Supporting Information

Fabbri et al. 10.1073/pnas.1702564114

a. b.

4X GCs

20x

4X GCs

4X Tonsil epithelium

ICN1 CD20 DAPI

Fig. S1. ICN1 is expressed in the B-cell fraction populating the mantle zone (M) of the germinal centers (GCs). (A) Double IF staining of ICN1 and AID in a representative GC in a human tonsil section. (B) Double IF staining of ICN1 and the B-cell–specific surface antigen CD20 at lower magnification (4×) in a human tonsil section.

Fabbri et al. www.pnas.org/cgi/content/short/1702564114 1of10 Fig. S2. ICN1 expression analysis in a panel of primary CLL cases and PBMC. (A) IB analysis of ICN1 and control β-actin in a panel of 124 CLL PB primary CLL cases, (B) in primary NOTCH1–wild-type CLL cells treated with the γ-secretase inhibitor Compound E (CpE, 500 nM, 8 h) or control DMSO, and (C) in PBMC protein extracts and representative primary CLL cases expressing ICN1. Samples are color-coded based on the NOTCH1 mutational status [red, clonal NOTCH1 PEST-truncating events; orange, subclonal NOTCH1 PEST-truncating events; blue, RAG-mediated NOTCH1 translocation (83); and black, NOTCH1–wild-type]. Samples in gray were excluded from the analysis because of low quality of the protein lysate, low viability, or low leukemic representation. Color-coded arrows indicate cases subjected to RNA-Seq analysis: dark red denotes NOTCH1-mutated cases expressing ICN1; blue, NOTCH1–wild-type cases expressing ICN1; and green, ICN1− NOTCH1–wild-type cases. Abbreviations: MO+DL1, MO1043 cells cocultured on OP9-DL1 cells (54); s.e., short exposure; l.e., long exposure.

Fabbri et al. www.pnas.org/cgi/content/short/1702564114 2of10 a. T-ALL (+) CLL CUTLL1 CEM MO1043 HD-M - + - PEST-M - + + t(7;9) + - - CpE (GSI) 72hr - + - + - +

IB:ICN1

IB:β-actin

5'LTR TRE2 ICN1-HA Ubc rtTA3 IRES Puro 3'LTR ICN1-HA

5'LTR TRE2 eGFP Ubc rtTA3 IRES Puro 3'LTR eGFP mir30 mir30 Xhol c. d. e. WCE CE NE +Doxy +Doxy - abcdef

IB:HA eGFP ICN1-HA ICN1-HA (ICN1) eGFP

IB:HA (ICN1) IB:β-actin mRNA expression mRNA IB:β-tub relative to eGFP (fold) relative to eGFP IB:Lamin-B1 DTX1 abcdef

Fig. S3. Establishment of ICN1-HA inducible MO1043 CLL cell line. (A) IB analysis of ICN1 and control β-actin in T-ALL cell lines (CUTLL1 and CEM) and MO1043 CLL cells treated with the γ-secretase inhibitor Compound E (CpE, 500 nM, 72 h) or control DMSO. (B) Diagrams of the pINDUCER vectors used to express ICN1- HA or control eGFP upon doxycycline addition (30). (C) IB analysis of HA (ICN1) and β-actin in MO1043-ICN1-HA whole cellular lysates (WCE) obtained upon doxycycline addition (1 μg/mL for 24 h, six independent inductions, a–f). (D) HA (ICN1) IB analysis of cytoplasmic (CE) and nuclear (NE) fractions of MO1043- ICN1-HA and control eGFP cells upon doxycycline addition (1 μg/mL for 24 h). β-Tubulin (β-tub) and Lamin-B1 IBs serve as controls for the purity of the sub- cellular fractions. (E) qRT-PCR analysis of DTX1 mRNA in MO1043-ICN1-HA cells upon doxycycline addition (1 μg/mL for 24 h, six independent inductions, a–f). Results are presented relative to those of induced MO1043-eGFP cells, set as 1. The bar graphs show the mean values, and the error bars represent the SD.

Fabbri et al. www.pnas.org/cgi/content/short/1702564114 3of10 a. TSS peaks All peaks H3K4me3 (n=27696) (-5/+4kb) Region % -2/+1 kb from TSS 50.7 -5/-2 kb from TSS 13.6 Exons 0.9 Introns 11.8 Intergenic 23.0 H3K4me3

H3K4me (n=105240) Region % -2/+1 kb from TSS 13.9 -5/-2 kb from TSS 17.6 Exons 1.1 Introns 29.6 Intergenic 37.8 H3K4me

H3K27Ac (n=57617) Region % -2/+1 kb from TSS 23.3 -5/-2 kb from TSS 9.5 Exons 1.1 Introns 30.6 Intergenic 35.5 H3K27Ac

H3K27me3 (n=53913) Region % -2/+1 kb from TSS 3.3 -5/-2 kb from TSS 5.1 Exons 0.6 Introns 21.0 Intergenic 70.0 H3K27me3 TSS Distance TSS Absolute Distance (kb) (bp) b.Percentage (%) c. (n=20417 genes) 18.5

23 49.2 9.3

Active promoter Silenced promoter Poised promoter Active Poised Silenced Other Other (n=10051) (n=1890) (n=4689) (n=3787) Promoters

Fig. S4. Distribution of histone modifications across the genome of MO1043-ICN1-HA cells. (A) Genomic distribution of histone marks-decorated regions with respect to the closest TSS (Left) and their distribution in the genome (Right). (B) Functional promoter status of genes based on the chromatin configuration of their TSSs and gene-expression levels (FPKM) of the corresponding transcripts (C) in MO1043-ICN1-HA CLL cells upon doxycycline addition (1 μg/μL for 24 h).

Fabbri et al. www.pnas.org/cgi/content/short/1702564114 4of10 a. SEs (n=917) H3K27me3 H3K4me1 P<0.0001 2%

100% 98%

Overlapping Not overlapping

b. P<0.0001

c. NOTCH1-BS (n=4737) Overlapping with SEs Other (n=698) (n=4039) P<0.001

29% 4%8% 52 71%

Up in MO1043-ICN1-HA cells Other

Fig. S5. CLL superenhancers features. (A) Overlap of MO1043-ICN1-HA superenhancers with the H3K27me3 and H3K4me1 chromatin marks. (B) Expression levels (log2 FPKM) of genes associated with superenhancers (SE) or regular enhancers (E) identified in MO1043-ICN1-HA cells. (C) Differential up-regulation of genes associated with NOTCH1 binding sites (BS) overlapping with superenhancer regions or located elsewhere in the genome (“other”) of MO1043-ICN1-HA cells. In A and C, P values are reported according to a two-tailed Fisher’s exact test. In B,theP value was calculated based on an unpaired unequal variance two- tailed Student’s t test.

Fabbri et al. www.pnas.org/cgi/content/short/1702564114 5of10 a.Top NOTCH1-bound genes b. Leading edge genes (n=400) (n=62) ICN1 eGFP HES4 NES=1.51 P2RY2 P DTX1 =0.00 C12orf42 FBN2 PDP1 IRAK2 ICN1 eGFP GLIPR1 ASCL1 n=4 n=4 AHCYL2 IKZF1

(RES) FSTL5 APLF DUSP16 MDGA1 AKT3 NCALD

Running Enrichment Score SSH1 EBI3 ENTPD1 ZNF425 HIRA OXR1 MALT1 CALCOCO1 ZNF554 IL15 EXD3 PAX5 AKAP13 KCTD21 GHR TCP11L2 PXDNL ATXN7L2 ROBO1 C2orf42 TMEM140 TSNAXIP1 BCL2 LILRB1 NLRC5 GRAP CCDC146 TGM5 PPP1R9B DLK2 ZNF398 GRAPL IL2RB C8orf44-SGK3 ZFYVE1 KCNRG NOTCH2NL CPOX CAPNS1 TFEB BACE2 COQ2 APBA3 ST3GAL2 BAZ2A -3 3

Fig. S6. Significant enrichment of top NOTCH1-bound genes in MO1043-ICN1-HA compared with control -eGFP cells. (A) GSEA enrichment plot depicting significant enrichment of a geneset composed by the top 400 NOTCH1-bound genes (i.e., top genes ranked based on ChIP-Seq P values) in MO1043-ICN1-HA CLL cells compared with -eGFP controls and (B) corresponding leading edge genes.

Fabbri et al. www.pnas.org/cgi/content/short/1702564114 6of10 BCR-NF-κB chr8:11334764-11379820 chr18:56296846-56433000 4 4

0 0 5 8

0 0 2 2 rpm 0 rpm 0 1 1

0 0 1 1

0 0 BLK MALT1

Chemokine Cell Cycle

chr2:136820809-136926834 chr6:41904064-42053263 2 3

0 0 6 8

0 0 2 2 rpm rpm 0 0 1 1

0 0 0.5 0.4

0 0 CXCR4 CCND3 TAF8

Apoptosis NOTCH chr1:150522456-150576784 chr20:10609037-10663989 5 5

0 0 9 8

0 0 2 2 rpm rpm 0 0 1 1

0 0 0.4 0.4

0 0 MCL1 JAG1

SEs NOTCH1-BS RBPJ motif H3K27Ac H3K4me3 H3K4me H3K27me3 NOTCH1

Fig. S7. Representative examples of NOTCH1-direct target genes. Representative ChIP-Seq plots depicting NOTCH1 binding and histone marking patterns at genes that are bound by NOTCH1 and up-regulated in MO1043-ICN1-HA CLL cells. The y axes in the ChIP-Seq plots indicate fragment density in reads per million (rpm).

Fabbri et al. www.pnas.org/cgi/content/short/1702564114 7of10 a. b. Tissue SE1 SE2 Tissue SE1 SE2 CD20 DHL6 Panel 1 (n=452) Panel 2 (n=353) CD19 DND41 Ly3 Duodenum Smooth Muscle Tonsil Esophagus VACO 400 Fetal Intestine HMEC Fetal Intestine Large HCT-116 Fetal Muscle GM12878 Fetal Thymus HeLa Gastric HCC1954 H1 CD4p CD25- Il17- PMAstim Th H2171 Adipose Nuclei HBL1 Adipose Tissue HepG2 Adrenal Gland HSMMtube Aorta HUVEC c. Astrocytes IMR90 Bladder Jurkat Chr8 Brain Angular Gyrus K562 128.0Mb 998kb 128.9Mb Brain Anterior Caudate Left Ventricle Brain Cingulate Gyrus LNCaP n=30/805 Brain Hippocampus Middle Lung Panel 1 and 2 Brain Hippocampus Middle 150 Ly1 Brain Inferior Temporal Lobe Ly4 Brain Mid Frontal Lobe MCF-7 CN gains CD14 MM1S MYC CD3 NHDF-Ad CD34 adult NHLF MCR CD34 fetal Osteoblasts NOTCH1 CD34 Primary RO01480 Ovary V$RBPJK_01 CD34 Primary RO01536 Panc1 V$RBPJK_04 Motifs CD34 Primary RO01549 Pancreas SE SE1 SE2 CD4 Memory Primary 7pool Psoas Muscle CD4 Memory Primary 8pool Right Atrium CD4 Naive Primary 7pool Right Ventricle CD4 Naive Primary 8pool RPMI-8402 d. CD4p CD225int CD127p Tmem Sigmoid Colon Panel 1 n CD4p CD25- CD45RAp Naive Skeletal Muscle ( =71/452 altered cases) CD4p CD25- CD45ROp Memory Skeletal Muscle Myoblast UM CD4p CD25- Il17p PMAstim Th17 Small Intestine IGHV M CD56 Spleen NA CD8 Memory 7pool Stomach Smooth Muscle NOTCH1 CD8 Naive 7pool Thymus -M Altered CD8 Naive 8pool Toledo WT CD8 primiary u87 MYC-act Colon B49:D78Crypt 1 VACO 503 Colon Crypt 2 VACO 9m Colon Crypt 3 Present Absent

Fig. S8. Focal copy number gains recurrently affect the NOTCH1-bound 8q24 B-cell–specific superenhancer region in CLL. (A) Overlap between NOTCH1- bound 8q24 superenhancers observed in CLL and other tissues. In the heatmap, rows correspond to different tissues (normal and malignant) reported in the dbSUPER database (84) and columns represent the two superenhancers identified in the 8q24 region encompassing the MYC locus, color-coded based on their presence or absence in the displayed cell type (light gray, absent; black, present). (B) Frequency of NOTCH1 mutations and MYC CN gains (including gains encompassing only the MYC-associated superenhancer region) in a panel of 452 primary CLL cases, as reported in Puente et al. (7) and of MYC CN gains (including gains encompassing only the MYC-associated superenhancer region) in a panel of 353 primary CLL cases (53). (C) Graphic display of CN data from 30 patients harboring CN gains involving the 8q24 region encompassing the newly identified MYC-associated superenhancer regions in CLL. Segmentation data were visualized using IGV (2.3.59), where each track represents one sample, and white denotes a normal (diploid) CN, red a region of CN gain and bluea CN loss. Individual genes in the region are aligned in the Bottom panel, and the red boxed area highlights the minimal common region (MCR) of CN gain. In the bottom are highlighted the locations of NOTCH1 binding sites, RBPJK motifs (RBP_Jkappa V$RBPJK_Q4 and V$RBPJK_01 from the TRANSFAC database) and the superenhancers identified in CLL. (D) Heatmap showing the distribution of NOTCH1 mutations and MYC CN gains identified in n = 71/452 primary CLL cases, as reported in Puente et al. (7). In the heatmap, each column corresponds to a different case, and the two Bottom rows represent NOTCH1 mutations (M) and MYC alterations (act), color-coded based on their presence or absence in the displayed case (light gray, absent; black, present). The Top row shows the IGHV mutational status of the displayed cases (M, mutated; NA, not available; UM, unmutated).

Fabbri et al. www.pnas.org/cgi/content/short/1702564114 8of10 fCLclscclue nO9srmlcls nO9D1srmlclsi h rsneo p,o ihvhceDS,sta .IN-oiiecssdepict cases ICN1-positive 1. as set DMSO, vehicle with or CpE, of presence the in cells stromal the OP9-DL1 of on absence cells, or stromal presence OP9 the Bottom in on cells cocultured OP9-DL1 cells stromal CLL on of coculture via induction 1 ICN1 upon (CpE, cases CLL primary in expression S9. Fig. abie al. et Fabbri μ ae ( panel ,2 ,tpfv rps,o pnbslNTH inln niiini h rsneo p ( CpE of presence the in inhibition signaling NOTCH1 basal upon or graphs), five top h, 24 M, MYC www.pnas.org/cgi/content/short/1702564114 n N eesaersosv omdlto fNTH inln ciaini rmr L ae.qTPRaayi of analysis qRT-PCR cases. CLL primary in activation signaling NOTCH1 of modulation to responsive are levels RNA = )icue3 include 6) NOTCH1 mttdad3 and -mutated % of mRNA reduction upon CpE Relative mRNA expression (fold) NOTCH1 n =6 – idtp cases. wild-type n =6 n =6 n =6

Bottom

in ICN1-positive in rp) eut r ersne eaiet those to relative represented are Results graph). CpE) + OP9DL1 or OP9 to (compared ICN1-negative/low in

ICN1 inhibition ICN1 ICN1 induction ICN1 γ sceaeihbtrCmon E Compound inhibitor -secretase MYC and HES1 di the in ed mRNAs 9of10 chr9

NOTCH1-WT ICN1-pos H3K27Ac

NOTCH1-M ICN1-pos

NOTCH1-WT ICN1-neg

NOTCH1 MIR4673 MIR4674 NALT1

Fig. S10. H3K27Ac ChIP-Sequencing plots at the NOTCH1 locus in nine primary CLL cases. The y axes in the ChIP-Seq plots indicate fragment density in reads per million. M, mutated; WT, wild-type.

Other Supporting Information Files

Dataset S1 (XLSX) Dataset S2 (XLSX) Dataset S3 (XLSX) Dataset S4 (XLSX) Dataset S5 (XLSX) Dataset S6 (XLSX) Dataset S7 (XLSX) Dataset S8 (XLSX) Dataset S9 (XLSX) Dataset S10 (XLSX) Dataset S11 (XLSX) Dataset S12 (XLSX) Dataset S13 (XLSX)

Fabbri et al. www.pnas.org/cgi/content/short/1702564114 10 of 10