Valls et al. Targeting BCL6 in FL Supplemental Information for: BCL6 antagonizes NOTCH2 to maintain survival of human follicular lymphoma cells Ester Valls1#, Camille Lobry2,7#, Huimin Geng1,3,8, Ling Wang1, Mariano Cardenas1, Martín Rivas1, Leandro Cerchietti1, Philmo Oh2, Shao Ning Yang1, Erin Oswald1, Camille W. Graham4, Yanwen Jiang1, Katerina Hatzi1,9, Xabier Agirre1,10, Eric Perkey5,11, Zhuoning Li1, Wayne Tam6, Kamala Bhatt2, John P. Leonard1, Patrick A. Zweidler-McKay4, Ivan Maillard5, Olivier Elemento3, Weimin Ci1,12*, Iannis Aifantis2* and Ari Melnick1* 1Division of Hematology/Oncology, Department of Medicine; Weill Cornell Medical College, New York, NY, 10065, USA 2Department of Pathology and Perlmutter Cancer Center, New York University School of Medicine, New York, New York 10016, USA 3Institute for Computational Biomedicine, Weill Cornell Medical College, New York, NY, 10065, USA 4Division of Pediatrics, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA 5Life Sciences Institute, Division of Hematology-Oncology, Department of Internal Medicine; University of Michigan, Ann Arbor, MI, USA 6Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, 10065, USA 7Institut Gustave Roussy, INSERM U1170, Villejuif and Université Paris Sud, Orsay, France 8Department of laboratory Medicine, University of California San Francisco, San Francisco, CA, 94143, USA 9Department of Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA 10Division of Hematology/Oncology, Centre for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain 11Graduate Program in Cellular and Molecular Biology 12Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China 1 Valls et al. Targeting BCL6 in FL Inventory of Supplemental Information Supplementary Figure S1. Related to Figure 1 Supplementary Figure S2. Related to Figure 2 Supplementary Figure S3. Related to Figure 3 Supplementary Figure S4. Related to Figure 3 Supplementary Figure S5. Related to Figure 4 Supplementary Figure S6. Related to Figure 5 Supplementary Figure S7. Related to Figure 5 Supplementary Figure S8. Related to Figure 6 Supplementary Table S1. Related to Figure 1 and Supplementary Figure S1 Supplementary Table S2. Related to Figure 1 Supplementary Table S3. Related to Figure 1 Supplementary Table S4. Related to Figure 1 Supplementary Table S5. Related to Figures 1, 2, 3, 4, 6, Supplementary Figures S1, S3, S4, S6 and S7. Supplementary Table S6. Related to Supplementary Figure S1 Supplementary Table S7. Related to Supplementary Figure S1 Supplementary Table S8. Related to Supplementary Figure S1. Supplementary Table S9. Related to Figure 1 and Supplementary Figure S1 Supplementary Table S10. Related to Figure 2 2 Valls et al. Targeting BCL6 in FL Supplementary Figure S1 A GENE FUNCTIONS B tyrosine-specific YES1, HCK protein kinase tyrosine-specificTyrosine-specific protein protein kinase kinasecell cycle CDK8, EIF4G2, FH, HERC5, NOTCH2 cellCell cyclecycle apoptosis ID3, IFIH1, NOTCH2, SOCS2 cellular structure ARF6, ARHGAP15, BLZF1, DDX5, ERBB2IP, NOTCH2, apoptosisApoptosis morphogenesis SOCS2, UBB cellularCellular structure structure morphogenesis morphogenesis lymphoid organ AOF2, BAZ2A, CBX3, CCL3, CCR6, ECGF1, ID2, NOTCH2, lymphoidLymphoid organ organ development development development RPS19, SETMAR, SMARCE1, TSPYL1 transcription AOF2, ARID3B, BAZ2A, BLZF1, CBX3, CDK8, CTNND1, transcriptionTranscription HIPK1, ID2, ID3, MEIS2, MLLT3, NOTCH2, NR4A2, NR4A3, RPS14, SMARCE1, SUPT3H, THAP7, TRIM33, TXNIP, UBB, othersOther functions functions ZBTB3, ZNF133, ZNF140, ZNF184, ZNF304, ZNF331, ZNF335, ZNF432, ZNF473, ZNF524, ZNF573, ZNF74, ZNF79 C 3 1kb 2kb 2kb 2kb 1kb 1.30 0 3.5 1.17 0 2 1.20 0 2 1.36 0 BCL6 binding enrichment in FL FL in enrichment binding BCL6 NOTCH2 BCL6 TP53 HPRT1 COX6B D GCB E BCL6 targets in DLBCL BCL6_targets_CHIPCHIP NEG BCL6 Proliferation_DLBCL IgG Cell_cycle_Whitfield Proliferation RBP-Jk Myc_ChIP_PET_Expr_Up Glutamine_Glucose_starve_both_down Serum_response_Fb_down MAML2 Glutamine_starve_down Tcell_cytokine_induced_proliferation Pan_B_U133plus NOTCH2 Resting_monocyte_GNF Myc_RNAi_OCILy3 0.0 0.5 1.0 1.5 2.0 IL6_Ly10_Up_group1 % enrichment vs. input Germinal_center_Bcell_DLBCL Myc_overexpression_1.5x_up F BCL6 expression BCL6 G GCB DLBCL (n=71) low high -3 BCL6 expression relative expression expression relative -2 -2 BCL6_Ave -1 -1 BCL6_1 BCL6_2 0 0 BCL6_3 +1 +1 NOTCH2_1 NOTCH2_2 +2 +2 NOTCH2_3 +3 +3 RBPJ_1 RBPJ_2 CDKN1A_1 MAML2_1 NOTCH1_1 MAML2_2 CCND1_1 NRARP_1 HEY1_1 NOTCH1_2 BCL6 targets in GCB-DLBCL expression arrays arrays expression GCB-DLBCL in targets BCL6 p<0.05 in GCB- DLBCL 3 Valls et al. Targeting BCL6 in FL Supplementary Figure S1: BCL6 displays a specific genomic localization pattern in FL. (A) A pie chart representation of the involved gene functions of the 184 target genes. Gene function analysis was performed by David Gene Functional Annotation Tool [1, 2]. (B) NOTCH2 is highlighted on the classification of BCL6 bound-genes on involved gene functions from previous panel A. (C) BCL6 binding enrichment in FL. From left to right: NOTCH2, BCL6, TP53 (the last 2 as positive control targets), and, HPRT1 and COX6B as negative control targets. (D) QChIP was performed in primary GCB cells to validate BCL6 binding to direct target genes repressed in FL specimens. BCL6 enrichment is represented as % of input. Black bars represent BCL6 and gray bars IgG control. The graph shows the mean of three independent experiments and error bars are the standard error of the mean (SEM, for primers see Supplementary Table S5). (E) The relative enrichment of specific gene signatures on DLBCL BCL6 target gene sets summarized in a heat map. The statistical significance (BH adjusted p values) is provided in color key. (F) A heatmap representation of the relative transcript abundance of BCL6 target genes in GCB-DLBCLs that display inverse correlation (p<0.05, Spearman correlation) with BCL6 expression, from a publicly available dataset of 71 primary DLBCL expression profiles. The color key indicates the relative expression values. (G) Primary GCB-DLBCL gene expression profiles were sorted by BCL6 expression from low to high (top row of heatmap), and the relative expression values of a set of Notch complex and target genes displayed in subsequent rows, indicating their degree of inverse correlation (p values are all p<0.05, Spearman correlation, n=71) with BCL6. Details are provided in Supplementary Tables S5, S7 and S8. (See Figure 1). 4 Valls et al. Targeting BCL6 in FL Supplementary Figure S2 A B Naïve B-cells GC B-cells 100100 100100 MW (KDa) 100 8080 8080 BCL6 100 6060 6060 ICN2 75 5.71 94.594.5 Max % of % of Max 4.354.35 95.795.2 50 4040 4040 Actin 37 Relative cellnumber Relative cell number number Relative cell 2020 2020 Naive GC B-cells 0 0 2 3 4 5 0 2 3 4 5 0 2 3 4 5 0102 310 410 5 10 0 10010 1010 1010 1010 0 10 10 PE-A10 10 FITC-A IgD CD38 C Murine splenocytes 100100 8080 6060 % of Max 0.39 99.699.6 4040 Relative cell number 2020 0 0 2 3 4 5 0102 10 3 10 4 10 5 0 10 10APC-A 10 10 CD45R / B220 Supplementary Figure S2: Inverse correlation between BCL6 and NOTCH2 complex genes in primary GC B-cells. (A) Naïve B-cells (red) and GC B-cells (green) were purified as detailed in methods section. Purity was >95% and >94% purity for naïve and GC B-cells respectively based IgD and CD38 B-cell markers. The histogram in the left panel shows the relative cell number of IgD staining for Naïve B-cells; the right panel shows the same for CD38 positive GC B-cells. (B) Immunoblots were conducted using BCL6 and ICN2 antibodies in lysates from purified human Naïve B-cells and GC B- cells. Actin immunoblot is included as loading control. (D) Murine splenocytes (blue) were isolated as detailed in methods section. Purity was >95% in all experiments as verified with staining for CD45R/B220+ B-cell marker. Three independent experiments and representative cell purification is shown. (See Figure 2). 5 Valls et al. Targeting BCL6 in FL Supplementary Figure S3 PNA A B μ μ Genotype Day Tissue Experiment 40 m 20 m WT ICN2 -1 Serum WT WT ICN2 0 NP65-CGG/SRBC WT ICN2 1 ICN2 Tamoxifen BCL6/ B220 C 40 μm 20 μm WT ICN2 7 Serum WT WT Serum WT ICN2 14 FACS Spleen ICN2 IHC D E 20 PNA 20 BCL6/ B220 p= 0.0001 p= 0.0001 15 15 10 10 5 5 Number of GC/ Spleen 0 Number of GC/ Spleen 0 WT ICN2 WT ICN2 n=5 n=8 n=5 n=8 H F G P < 0.0001 P = 0.0002 PNA BCL6/ B220 300 p < 0.0001 p = 0.0002 ) 2 WT 1000 1000 m 750 μ ICN2 ) 750 ) 2 2 m m 500 200 μ 500 μ 400 400 300 300 GC area( 200 GC area( 200 100 100 100 GC area ( Average 0 0 WT ICN2 WT ICN2 0 PNA BCL6/ B220 6 Valls et al. Targeting BCL6 in FL Supplementary Figure S3: GC reaction is impaired in Notch2 knock-in mice. (A) Schematic representation of GC induction experiments in ICN2 inducible mice. The first row reflects the alleles present in the experiment that include ROSA26 WT (in black) and ROSA26-ICN2-IRES-YFP or ROSA26-ICN2-YFP/CγCre (in green); the second row is the day; the third is the tissue or serum harvest and the fourth column is the assay conducted. GC immunization used was NP65-CGG for mouse strain engineered to contain an ICN2-IRES-YFP (ICN2) cassette with a loxP flanked NEO-ATG knocked in to the ROSA26 locus, crossed with a tamoxifen inducible ROSA26-Cre-ERT2 strain or in ROSA26-WT (WT) – Cre-ER T2, or Sheep Red Blood Cells (SRBC) for CγCre-WT or -ICN2 mice. (B) Representative images of spleen sections from CγCre-WT (control) and -ICN2 knock-in mice stained for GC markers PNA 14 days after immunization with SRBC.