 ATL gene expression data (Expression array) Kobayashi et al., 2014 GEO: GSE55851 H3K27me3 data in ATL (ChIP-on-chip data) Fujikawa et al., 2016 GEO: GSE71450 PTCL gene expression data (Expression array) Iqbal et al., 2010 GEO: GSE19069 DLBCL gene expression data (Expression array) Tagawa et al., 2005 GEO: GSE16920 RNA-seq data of normal cells and tissues Roadmap Epigenomics project www.roadmapepigenomics.org/

Techniques: Activity Assay, Functional Assay, Co-Immunoprecipitation Assay, Cell Culture