The transcription factor E2A is essential for lymphocyte development. In this study, we describe a recurrent E2A gene deletion in at least 70% of patients with Sézary syndrome (SS), a subtype of T cell lymphoma. Loss of E2A results in enhanced proliferation and cell cycle progression via derepression of the protooncogene MYC and the cell cycle regulator CDK6. Furthermore, by examining the gene expression profile of SS cells after restoration of E2A expression, we identify several E2A-regulated genes that interfere with oncogenic signaling pathways, including the Ras pathway. Several of these genes are down-regulated or lost in primary SS tumor cells. These data demonstrate a tumor suppressor function of E2A in human lymphoid cells and could help to develop new treatment strategies for human lymphomas with altered E2A activity.

E-proteins define a distinct class of basic helix-loop-helix transcription factors that are central regulators of cellular differentiation in various cell types and are essential for the development of B and T lymphocytes (Kee, 2009). There are three known E-protein coding genes in mammals, namely E2A (also called TCF3), E2-2 (also called TCF4) and HEB (HeLa E-box binding protein; also called TCF12), which all bind to a DNA sequence motif called E-box (CANNTG). The E2A gene, which is located on chromosome 19p13.3, encodes for two different basic helix-loop-helix transcription factors, E12 and E47, which are generated by alternative splicing (Mellentin et al., 1989; Murre et al., 1989). E2A proteins form homodimers and heterodimers with other HLH proteins to conduct their tissue- or cell type–specific functions (Kee, 2009).

Alterations of E2A expression and activity have been suggested to support malignant transformation of lymphoid cells. In mice, deletion of E2A, as well as enforced expression of its inhibitors, e.g., the inhibitor of DNA binding (Id) proteins, lead to rapid development of aggressive T cell lymphomas and T cell hyperproliferations (Bain et al., 1997; Yan et al., 1997; Morrow et al., 1999). In humans, diminished E2A activity has been proposed as a pathogenetic mechanism in TAL1/SCL- or E2A-PBX1–induced leukemias (Park et al., 1999; Aspland et al., 2001; O’Neil et al., 2004), and functional blockade of E2A is involved in the pathogenesis of human lymphomas (Mathas et al., 2006, 2009; Lietz et al., 2007). However, a frequent genomic loss of E2A has not been identified in human lymphoid malignancies so far. Arguing for a function as tumor suppressor in human cells, our study now demonstrates a recurrent deletion of E2A in leukemic cells of patients suffering from Sézary syndrome (SS), an aggressive variant of primary cutaneous T cell lymphoma characterized by the presence of neoplastic T cells in skin, lymph nodes, and peripheral blood (Willemze et al., 2005).

RESULTS AND DISCUSSION

In a genome-wide analysis of peripheral blood mononuclear cells from 20 SS patients (Table S1) by array comparative genomic hybridization (array CGH), we identified a minimal common region of chromosomal loss on chromosome 19p13.3 in 70% (14/20) of patients (Fig. 1 A, Table I, Fig. S1, and Table S2). This region of ∼1.4 Mb ranging from chr19:1368087 to chr19:2824434 (HG18) included the E2A gene locus. Fluorescence in situ hybridization (FISH) analysis on highly enriched tumor cells using an E2A-specific probe confirmed a heterozygous loss of E2A in 8/12 analyzed SS patient samples (Fig. 1 B and Table I; for details on tumor cell enrichment see Materials and methods, Fig. S2, and Table S3). The number of cases with E2A deletion might even be underestimated because in two cases without deletion in array CGH analysis, a deletion of E2A was detected by FISH (Table I). Concomitant with the genomic loss of E2A, the E2A mRNA expression level in enriched leukemic cells of SS patients was significantly reduced compared with purified CD4+ T cells from healthy volunteers (Fig. 1 C and Fig. S3 A; note, that the ΔCt of E12/E47 or E47, respectively, is significantly lower in CD4+ controls compared with SS patient samples. Hence, relative to GAPDH, SS patient samples express less E12/E47 or E47 mRNA, respectively, than the control CD4+ T lymphocytes), and immunohistochemistry showed weak or absent E2A protein expression in skin-infiltrating tumor cells in 15/15 patient samples (Fig. 1 D).

Among cutaneous T cell lymphomas, SS is unique in respect to the presence of a high load of lymphoma cells in the peripheral blood. Because E2A interferes with cell cycle control (Park et al., 1999; Murre, 2005), we first investigated the impact of reduced E2A expression on the growth of malignant SS cells. To this end, we chose the SS-derived Se-Ax cell line, which is associated with a heterozygous loss of E2A (Fig. 1 B and Table I) and is characterized by reduced E2A mRNA and protein levels and impaired E-box DNA binding activity (Fig. 1, E and F and Fig. S3 B). After transient transfection with a Myc-tagged E47 construct and, alternatively, a construct coding for two covalently linked E47 molecules (E47-forced dimer, E47-FD), Se-Ax cells showed a pronounced reduction of proliferation (Fig. 2 A). No significant effect on apoptosis induction was observed (unpublished data). To prove the biological significance of our transfection approach, we investigated transgene expression as well as the resulting E2A-DNA binding activity by immunoblotting and electrophoretic mobility shift assay. In both analyses, we reached levels comparable to endogenous ones in other T cell leukemia–derived cell lines (Fig. S3 C). To substantiate our finding of reduced proliferation after E2A reconstitution, we measured DNA synthesis (determined by BrdU incorporation) and the respective cell cycle phases (determined by 7-aminoactinomycin D [7-AAD] staining) in parallel by a two-color flow cytometric analysis (Fig. 2 B). This experimental approach revealed that reexpression of E2A in Se-Ax cells significantly increased the fraction of cells in the G0/G1 phase at the expense of cells in the S phase of the cell cycle, suggesting that the reduced proliferation of Se-Ax cells after E2A reconstitution is caused by a G0/G1 cell cycle arrest.

To establish a mechanistic link between E2A reduction and deregulated cell cycle control, we analyzed mRNA expression of the protooncogene MYC and the cell cycle regulator CDK6 in Se-Ax cells. After E2A reconstitution, we observed significantly reduced mRNA levels of both genes (Fig. 2 C), suggesting a pathogenetically relevant link in these human T cell–derived lymphoma cells. In line with these in vitro data, we observed robust protein expression of CDK6 in 7 out of 7 and, in accordance with previously published data (Vermeer et al., 2008), of MYC in 5 out of 6 primary SS tumor samples (Fig. 2 D). The high level of MYC expression in the SS tumor cells is in the majority of our SS cases most likely supported by genomic gains of chromosome 8q, which also includes the MYC locus (Fig. S1 and Table S4). Notably, the MYC-induced apoptotic program (Eischen et al., 1999) might be counteracted by the loss of 17p, including the tumor suppressor gene TP53, as demonstrated in a large number of our SS patient samples (Table S4).

To further investigate the impact of diminished E2A expression on SS tumor cells, we characterized E2A-dependent transcriptional changes in Se-Ax cells by microarray gene expression analyses. A highly overlapping, limited number of genes were responsive to E2A reconstitution with either E47 or E47-FD (Fig. S3 D and Table S5). Overall, far more genes were induced than repressed. Arguing for the biological relevance of our microarray analyses, the E2A-regulated genes in our data showed a significant overlap with E2A-dependent genes identified in E2A-deficient murine T cell lymphoma (Schwartz et al., 2006) and in a murine E2A-deficient hematopoietic progenitor cell line (Ikawa et al., 2006; Fig. S3 E and Table S6). In our dataset, among the E2A-induced genes were proapoptotic genes like BCL2L11 and BIK, genes known to modulate T cell–specific signaling pathways and differentiation (DTX1, MAL), as well as negative regulators of oncogenic signaling pathways including the Ras signaling pathway (RASSF4, DAB2IP, RASA4, RGS16). The E2A-dependent up-regulation of these genes was confirmed by quantitative PCR (Fig. 3 A). In view of their dependency on E2A, expression would be expected to be down-regulated or lost in SS tumor cells because of their reduced E2A expression. Accordingly, BCL2L11 and MAL were previously described to be specifically down-regulated in SS cells (Kari et al., 2003; van Doorn et al., 2004). In our samples, BCL2L11, DTX1, and RASSF4 were found to be down-regulated in 4/4 of our SS patient samples, whereas reduced levels of RGS16 were observed in 2/4 SS samples compared with normal CD4+ T cells (Fig. 3, B and C).

Apart from their central role in B and T lymphocyte development, E2A proteins have been suggested to act as tumor suppressors (Bain et al., 1997; Yan et al., 1997). Although other genes located at 19p13.3 might also contribute to the pathogenesis of SS, our data identify the loss of E2A as a pathogenetically important defect of SS tumor cells and strongly support a role of E2A as a tumor suppressor in human lymphoid cells. Mechanistically, we provide evidence that E2A controls a whole set of genes known to promote tumorigenesis. Concomitant with an increased proliferation rate, most likely caused by an impaired G0/G1 cell cycle checkpoint, loss of E2A resulted in an up-regulation of MYC and CDK6. Both genes have been described as E2A target genes in lymphomas that emerge in E2A-deficient mice (Schwartz et al., 2006) and are known to be involved in lymphocyte proliferation, survival, and differentiation (Herold et al., 2009; Hu et al., 2009). Furthermore, aggressive T cell lymphomas in E2A-deficient mice are characterized by high-level myc expression (Bain et al., 1997). This suggests that up-regulation of the MYC oncogene is a common phenomenon after loss of E2A, most likely by loss of transcriptional repression. Speculations on the interconnection of MYC, E2A, and TP53 and their synergistic influence on lymphoma development are substantiated by the high number of cases with simultaneous DNA copy number changes affecting these genes in our SS samples, as well as in E2A-deficient lymphoma cells in mice, a fact that most likely reflects the selective advantage provided by the combination of these aberrations (Fig. S1 and Table S4; Bain et al., 1997). Up-regulation of CDK6 as a consequence of reduced E2A levels could represent another strategy of SS cells to provide a growth and survival advantage. Higher levels of CDK6 in SS cells might cooperate with an aberrant activation of the NOTCH signaling pathway, because CDK6 is necessary to exert the proliferative and anti-apoptotic function of NOTCH (Hu et al., 2009). NOTCH itself promotes T lymphomagenesis (Koch and Radtke, 2007) and is required for the survival of E2A-deficient lymphoma cells in mice (Reschly et al., 2006). In line with this concept, NOTCH activation has been reported in SS cells (Kamstrup et al., 2010). In addition, our data show that E2A controls negative regulators of the oncogenic Ras signaling pathway (RASSF4, RASA4, and DAB2IP). Although altered expression of each of these genes alone might result in Ras activation (Eckfeld et al., 2004; Lockyer et al., 2001; Min et al., 2010), activation of the Ras signaling pathway has still to be proven formally in SS cells. Therefore, the therapeutic potential of Ras inhibition in lymphomas with reduced E2A activity has to be investigated in future studies.

By FISH analysis, no homozygous deletion of E2A was observed. Direct sequencing of the coding regions of all E2A exons in leukemic SS cells from 13 patients did not reveal deleterious mutations or deletions (Table S7). Furthermore, the investigated E2A promoter regions in enriched SS cells compared with normal CD4+ T cells did not show altered methylation patterns Fig. S4. These data suggest on the one hand that the reduction of E2A expression after monoallelic E2A deletion might be sufficient for lymphoma progression, and on the other hand that selective pressure exists to retain one E2A copy to ensure a certain level of E protein activity, which is probably necessary for cellular survival at some time point during lymphomagenesis. Lowering the E2A dosage might facilitate the posttranslational degradation of E2A by NOTCH1 (Nie et al., 2003), which is aberrantly activated in SS cells (Kamstrup et al., 2010). Such a model, i.e., that lowering of wild-type E2A expression contributes to human lymphomagenesis, is supported by the finding of aberrant up-regulation of E2A antagonists like inhibitory HLH proteins (O’Neil et al., 2004; Lietz et al., 2007; Mathas et al., 2006, 2009) or the disruption of one E2A allele and maintenance of one wild-type allele as a result of translocations involving the E2A locus (Aspland et al., 2001) in human lymphoma cells. That even a subtle dosage reduction of a putative tumor suppressor is sufficient for tumor formation has recently been formally shown for, e.g., PTEN (Alimonti et al., 2010).

Together, our data highlight the genomic loss of 19p13.3 including the E2A locus as a pathogenetically important defect of human T cell–derived SS lymphoma cells. Our results provide insights into how E2A acts as a tumor suppressor in human lymphoid cells and might help to develop new treatment strategies for human lymphomas with lost or reduced E2A activity.

MATERIALS AND METHODS

Patient samples.

20 clinically well-characterized patients with SS were included in our study. Diagnoses were established according to the World Health Organization-European Organization for Research and Treatment of Cancer classification for cutaneous lymphomas (Willemze et al., 2005). Detailed clinical information on all patients and blood samples are summarized in Table S1. The use of human material was approved by the Local Ethics Committee of the Charité-Universitätsmedizin Berlin and performed in accordance with the Declaration of Helsinki.

Clonality analysis and sequencing of tumor cell–specific TCRβ rearrangements.

PBMC samples were analyzed for the presence of a clonally expanded tumor cell population by TCRβ-rearrangement PCR analysis using primers and protocols developed within the BIOMED-2 BMH4-CT98-3936 Concerted Action initiative (van Dongen et al., 2003). Only samples that showed almost single clonal product peaks in the fluorescent fragment analysis (FFA) were directly used for subsequent CGH analysis. To identify the Vβ chain expressed by the respective tumor cells, amplification products were purified and complete TCRβ rearrangements were sequenced using the BigDye Terminator V1.1 cycle sequencing kit (Applied Biosystems) and subsequent analysis by high resolution electrophoresis on an ABI PRISM 310 Genetic Analyzer. Identification of the involved V, D, and J segments was done by submitting the received TCRβ sequence to the International Immunogenetics Information System V-QUEST tool (http://imgt.cines.fr/IMGT_vquest/share/textes/; Brochet et al., 2008; Lefranc et al., 2009). Results for each patient are presented in Table S3.

Enrichment of primary SS tumor cells from PBMCs.

PBMCs were isolated from whole blood or leukapheresis samples by density gradient centrifugation using Ficoll-Paque-Plus (GE Healthcare). For enrichment of tumor cells, PBMCs were incubated for 30 min with the respective fluorochrome-conjugated Vβ antibody (Beckman Coulter; see Table S3 for details) at a concentration of 5–20 µl/106 cells in 100 µl of MACS buffer, followed by incubation with anti-fluorochrome MicroBeads (Miltenyi Biotec). Cells were magnetically separated by use of LS columns (Miltenyi Biotec) according to the manufacturer’s recommendations. Where indicated, tumor cells were enriched after incubation with anti-CD4 microbeads (Miltenyi Biotec). The purity of enriched cells was determined by flow cytometry.

Flow cytometric analysis of tumor cell samples.

PBMCs and enriched tumor cells were analyzed on a FACSCalibur flow cytometer (BD) using CellQuest Pro software (BD) and WinMDI version 2.9 (The Scripps Research Institute). Antibodies directed against CD3, CD4, and CD8 were purchased from BD. All Vβ−chain specific antibodies were from Beckman Coulter (see Table S3 for details).

Cell lines, culture conditions, proliferation, and cell cycle analysis.

The human SS-derived cell line Se-Ax (Kaltoft et al., 1987); the T cell acute lymphoblastic leukemia (T-ALL) cell lines Molt-14, Jurkat, and KE-37; and H9 cells were cultured as previously described (Mathas et al., 2009), apart from adding 100 U/ml recombinant human IL-2 (Sigma-Aldrich) to Se-Ax cell culture medium. Se-Ax cells were electroporated in OPTIMEM I (Invitrogen) using a Gene-Pulser II (Bio-Rad Laboratories) with 500 μF and 0.24 kV. Transfection efficiency was determined by pEGFP-N3 (Takara Bio Inc.) co-transfection and subsequent flow cytometry. Cells were transfected with 30 µg myc-tagged E47 or E47-FD expression constructs (Sigvardsson et al., 1997; Lietz et al., 2007) or 30 µg control plasmid pcDNA3.1 (Invitrogen), along with 10 µg of pEGFP-N3. 48–72 h after transfection, EGFP+ cells were enriched by FACS sorting, and sorted cells were used for proliferation assays as well as RNA and protein preparation. Proliferation of cells was determined by measurement of DNA synthesis after [3H]thymidine incorporation using standard protocols. Parallel measurement of BrdU incorporation for determination of proliferation and of 7-AAD staining for determination of cell cycle position was performed by use of the APC BrdU Flow kit (BD). In brief, 48 h after transfection, cells were pulsed for 30 min with BrdU, and BrdU incorporation and 7-AAD staining in gated EGFP+ cells was determined by flow cytometry.

Array CGH.

Initial array CGH analysis was done by means of a submegabase resolution BAC array, as described previously (Erdogan et al., 2006). DNA copy number changes were defined by circular binary segmentation (Olshen et al., 2004) in combination with a log2 threshold of 0.2/−0.2. Aberrations encompassing chromosome arm 19p were further verified and fine-mapped by hybridizations onto a 400k whole genome (Agilent; Gene Expression Omnibus [GEO] accession no. GPL9777) and a customized 60k chromosome 19p oligonucleotide array (Agilent; GEO accession no. GPL10304), respectively, following the manufacturer’s recommendations (Table S8). The customized array comprised 52,828 oligonucleotides evenly covering chromosomal region chr19: 1–28,500,000 (NCBI36; HG18). Array CGH data discussed in this work are available (Barrett and Edgar, 2006) under GEO accession no. GSE19000.

FISH analysis of E2A.

FISH analysis was performed on enriched tumor cells using the specific DNA-probes spanning the E2A gene (RP11-690N6) and control probes for 19qter (D19S989; Kreatech) to judge the copy number of chromosome 19. Hybridization, detection, dual color image acquisition, and image analysis were performed as previously described (Schröck and Padilla-Nash, 2000).

RNA preparation, semiquantitative, and real-time PCR analyses.

Total RNA was prepared using the RNeasy kit (QIAGEN). First strand cDNA-synthesis was performed by use of the first-strand cDNA synthesis kit (AMV; Roche) adding oligo-p(dT)15 primer according to the manufacturer’s recommendation. For semiquantitative PCR analyses primers were as follows: GAPDH, sense (s) 5′-ATGCTGGCGCTGAGTAC-3′ and antisense (as) 5′-TGAGTCCTTCCACGATAC-3′; E2A, s 5′-CACCCTCCCTGACCTGTCT-3′ and as 5′-GGAGCTGAAAGCACCATCTG-3′; RGS16, s 5′-TGGAGAGAGTCGTTCGACCTG-3′ and as 5′-TGTCCTCTTGCACTTGCTTTGC-3′; RASSF4, s 5′-GGTGGGGATGACTTTCAATG-3′ and as 5′-GTGGCTTCCAAGCTATGCTC-3′; BCL2L11 s 5′-GGGTGACTGGAGAGCTCATT-3′ and as 5′-AAAGCACAGGAAGTTGCACA-3′; DTX1, s 5′-CTGGTCACAGCATCAGGCTA-3′ and as 5′-GGTCTTGTGGTGGATCTCGT-3′. Real-time PCR analyses were performed using Power SYBR Green Mastermix and the ABI StepOnePlus real-time PCR system (Applied Biosystems). Three technical replicates were performed for each reaction and specificity of PCR products was confirmed by melting curve analysis and subsequent agarose gel electrophoresis. Relative expression values were determined using the 2−ΔΔCt method. Calculations were done with the StepOnePlus software, taking into account the determined primer efficiency of each primer set used. Primers used for real-time PCR analyses were as follows: myc, s 5′-TCAAGAGGTGCCACGTCTCC-3′ and as 5′-TCTTGGCAGCAGGATAGTCCTT-3′; CDK6, s 5′-GGCTGTGTGAACCAGCCCAAG-3′ and as 5′-TGGCCAGGCCTAGACAGGCA-3′; DTX1, s 5′-TCAGGCTACGAGGGCGTGCT-3′ and as 5′-CCACGAGGCACAGCAGGTGG-3′; BIK, s 5′-AGCTCCTGGAACCCCCGACC-3′ and as 5′-CGCAGGGCCAATGCGTCACT-3′; MAL, s 5′-GGTGGGAAGTGGCGACCGTG-3′ and as 5′-ACAAGATGGGGCGCTCGGGA-3′; RASSF4, s 5′-TTGGGCGTGGAAGTCCCCCA-3′ and as 5′-AGGCGCTGCAGCATCGTCAG-3′; BCL2L11, s 5′-TGCCAGCCCTGGCCCTTTTG-3′ and as 5′-GGCCTGGCAAGGAGGACTTGG-3′; RGS16, s 5′-TCAGCCGCCTCTGCCACTCT-3′ and as 5′-CGGCTGGCTTCCTCACTGCC-3′; RASA4, s 5′-TTTGGCGGCTCGCACGTCAT-3′ and as 5′-CACCTTCCGCAGCGCAGACA-3′; DAB2IP, s 5′-CTGTGTGCAGCCCTCGAGCC-3′ and as 5′-GAGGTGCTCGTTGTCCCCGC-3′; TBP, s 5′-CAGGAGCCAAGAGTGAAGAACA-3′ and as 5′-AGCTGGAAAACCCAACTTCTGT-3′; GAPDH, s 5′-CTCTGCTCCTCCTGTTCGAC-3′ and as 5′-TTAAAAGCAGCCCTGGTGAC-3′; TaqMan Gene Expression Assays Hs01012686_m1 (E47), Hs0102692_g1 (E12/E47), and Hs03929097_g1 (GAPDH). For statistical analyses, independent Student’s t tests were used. All PCR products were verified by sequencing.

Direct sequencing of E2A coding regions.

All 18 coding exons of E2A (E12 and E47) were amplified from 10–20 ng of genomic DNA isolated from enriched tumor cells using AccuPrime GC-Rich Polymerase (Invitrogen) according to the manufacturer’s recommendations. PCR products were directly sequenced by using the BigDye Terminator V1.1 cycle sequencing kit (Applied Biosystems) and subsequent analysis on an ABI PRISM 310 Genetic Analyzer. Primer sequences for amplification and sequencing were as follows: Exon 1, s 5′-CCTCCCTGTTTCTCCCTGTC-3′ and as 5′-GAAAACCTTCCCGTGAACTG-3′; Exon 2, s 5′-TGTAGGGTTTGGTTTGCCAC-3′ and as 5′-CACTGTCTTCAACAGACCCTTG-3′; Exon 3, s 5′-AGGTTTGCCCCTGAGATGAT-3′ and 3 as 5′-CAGGACTCAAACCCATGTCC-3′; Exon 4, s 5′-GGACATGGGTTTGAGTCCTG-3′ and as 5′-GTGAACGCTGGGGACTTG-3′; Exon 5, s 5′-CCCTCAGGATGTTCCTTGGG-3′ and as 5′-GAGAGGGTGGGTGACAGATTTG-3′; Exon 6, s 5′-AACAGCCTGGGGTCTGGATG-3′ and as 5′-CCTACCTCCCTTTGCAGGCT-3′; Exon 7, s 5′-GCAGGGGTTGTTCTCATGGC-3′ and as 5′-AAGCTTCGCCAGGACACAGG-3′; Exon 8, s 5′-GAAACGGGGTGGTAGATGTG-3′ and as 5′-GAGGGGAGCTGGTAAGGTG-3′; Exon 9, s 5′-CTATCCCGCCCCCTTCTAC-3′ and as 5′-GTTCACCTCTGCTCCATGC-3′; Exon 10, s 5′-GCACGAGCGTATGGTAGGAC-3′ and as 5′-CTCTCAGGGCCAGCAGAC-3′; Exon 11+12, s 5′-CTGTGCAGGACGGCAGAAC-3′ and as 5′-CCGCAAAGCCTTCACAGA-3′; Exon 13, s 5′-ATCAGGCCATGCTCACACCC-3′ and as 5′-TCTGTCCTGCAAATTCTGTCGG-3′; Exon 14, s 5′-CATGCGGAAGGGACATGA-3′ and as 5′-GGTGGCTGCCTCCAACCT-3′; Exon 15, s 5′-GCTGGCCTCAGGTTTCAC-3′ and as 5′-ACCCTGACCCCCACCACTA-3′; Exon 16a, s 5′-CCAAGACCTGGTTTTTCCAG-3′ and as 5′-GAGGGAGACAGTGAGGTTGG-3′; Exon 16b, s 5′-ACCCACACTGGGAGGCCGT-3′ and as 5′-CAGTCATGGCAATGCGGTCA-3′; Exon 17, s 5′-TACCCTCTCCACAACCCAGC-3′ and as 5′-AGCATCTGCACCTGGGTGTG-3′; Exon 18, s 5′-ACATCTTTCTCCTCCCTGGG-3′ and as 5′-GTGTGGATGTGGATGAAGCC-3′.

Bisulfite pyrosequencing.

Bisulfite pyrosequencing of two amplicons located in the E2A promoter region was performed according to Lamprecht et al., 2010 with few modifications. In brief, genomic DNA was bisulfite converted using the EpiTect Bisulfite Conversion kit (QIAGEN). In a post-PCR amplification, locus-specific primers were used with one primer biotinylated at the 5′ end (PCR and sequencing primer sequences and analyzed region are shown in the following paragraph). For E2A amplification reactions, PyroMark PCR kit (QIAGEN) was used according to standard protocols. After initial denaturation (95°C for 15 min), PCR consisted of 45 cycles of 94°C for 30 s, annealing temperature for 30 s, and 72°C for 30 s, followed by a final synthesis at 72°C for 10 min. Pyrosequencing was performed using the Pyrosequencer ID and the DNA methylation analysis software Pyro Q-CpG 1.0.9 (Biotage), which was also used to evaluate the ratio T:C (mC:C) at the CpG sites analyzed. All assays were optimized and validated using commercially available completely methylated DNA (Millipore) and pooled DNA isolated from peripheral blood of 10 healthy male and female controls, respectively.

Primer sequences and conditions used for bisulfite-pyrosequencing were as follows: E2A_promA, s 5′-TTAGTTTATGGAGGGTAGGTA-3′ (5′ modification, biotin) and as 5′-AAACCCCAACAATATTCA-3′ (annealing temperature, 55°C; amplicon length, 126 bp; analyzed region [ucsc, HG18], chr19:1,601,941-1,602,066); E2A_promA_seq, 5′-CCCTAAATTACTTTACTAT-3′; E2A_promB, s 5′-TAAGGGGGAAATTGAGGT-3′ and as 5′-CCCTAATACTAAACCCTACATACAA-3′ (5′ modification, biotin; annealing temperature, 55°C; amplicon length, 139 bp; analyzed region [ucsc, HG18], chr19:1,596,870-1,597,008); E2A_promB_seq, 5′-ATTGAGGTTTTGGAG-3′.

Electrophoretic mobility shift assay (EMSA) and immunoblotting.

Preparation of whole-cell and nuclear extracts, as well as EMSA, was performed as previously described (Mathas et al., 2006). The following double-stranded oligonucleotides were used for EMSA: E2A (μE5) sense 5′-AGCTCCAGAACACCTGCAGCAG-3′ and E2A (μE5) antisense, 5′-AGCTCTGCTGCAGGTGTTCTGG-3′; Sp1 sense, 5′-AGCTATTCGATCGGGGCGGGGCGAGC-3′ and Sp1 antisense, 5′-AGCTGCTCGCCCCGCCCCGATCGAAT-3′. After annealing, oligonucleotides were end-labeled with [α-32P]dCTP using Klenow fragment. Positions of protein–DNA complexes were visualized by autoradiography. For supershift analyses, mouse monoclonal antibody to E12/E47 (clone G98-271; BD) was used. For immunoblot analyses, the following primary antibodies were used: mouse monoclonal antibody to E12/E47 (clone G98-271; BD), mouse monoclonal antibody to E47 (clone G127-32; BD), goat polyclonal antibody to PARP-1 (sc-1561; Santa Cruz Biotechnology, Inc.), and mouse monoclonal antibody to β-actin (A5316; Sigma-Aldrich). Filters were incubated with horseradish peroxidase–conjugated secondary antibodies. Bands were visualized with the enhanced chemiluminescence system (GE Healthcare).

Immunohistochemistry.

Immunohistochemistry was performed on 4-µm sections obtained from formalin-fixed and paraffin-embedded material, and done according to Mathas et al., 2009. The primary antibodies used for evaluation of various proteins were monoclonal antibody to E12/E47 (clone G98-271; BD), CDK6 (clone DCS-83; Progen Biotechnik), myc (clone Y69; Epitomics), and CD3 (clone LN10; Novocastra Laboratories).

Gene expression analysis.

One-color microarray-based gene expression analysis was performed following the Quick Amp Labeling protocol from Agilent (G4140-90040v5.7; Agilent). In brief, 500 ng of total RNA was reverse transcribed, and the cDNAs were used as a template for cRNA synthesis and Cy3 labeling by in vitro transcription. Hybridization was performed on whole human genome 4 × 44k microarrays (G4112F; Agilent; GEO accession no. GPL6480). After washing, slides were scanned using an Agilent DNA Microarray Scanner G2565BA with the following settings: scan region, 61 × 21.6 mm; scan resolution, 5 µm; extended dynamic range, selected; TIFF, 16 bit; dye channel, green (with Green PMT XDR Hi 100% and Green PMT XDR Lo 10%). The resulting TIFF images were processed with Agilent Feature Extraction Software v10.5.1.1 using the GE1_105_Dec08 protocol. Gene expression data discussed in this work (Barrett and Edgar, 2006) are available under GEO accession no. GSE21730.

Statistical analyses.

All statistical analyses were done in R v2.9.1 (http://www.r-project.org/). Independent, one-tailed Student’s t test was used to analyze data from real-time PCR experiments. For analyses of proliferation assays, one-way analysis of variance was done before applying Tukey’s Honestly Significant Difference test with 95% family-wise confidence level. Two-sided Welch’s t test was applied to determine significance of cell cycle phase differences between Mock- and E47-FD– or E47-transfected cells, respectively.

Online supplemental material.

In Fig. S1, the complete copy number changes detected by array CGH analysis of all analyzed patients are depicted. Fig. S2 shows a clonality analysis of a leukemic SS patient before and after tumor cell enrichment. Fig. S3 shows E47 mRNA expression levels in SS patient samples. Fig. S4 shows methylation of two regions within the promoter region of E2A in SS patient samples compared with purified CD4+ T lymphocytes from healthy volunteers. Table S1 shows detailed information on SS patient samples. Table S2 shows the exact positions of the 19p13.3 deletion in every individual patient. Table S3 presents the TCRβ rearrangements of the clonal tumor cell population and the antibodies used for detecting/enrichment of the tumor cells. Table S4 shows chromosomal copy number alterations of TP53 and myc in our SS samples. Table S5 gives a complete list of E2A-regulated genes in Se-Ax cells. Table S6 shows the overlap between our dataset and datasets derived from murine E2A-deficient cell types. Table S7 presents all detected sequence alterations within the E2A coding region. Table S8 gives an overview of the used CGH array platforms.

Acknowledgments

Funding was provided in part by Framework VII EU (European Union/BMBF-0315207A) grant, the Deutsche Forschungsgemeinschaft (TRR54), the Berliner Krebsgesellschaft, and the Max Planck Innovation Fonds. We thank Linda El-Ahmad, Franziska Hummel, Simone Kressmann, Katrin Tebel, and Arleta Frensel for excellent technical assistance, and Peter Rahn for cell sorting.

A. Steininger and M. Möbs designed and performed experiments and contributed to writing of the paper. I. Anagnostopoulos, M. Hummel, and H. Stein provided material and performed and interpreted immunohistochemistry analyses. K. Köchert, B. Lamprecht, and S. Kreher performed experiments and interpreted data. M. Beyer and C.D. Klemke provided material. E. Schrock designed and interpreted FISH analyses. J. Richter designed and interpreted bisulfite pyrosequencing analyses. B. Dörken, W. Sterry, E. Schrock, and M. Janz interpreted data and contributed to writing of the manuscript. R. Ullmann, S. Mathas, and C. Assaf designed the study, interpreted data, and wrote the manuscript.

The authors declare no competing financial interests.

References

References
Alimonti
A.
,
Carracedo
A.
,
Clohessy
J.G.
,
Trotman
L.C.
,
Nardella
C.
,
Egia
A.
,
Salmena
L.
,
Sampieri
K.
,
Haveman
W.J.
,
Brogi
E.
et al
.
2010
.
Subtle variations in Pten dose determine cancer susceptibility
.
Nat. Genet.
42
:
454
458
.
Aspland
S.E.
,
Bendall
H.H.
,
Murre
C.
.
2001
.
The role of E2A-PBX1 in leukemogenesis
.
Oncogene.
20
:
5708
5717
.
Bain
G.
,
Engel
I.
,
Robanus Maandag
E.C.
,
te Riele
H.P.
,
Voland
J.R.
,
Sharp
L.L.
,
Chun
J.
,
Huey
B.
,
Pinkel
D.
,
Murre
C.
.
1997
.
E2A deficiency leads to abnormalities in alphabeta T-cell development and to rapid development of T-cell lymphomas
.
Mol. Cell. Biol.
17
:
4782
4791
.
Barrett
T.
,
Edgar
R.
.
2006
.
Gene expression omnibus: microarray data storage, submission, retrieval, and analysis
.
Methods Enzymol.
411
:
352
369
.
Brochet
X.
,
Lefranc
M.P.
,
Giudicelli
V.
.
2008
.
IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis
.
Nucleic Acids Res.
36
(
Web Server issue
):
W503-W508
.
Eckfeld
K.
,
Hesson
L.
,
Vos
M.D.
,
Bieche
I.
,
Latif
F.
,
Clark
G.J.
.
2004
.
RASSF4/AD037 is a potential ras effector/tumor suppressor of the RASSF family
.
Cancer Res.
64
:
8688
8693
.
Eischen
C.M.
,
Weber
J.D.
,
Roussel
M.F.
,
Sherr
C.J.
,
Cleveland
J.L.
.
1999
.
Disruption of the ARF-Mdm2-p53 tumor suppressor pathway in Myc-induced lymphomagenesis
.
Genes Dev.
13
:
2658
2669
.
Erdogan
F.
,
Chen
W.
,
Kirchhoff
M.
,
Kalscheuer
V.M.
,
Hultschig
C.
,
Müller
I.
,
Schulz
R.
,
Menzel
C.
,
Bryndorf
T.
,
Ropers
H.H.
,
Ullmann
R.
.
2006
.
Impact of low copy repeats on the generation of balanced and unbalanced chromosomal aberrations in mental retardation
.
Cytogenet. Genome Res.
115
:
247
253
.
Herold
S.
,
Herkert
B.
,
Eilers
M.
.
2009
.
Facilitating replication under stress: an oncogenic function of MYC?
Nat. Rev. Cancer.
9
:
441
444
.
Hu
M.G.
,
Deshpande
A.
,
Enos
M.
,
Mao
D.
,
Hinds
E.A.
,
Hu
G.F.
,
Chang
R.
,
Guo
Z.
,
Dose
M.
,
Mao
C.
et al
.
2009
.
A requirement for cyclin-dependent kinase 6 in thymocyte development and tumorigenesis
.
Cancer Res.
69
:
810
818
.
Ikawa
T.
,
Kawamoto
H.
,
Goldrath
A.W.
,
Murre
C.
.
2006
.
E proteins and Notch signaling cooperate to promote T cell lineage specification and commitment
.
J. Exp. Med.
203
:
1329
1342
.
Kaltoft
K.
,
Bisballe
S.
,
Rasmussen
H.F.
,
Thestrup-Pedersen
K.
,
Thomsen
K.
,
Sterry
W.
.
1987
.
A continuous T-cell line from a patient with Sézary syndrome
.
Arch. Dermatol. Res.
279
:
293
298
.
Kamstrup
M.R.
,
Gjerdrum
L.M.
,
Biskup
E.
,
Lauenborg
B.T.
,
Ralfkiaer
E.
,
Woetmann
A.
,
Ødum
N.
,
Gniadecki
R.
.
2010
.
Notch1 as a potential therapeutic target in cutaneous T-cell lymphoma
.
Blood.
116
:
2504
2512
.
Kari
L.
,
Loboda
A.
,
Nebozhyn
M.
,
Rook
A.H.
,
Vonderheid
E.C.
,
Nichols
C.
,
Virok
D.
,
Chang
C.
,
Horng
W.H.
,
Johnston
J.
et al
.
2003
.
Classification and prediction of survival in patients with the leukemic phase of cutaneous T cell lymphoma
.
J. Exp. Med.
197
:
1477
1488
.
Kee
B.L.
2009
.
E and ID proteins branch out
.
Nat. Rev. Immunol.
9
:
175
184
.
Koch
U.
,
Radtke
F.
.
2007
.
Notch and cancer: a double-edged sword
.
Cell. Mol. Life Sci.
64
:
2746
2762
.
Lamprecht
B.
,
Walter
K.
,
Kreher
S.
,
Kumar
R.
,
Hummel
M.
,
Lenze
D.
,
Köchert
K.
,
Bouhlel
M.A.
,
Richter
J.
,
Soler
E.
et al
.
2010
.
Derepression of an endogenous long terminal repeat activates the CSF1R proto-oncogene in human lymphoma
.
Nat. Med.
16
:
571
579: 1p: 579
.
Lefranc
M.P.
,
Giudicelli
V.
,
Ginestoux
C.
,
Jabado-Michaloud
J.
,
Folch
G.
,
Bellahcene
F.
,
Wu
Y.
,
Gemrot
E.
,
Brochet
X.
,
Lane
J.
et al
.
2009
.
IMGT, the international ImMunoGeneTics information system
.
Nucleic Acids Res.
37
(
Database issue
):
D1006
D1012
.
Lietz
A.
,
Janz
M.
,
Sigvardsson
M.
,
Jundt
F.
,
Dörken
B.
,
Mathas
S.
.
2007
.
Loss of bHLH transcription factor E2A activity in primary effusion lymphoma confers resistance to apoptosis
.
Br. J. Haematol.
137
:
342
348
.
Lockyer
P.J.
,
Kupzig
S.
,
Cullen
P.J.
.
2001
.
CAPRI regulates Ca(2+)-dependent inactivation of the Ras-MAPK pathway
.
Curr. Biol.
11
:
981
986
.
Mathas
S.
,
Janz
M.
,
Hummel
F.
,
Hummel
M.
,
Wollert-Wulf
B.
,
Lusatis
S.
,
Anagnostopoulos
I.
,
Lietz
A.
,
Sigvardsson
M.
,
Jundt
F.
et al
.
2006
.
Intrinsic inhibition of transcription factor E2A by HLH proteins ABF-1 and Id2 mediates reprogramming of neoplastic B cells in Hodgkin lymphoma
.
Nat. Immunol.
7
:
207
215
.
Mathas
S.
,
Kreher
S.
,
Meaburn
K.J.
,
Jöhrens
K.
,
Lamprecht
B.
,
Assaf
C.
,
Sterry
W.
,
Kadin
M.E.
,
Daibata
M.
,
Joos
S.
et al
.
2009
.
Gene deregulation and spatial genome reorganization near breakpoints prior to formation of translocations in anaplastic large cell lymphoma
.
Proc. Natl. Acad. Sci. USA.
106
:
5831
5836
.
Mellentin
J.D.
,
Murre
C.
,
Donlon
T.A.
,
McCaw
P.S.
,
Smith
S.D.
,
Carroll
A.J.
,
McDonald
M.E.
,
Baltimore
D.
,
Cleary
M.L.
.
1989
.
The gene for enhancer binding proteins E12/E47 lies at the t(1;19) breakpoint in acute leukemias
.
Science.
246
:
379
382
.
Min
J.
,
Zaslavsky
A.
,
Fedele
G.
,
McLaughlin
S.K.
,
Reczek
E.E.
,
De Raedt
T.
,
Guney
I.
,
Strochlic
D.E.
,
Macconaill
L.E.
,
Beroukhim
R.
et al
.
2010
.
An oncogene-tumor suppressor cascade drives metastatic prostate cancer by coordinately activating Ras and nuclear factor-kappaB
.
Nat. Med.
16
:
286
294
.
Morrow
M.A.
,
Mayer
E.W.
,
Perez
C.A.
,
Adlam
M.
,
Siu
G.
.
1999
.
Overexpression of the Helix-Loop-Helix protein Id2 blocks T cell development at multiple stages
.
Mol. Immunol.
36
:
491
503
.
Murre
C.
2005
.
Helix-loop-helix proteins and lymphocyte development
.
Nat. Immunol.
6
:
1079
1086
.
Murre
C.
,
McCaw
P.S.
,
Baltimore
D.
.
1989
.
A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins
.
Cell.
56
:
777
783
.
Nie
L.
,
Xu
M.
,
Vladimirova
A.
,
Sun
X.H.
.
2003
.
Notch-induced E2A ubiquitination and degradation are controlled by MAP kinase activities
.
EMBO J.
22
:
5780
5792
.
O’Neil
J.
,
Shank
J.
,
Cusson
N.
,
Murre
C.
,
Kelliher
M.
.
2004
.
TAL1/SCL induces leukemia by inhibiting the transcriptional activity of E47/HEB
.
Cancer Cell.
5
:
587
596
.
Olshen
A.B.
,
Venkatraman
E.S.
,
Lucito
R.
,
Wigler
M.
.
2004
.
Circular binary segmentation for the analysis of array-based DNA copy number data
.
Biostatistics.
5
:
557
572
.
Park
S.T.
,
Nolan
G.P.
,
Sun
X.H.
.
1999
.
Growth inhibition and apoptosis due to restoration of E2A activity in T cell acute lymphoblastic leukemia cells
.
J. Exp. Med.
189
:
501
508
.
Reschly
E.J.
,
Spaulding
C.
,
Vilimas
T.
,
Graham
W.V.
,
Brumbaugh
R.L.
,
Aifantis
I.
,
Pear
W.S.
,
Kee
B.L.
.
2006
.
Notch1 promotes survival of E2A-deficient T cell lymphomas through pre-T cell receptor-dependent and -independent mechanisms
.
Blood.
107
:
4115
4121
.
Schröck
E.
,
Padilla-Nash
H.
.
2000
.
Spectral karyotyping and multicolor fluorescence in situ hybridization reveal new tumor-specific chromosomal aberrations
.
Semin. Hematol.
37
:
334
347
.
Schwartz
R.
,
Engel
I.
,
Fallahi-Sichani
M.
,
Petrie
H.T.
,
Murre
C.
.
2006
.
Gene expression patterns define novel roles for E47 in cell cycle progression, cytokine-mediated signaling, and T lineage development
.
Proc. Natl. Acad. Sci. USA.
103
:
9976
9981
.
Sigvardsson
M.
,
O’Riordan
M.
,
Grosschedl
R.
.
1997
.
EBF and E47 collaborate to induce expression of the endogenous immunoglobulin surrogate light chain genes
.
Immunity.
7
:
25
36
.
van Dongen
J.J.
,
Langerak
A.W.
,
Brüggemann
M.
,
Evans
P.A.
,
Hummel
M.
,
Lavender
F.L.
,
Delabesse
E.
,
Davi
F.
,
Schuuring
E.
,
García-Sanz
R.
et al
.
2003
.
Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936
.
Leukemia.
17
:
2257
2317
.
van Doorn
R.
,
Dijkman
R.
,
Vermeer
M.H.
,
Out-Luiting
J.J.
,
van der Raaij-Helmer
E.M.
,
Willemze
R.
,
Tensen
C.P.
.
2004
.
Aberrant expression of the tyrosine kinase receptor EphA4 and the transcription factor twist in Sézary syndrome identified by gene expression analysis
.
Cancer Res.
64
:
5578
5586
.
Vermeer
M.H.
,
van Doorn
R.
,
Dijkman
R.
,
Mao
X.
,
Whittaker
S.
,
van Voorst Vader
P.C.
,
Gerritsen
M.J.
,
Geerts
M.L.
,
Gellrich
S.
,
Söderberg
O.
et al
.
2008
.
Novel and highly recurrent chromosomal alterations in Sézary syndrome
.
Cancer Res.
68
:
2689
2698
.
Willemze
R.
,
Jaffe
E.S.
,
Burg
G.
,
Cerroni
L.
,
Berti
E.
,
Swerdlow
S.H.
,
Ralfkiaer
E.
,
Chimenti
S.
,
Diaz-Perez
J.L.
,
Duncan
L.M.
et al
.
2005
.
WHO-EORTC classification for cutaneous lymphomas
.
Blood.
105
:
3768
3785
.
Yan
W.
,
Young
A.Z.
,
Soares
V.C.
,
Kelley
R.
,
Benezra
R.
,
Zhuang
Y.
.
1997
.
High incidence of T-cell tumors in E2A-null mice and E2A/Id1 double-knockout mice
.
Mol. Cell. Biol.
17
:
7317
7327
.

    Abbreviations used:
     
  • AAD

    aminoactinomycin D

  •  
  • CGH

    comparative genomic hybridization

  •  
  • EMSA

    electrophoretic mobility shift assay

  •  
  • FISH

    fluorescence in situ hybridization

  •  
  • SS

    Sézary syndrome

Author notes

A. Steininger and M. Möbs contributed equally to this paper.

S. Mathas and C. Assaf contributed equally to this paper.

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