SUPPLEMENTARY APPENDIX CONTENTS
THE KRÜPPEL-LIKE FACTOR 2 TRANSCRIPTION FACTOR GENE IS
RECURRENTLY MUTATED IN SPLENIC MARGINAL ZONE LYMPHOMA
Roberto Piva
1§
, Silvia Deaglio
2§ , Rosella Famà 3 , Roberta Buonincontri 2 , Irene Scarfò 1 , Alessio Bruscaggin 3 , Elisabetta Mereu 1 ,
Sara Serra 2 , Valeria Spina 3 , Davide Brusa 2 , Giulia Garaffo 1 , Sara Monti 3 , Michele Dal Bo, 4 Roberto Marasca 5 , Luca Arcaini 6 ,
Antonino Neri 7 , Valter Gattei 4 , Marco Paulli 8 , Enrico Tiacci 9 , Francesco Bertoni 10 , Stefano A. Pileri 11 , Robin Foà 12 , Giorgio
Inghirami 2,13,14 , Gianluca Gaidano 3 , Davide Rossi 3*
§
R.P. and S.D. equally contributed
1 Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences Biology and Biochemistry and
2 Department of Medical Sciences, University of Turin and Human Genetics Foundation, Turin, Italy; 3 Division of Hematology,
Department of Translational Medicine, Amedeo Avogadro University of Eastern Piedmont, Novara, Italy; 4 Clinical and
Experimental Onco-Hematology, CRO, IRCCS, Aviano, Italy; 5 Division of Hematology, University of Modena and Reggio
Emilia, Modena, Italy; 6 Divisions of Hematology and 8 Pathology, Fondazione IRCCS Policlinico San Matteo & Department of
Molecular Medicine, University of Pavia, Pavia, Italy; 7 Department of Clinical Sciences and Community Health, University of
Milano and Hematology 1 CTMO, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy;
Hematology, University of Perugia, Perugia, Italy;
9 Institute of
10 Lymphoma and Genomics Research Program, IOR Institute of Oncology
Research and Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; 11 Haematopathology, L. & A. Seragnoli
Institute, University of Bologna, Bologna, Italy; 12 Division of Hematology, Department of Cellular Biotechnologies and
Hematology, Sapienza University, Rome, Italy; 13 Department of Pathology and New York University Cancer Center, New York
University School of Medicine, and 14 Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New
York, NY.
1

SUPPLEMENTARY METHODS
Samples
The study population was based on 756 lymphoid tumors, including 96 splenic marginal zone lymphomas, 154 diffuse large Bcell lymphomas, 100 chronic lymphocytic leukemias, 61 extranodal marginal zone lymphomas, 56 nodal marginal zone lymphomas, 56 Burkitt lymphomas, 27 mantle cell lymphomas, 24 BRAF p.V600E mutation positive hairy cell leukemias, 22 multiple myelomas, 21 follicular lymphomas, 11 BRAF p.V600E mutation negative variant hairy cell leukemias, 15 marginal zone lymphoma-like monoclonal B-cell lymphocytosis, 37 peripheral T-cell lymphomas and 76 T-cell acute lymphoblastic leukemias.
Consistent with the pathological diagnosis of SMZL, all cases lacked the t(11;18) and the t(14;18) translocations, and the p.V600E
BRAF mutation. All samples had been obtained at diagnosis from the involved site (lymph nodes or extra-nodal sites in the case of lymphoma; CD138+ cells purified from bone marrow aspirates in the case of multiple myeloma; peripheral blood purified B-cells in the case of hairy cell leukemia; peripheral blood mononuclear cells in the case of chronic lymphocytic leukemia, variant hairy cell leukemia, marginal zone lymphoma-like monoclonal B-cell lymphocytosis and T-cell acute lymphoblastic leukemia). In all cases, the fraction of tumor cells in the sample used for molecular studies was estimated to be >70% by morphology, immunohistochemistry and/or flow cytometry. Matched normal DNA was obtained from saliva or peripheral blood granulocytes in 260 patients. Patients provided informed consent in accordance with local IRB requirements and the Declaration of Helsinki.
The study was approved by the Ethical Committee of the Ospedale Maggiore della Carità di Novara affiliated with the Amedeo
Avogadro University of Eastern Piedmont (Protocol Code CE 116/12). Normal tonsil samples were obtained through the Servizio di Immunologia dei Trapianti, Città della Salute e della Scienza Hospital (Turin, Italy).
Cell lines and culture conditions
The HEK293T (ATCC), human T-cell acute lymphoblastic leukemia (T-ALL) Jurkat T-REx (Life Technologies), diffuse large Bcell lymphoma (DLBCL) OCY-Ly8, 1 Burkitt lymphoma AS283A, 2 and putative SMZL VL51, 3 SSK41 4 and KARPAS 1718 5
(Sigma-Aldrich) cell lines were cultured in in DMEM or RPMI-1640 media (Sigma-Aldrich) supplemented with 10% fetal bovine serum.
DNA extraction
High-molecular-weight (HMW) genomic DNA was extracted from tumor and normal samples according to standard procedures.
DNA was quantified by the NanoDrop 2000C spectrophotometer (Thermo Scientific). All DNA samples were verified for integrity by 1% agarose gel electrophoresis. Tumor cell clonality was established by amplification of the rearranged IGHV genes as previously described.
6 Analysis of patient-specific IGHV-IGHD-IGHJ rearrangements was also performed in the paired normal
DNA to exclude contamination from tumor cells.
Mutation analysis
The complete coding sequences and exon-intron junctions of KLF2 (NM_016270.2), SMURF1 (NM_020429.2), WWP1
(NM_007013.3), KLF3 (NM_016531.5), KLF4 (NM_004226.3), KLF6 (NM_001300.5), KLF8 (NM_007250.4), KLF9
(NM_001206.2), KLF14 (NM_138693.2), MAML1 (NM_014757.4), IKZF3 (NM_012481.4), S1PR1 (NM_001400.4), S1PR2
(NM_004230.3), S1PR3 (NM_005226.3), GNA12 (NM_007353.2), GNA13 (NM_006572.5), RBPJ (NM_005349.3), CXCR7
(NM_020311.2), IRF8 (NM_002163.2), FLI1 (NM_002017.4), CXCR5 (NM_001716.4), AIRE (NM_000383.3), TCF3
(NM_003200.3), ID3 (NM_002167.4), FOXO1 (NM_002015.3), DLK1 (NM_003836.5), CDKN1A (NM_000389.4), SELL
(NM_000655.4), and ITGB7 (NM_000889.2) were analyzed by PCR amplification and direct sequencing of whole-genomeamplified DNA obtained using the Repli-g Mini kit (QIAGEN).
7-22 Sequences for all annotated exons and flanking splice sites were retrieved from the UCSC Human Genome database using the corresponding mRNA accession number as a reference. PCR primers, located ~50 bp upstream or downstream to target exon boundaries, were designed in the Primer 3 program
(http://frodo.wi.mit.edu/primer3/) and filtered using UCSC in silico PCR to exclude pairs yielding more than a single product. All
PCR primers and conditions are available upon request. Purified amplicons were subjected to conventional Sanger sequencing using the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems), and compared to the corresponding germline sequences using the Mutation Surveyor Version 3.97 software package (SoftGenetics), after automated and/or manual curation. Candidate mutations were confirmed from both strands on independent PCR products obtained from HMW genomic DNA. Synonymous mutations, previously reported germline polymorphisms, and changes present in the matched normal DNA (when available) were removed from the analysis. To exclude known germline variants in cases for which paired normal DNA was not available we used the following databases: Human dbSNP Database at NCBI (Build 139) (http://www.ncbi.nlm.nih.gov/snp); Ensembl Database
(http://www.ensembl.org/index.html); The 1000 Genomes Project (http://www.1000genomes.org/); five single-genome projects available at the UCSC Genome Bioinformatics resource (http://genome.ucsc.edu/).
Deep next generation sequencing
Amplicons known to harbor KLF2 mutations were re-amplified from genomic DNA by oligonucleotides containing the genespecific sequences, along with 10-bp MID tag for multiplexing and amplicon library A and B sequencing adapters, using a high fidelity Taq polimerase (FastStart High fidelity PCR System, Roche Diagnostics). PCR products were then individually purified using Agencourt AMPure XP beads (Beckman Coulter) and quantified using the Quant-iT PicoGreen dsDNA kit (Invitrogen).
Corresponding patient-specific amplicon pools were generated by combining each of the amplicons in an equimolar ratio for each patient sample. The pools were diluted to a concentration of 1×10 6 molecules per μl and processed using the GS Junior Series Lib-
2

A method (Roche Diagnostics). Forward (A beads) and reverse (B beads) reactions were carried out using 5,000,000 beads per emulsion oil tube. The copy per bead ratio used was 1.1:1. The amplification reaction, breaking of the emulsions and enrichment of beads carrying amplified DNA were performed using the workflow as recommended by the manufacturer. Finally, the obtained amplicon library was loaded on a PicoTiterPlate (PTP) and subjected to ultra-deep-NGS on the Genome Sequencer Junior instrument (454 Life Sciences). The obtained sequencing reads were mapped to reference sequences and analyzed by the
Amplicon Variant Analyzer software (Roche) to establish the mutant allele frequency.
23
Copy number analysis by FISH
Copy number abnormalities of KLF2 were analyzed by FISH (probes: BAC RP11-1136A9, Fosmid G248P8818F11, Fosmid
G248P86655B5).
24 Labeled KLF2 probes were tested against normal control metaphases to verify the specificity of the hybridization. For each probe, at least 200 interphase cells with well-delineated fluorescent spots were examined. Nuclei were counterstained with 4',6'-diamidino-2-phenylindole (DAPI) and antifade reagent, and signals were visualized using an Olympus
BX51 microscope (Olympus Italia, Milan, Italy). The presence of copy number abnormalities was scored when the percentage of nuclei showing the abnormality was above 10%.
24
Copy number analysis by SNP array
Genome-wide DNA profiles were obtained from high molecular weight genomic DNA of SMZL patients using the GeneChip
Human Mapping 250K NspI Affymetrix) (GEO accession number: GSE24881) (Affymetrix, Santa Clara, CA, USA) as previously reported.
25
Modeling of the KLF2 zinc finger domain
The structural view of the KLF2 zinc finger domain was generated in DeepView-Swiss-PdbViewer (http://spdbv.vital-it.ch/) using the coordinates of the crystal structure of the KLF4 zinc finger domain (94% identity with KLF2) complexed with DNA (PDB
2wbs).
Production of doxycycline-inducible cell lines
To achieve inducible KLF2 expression, HEK293T cells were first transduced with pLV-DsRed-tTRKRAB vector (kindly provided by Dr. D. Trono, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland), 26 expanded, and sorted for high
DS-Red expression using FACS ARIA III (BD Biosciences), and used for transfection with pLenti CMV/TO puro DEST lentiviral particles carrying flag-tagged KLF2 variants. KLF2 expression was induced by doxycycline (1 μg/ml) and measured by RT-qPCR and western blotting at 72 and 96 hours, respectively.
Plasmid constructs, mutagenesis and transfection
Human KLF2 ORF was generated by PCR from SC127849 cDNA (Origene) using KLF2_EcoRI_FLAG85F
CTAGGAATTCGCCACCATGGACTACAAAGACGATGACGACAAGATGGCGCTGAGTGAACCCAT and
KLF2_XhoI_1152R CTAGCTCGAGCTACATGTGCCGTTTCATGTGC primers, tagged at the N-terminal with flag, and cloned into EcoRI/XhoI sites of pENTR1A no ccdB vector. Mutagenesis of wild type KLF2 was performed using QuickChange® II Site-
Directed Mutagenesis kit (Stratagene), according to the manufacturer's instructions (KLF2_A772T_F
GGCCAAGCCATAGCGCGGCCGCCG; KLF2_A772T_R CGGCGGCCGCGCTATGGCTTGGCC; KLF2_C862T_F
CAAGAGTTCGTATCTGAAGGCGCA; KLF2_C862T_R TGCGCCTTCAGATACGAACTCTTG). Wild type and mutant flagtagged KLF2 were verified by Sanger sequencing and recombined into the tetracyclin inducible lentiviral vector pLenti CMV/TO puro DEST using Gateway’s LR reaction (Invitrogen). pENTR1A and pLenti CMV/TO puro DEST were kindly provided by Eric
Campeau (Resverlogix Corp).
27 the manufacturer’s instructions.
Transfections of HEK293T cells were performed with Effectene reagent (Qiagen), according to
Purification of total RNA and cDNA synthesis
RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Milan Italy) and retro-transcribed using the Reverse Transcription
Kit (Applied Biosystems). Quantitative real-time PCR (qRT-PCR) was conducted with the 7900 HT Fast Real-Time PCR System
(SDS2.3 software) using commercially available primers (Applied Biosystems): Hs00360439_g1 ( KLF2 ) Reactions were done in triplicate from the same cDNA (technical replicates). The comparative CT method was used to calculate the relative expression of the gene under analysis. For each gene, expression levels were computed as the difference (ΔCT) between the CT of the target gene and those of ACTB CT (Hs99999903_m1).
Immunoblotting
Whole cell extracts were prepared as described, 28 while cytoplasmic and nuclear fractions were prepared with the Qproteome
Nuclear Protein kit (Qiagen). Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membranes, blocked (1 hour, room temperature) with 5% low-fat milk and incubated with the indicated antibodies. Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit or donkey anti-goat antibodies
(Amersham) were used to highlight binding by enhanced chemiluminescence (Amersham). Primary antibodies were: anti-KLF2
(Santa Cruz, N-13), anti-flag (Sigma, F1804), anti-β-tubulin (Sigma, T-4026), anti-BRG-1 (Santa Cruz, sc-17796), anti

-actin
(Millipore, MAB1501R), Image acquisition and densitometric analyses were performed using ImageQuant LAS4000 and TL
Version 7.0 software (GE Healthcare). The anti-KLF2 antibody for immunoblotting was validated using a blocking peptide (Santa
Cruz, sc-18690 P).
3

Luciferase assay
The pLightSwitch TM CDKN1A/p21 promoter (SwitchGear Genomics, #S721724) and the pLenti CMV/TO Puro DEST vector expressing wild type and mutant KLF2 were co-transfected in HEK293T tTR-KRAB cells with a 5:1 ratio. pGL3-Control Vector
(Promega) was used as positive control. KLF2 expression was induced by doxycycline (1 μg/ml) 24 hours post-transfection. Cell lysis was performed at 48 hours using 1X Passive Lysis Buffer (PLB). Firefly and renilla luciferase reporter activities were measured by automatic dispensing Stop & Glo Reagents according to the manufacturer’s instructions (Promega, #TM058) into the
Microplate Reader Synergy2 (Biotek).
Immunofluorescence
Expression of endogenous KLF2 was determined by using a mouse anti-KLF2 antibody (R&D Biosystems, MAB5466), 29 followed by Alexa-488 conjugated goat anti-mouse Ig. Actin filaments were visualized using Alexa-568 conjugated phallodin
(Life Technologies). Nuclei were counterstained with DAPI. HEK293T cells transiently transfected with flag-tagged wild type or mutant KLF2 constructs were seeded on coverslips, fixed (4% paraformaldehyde), permeabilized (0,1% saponin) and saturated
(pre-immune goat serum). Coverslips were then incubated with anti-flag followed by Alexa-633-conjugated goat anti-mouse IgG
(Life Technologies). DAPI (4,6 diamidino-2-phenylindole, Life Technologies) was used to counterstain. Twenty-four hours later, nucleofected cells were stained with anti-flag followed by Alexa-488-conjugated goat anti-mouse IgG (Life Technologies). Flagpositive cells were sorted and allowed to adhere on polylysine-coated coverslips. DAPI was used to counterstain. Slides were analyzed using a TCS SP5 laser scanning confocal microscope with four lasers (Leica Microsystems). Images were acquired with the LAS AF software (version Lite 2.4) and processed with Adobe Photoshop (Adobe Systems, San Jose, CA, USA).
Statistical analysis
Categorical variables were compared by chi-square test and Fisher’s exact test when appropriate. Continuous variables were compared by t-test and Mann-Whitney test when appropriate. All statistical tests were two-sided. Statistical significance was defined as p value <.05. The analysis was performed with the Statistical Package for the Social Sciences (SPSS) software v.21.0 or with the Graphpad software version 6.
4

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6

SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1. Missense KLF2 mutations of the zinc finger domain targets evolutionarily conserved codons. (A)
Sequencing traces of representative mutated tumor samples and paired normal DNA documenting the somatic origin of the two most recurrent missense substitutions affecting the zinc finger domain of KLF2 ; arrows point to the position of the nucleotide change (amino acid change shown at the bottom). (B) Crystal structure of the KLF2 zinc finger domain in complex with DNA.
Residues targeted by somatic point mutations are shown and color coded in red if recurrently mutated. Note the clustering of mutations around the KLF2 pocket that accommodates DNA. (C) Multiple alignment of the zinc finger domain amino acid sequence of the human KLF2 protein with orthologous KLF2 proteins. Amino acids conserved across species are highlighted.
Missense substitutions are mapped as red circles. Functionally relevant codons of the KLF2 zinc finger domain are color coded.
Supplementary Figure 2. Analysis of the expression of KLF2 mutant alleles. The cDNA from KLF2 mutated samples (n=16;
21 mutations) was amplified using primers that crossed intron-exon boundaries and sequences. This analysis confirmed that KLF2 mutations were always expressed at the transcript level.
Supplementary Figure 3. KLF2 is recurrently deleted in SMZL and DLBCL. (A) Graphic representation of segmentation data from SMZL carrying KLF2 deletions, visualized with Integrative Genomics Viewer (IGV) software
(http//www.broadinstitute.org/igv). Each track represents one sample, where blue indicates region of a copy number loss.
Individual genes in the region are aligned in top panel. (B) FISH validation of KLF2 focal deletion in the KARPAS 1718 cell line using the G248P8818F11 (size: 43 Kb) and RP11-1136A9 (size: 147 Kb) probes that cover the sole KLF2 locus. Green spectrum probe: KLF2 ; Orange spectrum probe: CEP19.
(C) Allelic (A or B) distribution of KLF2 genetic lesions in individual SMZL and
DLBCL samples. Chr19 indicates chromosome 19. (D) Overall frequency of KLF2 structural alterations in SMZL and DLBCL subtypes (mutations and deletions, combined).
Supplementary Figure 4. Associations between KLF2 molecular lesions and other biological features in SMZL and
DLBCL. (A) In the heatmaps, rows correspond to a molecular feature, and columns represent individual SMZL patients, colorcoded based on the status of the molecular feature ( KLF2 , white: wild type; red: mutated or deleted; NOTCH2 , white: wild type; red: mutated; 7q32q32, red: deleted; white: not deleted; IGHV1-2*04 , red: utilized; white not utilized; gray represent cases for which a specific molecular feature was not assessable). (B) Prevalence of KLF2 lesions (i.e. mutation or deletion) in DLBCL cases stratified according to the cell of origin as defined by the Hans immunohistochemical classifier in primary samples. GC, germinal center. p value according to chi-square test.
Supplementary Figure 5. Constitutive expression of KLF2 in a panel of lymphoid tumor cell lines and in primary SMZL samples . ( A ) KLF2 mRNA and protein expression were analyzed in the DLBCL cell line OCY-Ly8, the BL cell line AS283A, the putative SMZL cell lines VL51, SSK41, KARPAS 1718, and the T-ALL cell line Jurkat. For each cell line, the status of the two
KLF2 alleles is indicated. Purified tonsil B-cell preparations were included as representative of the normal B-cell compartment.
(B) KLF2 mRNA and protein expression were analyzed in three primary SMZL samples. CD19+ cells purified from spleen samples were utilized for the experiments. For each primary SMZL sample, the status of the two KLF2 alleles is indicated. RE, relative expression.
Supplementary Figure 6. Endogenous KLF2 protein expression (anti-KLF2 antibody, Santa Cruz, N-13) in the HEK293T cells.
NT, untransfected; EV, transfected with an empty vector construct; WT, transfected with a wild type KLF2 construct.
7

Supplementary Table 1. Mutation analysis of the 16 candidate marginal zone differentiation genes (other than KLF2), of KLF2 paralogs and of KLF2 pathway genes
13444
13446
13447
13448
13449
13450
13451
13452
13453
13458
ID
4044
4625
4687
4689
5940
7427
10032
11731
KLF3 ab IKZF3 a MAML1 a S1PR1 ac S1PR3 ac IRF8 a FOXO1 a TCF3 a ID3 a FLI1 a CXCR5 ac AIRE a RPBJ a CXCR7 ac DLK1 a KLF4 b KLF6 b KLF8 b KLF9 b KLF14 b S1PR2 c GNA12 c GNA13 c SMURF1 c WWP1 c CDKN1A c SELL c ITGB7 c wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt
13460 wt
13462 wt
KARPAS1718 wt
13465
13468 wt wt wt wt wt wt wt
13470
13710
13711 wt wt wt wt wt wt wt wt wt
13713
13714
13716
13717 wt wt wt wt wt wt wt wt wt wt wt wt
13718 wt wt wt a Marginal zone differentiation genes b
KLF2 paralogs c
KLF2 pathway genes wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt
8

Supplementary Table 2. Molecular features of the SMZL panel
Variable n
IGHV1-2*04 usage
IGHV homology 100%/97-99%/<97%
28
4/19/64
NOTCH2 mutation
TP53 mutation
TP53 deletion
30
13
10
7q31-q32 deletion
BRAF p.V600E
27
0 t(11;14)(q13;q32) 0 t(14;18)(q32;q21) 0
Abbreviations: IGHV , immunoglobulin heavy variable gene
Total
87
87
96
96
90
90
96
96
96
%
32%
5%/22%/73%
31%
13%
11%
30%
0%
0%
0%

Supplementary Table 3. KLF2 mutations
730
4627
5034
14228
14833
16189
16189
16215
16215
16973
13470
13477
16235
16235
16235
16235
16235
16165
5450/07
5450/07
6946/08
14228
ID
11236
11278
13444
13464
4625
11221
11221
11235
11242
11272
11278
11937
13711
13451
13471
VL51
11234
13711
13713
13460
3919/08
3919/08
50/07
5450/07
7013/08
OCI-Ly8
RCK8
RCK8
RCK8
SMZL
SMZL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
Diagnosis
SMZL
SMZL
SMZL
SMZL
SMZL
SMZL
SMZL
SMZL
SMZL
SMZL
Nucleotide Change c.825_delC c.770_786del17bp c.889_892+15del19bp c.770_816_del47bp c.817_828del12bp c.C862T c.G548A c.C862T c.G821A c.A863T
SMZL
SMZL
SMZL
SMZL c.C862G c.C95T c.C872T c.G871A
SMZL c.T876A
SMZL (putative) c.C812T
SMZL
SMZL
SMZL
SMZL c.C70T c.C70T c.76-1G>C c.76-1G>A
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL c.76-1G>C c.892_892+2delTGG c.856_861del6bp c.C851T
c.G871A c.C740A c.G803T c.503delC c.495_520del26bp c.1051delA c.701_707del7bp c.239_397del159bp c.G398A c.C580T c.C743T c.G824A c.G774C + c.A772T c.G824A c.G871A c.G563C c.G824A c.C110T c.C851T c.G857A c.C322G c.C862G c.C44T c.C653A c.C110T c.C619T c.C802T p.S275N p.A291T p.G188A p.S275N p.T37I p.T284I p.S286N p.L108V p.H288D p.A15V p.A218D p.T37I p.P207S p.R268C p.S286delSS p.T284I p.A291T p.A247E p.R268L p.P168fs*122 p.G165fs*126 p.M351fs*1 p.L234fs*54 p.A80del53AA p.G133D p.P194S p.S248F p.S275N p.K258Y
AA Change p.S275fs*15 p.P257fs*37 p.T297fs*118 p.P257fs*27 p.T273delTCSY p.H288Y p.R183H p.H288Y p.C274Y p.H288L p.H288D p.P32L p.A291V p.A291T p.H292Q p.T271I p.Q24* p.Q24*
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Somatic status
Unknown
Unknown
Unknown
Unknown
Confirmed
Confirmed
Unknown
Confirmed
Unknown
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Unknown
Confirmed
Confirmed
Confirmed
Unknown
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Unknown
Unknown
Unknown
Unknown
Splice site
Splice site
In frame
Missense
Missense
Missense
Missense
Frameshift
Frameshift
Frameshift
Frameshift
In frame
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Mutation type
Frameshift
Frameshift
Frameshift
Frameshift
In frame
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Nonsense
Nonsense
Splice site
Splice site
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Affected domain
CH2 type 1
Inhibitory domain
Interdomain region
Inhibitory domain
CH2 type 1
CH2 type 1
Inhibitory domain
CH2 type 1
CH2 type 1
CH2 type 1
CH2 type 1
Activation Domain
CH2 type 1
CH2 type 1
CH2 type 1
Inhibitory domain
Activation Domain
Activation Domain
Activation Domain
Activation Domain
Activation Domain
Interdomain region
CH2 type 1
CH2 type 1
CH2 type 1
Inhibitory domain
Interdomain region
Inhibitory domain
Inhibitory domain
CH2 type 3
Inhibitory domain
Activation Domain
Inhibitory domain
Inhibitory domain
Inhibitory domain
CH2 type 1
Inhibitory domain
CH2 type 1
CH2 type 1
Inhibitory domain
CH2 type 1
Activation Domain
CH2 type 1
CH2 type 1
Activation Domain
CH2 type 1
Activation Domain
Inhibitory domain
Activation Domain
Inhibitory domain
Interdomain region
PolyPhen-2
Possibly damaging
Benign
Possibly damaging
Possibly damaging
Probably damaging
Probably damaging
Benign
Probably damaging
Probably damaging
Probably damaging
Benign
Probably damaging
Probably damaging
Benign
Possibly damaging
Benign
Benign
Probably damaging
Possibly damaging
Probably damaging
Possibly damaging
Probably damaging
Benign
Possibly damaging
Benign
Probably damaging
Probably damaging
Benign
Probably damaging
Probably damaging
Benign
Benign
Benign
Probably damaging
10

SUDHL8
SUDHL8
174
174
174
11863
11898
11900
11925
DLBCL
DLBCL
EMZL
EMZL
EMZL
EMZL
EMZL
EMZL
EMZL c.C731T c.C806T c.770_798del29bp c.818_838del20ins3 c.G469C c.G926A c.G821A c.C506T c.C208T c.299_307del9ins2 p.T244M p.T269I p.P257fs*33 p.T273fs*21 p.A157P p.C309Y p.C274Y p.P169L p.P70S p.L100fs*39
Missense
Missense
Frameshift
Frameshift
Missense
Missense
Missense
Missense
Missense
Frameshift
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Inhibitory domain
Interdomain region
Inhibitory domain
Inhibitory domain
Inhibitory domain
CH2 type 2
CH2 type 1
Inhibitory domain
Activation Domain
Probably damaging
Benign
Benign
Probably damaging
Possibly damaging
Benign
Benign
17183
14054
7279
17183
14054
13997
17176
7221
7221
11817
11824
17417
4262
4380
5141
NMZL
NMZL
NMZL
NMZL
NMZL
NMZL
NMZL
HCL
HCL
HCL
HCL
HCL
CLL
CLL
CLL c.A863G c.C862G c.C862G c.C70T c.G97T c.C955T c.G332A c.G437C c.G824A c.C872T c.A853C c.A953T c.C862T p.H288R p.H288D p.H288D p.Q24* p.E33* p.L319F p.R111H p.G146A p.S275N p.A291V p.K285Q p.E318V p.H288Y
Missense
Missense
Missense
Nonsense
Nonsense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Unknown
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Confirmed
Unknown
Confirmed
Confirmed
Unknown
Confirmed
Confirmed
Activation Domain
Inhibitory domain
CH2 type 1
CH2 type 1
CH2 type 1
Activation Domain
Activation Domain
CH2 type 2
Inhibitory domain
Inhibitory domain
CH2 type 1
CH2 type 1
CH2 type 1
CH2 type 2
CH2 type 1
Probably damaging
Probably damaging
Probably damaging
Probably damaging
Probably damaging
Benign
Possibly damaging
Probably damaging
Probably damaging
Probably damaging
Possibly damaging
AS283A
AS283A
4351
BL
BL
FL c.C110G c.C481T c.868_879del12bp p.T37S p.R161* p.K290delKAHL
Missense
Nonsense
In frame
Unknown
Unknown
Confirmed
Activation Domain
Inhibitory domain
CH2 type 1
Benign
4087
Jurkat
MCL
T-ALL c.C976T c.C704T p.H326Y p.A235V
Missense
Missense
Unknown
Unknown
CH2 type 2
Inhibitory domain
Probably damaging
Possibly damaging
Abbreviations: SMZL, splenic marginal zone lymphoma; DLBCL, diffuse large B-cell lymphoma; EMZL, extranodal marginal zone lymphoma; NMZL, nodal marginal zone lymphoma; HCL, BRAF p.V600E mutation positive hairy cell leukemia; CLL, chronic lymphocytic leukemia; BL, Burkitt lymphoma; FL, follicular lymphoma; MCL, mantle cell lymphoma; T-ALL, T-cell acute lymphoblastic leukemia.
11

Supplementary Table 4. Copy number abnormalities of the KLF2 gene in SMZL and DLBCL
ID Diagnosis KLF2 deletion by FISH a
10056
10032
SMZL
SMZL
12% (monoallelic)
16% (monoallelic)
13452
9641
13449
13475
SMZL
SMZL
SMZL
SMZL
18% (monoallelic)
21% (monoallelic)
25% (monoallelic)
29% (monoallelic)
13715
11731
KARPAS 1718
SMZL
SMZL
SMZL (putative)
10% (monoallelic)
10% (monoallelic)
93% (monoallelic)
13461
13455
SMZL
SMZL
NA
NA
11455
10235
16962
16967
DLBCL
DLBCL
DLBCL
DLBCL
16% (monoallelic)
19% (monoallelic)
20% (monoallelic)
30% (monoallelic)
14123
16165
DLBCL
DLBCL
45% (monoallelic)
60% (monoallelic)
14228 DLBCL 62% (monoallelic)
Abbreviations: SMZL, splenic marginal zone lymphoma; DLBCL, diffuse large B-cell lymphoma; NA, not assessable a Percentage of nuclei showing the deletion
KLF2 deletion by SNP array
No
No
No
No
No
No
No
No chr19:16,371,442-16,546,155 (monoallelic) chr19:16,355,163-42,054,098 (monoallelic) chr19:38,371-20,758,455 (monoallelic)
NA
NA
NA
NA
NA
NA
NA
12

13

A
852
852
#5141
862 872 861
862 p.H288Y
872 861
#16235
871 881
B
871 881
H288Y/D/L
K285Q
A291T/V
H292Q
S286N
T284I
T271I
T269I
S275N
C274Y E318V
C309Y
L319F
H326Y p.A291T
C
H. sapiens
P. troglodytes
M. musculus
R. norvegicus
B. taurus
G. gallus
X. laevi
C. elegans
D. melanogaster
Znf domain
DNA recognition
Fold stabilization
Zn coordination
T A T H T C S Y A G C G K T Y T K S S H L K A H L R T H T G E K P Y H C N W D G C G W K F A R S D E L T R H Y R K H T G H R P F Q C H L C D R A F S R S D H L A L H M K R H M
T A T H T C S Y A G C G K T Y T K S S H L K A H L R T H T G E K P Y H C N W D G C G W K F A R S N E L T R H Y R K H T G H R P F Q C H L C D R A F S R S D H L A L H M K R H M
A A T H T C S Y T N C G K T Y T K S S H L K A H L R T H T G E K P Y H C N W E G C G W K F A R S D E L T R H Y R K H T G H R P F Q C H L C D R A F S R S D H L A L H M K R H M
A A T H T C S Y T N C G K T Y T K S S H L K A H L R T H T G E K P Y H C N W D G C G W K F A R S D E L T R H Y R K H T G H R P F Q C H L C D R A F S R S D H L A L H M K R H M
T A T H T C S Y A G C G K T Y T K S S H L K A H L R T H T G E K P Y H C N W E G C G W K F A R S D E L T R H Y R K H T G H R P F Q C H L C D R A F S R S D H L A L H M K R H M
T A T H T C T Y A G C G K T Y T K S S H L K A H L R T H T G E K P Y H C N W E G C G W K F A R S D E L T R H Y R K H T G H R P F Q C H L C D R A F S R S D H L A L H M K R H M
T A S H T C S Y A G C G K T Y T K S S H L K A H L R T H T G E K P Y H C N W E G C G W K F A R S D E L T R H F R K H T G H R P F Q C H L C D R A F S R S D H L A L H M K R H M
L R V H K C F Y Q G C G K V Y T K S S H L T A H E R V H S G E K P Y P C E W P G C S W R F A R S D E L T R H Y R K H T G A K P F A C K E C S R K F S R S D H L Q L H M K R H E
- K V H K C D T E G C D K V Y T K S S H L K A H K R T H T G E K P Y V C T W E G C I W R F A R S D E L T R H Y R K H T G V K P F R C Q L C T R S F S R S D H L S L H M R R H -
Codon
2
6
9
2 2 2 2 2 2
7
0
7
1
7
2
7
3
7
4
7
5
2 2 2 2 2 2 2 2 2 2 2
7
6
7
7
7
8
7
9
8
0
8
1
8
2
8
3
8
4
8
5
8
6
2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3
8
7
8
8
8
9
9
0
9
1
9
2
9
3
9
4
9
5
9
6
9
7
9
8
9
9
0
0
0
1
0
2
0
3
0
4
0
5
0
6
0
7
0
8
0
9
1
0
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
1
1
1
2
1
3
1
4
1
5
1
6
1
7
1
8
1
9
2
0
2
1
2
2
2
3
2
4
2
5
2
6
2
7
2
8
2
9
3
0
3
1
3
2
3
3
3
4
3
5
3
6
3 3 3 3 3 3 3 3 3 3 3 3
3
7
3
8
3
9
4
0
4
1
4
2
4
3
4
4
4
5
4
6
4
7
4
8
3 3 3 3 3 3
4
9
5
0
5
1
5
2
5
3
5
4
3
5
5
14

943
852
945
85
511
811
34
953
862
955
95
521
821
44
963
# 4380 c.A953T
p.E318V
872
# 5141 c.C862T
p.H288Y
965
# 7221 c.C955T
p.L319F
105
# 11937 c.C95T
p.P32L
807
843
811
858
531
#5450/07 c.496_521del26bp
p.G165fs*125
322
831
# 11898 c.G821A p.C274Y
54
#7013/08 c.C44T
p.A15V
694
694
817
853
821
868
332
704
704
827
# 4625 c.817_828del12bp
p.H273delTCSY
863
# 4262 c.A853C
p.K285Q
831
# 13444 c.889_892+15del19 p.T297fs*118
878
# 4351 c.868_879del12bp
p.K290KAHL
342
# 7221 c.G332A
p.R111H
714
# 6946/08 c.701_707del7bp p.L234fs*54
714
# Jurkat c.C704T p.A235V
802
852
312
1041
847
841
643
812
862
322
1051
857
851
653
822
# VL51 c.C812T
p.T271I
872
#5450/07 c.C862G
p.H288D
332
#5450/07 c.C322G p.L108V
1061
#5450/07 c.1051delA
p.M351fs*1
867
# 3919/08 c.G857A
p.S286N
861
# 3919/08 c.C851T
p.T284I
663
# OCI-Ly8 c.C653A
p. A218D
15

A chr19 (p13.11)
B
KLF2
CEP19
KARPAS 1718
13455
13461
KARPAS 1718
G248P8818F11 (19p13.11) RP11-1136A9 (19p13.11)
C
Chr19
SMZL DLBCL
A B A B
D
19p13.11
31%
SMZL (n=96)
11%
18%
2%
69%
26%
DLBCL (n=74)
3%
7%
14%
3%
73%
M/M M/wt Δ/wt Δ/M wt/wt
Non-sense/Frameshift indel
Missense/In frame indel
Splice-site
Deletion
Fig. 3S
16

A
B
KLF2
NOTCH2
7q31q32
IGVH1-2*04
15/51
(29%)
SMZL=96 p=.002
40%
30%
20%
10%
0% non-GC
DLBCL
0/33
GC
17

A
2000
1000
0
B
300
200
100
0
Mutated
Deleted wt
KLF2 gene
KLF2 actin p.T271I
p.A218D
p.A235V
A
B
37 kDa
37 kDa
KLF2 gene p.P32L
KLF2 actin
A
B
37 kDa
37 kDa
18

KLF2
β-tubulin
37 kDa
50 kDa
19