Accepted Article Preview: Published ahead of advance online publication The kru¨ppel-like factor 2 transcription factor gene is recurrently mutated in splenic marginal zone lymphoma R Piva, S Deaglio, R Fama`, R Buonincontri, I Scarfo`, A Bruscaggin, E Mereu, S Serra, V Spina, D Brusa, G Garaffo, S Monti, M Dal Bo, R Marasca, L Arcaini, A Neri, V Gattei, M Paulli, E Tiacci, F Bertoni, S A Pileri, R Foa`, G Inghirami, G Gaidano, D Rossi
Cite this article as: R Piva, S Deaglio, R Fama`, R Buonincontri, I Scarfo`, A Bruscaggin, E Mereu, S Serra, V Spina, D Brusa, G Garaffo, S Monti, M Dal Bo, R Marasca, L Arcaini, A Neri, V Gattei, M Paulli, E Tiacci, F Bertoni, S A Pileri, R Foa`, G Inghirami, G Gaidano, D Rossi, The kru¨ppel-like factor 2 transcription factor gene is recurrently mutated in splenic marginal zone lymphoma, Leukemia accepted article preview 6 October 2014; doi: 10.1038/leu.2014.294. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Accepted article preview online 6 October 2014
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2014 Macmillan Publishers Limited. All rights reserved.
THE KRÜPPEL-LIKE FACTOR 2 TRANSCRIPTION FACTOR GENE IS RECURRENTLY MUTATED IN SPLENIC MARGINAL ZONE LYMPHOMA
Running head: KLF2 mutations in SMZL
Splenic marginal zone lymphoma (SMZL) is an indolent B-cell tumor involving the spleen, and is characterized by recurrent deletion of chromosome 7q and biased usage of the immunoglobulin heavy variable (IGHV) allele 1-2*04.1 Genomic studies have partially unraveled the typical SMZL coding genome, which is characterized by lesions affecting genes involved in the physiological homeostasis of marginal zone (MZ) B-cells, including mutations of NOTCH2.2-5 However, the full spectrum of lesions that contribute to the malignant transformation of SMZL remains unknown. In order to identify novel genetic lesions recurrently associated with SMZL, we extended mutation analysis to genes that, though known to be well-established regulators of MZ B-cell differentiation, had not emerged as significantly mutated from genomic studies of SMZL (Table 1S2S). This analysis disclosed recurrent mutations of the Krüppel-like factor 2 (KLF2) zinc finger gene, a transcription factor important for the homeostasis and differentiation of peripheral B-cell subsets,6,7 in 20% (19/96) of SMZL (Figure 1A). To investigate the full complement of KLF2 mutations in the spectrum of lymphoid neoplasia, we extended the analysis to 547 mature B- and 113 T-cell tumors. In addition to SMZL, KLF2 mutations recurred also in 16% (4/24) of hairy cell leukemia, 11% (17/154) of diffuse large B-cell lymphoma (DLBCL), 9% (5/56) of nodal marginal zone lymphoma, and 8% (5/61) of extranodal marginal zone lymphoma, while they were rare or absent (frequency=0-4%) in the remaining entities (Figure 1A). Overall, 80 KLF2 mutations were identified (Table 3S). Of these, 26% (n=21) were truncating mutations (frameshift indels=12; non-sense substitutions=5; splice-site variants=4) (Figure 1B). By cDNA analysis, splice-site mutations caused aberrant transcripts that retained ©
2014 Macmillan Publishers Limited. All rights reserved.
intronic sequences and lost their coding potential. Based on their distribution along KLF2, truncating mutations were predicted to either disrupt the entire protein or to remove the zing finger (ZnF) domains of KLF2, including the putative nuclear localization signal (NLS) sequences (Figure 1C).8-10 Missense mutations (n=55) and in frame indels (n=4) accounted for 74% of the variants (Figure 1B). According to PolyPhen-2, the majority of KLF2 missense substitutions were predicted to be deleterious (Table 3S). A large fraction of the missense mutations (n=27) clustered in the region encoding the first two ZnFs of KLF2, suggesting that they may affect its nuclear localization as well as the transcriptional function of the protein (Figure 1C).8-11 Notably, amino acid changes recurrently affected evolutionarily conserved codons of the first ZnF, including recurrent substitutions at positions 288 (n=9) and 291 (n=5), which are involved in DNA recognition by KLF2 (Figure 1S).11 The 5' basic NLS of KLF2, which maps before the ZnF, was also recurrently targeted by missense substitutions (n=7) (Figure 1C).8-11 The remaining missense mutations distributed in the N-terminal activation domain (n=7, including 3 somatic mutations targeting a hotspot at codon 37) and in the inhibitory domain (n=14) (Figure 1C).9,10 Targeted next generation sequencing analysis revealed that KLF2 mutations (assessable=42) were clonally represented in the tumor (median variant allele frequency=35%) (Figure 1D). Whenever possible, cDNA sequencing confirmed that KLF2 mutations (n=21) were always expressed at the transcript level (Figure 2S). The somatic origin of mutations was confirmed in all tested cases by analysis of paired normal DNA, which was available for most of the variants (n=52; including 36 missense substitutions) (Figure 1C; Table 3S). Most (72%; 41/57) lymphoma cases harbored one single monoallelic KLF2 mutation. Among cases displaying more than one mutational event, sequencing analysis of KLF2 transcripts after subcloning demonstrated that the mutations were generally (60%) located on separate alleles. Beside KLF2, no other KLF family member homologous to KLF2, nor genes belonging to the KLF2 pathway were mutated in SMZL (Table 1S). Copy number abnormalities of the KLF2 locus were examined by a combination of SNP array (n=58 samples) and FISH (n=112 samples) in 96 SMZL and 74 DLBCL cases. Monoallelic 2 ©
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KLF2 deletions occurred in 11% (11/96) SMZL and in 9% (7/74) DLBCL (Table 4S). One 174 kb focal loss of the KLF2 locus identified KLF2 as the specific target of the deletion (Figure 3S). KLF2 mutations and deletions were mutually exclusive in most samples, revealing a predominant monoallelic distribution (Figure 3S). Thus, when considering both mutations and copy number aberrations in cases in which both types of alterations could be assessed, 31% (30/96) of SMZL and 26% (19/74) of DLBCL harbored KLF2 structural alterations (i.e. mutations or deletions) (Figure 3S). In SMZL, KLF2 lesions were enriched among cases harboring 7q deletion (61%, 11/18 vs 22%, 16/72; p=.0013), NOTCH2 mutations (55%, 10/18 vs 25%, 20/78; p=.013), and IGHV1-2*04 allele usage (47%, 10/21 vs 27%, 18/66; p=.082) (Figure 4S). In DLBCL, KLF2 lesions were restricted to cases showing a non-germinal center phenotype (Figure 4S). Cell lines and primary tumors carrying KLF2 heterozygous mutations retained expression of the unmutated allele at transcriptional (Figure 2S) and protein (Figure 5S) levels. Future experiments will be required to clarify whether KLF2 mutations act as a dominant negative or whether loss of one copy has functional impact. Western blot analysis after fractionation of cytoplasmic and nuclear proteins and confocal microscopy analysis were utilized to determine KLF2 distribution/localization across the subcellular compartments. In the SSK41 cell line, which harbors a wild type gene, KLF2 was predominantly expressed in the nucleus (Figure 2A). In contrast, the protein was primarily cytoplasmic (Figure 2A) in cell lines harboring mutations that truncated or changed the amino acid composition of the ZnFs, or targeted codons within or flanking the 5' basic NLS of KLF2. Cytoplasmic KLF2 expression was also observed in primary tumor cells harboring a monoallelic KLF2 mutation of the ZnF (Figure 2A). The near complete cytoplasmic expression of KLF2 observed in cases with monoallelic mutations warrants investigations aimed at testing whether wild type KLF2 is sequestered by the mutant protein. To provide a proof of principle of the functional significance of KLF2 mutations, we transfected HEK293T cells with flag-tagged wild type KLF2 constructs and two mutants: i) the p.P257* variant, representative of tumor cell mutations that truncate the carboxy-terminal portion of 3 ©
2014 Macmillan Publishers Limited. All rights reserved.
KLF2, including its 5' basic NLS sequence and the ZnFs; ii) the p.H288Y variant, the most recurrent substitution affecting a hot spot codon that is physiologically involved in DNA recognition by KLF2 and in its nuclear localization.8-10 Confocal microscopy showed predominant nuclear localization of wild type KLF2. In contrast, p.P257* and p.H288Y mutants were displaced from the nucleus (Figure 2B). We then assessed the relative transactivation activity of the mutant KLF2 proteins by measuring their ability to upregulate the expression of a luciferase reporter gene driven by the CDKN1A/p21 promoter, a known direct target of KLF2,12,13 in HEK293T cells that lack endogenous KLF2 (Figure 6S). Expression of wild type, but not mutant KLF2, strongly induced the reporter (Figure 2C), a finding consistent with the predicted loss of interaction between KLF2 mutants and DNA. KLF2 mutations represent one of the most frequent genomic abnormalities of SMZL, and unravel the disruption in human tumors of a previously unidentified molecular pathway. Given the involvement of KLF2 in the transcriptional regulation of an array of genes, it is difficult to predict which cellular targets/pathways may be critically affected by its mutations in lymphomagenesis. In normal lymphocytes, KLF2 binds the promoter and regulates the expression of genes involved in cell cycle/apoptosis (i.e. CDKN1A/p21) and cell trafficking (i.e. S1PR1, SELL/CD62L, ITGB7/β7integrin, CXCR5).6,7,12-13 A limited set of KLF2 domains is necessary to exploit its transcriptional activities, including three highly conserved ZnFs, which allow protein contact with DNA, and two potent, independent NLS that mediate the localization of KLF2 in the nucleus.8-11 Consistent with the critical roles of the ZnFs and the NLS, ~60% of KLF2 mutations disrupt or are predicted to modify these structures of the KLF2 protein. Our data confirm that KLF2 mutants lacking the NLS and some of those harboring missense substitutions of the first ZnF (i.e. codon 288 substitutions) are displaced from the nucleus and transcriptionally defective. Due to the large number of somatic missense KLF2 mutations disclosed by our molecular investigations, additional analyses are needed to comprehensively characterize the effects of the full spectrum of these variants. According to their distribution and predicted functional consequences, it is conceivable that, in addition to nuclear 4 ©
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localization, missense substitutions might also disturb other KLF2 functions, including DNA binding, transcriptional activation or protein interactions. As is the case for most cancer-associated genetic lesions, KLF2 inactivation may not be sufficient for malignant transformation. In fact, transgenic mice engineered to lack KLF2 in mature B-cells display an expansion of the MZ at the expense of the follicular compartment, but do not develop lymphoma.6,7 It is important to note, however, that lymphoma development may require longer times than those observed so far in mice,6,7 in line with the indolent course of SMZL and the elderly age of patients affected by this lymphoma. Consistent with a multistep process of lymphomagenesis, KLF2 lesions frequently co-occur with IGHV1-2*04 usage, NOTCH2 mutations, and 7q deletion in SMZL, suggesting a possible cooperation between genetic abnormalities and Bcell receptor configuration in promoting transformation. Emerging evidence points to the role of KLF transcription factors in human cancers.14 While KLF family gene expression and function are altered in a large number of neoplasms, including Bcell tumors, recurrent structural alterations of these genes are exceedingly rare and sporadically restricted to solid cancers.14,15 Our data provide the first evidence of the molecular deregulation of KLF family genes in hematologic malignancies, and suggest that selection of KLF2 mutations plays a role in transformation common to several lymphoma subtypes.
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Roberto Piva1§, Silvia Deaglio2§, Rosella Famà3, Roberta Buonincontri2, Irene Scarfò1, Alessio Bruscaggin3, Elisabetta Mereu1, Sara Serra2, Valeria Spina3, Davide Brusa2, Giulia Garaffo1, Sara Monti3, Michele Dal Bo,4 Roberto Marasca5, Luca Arcaini6, Antonino Neri7, Valter Gattei4, Marco Paulli8, Enrico Tiacci9, Francesco Bertoni10, Stefano A. Pileri11, Robin Foà12, Giorgio Inghirami2,13,14, Gianluca Gaidano3, Davide Rossi3 §
R.P. and S.D. equally contributed
1
Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences Biology and Biochemistry and 2Department of Medical Sciences, University of Turin and Human Genetics Foundation, Turin, Italy; 3Division of Hematology, Department of Translational Medicine, Amedeo Avogadro University of Eastern Piedmont, Novara, Italy; 4Clinical and Experimental Onco-Hematology, CRO, IRCCS, Aviano, Italy;5Division of Hematology, University of Modena and Reggio Emilia, Modena, Italy; 6Divisions of Hematology and 8Pathology, 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; 9Institute of Hematology, University of Perugia, Perugia, Italy; 10Lymphoma and Genomics Research Program, IOR Institute of Oncology Research and Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; 11Haematopathology, L. & A. Seragnoli Institute, University of Bologna, Bologna, Italy; 12Division of Hematology, Department of Cellular Biotechnologies and Hematology, Sapienza University, Rome, Italy; 13Department of Pathology and New York University Cancer Center, New York University School of Medicine, and 14Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY. Correspondence: Davide Rossi, MD, Division of Hematology, Department of Translational Medicine, Amedeo Avogadro University of Eastern Piedmont, Via Solaroli 17, 28100 Novara, Italy; Ph +39-0321-660698; Fax +39-0321-620421; E-mail [email protected]. Conflict-of-interest disclosure: The authors have no conflict of interest to disclose. Keywords: KLF2, mutation, splenic marginal zone lymphoma, diffuse large B-cell lymphoma
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Acknowledgment: This study was supported by: Special Program Molecular Clinical Oncology 5 x 1000 No. 10007, My First AIRC Grant No. 13470, and Investigator Grant IG-13227, Associazione Italiana per la Ricerca sul Cancro (AIRC) Foundation Milan, Italy; Fondazione Cariplo, Grant No. 2012-0689; Progetto Giovani Ricercatori 2010, Grant No. GR-2010-2317594, Ministero della Salute, Rome, Italy; Compagnia di San Paolo, Grant No. PMN_call_2012_0071, Turin, Italy; Futuro in Ricerca 2012 Grant No. RBFR12D1CB, Ministero dell'Istruzione, dell'Università e della Ricerca, Rome, Italy; Nelia et Amadeo Barletta Foundation, Lausanne, Switzerland. Contributions: R.P., S.D., G. Gaidano, and D.R. designed the study, interpreted data and wrote the manuscript; R. Famà. A.B., V.S. and S.M. performed and interpreted molecular studies; R.B., I.S., E.M., S.S., D.B., G. Garaffo, and M.D.B. performed and interpreted functional experiments; R.M., L.A., A.N., V.G., M.P., E.T., F.B., S.A.P., R. Foà and G.I. contributed to data interpretation. Supplementary information is available at Leukemia's website.
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REFERENCES
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Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW. WHO classification of tumours of haematopoietic and lymphoid tissues, Fourth Edition. Lyon, France: IARC; 2008.
2.
Rossi D, Trifonov V, Fangazio M, Bruscaggin A, Rasi S, Spina V, et al. The coding genome of splenic marginal zone lymphoma: activation of NOTCH2 and other pathways regulating marginal zone development. J Exp Med 2012;209:1537-1551.
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Kiel MJ, Velusamy T, Betz BL, Zhao L, Weigelin HG, Chiang MY, et al. Whole-genome sequencing identifies recurrent somatic NOTCH2 mutations in splenic marginal zone lymphoma. J Exp Med 2012;209:1553-1565.
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Martínez N, Almaraz C, Vaqué JP, Varela I, Derdak S, Beltran S, et al. Whole-exome sequencing in splenic marginal zone lymphoma reveals mutations in genes involved in marginal zone differentiation. Leukemia Prepublished on December 3, 2013, as doi: 10.1038/leu.2013.365.
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Parry M, Rose-Zerilli MJ, Gibson J, Ennis S, Walewska R, Forster J, et al. Whole exome sequencing identifies novel recurrently mutated genes in patients with splenic marginal zone lymphoma. PLoS One 2013;8:e83244.
6.
Hart GT, Wang X, Hogquist KA, Jameson SC. Krüppel-like factor 2 (KLF2) regulates B-cell reactivity, subset differentiation, and trafficking molecule expression. Proc Natl Acad Sci U S A 2011;108:716-721.
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Winkelmann R, Sandrock L, Porstner M, Roth E, Mathews M, Hobeika E, et al. B cell homeostasis and plasma cell homing controlled by Krüppel-like factor 2. Proc Natl Acad Sci U S A 2011;108:710-715.
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8.
Shields JM, Yang VW. Two potent nuclear localization signals in the gut-enriched Krüppellike factor define a subfamily of closely related Krüppel proteins. J Biol Chem 1997;272:18504-18507.
9.
Kozyrev SV, Hansen LL, Poltaraus AB, Domninsky DA, Kisselev LL. Structure of the human CpG-island-containing lung Kruppel-like factor (LKLF) gene and its location in chromosome 19p13.11-13 locus. FEBS Lett 1999;448:149-152.
10. Wani MA, Conkright MD, Jeffries S, Hughes MJ, Lingrel JB. cDNA isolation, genomic structure, regulation, and chromosomal localization of human lung Kruppel-like factor. Genomics 1999;60:78-86. 11. Swamynathan SK. Krüppel-like factors: three fingers in control. Hum Genomics 2010;4:263270. 12. Wu J, Lingrel JB. KLF2 inhibits Jurkat T leukemia cell growth via upregulation of cyclindependent kinase inhibitor p21WAF1/CIP1. Oncogene 2004;23:8088-8096. 13. Taniguchi H, Jacinto FV, Villanueva A, Fernandez AF, Yamamoto H, Carmona FJ, et al. Silencing of Kruppel-like factor 2 b y the histone methyltransferase EZH2 in human cancer. Oncogene 2012;31:1988-1994. 14. Tetreault MP, Yang Y, Katz JP. Krüppel-like factors in cancer. Nat Rev Cancer 2013;13:701713. 15. Pasqualucci L, Trifonov V, Fabbri G, Ma J, Rossi D, Chiarenza A, et al. Analysis of the coding genome of diffuse large B-cell lymphoma. Nat Genet 2011;43:830-7.
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FIGURE LEGENDS
Figure 1. KLF2 mutations in lymphoid tumors. (A) Prevalence of KLF2 mutations in lymphoid tumors. SMZL, splenic marginal zone lymphoma; HCL, hairy cell leukemia; DLBCL, diffuse large B-cell lymphoma; NMZL, nodal marginal zone lymphoma; EMZL, extranodal marginal zone lymphoma; FL, follicular lymphoma; MCL; mantle cell lymphoma; CLL, chronic lymphocytic leukemia; BL, Burkitt lymphoma; MM, multiple myeloma; vHCL, variant hairy cell leukemia; MZL-like MBL, marginal zone-like monoclonal B-cell lymphocytosis; PTCL, peripheral T-cell lymphoma; T-ALL, T-cell acute lymphoblastic leukemia. (B) Prevalence of the various types of non-silent KLF2 mutations identified in lymphoid tumors. (C) Schematic representation of the human KLF2 protein, with its key functional domains and nuclear localization signals (one within the zinc finger domains and the other in a cluster of basic amino acids immediately before the first zinc finger). Symbols depict distinct types of KLF2 mutations. Non-silent mutations of confirmed somatic origin are color-coded in red. Non-silent mutations of unknown somatic origin are colorcoded in black. Znf, zinc finger; NLS, nuclear localization signal. (D) Distribution of the variant allele frequency of KLF2 mutations (blue circles).
Figure 2. Nuclear localization signal (NLS) mutations of KLF2 cause nuclear displacement of the KLF2 protein. (A) Cell fractionation experiments and confocal microscopy analyses performed in a panel of cell lines with known KLF2 mutation status (A and B represent the two alleles). In the wild type SSK41 line, KLF2 is preferentially expressed in the nucleus, while the presence of mutations in the VL51, AS283A, OCI-Ly8 cell lines cause a predominant accumulation of the molecule in the cytosolic compartment. Primary cells from a patient with a KLF2 missense mutation (#5141) confirm predominant localization of the mutated protein in the cytosol. The nuclear/cytoplasmic expression ratio was calculated by dividing the mean pixel intensity of the green fluorescence in the nuclear area (defined by the DAPI blue fluorescence) by the mean pixel 10 ©
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intensity of the green fluorescence in the rest of the cell. Confocal images were acquired with a 63x oil immersion objective. Wt, wild type; WCL, whole cell lysate; N, nuclear lysate; C, cytoplasmic lysate. (B) HEK293T cells were transiently transfected with flag-tagged KLF2-GFP constructs and grown on glass coverslips. After 24 hours, cells were washed, fixed, stained with anti-flag (red) and counterstained with DAPI (blue). Cells were analyzed using a confocal microscope with a 63x oil immersion
objective.
Representative
images
from
3
independent
experiments.
The
nuclear/cytoplasmic expression ratio was calculated by dividing the mean pixel intensity of the red fluorescence in the nuclear area (defined by the DAPI blue fluorescence) by the mean pixel intensity of the red fluorescence in the rest of the cell. (C) Luciferase activity of HEK293T cells transfected to express a luciferase reporter driven by the CDKN1A/p21 promoter encompassing the KLF2-binding region together with the flag-tagged wild type or mutants KLF2 constructs, and with the empty vector as control. KLF2 expression was induced by doxycycline (1 μg/ml) 24 hours posttransfection. Transactivation activity was detected at 48 hours and expressed as relative luciferase intensity compared to wild type (WT) KLF2 (referred as 100%), in five independent experiments (mean: top of the bar; standard deviation: whiskers; p value by t-test). Untransfected cells (NT) or transfected with an empty vector construct (EV) were used as negative controls. qRT-PCR and Western blot at 48 hours, showing comparable expression of the constructs, are also shown. βtubulin expression is included as protein loading control. Densitometry analysis of 5 independent Western blots shows quantitation of KLF2 levels after normalization over β-tubulin. KLF2 mRNA expression was assessed in 5 independent experiments and calculated relative to ACTB using the ΔCT method.
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2014 Macmillan Publishers Limited. All rights reserved.
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2014 Macmillan Publishers Limited. All rights reserved.
Cite this article as: R Piva, S Deaglio, R Fama`, R Buonincontri, I Scarfo`, A Bruscaggin, E Mereu, S Serra, V Spina, D Brusa, G Garaffo, S Monti, M Dal Bo, R Marasca, L Arcaini, A Neri, V Gattei, M Paulli, E Tiacci, F Bertoni, S A Pileri, R Foa`, G Inghirami, G Gaidano, D Rossi, The kru¨ppel-like factor 2 transcription factor gene is recurrently mutated in splenic marginal zone lymphoma, Leukemia accepted article preview 6 October 2014; doi: 10.1038/leu.2014.294. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Accepted article preview online 6 October 2014
©
2014 Macmillan Publishers Limited. All rights reserved.
THE KRÜPPEL-LIKE FACTOR 2 TRANSCRIPTION FACTOR GENE IS RECURRENTLY MUTATED IN SPLENIC MARGINAL ZONE LYMPHOMA
Running head: KLF2 mutations in SMZL
Splenic marginal zone lymphoma (SMZL) is an indolent B-cell tumor involving the spleen, and is characterized by recurrent deletion of chromosome 7q and biased usage of the immunoglobulin heavy variable (IGHV) allele 1-2*04.1 Genomic studies have partially unraveled the typical SMZL coding genome, which is characterized by lesions affecting genes involved in the physiological homeostasis of marginal zone (MZ) B-cells, including mutations of NOTCH2.2-5 However, the full spectrum of lesions that contribute to the malignant transformation of SMZL remains unknown. In order to identify novel genetic lesions recurrently associated with SMZL, we extended mutation analysis to genes that, though known to be well-established regulators of MZ B-cell differentiation, had not emerged as significantly mutated from genomic studies of SMZL (Table 1S2S). This analysis disclosed recurrent mutations of the Krüppel-like factor 2 (KLF2) zinc finger gene, a transcription factor important for the homeostasis and differentiation of peripheral B-cell subsets,6,7 in 20% (19/96) of SMZL (Figure 1A). To investigate the full complement of KLF2 mutations in the spectrum of lymphoid neoplasia, we extended the analysis to 547 mature B- and 113 T-cell tumors. In addition to SMZL, KLF2 mutations recurred also in 16% (4/24) of hairy cell leukemia, 11% (17/154) of diffuse large B-cell lymphoma (DLBCL), 9% (5/56) of nodal marginal zone lymphoma, and 8% (5/61) of extranodal marginal zone lymphoma, while they were rare or absent (frequency=0-4%) in the remaining entities (Figure 1A). Overall, 80 KLF2 mutations were identified (Table 3S). Of these, 26% (n=21) were truncating mutations (frameshift indels=12; non-sense substitutions=5; splice-site variants=4) (Figure 1B). By cDNA analysis, splice-site mutations caused aberrant transcripts that retained ©
2014 Macmillan Publishers Limited. All rights reserved.
intronic sequences and lost their coding potential. Based on their distribution along KLF2, truncating mutations were predicted to either disrupt the entire protein or to remove the zing finger (ZnF) domains of KLF2, including the putative nuclear localization signal (NLS) sequences (Figure 1C).8-10 Missense mutations (n=55) and in frame indels (n=4) accounted for 74% of the variants (Figure 1B). According to PolyPhen-2, the majority of KLF2 missense substitutions were predicted to be deleterious (Table 3S). A large fraction of the missense mutations (n=27) clustered in the region encoding the first two ZnFs of KLF2, suggesting that they may affect its nuclear localization as well as the transcriptional function of the protein (Figure 1C).8-11 Notably, amino acid changes recurrently affected evolutionarily conserved codons of the first ZnF, including recurrent substitutions at positions 288 (n=9) and 291 (n=5), which are involved in DNA recognition by KLF2 (Figure 1S).11 The 5' basic NLS of KLF2, which maps before the ZnF, was also recurrently targeted by missense substitutions (n=7) (Figure 1C).8-11 The remaining missense mutations distributed in the N-terminal activation domain (n=7, including 3 somatic mutations targeting a hotspot at codon 37) and in the inhibitory domain (n=14) (Figure 1C).9,10 Targeted next generation sequencing analysis revealed that KLF2 mutations (assessable=42) were clonally represented in the tumor (median variant allele frequency=35%) (Figure 1D). Whenever possible, cDNA sequencing confirmed that KLF2 mutations (n=21) were always expressed at the transcript level (Figure 2S). The somatic origin of mutations was confirmed in all tested cases by analysis of paired normal DNA, which was available for most of the variants (n=52; including 36 missense substitutions) (Figure 1C; Table 3S). Most (72%; 41/57) lymphoma cases harbored one single monoallelic KLF2 mutation. Among cases displaying more than one mutational event, sequencing analysis of KLF2 transcripts after subcloning demonstrated that the mutations were generally (60%) located on separate alleles. Beside KLF2, no other KLF family member homologous to KLF2, nor genes belonging to the KLF2 pathway were mutated in SMZL (Table 1S). Copy number abnormalities of the KLF2 locus were examined by a combination of SNP array (n=58 samples) and FISH (n=112 samples) in 96 SMZL and 74 DLBCL cases. Monoallelic 2 ©
2014 Macmillan Publishers Limited. All rights reserved.
KLF2 deletions occurred in 11% (11/96) SMZL and in 9% (7/74) DLBCL (Table 4S). One 174 kb focal loss of the KLF2 locus identified KLF2 as the specific target of the deletion (Figure 3S). KLF2 mutations and deletions were mutually exclusive in most samples, revealing a predominant monoallelic distribution (Figure 3S). Thus, when considering both mutations and copy number aberrations in cases in which both types of alterations could be assessed, 31% (30/96) of SMZL and 26% (19/74) of DLBCL harbored KLF2 structural alterations (i.e. mutations or deletions) (Figure 3S). In SMZL, KLF2 lesions were enriched among cases harboring 7q deletion (61%, 11/18 vs 22%, 16/72; p=.0013), NOTCH2 mutations (55%, 10/18 vs 25%, 20/78; p=.013), and IGHV1-2*04 allele usage (47%, 10/21 vs 27%, 18/66; p=.082) (Figure 4S). In DLBCL, KLF2 lesions were restricted to cases showing a non-germinal center phenotype (Figure 4S). Cell lines and primary tumors carrying KLF2 heterozygous mutations retained expression of the unmutated allele at transcriptional (Figure 2S) and protein (Figure 5S) levels. Future experiments will be required to clarify whether KLF2 mutations act as a dominant negative or whether loss of one copy has functional impact. Western blot analysis after fractionation of cytoplasmic and nuclear proteins and confocal microscopy analysis were utilized to determine KLF2 distribution/localization across the subcellular compartments. In the SSK41 cell line, which harbors a wild type gene, KLF2 was predominantly expressed in the nucleus (Figure 2A). In contrast, the protein was primarily cytoplasmic (Figure 2A) in cell lines harboring mutations that truncated or changed the amino acid composition of the ZnFs, or targeted codons within or flanking the 5' basic NLS of KLF2. Cytoplasmic KLF2 expression was also observed in primary tumor cells harboring a monoallelic KLF2 mutation of the ZnF (Figure 2A). The near complete cytoplasmic expression of KLF2 observed in cases with monoallelic mutations warrants investigations aimed at testing whether wild type KLF2 is sequestered by the mutant protein. To provide a proof of principle of the functional significance of KLF2 mutations, we transfected HEK293T cells with flag-tagged wild type KLF2 constructs and two mutants: i) the p.P257* variant, representative of tumor cell mutations that truncate the carboxy-terminal portion of 3 ©
2014 Macmillan Publishers Limited. All rights reserved.
KLF2, including its 5' basic NLS sequence and the ZnFs; ii) the p.H288Y variant, the most recurrent substitution affecting a hot spot codon that is physiologically involved in DNA recognition by KLF2 and in its nuclear localization.8-10 Confocal microscopy showed predominant nuclear localization of wild type KLF2. In contrast, p.P257* and p.H288Y mutants were displaced from the nucleus (Figure 2B). We then assessed the relative transactivation activity of the mutant KLF2 proteins by measuring their ability to upregulate the expression of a luciferase reporter gene driven by the CDKN1A/p21 promoter, a known direct target of KLF2,12,13 in HEK293T cells that lack endogenous KLF2 (Figure 6S). Expression of wild type, but not mutant KLF2, strongly induced the reporter (Figure 2C), a finding consistent with the predicted loss of interaction between KLF2 mutants and DNA. KLF2 mutations represent one of the most frequent genomic abnormalities of SMZL, and unravel the disruption in human tumors of a previously unidentified molecular pathway. Given the involvement of KLF2 in the transcriptional regulation of an array of genes, it is difficult to predict which cellular targets/pathways may be critically affected by its mutations in lymphomagenesis. In normal lymphocytes, KLF2 binds the promoter and regulates the expression of genes involved in cell cycle/apoptosis (i.e. CDKN1A/p21) and cell trafficking (i.e. S1PR1, SELL/CD62L, ITGB7/β7integrin, CXCR5).6,7,12-13 A limited set of KLF2 domains is necessary to exploit its transcriptional activities, including three highly conserved ZnFs, which allow protein contact with DNA, and two potent, independent NLS that mediate the localization of KLF2 in the nucleus.8-11 Consistent with the critical roles of the ZnFs and the NLS, ~60% of KLF2 mutations disrupt or are predicted to modify these structures of the KLF2 protein. Our data confirm that KLF2 mutants lacking the NLS and some of those harboring missense substitutions of the first ZnF (i.e. codon 288 substitutions) are displaced from the nucleus and transcriptionally defective. Due to the large number of somatic missense KLF2 mutations disclosed by our molecular investigations, additional analyses are needed to comprehensively characterize the effects of the full spectrum of these variants. According to their distribution and predicted functional consequences, it is conceivable that, in addition to nuclear 4 ©
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localization, missense substitutions might also disturb other KLF2 functions, including DNA binding, transcriptional activation or protein interactions. As is the case for most cancer-associated genetic lesions, KLF2 inactivation may not be sufficient for malignant transformation. In fact, transgenic mice engineered to lack KLF2 in mature B-cells display an expansion of the MZ at the expense of the follicular compartment, but do not develop lymphoma.6,7 It is important to note, however, that lymphoma development may require longer times than those observed so far in mice,6,7 in line with the indolent course of SMZL and the elderly age of patients affected by this lymphoma. Consistent with a multistep process of lymphomagenesis, KLF2 lesions frequently co-occur with IGHV1-2*04 usage, NOTCH2 mutations, and 7q deletion in SMZL, suggesting a possible cooperation between genetic abnormalities and Bcell receptor configuration in promoting transformation. Emerging evidence points to the role of KLF transcription factors in human cancers.14 While KLF family gene expression and function are altered in a large number of neoplasms, including Bcell tumors, recurrent structural alterations of these genes are exceedingly rare and sporadically restricted to solid cancers.14,15 Our data provide the first evidence of the molecular deregulation of KLF family genes in hematologic malignancies, and suggest that selection of KLF2 mutations plays a role in transformation common to several lymphoma subtypes.
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Roberto Piva1§, Silvia Deaglio2§, Rosella Famà3, Roberta Buonincontri2, Irene Scarfò1, Alessio Bruscaggin3, Elisabetta Mereu1, Sara Serra2, Valeria Spina3, Davide Brusa2, Giulia Garaffo1, Sara Monti3, Michele Dal Bo,4 Roberto Marasca5, Luca Arcaini6, Antonino Neri7, Valter Gattei4, Marco Paulli8, Enrico Tiacci9, Francesco Bertoni10, Stefano A. Pileri11, Robin Foà12, Giorgio Inghirami2,13,14, Gianluca Gaidano3, Davide Rossi3 §
R.P. and S.D. equally contributed
1
Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences Biology and Biochemistry and 2Department of Medical Sciences, University of Turin and Human Genetics Foundation, Turin, Italy; 3Division of Hematology, Department of Translational Medicine, Amedeo Avogadro University of Eastern Piedmont, Novara, Italy; 4Clinical and Experimental Onco-Hematology, CRO, IRCCS, Aviano, Italy;5Division of Hematology, University of Modena and Reggio Emilia, Modena, Italy; 6Divisions of Hematology and 8Pathology, 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; 9Institute of Hematology, University of Perugia, Perugia, Italy; 10Lymphoma and Genomics Research Program, IOR Institute of Oncology Research and Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; 11Haematopathology, L. & A. Seragnoli Institute, University of Bologna, Bologna, Italy; 12Division of Hematology, Department of Cellular Biotechnologies and Hematology, Sapienza University, Rome, Italy; 13Department of Pathology and New York University Cancer Center, New York University School of Medicine, and 14Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY. Correspondence: Davide Rossi, MD, Division of Hematology, Department of Translational Medicine, Amedeo Avogadro University of Eastern Piedmont, Via Solaroli 17, 28100 Novara, Italy; Ph +39-0321-660698; Fax +39-0321-620421; E-mail [email protected]. Conflict-of-interest disclosure: The authors have no conflict of interest to disclose. Keywords: KLF2, mutation, splenic marginal zone lymphoma, diffuse large B-cell lymphoma
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Acknowledgment: This study was supported by: Special Program Molecular Clinical Oncology 5 x 1000 No. 10007, My First AIRC Grant No. 13470, and Investigator Grant IG-13227, Associazione Italiana per la Ricerca sul Cancro (AIRC) Foundation Milan, Italy; Fondazione Cariplo, Grant No. 2012-0689; Progetto Giovani Ricercatori 2010, Grant No. GR-2010-2317594, Ministero della Salute, Rome, Italy; Compagnia di San Paolo, Grant No. PMN_call_2012_0071, Turin, Italy; Futuro in Ricerca 2012 Grant No. RBFR12D1CB, Ministero dell'Istruzione, dell'Università e della Ricerca, Rome, Italy; Nelia et Amadeo Barletta Foundation, Lausanne, Switzerland. Contributions: R.P., S.D., G. Gaidano, and D.R. designed the study, interpreted data and wrote the manuscript; R. Famà. A.B., V.S. and S.M. performed and interpreted molecular studies; R.B., I.S., E.M., S.S., D.B., G. Garaffo, and M.D.B. performed and interpreted functional experiments; R.M., L.A., A.N., V.G., M.P., E.T., F.B., S.A.P., R. Foà and G.I. contributed to data interpretation. Supplementary information is available at Leukemia's website.
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REFERENCES
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Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW. WHO classification of tumours of haematopoietic and lymphoid tissues, Fourth Edition. Lyon, France: IARC; 2008.
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Rossi D, Trifonov V, Fangazio M, Bruscaggin A, Rasi S, Spina V, et al. The coding genome of splenic marginal zone lymphoma: activation of NOTCH2 and other pathways regulating marginal zone development. J Exp Med 2012;209:1537-1551.
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Kiel MJ, Velusamy T, Betz BL, Zhao L, Weigelin HG, Chiang MY, et al. Whole-genome sequencing identifies recurrent somatic NOTCH2 mutations in splenic marginal zone lymphoma. J Exp Med 2012;209:1553-1565.
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Martínez N, Almaraz C, Vaqué JP, Varela I, Derdak S, Beltran S, et al. Whole-exome sequencing in splenic marginal zone lymphoma reveals mutations in genes involved in marginal zone differentiation. Leukemia Prepublished on December 3, 2013, as doi: 10.1038/leu.2013.365.
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Parry M, Rose-Zerilli MJ, Gibson J, Ennis S, Walewska R, Forster J, et al. Whole exome sequencing identifies novel recurrently mutated genes in patients with splenic marginal zone lymphoma. PLoS One 2013;8:e83244.
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Hart GT, Wang X, Hogquist KA, Jameson SC. Krüppel-like factor 2 (KLF2) regulates B-cell reactivity, subset differentiation, and trafficking molecule expression. Proc Natl Acad Sci U S A 2011;108:716-721.
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Winkelmann R, Sandrock L, Porstner M, Roth E, Mathews M, Hobeika E, et al. B cell homeostasis and plasma cell homing controlled by Krüppel-like factor 2. Proc Natl Acad Sci U S A 2011;108:710-715.
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Shields JM, Yang VW. Two potent nuclear localization signals in the gut-enriched Krüppellike factor define a subfamily of closely related Krüppel proteins. J Biol Chem 1997;272:18504-18507.
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Kozyrev SV, Hansen LL, Poltaraus AB, Domninsky DA, Kisselev LL. Structure of the human CpG-island-containing lung Kruppel-like factor (LKLF) gene and its location in chromosome 19p13.11-13 locus. FEBS Lett 1999;448:149-152.
10. Wani MA, Conkright MD, Jeffries S, Hughes MJ, Lingrel JB. cDNA isolation, genomic structure, regulation, and chromosomal localization of human lung Kruppel-like factor. Genomics 1999;60:78-86. 11. Swamynathan SK. Krüppel-like factors: three fingers in control. Hum Genomics 2010;4:263270. 12. Wu J, Lingrel JB. KLF2 inhibits Jurkat T leukemia cell growth via upregulation of cyclindependent kinase inhibitor p21WAF1/CIP1. Oncogene 2004;23:8088-8096. 13. Taniguchi H, Jacinto FV, Villanueva A, Fernandez AF, Yamamoto H, Carmona FJ, et al. Silencing of Kruppel-like factor 2 b y the histone methyltransferase EZH2 in human cancer. Oncogene 2012;31:1988-1994. 14. Tetreault MP, Yang Y, Katz JP. Krüppel-like factors in cancer. Nat Rev Cancer 2013;13:701713. 15. Pasqualucci L, Trifonov V, Fabbri G, Ma J, Rossi D, Chiarenza A, et al. Analysis of the coding genome of diffuse large B-cell lymphoma. Nat Genet 2011;43:830-7.
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FIGURE LEGENDS
Figure 1. KLF2 mutations in lymphoid tumors. (A) Prevalence of KLF2 mutations in lymphoid tumors. SMZL, splenic marginal zone lymphoma; HCL, hairy cell leukemia; DLBCL, diffuse large B-cell lymphoma; NMZL, nodal marginal zone lymphoma; EMZL, extranodal marginal zone lymphoma; FL, follicular lymphoma; MCL; mantle cell lymphoma; CLL, chronic lymphocytic leukemia; BL, Burkitt lymphoma; MM, multiple myeloma; vHCL, variant hairy cell leukemia; MZL-like MBL, marginal zone-like monoclonal B-cell lymphocytosis; PTCL, peripheral T-cell lymphoma; T-ALL, T-cell acute lymphoblastic leukemia. (B) Prevalence of the various types of non-silent KLF2 mutations identified in lymphoid tumors. (C) Schematic representation of the human KLF2 protein, with its key functional domains and nuclear localization signals (one within the zinc finger domains and the other in a cluster of basic amino acids immediately before the first zinc finger). Symbols depict distinct types of KLF2 mutations. Non-silent mutations of confirmed somatic origin are color-coded in red. Non-silent mutations of unknown somatic origin are colorcoded in black. Znf, zinc finger; NLS, nuclear localization signal. (D) Distribution of the variant allele frequency of KLF2 mutations (blue circles).
Figure 2. Nuclear localization signal (NLS) mutations of KLF2 cause nuclear displacement of the KLF2 protein. (A) Cell fractionation experiments and confocal microscopy analyses performed in a panel of cell lines with known KLF2 mutation status (A and B represent the two alleles). In the wild type SSK41 line, KLF2 is preferentially expressed in the nucleus, while the presence of mutations in the VL51, AS283A, OCI-Ly8 cell lines cause a predominant accumulation of the molecule in the cytosolic compartment. Primary cells from a patient with a KLF2 missense mutation (#5141) confirm predominant localization of the mutated protein in the cytosol. The nuclear/cytoplasmic expression ratio was calculated by dividing the mean pixel intensity of the green fluorescence in the nuclear area (defined by the DAPI blue fluorescence) by the mean pixel 10 ©
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intensity of the green fluorescence in the rest of the cell. Confocal images were acquired with a 63x oil immersion objective. Wt, wild type; WCL, whole cell lysate; N, nuclear lysate; C, cytoplasmic lysate. (B) HEK293T cells were transiently transfected with flag-tagged KLF2-GFP constructs and grown on glass coverslips. After 24 hours, cells were washed, fixed, stained with anti-flag (red) and counterstained with DAPI (blue). Cells were analyzed using a confocal microscope with a 63x oil immersion
objective.
Representative
images
from
3
independent
experiments.
The
nuclear/cytoplasmic expression ratio was calculated by dividing the mean pixel intensity of the red fluorescence in the nuclear area (defined by the DAPI blue fluorescence) by the mean pixel intensity of the red fluorescence in the rest of the cell. (C) Luciferase activity of HEK293T cells transfected to express a luciferase reporter driven by the CDKN1A/p21 promoter encompassing the KLF2-binding region together with the flag-tagged wild type or mutants KLF2 constructs, and with the empty vector as control. KLF2 expression was induced by doxycycline (1 μg/ml) 24 hours posttransfection. Transactivation activity was detected at 48 hours and expressed as relative luciferase intensity compared to wild type (WT) KLF2 (referred as 100%), in five independent experiments (mean: top of the bar; standard deviation: whiskers; p value by t-test). Untransfected cells (NT) or transfected with an empty vector construct (EV) were used as negative controls. qRT-PCR and Western blot at 48 hours, showing comparable expression of the constructs, are also shown. βtubulin expression is included as protein loading control. Densitometry analysis of 5 independent Western blots shows quantitation of KLF2 levels after normalization over β-tubulin. KLF2 mRNA expression was assessed in 5 independent experiments and calculated relative to ACTB using the ΔCT method.
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2014 Macmillan Publishers Limited. All rights reserved.
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2014 Macmillan Publishers Limited. All rights reserved.
