GENES, CHROMOSOMES & CANCER 54:288–302 (2015)

A Novel Zinc Finger Gene, ZNF465, is Inappropriately Expressed in Acute Myeloid Leukaemia Cells Joseph F. Collin,1† James W. Wells,1‡ Barbara Czepulkowski,1 Linden Lyne,2 Patrick J. Duriez,3 Alison H. Banham,2 Ghulam J. Mufti,1 and Barbara-ann Guinn1,4* 1 Department of Haematological Medicine,Guy’s,King’s and St.Thomas’School of Medicine,King’s College London, The Rayne Institute,London,UK 2 Nuff|eld Department of Clinical Laboratory Science,Radcliffe Department of Medicine, University of Oxford,John Radcliffe Hospital,Oxford,UK 3 CRUKProtein Core Facility,Cancer Sciences Unit, Southampton University HospitalsTrust, Southampton,UK 4 Department of Life Sciences,University of Bedfordshire,Park Square,Luton,UK

To increase our knowledge of leukaemia-associated antigens, especially in acute myeloid leukaemia (AML) M4, we prepared a phage display cDNA library using mRNA from the bone marrow cells of a patient with AML M4 at diagnosis. We immunoscreened 106 pfu with autologous sera and identified an antigen which we named GKT-AML8. The cDNA showed more than 99% similarity to a sequence on 2q21.2 and 95% sequence similarity to a sequence on 19q13.3. These genes were named ZNF465 and ZNF466, respectively, following HUGO Gene Nomenclature Committee (HGNC) guidelines. Expressed sequence tag data suggests that both genes are transcriptionally active. ZNF465 and ZNF466 encode a 50 kr€uppel associated box domain typical of negative regulators of gene transcription. We have confirmed the translational start site in the 11 frame in a near-Kozak sequence that produces a 102 amino acid polypeptide from ZNF465. The high level of sequence similarity between ZNF465 and ZNF466 makes their transcripts almost indistinguishable by real-time polymerase chain reaction (RT-PCR). However, GKT-AML8 showed most sequence similarity to ZNF465 and no transcript matching the 30 ZNF466 sequence could be detected in patient samples or healthy volunteers. ZNF465/466 expression was detectable in 12/13 AML and 10/14 chronic myeloid leukaemia patients’ samples but not in normal donor peripheral blood (0/8) or 0/3 bone marrow samples which had been separated into CD341 and CD342 samples. The altered expression of ZNF465/466 in patients’ samples and its absence in healthy donor haematopoietic samples indicate that ZNF465 is overexpressed in early myeloid disease C 2015 Wiley Periodicals, Inc. V and as such may represent a promising target for immunotherapy.

INTRODUCTION

Acute myeloid leukaemia (AML) is characterized by a block in normal myeloid differentiation from stem cells to monocytes or granulocytes (Burnett and Eden, 1997). A common cause of this block in AML blasts has been shown to be the abnormal expression of transcription factors including PU.1 in erythroleukaemias (MoreauGachelin et al., 1988, 1996) or the expression of hybrid transcription factors (Nichols and Nimer, 1992; Tenen et al., 1997) such as the RUNX1/ RUNX1T1 fusion product in AML M2 (Miyoshi et al., 1991; Nucifora and Rowley, 1995) and the PML/RARA fusion protein in AML M3 (Grignani et al., 1998; Lin et al., 1998). This deregulation of transcription factor activity is thought to lead to the altered regulation of genes important for normal myeloid development (Takahashi et al., 1995; Look, 1997; Grignani et al., 1998; Lin et al., 1998; Westendorf et al., 1998). In addition, nonrandom C 2015 Wiley Periodicals, Inc. V

chromosomal loss and/or deletion suggest that tumour suppressor genes and oncogenes are also involved in AML (Caligiuri et al., 1997; Mrozek et al., 1997) examples including TP53 (Wattel et al., 1994), RAS (Radich et al., 1990; Neubauer et al., 1994), and FLT3 (Kiyoi et al., 1999). The most frequently expressed antigens in AML have

Additional Supporting Information may be found in the online version of this article. Supported by: Leukemia and Lymphoma Research. † Institute of Genetic Medicine, Newcastle University, International Centre for Life, Central Parkway, Newcastle upon Tyne, UK. ‡ The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, Australia. *Correspondence to: Barbara Guinn; Department of Life Sciences, University of Bedfordshire, Park Square, Luton, LU1 3JU, UK. E-mail: [email protected] Received 22 November 2014; Accepted 12 January 2015 DOI 10.1002/gcc.22242 Published online 23 February 2015 in Wiley Online Library (wileyonlinelibrary.com).

ZNF465 EXPRESSION IN ACUTE MYELOID LEUKAEMIA

been the cancer–testis antigens HAGE (Adams et al., 2002) and PASD1 (Guinn et al., 2005) along with the Wilms’ tumour (WT1) antigen (Krauth et al., in press) and mutated nucleophosmin 1 (NPM1; Thiede et al., 2006). However, the heterogeneous nature of AML (Kadia et al., in press) has made it difficult to identify aberrant antigens common to patients diagnosed with AML M4. In leukaemias, most tumour antigens had been found through methods that originally identified mutated genes or chromosomal translocations and whose products were later shown to be effective antigenic targets when presented on major histocompatibility complex (MHC; Molldrem et al., 1996; Pinilla-Ibarz et al., 2000). In 1995, Sahin and coworkers (Sahin et al., 1995) devised the serological analysis of tumor antigens by recombinant cDNA expression cloning (SEREX) method. SEREX has few limitations with regards to patient material and has been shown to be effective in the identification of a variety of tumour-associated antigens in a number of different tumour types including AML (Greiner et al., 2003; Niemeyer et al., 2003; Chen et al., 2005; Guinn et al., 2005; Takahashi et al., 2007). In AML, SEREX has helped to identify cancer–testis antigens such as PASD1 (Guinn et al., 2005) and leukaemiaassociated antigens (LAAs) such as SSX2IP (Guinn et al., 2005) and RHAMM (Greiner et al., 2002). These antigens have helped to provide insights into the roles of LAAs in cell cycle (Guinn et al., 2008), as markers of survival (Guinn et al., 2009), and as targets for immunotherapy (Hardwick et al., 2013). Differential regulation of gene expression in eukaryotes is mediated in part by specific binding of transcription factors to DNA sequences. DNA binding proteins can be classified according to the conserved structural motifs they have in common. It has been estimated there are 300–700 different zinc finger (ZNF) genes in the human genome (Bellefroid et al., 1989) accounting for 1–2% of all human genes. ZNFs such as WT1 and MECOM have been shown to play a role in the pathogenesis of AML, with expression levels being found to be indicative of prognosis (Hou et al., 2010; Vazquez et al., 2011; Krauth et al., in press). Indeed targeting of ZNFs such as WT1 have been shown to induce short-term immune responses (Rezvani et al., 2008; Uttenthal et al., 2014) which may reflect the mode of immunotherapy treatment rather than a limitation of the target. Here, we describe the identification of a novel pair of ZNF genes, one of which is aberrantly expressed in the early stages of AML.

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MATERIALS AND METHODS Sera, Tissues, and Cell Lines

Cell lines were obtained from the American Type Culture Collection (ATCC). Specimens of normal or tumour tissue were obtained following informed consent and local ethical committee approval in accordance with the declaration of Helsinki from patients attending the Department of Haematology at King’s College Hospital. All samples of peripheral blood and bone marrow were collected in ethylenediaminetetraacetic acid (EDTA). The tumour specimen used to construct the phage display expression cDNA library (referred to as AML006) was a bone marrow sample from a 61-year-old male taken at the time his disease transformed from myelodysplastic syndrome—refractory anaemia with ringed sideroblasts—to AML M4. The bone marrow sample showed no cytogenetic abnormalities at the time the sample was taken. Serum was collected from clotted peripheral blood following centrifugation at 1,200 rpm for 10 min. Red cells were lysed from bone marrow and peripheral blood samples using red cell lyses buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA), following a 30 min incubation at room temperature and white cells were pelleted by centrifugation for 10 min at 800g. The panel of normal organ cDNAs (Human MTCTM Panel I) was purchased from BD Biosciences (Oxford, UK). RNA Extraction and Construction of the cDNA Library

Direct isolation of mRNA from crude AML006 cell lysate was performed using Dynabeads Oligo (dT)25 (Dynal, Wirral, UK; Guinn et al., 2002). The cDNA library was constructed in a k-phage ZAP express vector using the cDNA library kit commercially available from Stratagene (Stratagene Europe, Amersterdam, The Netherlands) and following the manufacturer’s guidelines. Immunoscreening of the AML006 cDNA Library and Identification of Positive Clones

The AML006 library was amplified and plated at 104 pfu per 600 ml XL1 Blue MRF’ at OD600 5 0.5–0.7 in 8 ml of 0.7% NZY top agar. Sera were prepared following serial clearings with XL1 Blue MRF’ and XL1 Blue MRF’ cells infected with phage containing vector alone. Lysates were bound to CNBr activated sepharose 4B beads (Amersham Pharmacia Biotech, Buckinghamshire, Genes, Chromosomes & Cancer DOI 10.1002/gcc

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UK) and sera cleared using serial incubations with these and then applied to nitrocellulose membranes. Cleared autologous serum was diluted to a final concentration of 1:100 in tris-buffered saline (TBS)/ 0.05% sodium azide and used to screen at least 106 pfu as described previously (Sahin et al., 1995; Chen et al., 1998). All immunoreactive clones were immunoscreened second time to confirm positivity and positive plaques were isolated, eluted, in vivo excised and plated as phagemids in Escherichia coli on selective media plates. Single colonies were expanded in an overnight culture and plasmid DNA isolated using a QIAGEN plasmid mini kit (QIAGEN, Manchester, UK). Sequence Analysis of the Reactive Clones

T3 and T7 primers, which flanked regions of cDNA insert in the multiple cloning site, were used to sequence the full length cDNA insert (MWG Biotech, Milton Keynes, UK). Nucleotide and translated amino acid sequences were compared with known sequences in the gene, expressed sequence tag (EST) and protein databases using National Center for Biotechnology Information (NCBI) BLAST (Altschul et al., 1997), Baylor College of Medicine (BCM) Search launcher, UK human genome mapping project (hgmp) resource centre, and the institute for genomic research (TIGR) webbased facilities. Open reading frame (ORF) finder at NCBI was used to identify ORFs and hydrophobicity plots were prepared using the hydrophobicity indices of Kyte and Doolittle (1982). Immunoscreening of AML006 Reactive Clones with Sera from Unrelated AML and CML Patients and Normal Donors

Sera were obtained from the peripheral blood of patients with AML (n 5 22; median age of 51.2 6 18.8; male:female of 13:9; 2 3 M1; 1 3 M2; 1 3 M3; 4 3 M4; 3 3 M4/5; 1 3 M5; 4 3 unknown) in addition to those already mentioned: six samples were taken at the time of transformation from myelodysplastic syndrome of which two were to M4 and chronic myeloid leukaemia (CML) (n 5 19; median age 50 6 15.8; male:female of 11:8; nine presentation of disease; two chronic phase; one accelerated phase; one patient at transformation of CML to accelerated phase and a later sample of chimeric lymphoid and myeloid disease; six unknown). In addition, sera were obtained from normal donors (n 5 40; median age 48.4 6 10.8; male:female of 25:15). All clones were Genes, Chromosomes & Cancer DOI 10.1002/gcc

screened with the sera from patients and normal donors in duplicate. RT-PCR Analysis of ZNF465/466 Expression

To evaluate the expression of ZNF465/466 in normal and malignant tissues, we isolated mRNA from various bone marrow and peripheral blood samples using the QIAGEN RNeasy kit (QIAGEN). We prepared cDNA using the MBI Fermentas RevertAid First Strand cDNA synthesis kit (MBI Fermentas, Helena BioSciences, Sunderland, UK), which was DNase I treated (Roche Products, Herts, UK), cleaned using a RNeasy kit (QIAGEN), checked on a 1% agaroseTBE gel and quantified using a spectrophotometer. All mRNA samples were subjected to polymerase chain reaction (PCR) without the reverse transcriptase step to detect if there was any genomic DNA contamination; if so, the sample was DNAse treated and this step repeated. All RT-PCR analyses were repeated on independently isolated mRNA and to ensure the fidelity of the cDNA samples, they were subjected to b-actin PCR as described previously (van Baren et al., 1999) with slight modifications. Briefly, PCR amplifications were performed on 200 ng of the cDNA solution using ReadyMix (Sigma) and 1.5 mM MgCl2 in a final volume of 25 ml. The PCR conditions were 95 C for 5 min followed by 26 cycles of 95 C for 1 min, 60 C for 1 min, and 72 C for 2 min followed by 10 min final extension at 72 C. b-actin PCR was performed to show the competency of the mRNA for PCR amplification. Primers were 50 -GGCATCGTGAT GGACTCCG-30 and 50 -GCTGGAAGGTGGACA GCGA-30 . RT-PCR analysis on the 50 region of the ZNF465/466 cDNA was performed using primers identified with the Primer3 output MIT educational tool (www.genome.wi.mit.edu). ZNF465/ 466 forward primer 50 -GGCTCAAGTCTCACAT GCAG-30 and reverse 50 -GTGGAGTCCTTGTC GTCGTT-30 were used to amplify a 195 bp product. PCR conditions were 50 denaturation at 95 C, 35 cycles of 10 95 C, 10 52 C, and 10 72 C were followed by 100 at 72 C. All PCR products were electrophoresed on 1% agarose gels and assessed following staining with 10 mg/ml ethidium bromide. Bands of amplified transcripts were isolated from 1% agarose gels using the QIAquick gel isolation kit (QIAGEN) and sequenced as described previously (Complement Genomics, Sunderland, UK). The major difference in the two genes ZNF465 and ZNF466 occurred in the region of the

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TABLE 1. Epitopes Investigated for HLA-A2 Binding in the T2 Test

Flu M1 GKT-AML8 GKT-AML8 GKT-AML8 GKT-AML8 GKT-AML8 GKT-AML8

Peptide Peptide Peptide Peptide Peptide Peptide

1 2 3 4 5 6

a.a location

Sequence

58–66 66–74 91–100 65–74 31–40 91–100 58–67

GILGFVFTL ILPTTVPRM SAFKLKVTSL KILPTTVPRM VLPKRKCCRL SLFKLKVTSLc RLPPAWVKILd

Wild type (WT) or modified WT WT WT WT WT Modified Modified

SYPEITHI scorea

BIMAS scoreb

30 21 12 17 11 18 24

550.9 19.425 0.004 0.027 0.025 0.027 17.736

a

Ligation strength to a defined HLA type for a sequence of amino acids. Estimate of the half-time of disassociation of a molecule containing this sequence. c Peptide 5 is a modification of WT Peptide 2, with a change to the 2nd amino acid. d Peptide 6 at amino acid 2 is modified from a T in the WT sequence to L in the modified sequence. b

sequence extending beyond the 30 of the GKTAML8 cDNA sequence. Primers were designed using the only EST (AI904620) that mapped to this region and encoded sequence that was only on chromosome 19 making it specific for ZNF466. The ZNF466 30 EST forward primer 50 -TGG GAGACGGACTCCAGATA-30 and 30 EST reverse primer 50 -CCCATCTTCTGGGTTGTG AC-30 produced a 257 bp product when used on human testis cDNA but despite optimization at a range of annealing temperatures (65 6 10 C) we were unable to produce a PCR product when these primers were used to amplify template from haematological samples. Determination of the Frame of GKT-AML8 Expression

The GKT-AML8 cDNA was subcloned using BamHI and EcoRI into the pGEX2TK plasmid. The GST-GKT-AML8 protein was induced on a 500 ml scale with 0.6 mM IPTG for 3 hr at 37 C. Samples were collected before and after induction to check the levels of expression. The bacteria were lysed and sonicated and the lysate was centrifuged. The pellet containing insoluble inclusion bodies of GST-GKT-AML8 was washed five times in ice-cold Triton X-100 buffer (50 mM TrisHCl pH 8.0, 100 mM NaCl, 0.5% Triton X100 and 1 mM EDTA) and once in the same buffer but without Triton X100. The inclusion bodies were resuspended in phosphate buffered saline (PBS). Samples were analyzed on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and protein visualized with Coomasie blue staining. We subcloned GKT-AML8 cDNA into HisMax vectors (Invitrogen) and sequenced across the multiple cloning site to confirm correct insertion of the sequence. HisMaxA, B, and C vectors

enabled expression of XpressTM epitope and polyhistidine-tagged GKT-AML8 in the 11, 12, and 13 frames, respectively. We transiently transfected each vector into COS-1, COS-7, and 293T cells (all ATCC) using the FuGENE6 reagent (Roche). A ratio of 6:3 and 2:1 were found to give the most efficient expression in 3 3 106 cells in a volume of 1 ml diluted 1:10 in serum free media and incubated for 3 hr. Cells were then cultured in 5% foetal bovine serum (FBS) in DMEM for 24 hr and harvested by trypsinization. Transfected COS-1 cells were subjected to immunocytochemistry using standard methods and DAKO reagents and his-tagged proteins were detected using the anti-XPRESSTM antibody. Prediction of HLA-A*0201-Binding Wild-Type Peptides Within the ZNF465/466 Sequence and HLA-A2 Binding Assays

Six nonamers specific for ZNF465/466 were identified using SYFPEITHI (Rammensee et al., 1999) and bioinformatics and molecular analysis section (BIMAS; Parker et al., 1994) algorithms (Table 1). Four were wild type peptides and two were modified, Peptide 5 from the wild type Peptide 2 and Peptide 6 from the wild type peptide (RTPPAWVKIL) which had a low predicted binding score. Only those peptides with low similarity (95% purity (PPR, Southampton, UK). The HLA-A21 T2 cell line (Hosken and Bevan, 1990) was used to assess binding of peptides to HLA-A2. T2 cells were incubated overnight in complete media (RPMI1640, 1 mM Genes, Chromosomes & Cancer DOI 10.1002/gcc

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sodium pyruvate, 2 mM L-glutamine, 1% nonessential amino acids, 50 mM 2-mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin; all Invitrogen) with 10% FCS alone or with peptide (0.05–100 mM) prior to staining with antihuman HLA-A2-FITC antibody and flow cytometry (FACS) analysis. Antigen Presentation Assays

Full length GKT-AML8 cDNA was subcloned using BamHI and NotI into the pSPT18 vector. Green fluorescent protein (GFP) cDNA was subcloned using BamHI and SalI into the pSPT19 vector as a control. Each plasmid was linearized with KpnI prior to mRNA transcription using the SP6 transcription Kit (Roche, Welwyn Garden City, UK). mRNA products were electrophoresed in RNA loading dye and purified from agarose gels using the RNeasy mini kit (QIAGEN). Antigen presentation assays were initiated 24 hr after electroporation. Briefly, anonymized normal donor buffy coats were received from the North Thames National Blood Service, Tooting, UK, were Ficoll purified to isolate mononuclear cells, and washed after which CD141 cells were isolated using MACs beads and the autoMACs cell separator (both Miltenyi Biotec, Surrey, UK). CD141 cells were then placed in X-VIVO 15 (BioWhittacker Cell Biology, Berks, UK), 1% human AB sera from normal donor clotted blood (Sigma-Aldrich, UK), 800 U/ml recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) and 500 U/ml rhIL-4. Six days later immature monocyte-derived dendritic cells (moDCs) were washed in cell wash buffer A and 50 3 106 DCs resuspended in 600 ml cell wash buffer B (both Cell Projects % GENEFLOW, Staffs, UK). Twenty, 30, or 120 mg of mRNA were added to 100 ml cells and electroporation performed using EasyjecT Plus apparatus (Equibio, Maidstone, UK) at 300 V, 150 mF, and 99 X, resulting in a pulse time of approximately 5 sec. Cells were immediately returned to ice for 2 min and then warm X-VIVO 15, 1% huAB sera. Four hours later we added 800 U/ml rhGM-CSF, 400 U/ ml rhIL-4, 100 U/ml rhTNFa (all R&D Systems) and 1 mg/ml prostin (Pharmacy Department, King’s College Hospital) to mature the DCs. The CD14 negative cells, left over from the CD141 positive isolation, were subjected to CD31 isolation, aliquoted and frozen until required in X-VIVO 15, 1% human AB sera and 10% DMSO (Sigma). Five days later, cells were either harvested and analyzed or restimulated for a further 3 days with 3 3 104 moDCs (which were nonelectroporated, electroporated with Genes, Chromosomes & Cancer DOI 10.1002/gcc

GFP mRNA, or electroporated with GKT-AML8 mRNA) as previously described. Sixteen hours prior to harvesting, 1 mCi of [methyl-3H]thymidine (Amersham) was added to each well and cells were harvested using a Filtermate harvester (Packard Bioscience, Berks, UK) and [methyl-3H]thymidine incorporation determined using an TopCount NXT, microplate scintillation and luminescence counter (Packard Bioscience). We analyzed the T cells by flow cytometry for CD3, CD8, CD11c, CD14, CD15, CD19, CD25, and CD56 expression. DCs were analyzed by FACS for markers of maturation measuring the expression of CD80 (B7-1), CD86 (B7-2), CD1a, CD83, CD54 (ICAM1), CD40, HLA Class I and II (all from PharMingen % BD Biosciences, Oxford, UK), and CCR7 (R&D Systems). RESULTS Immunoscreening of the AML006 Phage Display Library

We had previously immunoscreened 1.17 3 106 plaques from the AML006 phage display library and identified 74 immunoreactive clones (Guinn et al., 2002). Sequencing of the clones showed we had isolated 17 independent cDNA inserts. All of these antigens were immunoscreened with sera from AML and CML patients and from normal donors to determine the disease specificity of their immunoreactivity, in at least duplicate. All scored data were reproducible. We found that 19/22 (86%) AML, 14/19 (74%) CML, and 29/40 (72%) age and sex-matched normal donor sera were reactive with the GKT-AML8 antigen (Guinn et al., 2002). There were no obvious differences between recognition of the antigen by males compared with females in either the patient or normal donor groups. Differences in recognition were not observed between French-American-British (FAB) subtypes and/or disease stage, probably reflecting the relatively small number of patient samples examined and heterogeneity within AML subgroups. Most notably recognition by healthy donor sera was almost as frequent as recognition by AML sera, however, the GKT-AML8 sequence showed features common to ZNF transcription factors, found to play a role in AML pathogenesis. So, we examined its expression in patient and normal donor samples. Analysis of the cDNA Encoding GKT-AML8

The GKT-AML8 cDNA (accession number: AY371499) encoded a 1,134 bp insert (Fig. 1A),

ZNF465 EXPRESSION IN ACUTE MYELOID LEUKAEMIA

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Figure 1. Sequence analysis of GKT-AML8. GKT-AML8 maps to two sequences in the human genome with only 3 bp difference in the 0 3 adenosine rich region between GKT-AML8 and the sequence on 2q21.2 (highlighted in gray), and high similarity with 19q13.3. (A) The translational start site is shown in bold, translational stop signal is denoted by an asterisk. Chromosome band 2q21.2 encodes ZNF465, a 102 amino acid long sequence, which is the longest ORF encoded by GKT-AML8. ZNF466 on 19q13 potentially encodes a shorter 89 amino

acid long protein due to a single base change creating a premature stop signal. All differences in the amino acid sequences between ZNF465 and ZNF466 are highlighted in black; (B) Schematic diagram of the GKT-AML8 cDNA showing a KRAB domain in the 50 region (denoted KD), a single tyrosine kinase phosphorylation site (denoted TK), two splice acceptor sites (denoted *), which are typical of KRAB zinc finger genes, and a 30 single zinc finger that shows similarity to the C2H2 zinc finger gene THC482099.

which was not found to match to any known genes in the NCBI BLAST, TIGR, UK MRC HGMPRC, or ensembl databases; however, the sequence was found to map to two genomic sites. NCBI

human contig database searches indicated that there were two GKT-AML8 related sequences in the human genome, one located on 2q21.2 (Accession number AC097532 or AC018688) and Genes, Chromosomes & Cancer DOI 10.1002/gcc

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the other on 19q33.3 (Accession number AC138473 or AC084239). The close sequence similarity between ZNF465 (which we assigned to 2q21.2) and ZNF466 (which we assigned to 19q13.3; Fig. 1A) based on sequence homology implies that these genes are paralogs (Remm et al., 2001), that is genes related by duplication within a genome. Analysis for sequence similarities between the ZNF465 and ZNF466 genes in other species did not identify any similar genes. Transcript data indicate that GKT-AML8 encodes ZNF465, although this does not exclude the possibility of immunoreactivity against ZNF466. The NCBI database indicated that ZNF465/466 were each encoded by a single exon. An ATG start site was found embedded in a Kozak-like sequence of ACATG (Fig. 1A) rather than the perfect Kozak sequence of ACCATGG (Kozak, 1986) with the methionine codon starting at base 19. EST analysis (EST BM545453) provided the upstream region to the translational start site that contained stop signals (TAA, TGA, or TAG) in all three frames. Three ORFs were present in the 11 frame, the first one starting at the proposed translational start site at base 19 (Fig. 1A), from bases 19–327, 712–849, and 922–1,056, which are 309, 138, and 135 bp’s in length, respectively. BCM search launcher indicated that the ZNF465/GKT-AML8 cDNA did not contain a polyadenylation signal and it would seem that first strand synthesis had primed off the A rich 30 region of GKT-AML8. Splice variants are common 30 of kr€ uppel associated box (KRAB) domains and allow a degree of heterogeneity and control of transcription by the variants. In ZNF465, we found two acceptor sites at bases 170 and 598 (Fig. 1B). Analysis of ZNF465/GKT-AML-8 using the BLASTP 1 BEAUTY (Worley et al., 1998) tool in the BCM search launcher database indicated that there was a tyrosine kinase phosphorylation site in the sequence between bases 319 and 326. EST Analysis of the GKT-AML8 cDNA Sequence

Database dependent EST walking indicated that there was one EST, BM545453, a 50 clone of 954 bp, that matched to the 50 end of the ZNF465/ GKT-AML8 cDNA. However, BM545453 was transcribed in the opposite direction to ZNF465/GKTAML8 and is not part of the expressed ZNF465/ 466 gene(s). BM545453 was shown to map to the same sequences as ZNF465/GKT-AML8 including RP11–725P16 on chromosome 2 (AC97532 and AC067980), CTC-512J12 on chromosome 19 Genes, Chromosomes & Cancer DOI 10.1002/gcc

(AC138473), and RP11–462H3 on chromosome 19 (AC018688), and allowed confirmation of the upstream sequence of GKT-AML8 for gene analysis. Analysis using the TIGR database indicated that BM545453 and ZNF465/GKT-AML8 mapped to a region of 2q21.2 assigned THC1273420. BU568117 and BQ429386 were another two ESTs transcribed near the 50 end of ZNF465/GKT-AML8 within the cDNA sequence, both transcribed in the same direction as ZNF465 and ZNF466. 50 RACE did not identify any further upstream transcribed sequence although positive controls used during RACE produced the expected band sizes. At the 30 end of ZNF465 and ZNF466, a number of ESTs were identified. In the region from base 1,039 to 1,109, 49 EST sequences were found (including CB998665 and AV761044), which all mapped with more than 95% sequence similarity and many of which indicated the presence of Alu repetitive sequences. It was around this region that most differences between the ZNF465 and ZNF466 sequences were found. The presence of ESTs mapping onto both ZNF465 and ZNF466 suggests that both of these genes are transcriptionally active; however, the cDNA we isolated from the AML006 library showed the most sequence similarity to the sequence on 2q21.2, confirming transcription from the ZNF465 gene. Unigene analysis (using the NCBI database) indicated that only the AA761044 EST had been analyzed further for its tissue expression and this sequence mapped to ZNF465 on 2q21.2. Analysis of human ESTs that matched to the 30 sequence extended our knowledge of this region through AA761044 which solely mapped on 2q21.2 or AI904620 which mapped solely onto 19q13.3 (Altschul et al., 1997). 30 RACE was performed on cell line samples known to express GKT-AML8 by RT-PCR, however, no further 30 sequence was identified despite the successful production of 30 transcripts from positive control samples using control primers. RT-PCR analyses of the region 30 of GKT-AML8 did not elucidate any further expressed sequences although genomic templates produced PCR products. Analysis of the GKT-AML8 Amino Acid Sequence

Using the ExPASy web site (www.us.expasy.org/ tools) and its ProtParam tool we analyzed the predicted amino acid sequence in each frame. The first methionine was found in a near-KOZAK sequence and would produce a 102 amino acid protein with a calculated molecular weight of 11498.3 Da, a formula

ZNF465 EXPRESSION IN ACUTE MYELOID LEUKAEMIA

295

Figure 2. Protein blot and immunocytochemical analysis of the ZNF465 protein. (A) Protein staining demonstrated the induction of protein expression from GKT-AML8 cDNA in the pGEX2TK plasmid to produce GST-ZNF465. Samples were collected before (T0) and after induction (T3) to check the level of expression. The bacteria were lysed and sonicated (Total extract) and the lysate was centrifuged to separate the pellet (Insoluble material containing inclusion bodies) and the supernatant (Soluble material). The pellet was washed (W1–W5) five times in ice-cold Triton X-100 buffer and once in the same buffer but without Triton X100 (W6). The inclusion bodies were resuspended in PBS. Protein concentration was estimated using a BioRad kit against bovine

serum albumin (BSA) and the concentration was adjusted to 1 mg/ml. The GST1ZNF465 on the gel is approximately 38 kDa which consists of an approximately 11.5 kDa ZNF465 protein product (indicated by the !) predicted from translation in the 11 frame (GST is approximately 26 kDa). (B) Immunocytochemical analysis of COS-1 cells transiently transfected with histadine-tagged GKT-AML8 indicated that a product was only made when the polyhistadine tagged GKT-AML8 was produced in the 11 frame and ran through the ZNF465 protein product. His-tagged LacZ was used as a positive control for transient transfection and anti-Xpress staining. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

of C515H795N145O139S8, 1,602 atoms and a theoretical pI of 9.69. Protein gel analysis of GST-tagged GKTAML8 expressed from the pGEX2TK plasmid in bacteria produced an approximately 38 kDa product which consisted of approximately 11.5 kDa GKTAML8 protein as predicted from translation in the 11 frame and the GST tag which is approximately 26 kDa (Fig. 2A).

Transient transfections of COS-7 failed to produce a protein product; however, we transiently transfected COS-1 or 293 T cells with XpressTM and polyhistidine-tagged GKT-AML8. We detected a protein product by virtue of binding of the anti-Xpress antibody to the histadine tag when the GKT-AML8 cDNA and Xpress epitope were produced in the 11 frame (Fig. 2B). Genes, Chromosomes & Cancer DOI 10.1002/gcc

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TABLE 2. Location of the Transmembrane Helices in ZNF465/466 Polypeptide when Translated in the 11 Frame as Determined by the SOUSI Programme of the BCM Database No. 1 2 3

N Terminal

Transmembrane region

C Terminal

Type

Length

153 182 253

RMPSTLVLPPSCSSFCWFSAFK RALGLFIFVIFFLKKKRACGNI TWFNSVASLYVPILITSAYAIWH

174 203 275

Secondary Secondary Primary

22 22 23

Figure 3. RT-PCR analyses of ZNF465 gene expression. The lanes marked as M contain Hyper Ladder I marker. The lane marked Bl. is the no template control reaction. Row A contains PCR products amplified from the leukemic cell lines K562, KG1, P39, and U937, and three normal donor samples (Nrml 10, Nrml 11, and Nrml 30); while row B shows products from AML patients (A32, A35, A37, A46, A59,

and A61); Row C shows ZNF465/466 products from healthy donor tissues as indicated. Amplification of ZNF465 led to a 195 bp length product while amplifications using b-actin primers led to products of 615 bp length. The size marker in lane M is 200 bp in the ZNF465/466 gels and 600 bp for the b-actin gel.

The protein is estimated to have a half-life of 20 hr in mammalian reticulocytes in vitro and an instability index of 54.05, which leads this protein to be classified as unstable. The grand average of hydrophobicity was suggested to be 20.363. The PSORT tool indicated that this protein was likely to have a nuclear subcellular localization due to its richness in basic residues as indicated by the presence of greater than 20% K and R amino acids in its composition (Reinhardt and Hubbard, 1998) and nuclear localization signals at amino acids 30 and 33. Using the BCM database, we found that the GKT-AML8 polypeptide, analyzed in the 11 frame, was also likely to encode a membrane protein that has three transmembrane helices (Table 2) and a hydrophobicity of 20.347 similar to that suggested by the ExPASy web-based program.

The strongly preferred model of topology predicted that the N-terminus would be inside and the three helices would go from inside to out over bases 155–174, outside to in with bases 178–195, and inside to out from bases 255 to 275, making this a Type II transmembrane protein. An alternative model, which was less favored predicted two transmembrane helices from 178 to 195 (outside to in) and 255–275 (inside to out).

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RT-PCR Analyses of ZNF465/ZNF466 Expression in Leukaemia Patient Samples

RT-PCR analysis was initially performed using the b-actin primers to indicate whether the cDNA was intact (Fig. 3) as demonstrated by successful amplification of a 615 bp product from the cDNA

ZNF465 EXPRESSION IN ACUTE MYELOID LEUKAEMIA

template. In addition, these primers give a larger product size of 820 bp if genomic DNA contamination was present. Amplification of the 50 ZNF465/466 template produced a 195 bp product from its single exon and a similarly sized product was amplified from genomic DNA (K562 and U937). All primers designed to amplify GKTAML8 cDNA were checked for homology with other regions of the human genome using the NCBI, TIGR, and BCM search launcher databases and were chosen because they were specific for ZNF465/466. RT-PCR analysis indicated the ZNF465/466 transcript was expressed in the AML006 patient sample used to make the AML006 phage display library from which it was originally identified (data not shown). Patients with AML (n 5 12/13) and CML (n 5 10/14) expressed ZNF465/466 transcripts while expression was not found in healthy donor peripheral blood samples (n 5 0/8) or CD341 or CD342 cells separated from a further three healthy allogeneic donor bone marrow samples (Supporting Information). We also tested leukaemia (n 5 8/8), lymphoma (3/3), and solid tumour cell lines (4/5) most of which expressed detectable ZNF465/466 transcripts (Fig. 3; Supporting Information) by RT-PCR. Sequence analysis of excised bands of amplified ZNF465/466 products from agarose gels demonstrated that the transcripts were identical to the ZNF465/466 template. One limitation of the RT-PCR analyses was that the primers for GKT-AML8 would detect products from either the ZNF465 or the ZNF466 gene. Using 30 EST primers unique to ZNF466 we found no expression in any of the cell lines, AML, CML, or normal donor samples, suggesting that the PCR products we had amplified using the 50 primers were from ZNF465 transcripts. This also fits with the slightly higher degree of sequence similarity between the GKT-AML8 sequence we isolated by SEREX and the ZNF465 gene sequence. Identification of HLA-A2 Binding Epitopes Within ZNF465/466

We identified four peptides (Peptides 1–4) with HLA-A*0201 binding motifs, which were specific to ZNF465/ZNF466 and no other known eukaryotic proteins (as determined by BLAST searches; Table 1). However, the SYFPEITHI binding scores for many of the possible peptides were low and so for two peptides (Peptide 5 and Peptide 6) we substituted a single anchor residue within the

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wild type peptides sequence to improve the predicted MHC Class I binding score. Two of the peptides (wild type Peptide 3 and analogue Peptide 6) consistently showed elevated stabilization of HLA-A2 molecules in T2 assays (Table 3; Fig. 4). However, it should be noted that patient AML006 was not HLA-A*0201 positive but had the haplotype A1;32 B7;37, Cw6;7 DRB1 1501, 0401 DRB4 01031 DRB5 01011 DQB1 0602, 0301. GKT-AML8 Antigen Stimulation Assays

To determine whether the GKT-AML8 transcript, when processed and presented by moDCs could stimulate T-cell responses, we electroporated moDCs with GKT-AML8 mRNA as part of their maturation process (Figs. 5A and 5B) and used them to stimulate autologous T cells. Like others, we found that both CD41 and CD81 cells were required for the occurrence of T cell stimulation. We found that moDCs that had been electroporated with mRNA derived from the GKT-AML8 (ZNF465/466) cDNA were able to stimulate T cells to proliferate more than GFP mRNA electroporated moDCs which had been similarly prepared (Fig. 5C). The response was found to be consistent between the three independent normal donor samples tested. DISCUSSION

As the first description of SEREX (Sahin et al., 1995) more than 2,000 antigens have been identified in a growing number of tumour types (www. licr.org/SEREX.html). Approximately, two-thirds of the SEREX-identified genes encode previously identified genes while one-third encode novel gene products. We have screened an AMLderived phage display library with autologous sera and identified a novel antigen, GKT-AML8, which could be potentially encoded by the ZNF465 and ZNF466 genes which mapped to different regions of the human genome. The 1.134 kb GKT-AML8 cDNA encoded an N-terminal KRAB box and a single C-terminal ZNF. KRAB domains, which are found in the amino-terminal region of proteins, act as transcriptional repressor domains’ while C2H2 zinc fingers, as found in the carboxy terminus of ZNF465/466, bind DNA. Functions proposed for members of the KRABdomain containing family include repression of transcription through the targeting of RNA polymerase promoters and family members have been Genes, Chromosomes & Cancer DOI 10.1002/gcc

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TABLE 3. Mean Fluorescence Intensity of HLA-A2 Expression on T2 Cells Following Incubation with Purified GKT-AML8 Derived Nonamers MFI score in T2 assay Cells only, DMSO no peptide

Flu M1 ZNF465/466 ZNF465/466 ZNF465/466 ZNF465/466 ZNF465/466 ZNF465/466

Peptide Peptide Peptide Peptide Peptide Peptide

1 2 3 4 5 6

1.7 2.79 2.69 Concentration of peptide incubated with cells 1 mM 10 mM 100 mM 3.59 7.43 18.77 2.55 2.53 2.74 2.69 2.62 2.46 2.76 2.64 10.99 2.62 2.89 2.71 2.69 2.57 2.53 2.23 2.76 3.55

2.59 500 mM 8.66 20.35 2.86 19.99 2.46 3.25 17.47

Bold text indicate values above background.

Figure 4. Stabilization of HLA-A2 molecules on the surface of T2 cells using WT (Peptide 1, Peptide 2, Peptide 3, or Peptide 4) or analogue (Peptide 5 or Peptide 6) peptides from ZNF465. A peptide from Flu was used as a positive control and HLA-A2 stabilization measured using

FITC-HLA-A2-specific antibody. X-axis indicates mean fluorescence intensity and Y-axis: counts from 0 to 200. Yellow line: 1 mM, Green line: 10 mM; Blue line: 100 mM, and Pink line: 500 mM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

shown to be involved in the maintenance of the nucleolus, cell proliferation and differentiation, apoptosis, and neoplastic transformation (Urrutia, 2003).

Using the NCBI BLASTP 2.2.6 database, we found that the ZNF465/466 amino acid sequence showed high sequence similarity between amino acid 6–35 with the human paternally expressed

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Figure 5. FACS analysis of marker expression of peripheral blood CD141 derived moDCs and measurement of T cell proliferation by [methyl-3H]thymidine incorporation following stimulation of allogeneic T cells with GKT-AML8 mRNA transduced autologous moDCs. (A) T cells were negatively isolated from a normal donor buffy coat using the StemSep human T cell enrichment cocktail including anti-CD11b custom made Ab on the AutoMACs; (B) (i) CD141 cells were isolated from a normal donor buffy coat using the Human CD14 positive selection cocktail from StemSep and the AutoMACs separation system; (ii) following mRNA electroporation and cytokine maturation moDCs show elevation in CD1a and CD83, markers of mature DCs, and

increased B7-1 (CD80) and CD40 costimulatory molecule expression. All flow cytometric analysis was performed using 1 mg Ab per 106 cells and the histograms show cell counts on the Y-axis and fluorescence on a log scale on the X-axis. (C) T cells were stimulated by two rounds of moDCs electroporated with GKT-AML8 mRNA and harvested two days after the initiation of the second round of stimulation. [Methyl-3H]thymidine incorporation is shown as counts per minute (cpm) on the Y-axis. Data showed that GKT-AML8 mRNA fed moDCs stimulated more proliferation by T cells than GFP mRNA or moDCs not fed with mRNA. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

gene 3 (PEG3), an imprinted gene involved in the TNF-NF-jB signal pathway (Relaix et al., 1998) that has been shown to exon share with the ZIM2 zinc finger (Kim et al., 2000). PEG3 is located in the 19q13.4 ZNF rich region of the genome (Kim et al., 1997) and the amino acids which are identical between PEG3 and ZNF465/466 are identical with ZIM2 (zinc-finger gene 2 from imprinted domain) which is located 25 kb downstream of PEG3. ZIM2 produces two different-size transcripts, 2.5 and 9.0 kb in length, with highest levels of expression in adult testis and modest levels in fetal kidney and brain. The 2.5 kb transcript of ZIM2 consists of 11 exons and encodes a Kruppeltype (C2H2) zinc-finger protein with a conserved

KRAB domain. ZIM2 and PEG3 transcripts share identical 50 -ends, composed of seven small exons. Alternative splicing events connect these seven exons either with the remaining two exons of PEG3 or with the remaining four exons of ZIM2. ZIM2, and PEG3 share identical transcription start sites and may also share upstream regulatory elements, although the two genes show distinct patterns of tissue-specific expression (Kim et al., 2000). It is of note that ZNF466 is located close to the ZNF rich region of 19q13.4 in 19q13.3 but did not appear to be transcribed in AML. A number of ZNFs have been found to be involved in the pathogenesis of AML including WT1 (Hou et al., 2010), MZF1 (Robertson et al., Genes, Chromosomes & Cancer DOI 10.1002/gcc

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1998), PLZF (Takahashi and Licht 2002), MLL (Meyer et al., 2006), and GATA2 mutations (Niimi et al., 2013). In addition, the PSORT tool indicated that ZNF465/466 was likely to have a nuclear subcellular localization due to its richness in basic residues (Reinhardt and Hubbard, 1998). This is typical of SEREX-defined proteins which are often nuclear proteins, transcription factors, and have low innate immunity, thus avoiding immune surveillance (Dunn et al., 2004) as the tumour grows. Although immunoscreening did not indicate a notable difference in the frequency of recognition of GKT-AML8 by patient compared with healthy volunteer sera, the transcription of ZNF465/466 in patients compared with healthy volunteer blood and bone marrow samples was starkly different. By RT-PCR, we demonstrated ZNF465/466 expression in AML and CML patient samples but no expression was found in healthy peripheral blood cells. This was maintained even when CD341 and CD342 cells from the bone marrow of patients and healthy volunteers were examined. However, ZNF465/466 is expressed in a range of healthy tissues indicating a key role despite being a paralog (similar copies of this genes are only found in the human genome). Despite the existence of tolerance, there are a number of studies that demonstrate that SEREXdefined antigens can act as effective targets for immunotherapy (Hardwick et al., 2013; Jager et al., 2000) suggesting that targeting of ZNF proteins, such as ZNF465/466 which are inappropriately expressed early in AML disease, may offer some therapeutic advantages. WT1 has already been used in clinical trials for the immunotherapy of AML with some success (Gaiger et al., 2000) although short peptide vaccines appear to stimulate short-term T cell responses (Rezvani et al., 2008; Uttenthal et al., 2014) that may be circumvented by the use of long peptides (Melief and van der Burg 2008), for example, in future clinical trials. Alternative ways in which tumour tolerance in AML has been broken using immunotherapy have included the use of donor leukocyte infusions also known as DLIs (Kolb et al., 1995), DC vaccination (Van Tendeloo et al., 2010), antibody therapy (Chevallier et al., 2008), natural killer cells (Miller et al., 2005), and DNA vaccines (Chaise et al., 2008) as reviewed in Barrett and Le Blanc (2010). Of note, we prepared pDOM-epitope DNA vaccines (Guinn, Stevenson, unpublished data) encoding either Peptide 3 (pDOM.pA3) or Peptide 6 (pDOM.pA6) from ZNF465/466 which Genes, Chromosomes & Cancer DOI 10.1002/gcc

were prepared using methodology described by Rice et al. (2001). Although Peptide 3 did not have very high predicted binding scores by SYFPEITHI or BIMAS it did stabilize HLA-A2 on T2 cells to a notable extent although the modified Peptide 6 was the most effective. We and others have previously shown that modification of peptides at anchor residues can enhance their binding to HLA-A2 and lead to effective CTL killing (Hardwick et al., 2013). However, it should be noted that patient AML006 was not HLAA*0201 positive and so none of the peptides examined is likely to ever have been presented to the adaptive immune system of this patient. In the antigen presentation assays, we used GFP as a control for responses to transfected mRNA. Although we know that GFP is immunogenic and can stimulate T cells in a number of different model systems (Re et al., 2004) we could still demonstrate an enhanced proliferative response by T cells to GKT-AML8 mRNA fed DCs. Of note, GKT-AML8 mRNA fed moDCs were far more effective than GFP mRNA similarly fed moDCs at stimulating the same autologous T cells. Our previous studies (Hardwick et al., 2013) have shown that CD81 or CD41 alone are not effectively stimulated, and this was also found in this study so all stimulated T cell populations were purified by virtue of CD31 expression. To summarize, we have identified two novel ZNF genes, ZNF465 which is transcribed from chromosome 2 and ZNF466 which is located on chromosome 19. These proteins were identified through humoral responses in an autologous AML patient cDNA library but do not appear to be notably recognized by AML patient sera. However, ZNF465 is overexpressed in the vast majority of AML patient samples, including 8/9 samples taken from patients at diagnosis. Of particular note is the absence of ZNF465/466 expression in healthy donor haematopoietic samples although these genes are expressed in a number of other healthy tissues including pancreas, brain, and testis. Thus, ZNF465 may act as a marker of early AML disease and potentially, based on its abnormal expression in AML, as an immunotherapeutic target for future treatments. ACKNOWLEDGMENTS

The authors are indebted to Dr. Geng Li and Professor Robert Rees for their help with the immunoscreening technique and Lynden Lyne, Elizabeth Bland, and Ramona Becker for

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technical assistance. We would also like to thank Professors Farzin Farzaneh, Andrew McKie, Shaun Thomas, and Dr. William Hirst for their suggestions, and members of the Department of Haematological Medicine, particularly Dr. Nigel Westwood and Mr. Simon Goodwin for assistance with the provision of samples. REFERENCES Adams SP, Sahota SS, Mijovic A, Czepulkowski B, Padua RA, Mufti GJ, Guinn BA. 2002. Frequent expression of HAGE in presentation chronic myeloid leukaemias. Leukemia 16:2238–2242. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. Barrett AJ, Le Blanc K. 2010. Immunotherapy prospects for acute myeloid leukaemia. Clin Exp Immunol 161:223–232. Bellefroid EJ, Lecocq PJ, Benhida A, Poncelet DA, Belayew A, Martial JA. 1989. The human genome contains hundreds of genes coding for finger proteins of the Kruppel type. DNA 8: 377–387. Burnett AK, Eden OB. 1997. The treatment of acute leukaemia. Lancet 349:270–275. Caligiuri MA, Strout MP, Gilliland DG. 1997. Molecular biology of acute myeloid leukemia. Semin Oncol 24:32–44. Chaise C, Buchan SL, Rice J, Marquet J, Rouard H, Kuentz M, Vittes GE, Molinier-Frenkel V, Farcet JP, Stauss HJ, DelfauLarue MH, Stevenson FK. 2008. DNA vaccination induces WT1-specific T-cell responses with potential clinical relevance. Blood 112:2956–2964. Chen YT, Gure AO, Tsang S, Stockert E, Jager E, Knuth A, Old LJ. 1998. Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library. Proc Natl Acad Sci USA 95:6919–6923. Chen G, Zhang W, Cao X, Li F, Liu X, Yao L. 2005. Serological identification of immunogenic antigens in acute monocytic leukemia. Leuk Res 29:503–509. Chevallier P, Delaunay J, Turlure P, Pigneux A, Hunault M, Garand R, Guillaume T, Avet-Loiseau H, Dmytruk N, Girault S, Milpied N, Ifrah N, Mohty M, Harousseau JL. 2008. Longterm disease-free survival after gemtuzumab, intermediate-dose cytarabine, and mitoxantrone in patients with CD33(1) primary resistant or relapsed acute myeloid leukemia. J Clin Oncol 26: 5192–5197. Dunn GP, Old LJ, Schreiber RD. 2004. The three Es of cancer immunoediting. Annu Rev Immunol 22:329–360. Gaiger A, Reese V, Disis ML, Cheever MA. 2000. Immunity to WT1 in the animal model and in patients with acute myeloid leukemia. Blood 96:1480–1489. Gotch F, Rothbard J, Howland K, Townsend A, McMichael A. 1987. Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2. Nature 326:881–882. Greiner J, Ringhoffer M, Taniguchi M, Schmitt A, Kirchner D, Krahn G, Heilmann V, Gschwend J, Bergmann L, Dohner H, Schmitt M. 2002. Receptor for hyaluronan acid-mediated motility (RHAMM) is a new immunogenic leukemia-associated antigen in acute and chronic myeloid leukemia. Exp Hematol 30: 1029–1035. Greiner J, Ringhoffer M, Taniguchi M, Hauser T, Schmitt A, Dohner H, Schmitt M. 2003. Characterization of several leukemia-associated antigens inducing humoral immune responses in acute and chronic myeloid leukemia. Int J Cancer 106:224–231. Grignani F, De Matteis S, Nervi C, Tomassoni L, Gelmetti V, Cioce M, Fanelli M, Ruthardt M, Ferrara FF, Zamir I, Seiser C, Grignani F, Lazar MA, Minucci S, Pelicci PG. 1998. Fusion proteins of the retinoic acid receptor-alpha recruit histone deacetylase in promyelocytic leukaemia. Nature 391:815–818. Guinn BA, Collin JF, Li G, Rees RC, Mufti GJ. 2002. Optimised SEREX technique for the identification of leukaemia-associated antigens. J Immunol Methods 264:207–214.

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A novel zinc finger gene, ZNF465, is inappropriately expressed in acute myeloid leukaemia cells.

To increase our knowledge of leukaemia-associated antigens, especially in acute myeloid leukaemia (AML) M4, we prepared a phage display cDNA library u...
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