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© 1999 American Society for Clinical Oncology Expression of Aberrantly Spliced Oncogenic Ikaros Isoforms in Childhood Acute Lymphoblastic LeukemiaFrom the Parker Hughes Cancer Center and Children's Cancer Group ALL Biology Reference Laboratory, Hughes Institute, St Paul, MN; Group Operations Center, Children's Cancer Group, Arcadia, and Department of Hematology-Oncology, Children's Hospital Los Angeles, Los Angeles, CA; Department of Pediatrics, University of Chicago, Chicago, IL; Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY; and Children's National Medical Center and George Washington University, Washington, DC. Address reprint requests to F.M. Uckun, MD, Hughes Institute, 2665 Long Lake Rd, Suite 330, St. Paul, MN 55113; email fatih_uckun{at}ih.org
PURPOSE: We sought to determine if molecular abnormalities involving the Ikaros gene could contribute to the development of acute lymphoblastic leukemia (ALL) in children. PATIENTS AND METHODS: We studied Ikaros gene expression in normal human bone marrow, normal thymocytes, normal fetal liver–derived immature lymphocyte precursor cell lines, eight different ALL cell lines, and leukemic cells from 69 children with ALL (T-lineage ALL, n = 18; B-lineage ALL, n = 51). Expression of Ikaros protein and its subcellular localization were examined by immunoblotting and confocal laser-scanning microscopy, respectively. Polymerase chain reaction (PCR) and nucleotide sequencing were used to identify the specific Ikaros isoforms expressed in these cells. Genomic sequencing of splice junction regions of the Ikaros gene was performed in search for mutations. RESULTS: In each of the ALL cases, we found high-level expression of a non–DNA-binding or aberrant DNA-binding isoform of Ikaros with abnormal subcellular compartmentalization patterns. In contrast, only wild-type Ik-1 and Ik-2 isoforms with normal subcellular localization were found in normal bone marrow cells and thymus-derived or fetal liver–derived normal lymphocyte precursors. In leukemic cells expressing the aberrant Ikaros coding sequences with the 30-base-pair deletion, genomic sequence analysis of the intron-exon junctions between exons 6 and 7 yielded the wild-type sequence. We identified a single nucleotide polymorphism (SNP) affecting the third base of the triplet codon for a proline (CCC or CCA) in the highly conserved bipartite activation region (viz, A or C at position 1002 numbering from the translation start site of Ik-1) within our Ikaros clones. Bi-allelic expression of truncated and/or non–DNA-binding isoforms along with wild-type isoforms was observed in leukemic cells, which implicates trans-acting factor(s) affecting splice site recognition. CONCLUSION: Our findings link specific molecular defects involving the Ikaros gene to childhood ALL. Posttranscriptional regulation of alternative splicing of Ikaros RNA seems to be defective in leukemic lymphocyte precursors from most children with ALL. Consequently, leukemic cells from ALL patients, in contrast to normal lymphocyte precursors, express high levels of non–DNA-binding Ikaros isoforms that are reminiscent of the non–DNA-binding Ikaros isoforms that lead to lymphoblastic leukemia in mice.
ACUTE LYMPHOBLASTIC leukemia (ALL) is the most common form of cancer in children.1-4 A better understanding of the biologic basis and predisposing leukemogenic events in this disease may lead to the development of more effective treatment programs as well as novel prevention strategies. The leukemic clones in ALL patients are thought to originate from normal lymphocyte precursors that have been arrested at various stages of T- or B-lymphocyte development2; hence, any critical regulatory network that controls normal lymphocyte development is a potential target for a leukemogenic event. One such regulatory network that is vital for normal lymphopoiesis involves Ikaros, a member of the Kruppel family "zinc finger" DNA-binding proteins. Ikaros acts as an evolutionarily conserved "master switch" of hematopoiesis that dictates the transcriptional regulation of the earliest stages of lymphocyte ontogeny and differentiation.5-16 In mice, absence of the normal Ikaros gene results in an early and complete arrest in the development of all lymphoid lineages during both fetal and adult hematopoiesis.5 Ikaros-deficient mice have a rudimentary thymus, lack peripheral lymph nodes, and are characterized by a complete absence of lymphocyte progenitor cells as well as mature B-lymphocytes, T-lymphocytes, and natural killer cells.5 Ikaros also has an important leukemia suppressor function that depends on its DNA binding ability: mice that are heterozygous for a germline mutation which results in loss of critical DNA-binding zinc fingers of Ikaros develop a very aggressive form of lymphoblastic leukemia with a concomitant loss of heterozygosity that occurs between 3 and 6 months after birth.10 Moreover, Ikaros has been localized to centromeric heterochromatin in immature lymphocyte precursors, and it has been proposed that Ikaros might play an important role in recruitment and centromere-associated silencing of potentially leukemogenic growth-regulatory genes.15,16 The programmed expression and function of the Ikaros gene is tightly controlled by alternative splicing of the Ikaros pre-mRNA, which results in production of eight different Ikaros isoforms.5-16 Therefore, splicing errors could have severe consequences for the lymphocyte compartment of the developing immune system. All eight Ikaros isoforms share a common carboxy(C)-terminal domain that contains a transcription activation motif and two zinc finger motifs that are required for hetero- and homodimerization among the Ikaros isoforms and for interactions with other proteins.7,8,12 Only three of the eight Ikaros isoforms, however, contain the requisite three or more amino (N)-terminal zinc fingers that confer high-affinity binding to an Ikaros-specific core DNA sequence motif in the promoters of target genes.12 The formation of homo- and heterodimers among the DNA-binding isoforms increases their affinity for DNA, whereas heterodimers between the DNA-binding isoforms and non–DNA-binding isoforms are unable to bind DNA. Therefore, Ikaros proteins with fewer than three N-terminal zinc fingers can interfere with the activity of Ikaros isoforms that can bind DNA.11,12 An abundance of the non–DNA-binding Ikaros isoforms that no longer bind DNA could result in significantly impaired expression of regulatory target genes that are essential for the orderly development and maturation of lymphocyte precursors and could interfere with centromeric recruitment and repression of potentially leukemogenic genes (see Discussion). Based on the critical regulatory function of Ikaros for normal lymphocyte development in mice, as well as the observation of rapid development of lymphoblastic leukemia in mice expressing Ikaros isoforms that lack critical amino-terminal zinc fingers, we hypothesized that normal Ikaros expression and function might be altered in childhood ALL. Our present study, which involved 18 children with T-lineage ALL and 51 children with B-lineage ALL, shows that the posttranscriptional regulation of alternative splicing of Ikaros pre-mRNA is defective in leukemic lymphocyte precursors from most patients. Consequently, leukemic cells from ALL patients, in contrast to normal lymphocyte precursors, express high levels of non–DNA-binding Ikaros isoforms that are reminiscent of the Ikaros isoforms which lead to lymphoblastic leukemia in germline mutant mice. Our findings link for the first time specific molecular defects involving Ikaros to a disease of the human immune system and thereby provide strong support for the long-suspected function of Ikaros as an indispensable regulatory gene. The detection of Ikaros abnormalities as leukemia-specific indicators of disease burden may facilitate the evaluation and monitoring of the quality of remission in children with ALL who are enrolled in contemporary treatment programs.
Patients and Cell Lines The patient population included 64 children (< 21 years of age) with newly diagnosed ALL who were enrolled on Children's Cancer Group (CCG) protocols CCG-1882 and CCG-1961 (for ALL patients 1 to 9 years of age with WBCs  50,000/µL or age 10 years), CCG-1901 (for ALL patients with lymphomatous features, including T-ALL), or CCG-107, CCG-1883, and CCG-1953 (for infants with ALL). Fifteen patients had T-lineage ALL and 49 patients had B-lineage ALL. Except for eight patients with B-lineage ALL, all other patients (87.5%) had high-risk ALL according to National Cancer Institute (NCI) risk classification.17 Five patients in first bone marrow relapse also were studied. Each protocol was approved by the NCI as well as the institutional review boards of the participating CCG-affiliated institutions. Informed consent was obtained from parents, patients, or both as deemed appropriate for treatment and laboratory studies according to United States Department of Health and Human Services guidelines.
Diagnosis of ALL was made on the basis of morphologic, biochemical, and immunologic features of the leukemic cells, including lymphoblast morphology as determined by Wright-Giemsa staining, positive nuclear staining for terminal deoxynucleotidyl transferase, negative staining for myeloperoxidase, and reactivity with monoclonal antibodies to lymphoid differentiation antigens, as described previously.18-20 All T-lineage ALL patients in the present study were classified as T-lineage ALL because
The presenting clinical features of the 64 newly diagnosed patients are listed in Table 1. Among the 15 newly diagnosed T-lineage ALL patients, all 15 had high-risk ALL according to NCI risk classification,17 nine (60%) were male, 14 (97%) had high WBC counts, 12 (80%) had hepatosplenomegaly, and 11 (73%) had a mediastinal mass (Table 1). Among the 49 newly diagnosed B-lineage patients, 41 (30 infants and 11 children; 84%) had high-risk ALL, 28 (57%) were male, 27 (55%) had high WBC counts, and 34 (69%) had hepatosplenomegaly. The preponderance of high-risk patients in this study was due to the availability of larger numbers of leukemic blasts from children with high-risk ALL. Normal bone marrow specimens were obtained from two children who were bone marrow donors in the context of sibling bone marrow transplantation. Normal thymuses were obtained from five children undergoing thoracic surgery for a cardiac defect. One fetal thymus was obtained from a prostaglandin-induced human abortus of 21 weeks' gestational age. These tissues were used according to the guidelines of the Hughes Institute Committee on the Use of Human Subjects. In addition, the human T-lineage ALL cell lines MOLT-3 and JK-E6-1 (ATCC TIB-152), as well as the B-lineage ALL cell lines LC1;19 (E2A-PBX1+), KM-3, HPB-NULL, NALM-6, ALL-1 (BCR-ABL+), and RS4;11 (MLL-AF4+), were also included in the analyses. Also included in our study were the fetal liver–derived immature lymphocyte precursor cell lines FL8.2+ (a CD2+CD19+CD10+CD34+ pro-B/T cell line with germline IgH and T-cell receptor (TCR)
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and Nucleotide Sequencing Purified RT-PCR products (QIAquickTM PCR purification kit, Qiagen) were cloned into the pCR II vector using the TA Cloning kit (Invitrogen, San Diego, CA). The cloned PCR products were purified with a Qiagen plasmid isolation kit and sequenced automatically with the Thermosequenase sequencing kit (Amersham, Arlington Heights, IL) and the ALF Sequencer (Pharmacia, LKB Biotech, Piscataway, NJ).22 Manual sequencing by the dideoxynucleotide chain termination method was performed using the T7 Sequenase Quick-denature Plasmid Sequencing kit (Amersham) according to the manufacturer's instructions. The sequences were compared with the published human Ikaros cDNA sequence obtained through GenBank (accession codes S80876 and U40462).
Genomic Sequence Analysis of the Ikaros Gene at the Two Alternative Splice Donor Sites In the amplification of the 3' splice site, the AdvanTAge PCR mix was replaced with the Expand Long Template PCR system (Roche Molecular Biochemicals, Indianapolis, IN), which contains a combination of Taq polymerase and Pwo polymerase, as the proofreading enzyme, along with precise reagent buffer formulations. Buffer 3, which is formulated for difficult templates and contains detergents, was used at the recommended dilution. For the first round, the gene-specific PCR primer from Ikaros exon 7 was 5'-AGC GGG CGC AGG GAC TC-3' (P7, position +989-973), whereas for the second round, the nested primer was 5'-GAC TCG GCC CCC AGG TAG TTG-3' (P6, position +977-957). Primers AP1 and AP2 were briefly described above. The PCR protocol was performed essentially as recommended in the GenomeWalker manual and as described above. The human tissue-type plasminogen activator (tPA) PCR primers were the positive control primers, PCP1 and PCP2, provided with the GenomeWalker kit. The tPA control cycling parameters were as described in the manufacturer's protocol using the genomic library digest, PvuII. Nested PCR products were cloned using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA). Plasmid minipreps of the cloned DNA were performed using the High Pure Plasmid Isolation Kit (Roche Molecular Biochemicals). Clones containing insert were sequenced using a Thermo Sequenase primer cycle sequencing kit (Amersham Pharmacia Biotech, Piscataway, NJ) and the ALFexpress automated DNA sequencer (Amersham Pharmacia). In some cases (3' splice junction), dimethyl sulfoxide was added to the cycle sequencing reactions at a final concentration of 5% volume-to-volume ratio. Ikaros cDNAs of GenBank accession nos. HSU40462 (human Ikaros mRNA, hIk-1) and S80876 (human Ikaros mRNA, alternatively spliced form, Jurkat) were used in sequence comparisons and mapping. A composite cDNA sequence was generated from these sequences.
Amplification of Genomic DNA and Genomic Sequence Analysis of the Ikaros Gene in ALL Cells PCR was performed using 500 ng of genomic DNA in a 50-µL reaction volume using the Expand Long Template PCR system (Roche Molecular Biochemicals) with buffer and component concentrations as recommended using buffer system 3 for difficult templates. The long-range PCR cycling parameters were as follows: 95°C for 2 minutes (complete denaturation), which is followed by 10 cycles at 94°C for 25 seconds, 65°C for 30 seconds, extension at 68°C for 2 minutes, with an additional 20 cycles of 94°C for 25 seconds, 65°C for 30 seconds, 68°C for 2 minutes (extension), in which 20 seconds is added per cycle to the extension step, and then a final extension at 68°C for 10 minutes. The resulting products were cloned using the TOPO TA Cloning Kit (Invitrogen). Plasmid minipreps of the cloned DNA were performed using the High Pure Plasmid Isolation Kit (Roche Molecular Biochemicals). Clones containing insert were sequenced using the Thermo Sequenase primer cycle sequencing kit (Amersham Pharmacia) and an ALFexpress automated sequencer (Amersham Pharmacia). For the 3' splice site, genomic PCR was performed as above with the Expand Long Template PCR system (Roche Molecular Biochemicals) but using buffer system 1 and 5% dimethyl sulfoxide. The sense primer was 5'-GTA GGT CCT GGC TCG GTG TCC C-3' (P5, intronic position -244 to -223 from the splice acceptor site), and the antisense primer was 5'-GAC TCG GCC CCC AGG TAG TTG-3' (P6, position in cDNA +977-957). In this case, the long-range PCR cycling parameters were 1 x 95°C for 3 minutes; 10 x 95°C for 30 seconds, 66°C for 45 seconds, 68°C for 2 minutes, 68°C for 2 minutes; 20 x 95°C for 30 seconds, 66°C for 45 seconds, 68°C for 2 minutes + 10 sec/cycle; and 1 x 68°C for 5 minutes. For this 3' fragment analysis, dimethyl sulfoxide was added to both the genomic PCR and the cycle sequencing reactions at a final concentration of 5% volume-to-volume ratio.
Western Blot Analysis of Ikaros Protein Expression
Subcellular Localization Studies Using Confocal Laser Scanning Microscopy
Ikaros Protein Expression in Leukemic Cells From Children With ALL Ikaros expression was studied in eight different ALL cell lines, normal tissues, and primary leukemic cells from 59 children with ALL by Western blot analysis (Fig 1 and Table 2). Normal fetal liver–derived human lymphocyte precursor cell lines FL8.2+ (pro-B/T) and FL8.2- (pro-B) (Fig 1A) as well as normal bone marrow cells and thymocytes (Fig 1B and 1C, and Table 2) expressed a 57-kd immunoreactive protein that corresponded in size to Ik-1 and a 47-kd immunoreactive protein that corresponded to either Ik-2 or Ik-3. In contrast, the T-lineage ALL cell lines MOLT-3 cells and JK-E6-1 (data not shown, see legend of Table 2), B-lineage ALL cell lines LC1;19, KM-3, HPB-NULL, NALM-6, ALL-1, and RS4;11 (Fig 1B, lanes 1 to 6), and primary leukemic cells from 16 of 17 (94%) T-lineage ALL patients (Table 2) and 42 of 42 (100%) B-lineage ALL patients (Table 2 and Fig 1C) primarily expressed a smaller immunoreactive protein band of approximately 37 to 40 kd, which corresponded in size and electrophoretic mobility to one or more of the small non–DNA-binding Ikaros isoforms of Ik-4, Ik-5, Ik-6, Ik-7, and/or Ik-8, all of which lack critical N-terminal zinc fingers that are involved in DNA binding.
Abnormal Subcellular Compartmentalization of Ikaros Proteins and Loss of Ikaros-Specific DNA Binding Activity in Leukemic Cells From Children With ALL
Molecular Characterization of Ikaros Transcripts in Leukemic Cells From Children With ALL
Expression of aberrant Ikaros isoforms in leukemic cells could result in cis from sequence alterations or from leukemia-associated alterations in trans-acting factors. Whereas cis activation of aberrant expression would cause mono-allelic expression of the aberrant isoforms, trans activation would be more likely to cause bi-allelic expression. We carefully examined the sequence of 128 Ikaros RT-PCR clones from 25 ALL cases for the presence of polymorphic sequence variations to determine whether the aberrant isoforms with the
Genomic Sequence Analysis of Splice Donor and Acceptor Site Regions in Leukemic Cells That Overexpress the
Subsequent amplification and genomic sequence analysis of the corresponding exon 6/exon 7 splice junction regions from leukemic patients and cell lines that express the deletion variant demonstrated no mutation in the region spanning the cryptic splice site, as well as at the predominant 5' (donor) or 3' (acceptor) splice sites. Bands of the predicted sizes were obtained in the genomic PCR from the patient and cell line DNAs of those samples expressing the alternative splice variant (Fig 4C and 4D [5' splice site] or 4E [3' splice site]). No size differences were detected in restriction analysis of numerous cloned isolates covering both the 5' and 3' splice sites (data not shown). A minimum of six clones from each sample were sequenced for mutational analysis. Sequencing results confirmed the presence of the region between the alternative splice sites in all genomic DNAs examined. There was no mutation within a 284-bp sequence at the normal splice donor site or the region directly surrounding the deleted sequence (ie, near the cryptic splice donor site) (Fig 5A and 5B). Similarly, no mutations were found within a 328-bp exonic + intronic sequence at the 3' splice acceptor site (Fig 6).
Ikaros is a master regulator of the earliest stages of lymphocyte ontogeny and differentiation.5-16 In the present study of 69 pediatric patients with ALL, we sought to determine if molecular abnormalities involving the Ikaros gene could contribute to the development of childhood ALL. Our findings provide unprecedented evidence that, in contrast with normal human thymocytes, bone marrow cells, and lymphocyte precursor cell lines, which express the DNA-binding Ikaros isoforms Ik-1 and Ik-2, leukemic cells from children with ALL preferentially express the alternatively spliced Ikaros mRNA species, which lack two or more of the exons 3, 4, and 5 that encode crucial DNA-binding zinc finger domains. Consequently, these leukemic cells primarily express non–DNA-binding isoforms of Ikaros (Ik-4, Ik-6, Ik-7, and Ik-8), which display abnormal subcellular compartmentalization patterns. These results indicate that the posttranscriptional regulation of alternative splicing of Ikaros pre-mRNA is defective in leukemic cells from children with ALL. The presented data are consistent with the evidence from previous studies in mice, which demonstrated that germline mutant mice that express dominant-negative isoforms of Ikaros develop lymphoblastic leukemia,10 as well as with that of our recent study in infants with ALL,25 and implicate the Ikaros gene in the leukemogenesis of childhood ALL. It is also noteworthy that other investigators have recently presented preliminary evidence that implicates the expression of the dominant-negative Ikaros isoforms in the development of lymphoid blast crisis in adult patients with chronic myelogenous leukemia.26 Our current model for the leukemia suppressor function of Ikaros and a role for non–DNA-binding Ikaros isoforms in the development of childhood ALL is illustrated in Fig 7: the DNA-binding isoforms of Ikaros form homo- and heterodimers with distinct DNA binding capabilities and specificities.12 Furthermore, other DNA binding proteins such as Aiolos27 and Helios,28,29 which can dimerize with all Ikaros isoforms via their shared C-terminal zinc finger domains to form stable multimeric complexes, act in concert with Ikaros and may partially complement its function. These different multimeric complexes are thought to control the transcription of developmentally important genes (Fig 7D) during lymphocyte ontogeny and thereby play pivotal roles for the orderly maturation of lymphocyte precursors.14 Non–DNA-binding Ikaros proteins with fewer than three N-terminal zinc fingers can act as dominant-negative regulators by interfering with the ability of DNA-binding Ikaros isoforms to form homo- and heterodimers or complexes with Aiolos and Helios.12 It is therefore conceivable that inappropriate expression of non–DNA-binding Ikaros isoforms during early lymphopoiesis may dysregulate normal lymphocyte development by impairing the expression of regulatory target genes that are essential for the orderly development and maturation of lymphocyte precursors. Such a developmental error could lead to a maturational arrest at discrete stages of lymphocyte ontogeny and predispose lymphocyte precursors to second hits and leukemic transformation. Recent studies have also indicated that Ikaros might play an important role in recruitment and centromere-associated silencing of growth regulatory genes in immature lymphocyte precursors.15,16 An abundance of non–DNA-binding or aberrant Ikaros isoforms could, therefore, interfere with centromeric recruitment and repression of potentially leukemogenic genes (Fig 7) during lymphocyte development.
A recurring 30-bp in-frame deletion at the 3' end of exon 6 was detected in PCR clones of leukemic cells from 15 of 21 cases sequenced and involved the Ik-2, Ik-4, Ik-7, and Ik-8 coding sequences. The deleted peptide in these aberrant Ikaros isoforms is located in close proximity to the conserved bipartite transcription activation domain12 within the first 81 amino acids of exon 7 adjacent to the C-terminal zinc finger dimerization motifs. The structural changes caused by deletion of this peptide could affect the accessibility of the Ikaros activation domain for the aberrant Ik-2 for interactions with members of the basal transcription machinery and stability of such interactions. These aberrant Ik-2 isoforms could also be impaired in their ability to form dimers or other higher-order complexes with other Ikaros isoforms or other proteins. Such impairments could lead to altered DNA-binding or altered subcellular localization of Ikaros, as we observed in the experiments described above. These possibilities will be examined further in future experiments. We hypothesize that a 30-bp deletion in exon 6 might have resulted from the selection of an alternative 5' splice site, because it starts with a GU sequence (ie, GT in cDNA) at the 5' junction, which could very well serve as a donor site recognition sequence. This hypothesis is strongly supported by our preliminary PCR analyses of the Ikaros genomic locus, which did not yield any evidence for a deletion mutation in leukemic cells with the aberrant mRNA species. Specifically, no mutations were found at the predominant 5' normal splice donor site or the region directly surrounding the deleted sequence near the cryptic splice donor site. Similarly, at the region of the 3' splice acceptor site, no mutation was found in either the exonic or intronic sequence. This region includes the 3' branch point30,31 and the polypyrimidine tract at the acceptor site,32 as well as the upstream, intronic sequence. Therefore, activation of the upstream cryptic splice donor site, which results in an in-frame deletion, is not due to mutation in this region, and this suggests that another unknown alternative splicing mechanism is used.33,34 We identified an SNP that affects the third base of the triplet codon for a proline (CCC or CCA) in the highly conserved bipartite activation region within our Ikaros clones. Bi-allelic expression of truncated and/or non–DNA-binding isoforms along with wild-type isoforms was observed in leukemic cells, implicating trans-acting factor(s) that affect splice site recognition in the generation of this aberrant Ikaros expression profile. It is possible that another region or protein is mutated, which results in cryptic splice site activation, such as a cis-acting regulatory element (ie, alternative splice enhancer35-37) or a trans-acting factor (ie, RNA-binding protein38). Separate detailed studies are required to determine the exact influence of both cis- and trans-acting elements on aberrant Ikaros expression in childhood leukemia. Our results suggest a new mechanism of activation of an oncogene in ALL. Specifically, our findings reveal a new mechanism that involves the aberrant regulation of splicing, which results in overexpression of oncogenic forms of the Ikaros protein that lack the DNA binding domain and may function as dominant interfering proteins in pathways that are normally regulated by the full-length Ikaros protein. Although most of the reported molecular abnormalities in leukemia involve chromosomal translocations, which sometimes result in the generation of chimeric fusion proteins and/or point mutations, there are a few examples of aberrant splicing. In two patients with adult T-cell leukemia, both intact and truncated (without exon 4) Fas gene transcripts were detected.39 This aberrantly spliced message was not found in normal tissues. The loss of exon 4 in the Fas gene resulted in premature termination and loss of Fas antigen expression. Aberrant IRF-1 transcripts lacking exon 2 and 3 and little or no intact IRF-1 mRNA were reported in bone marrow and peripheral-blood mononuclear cells from patients with myelodysplastic syndrome.40 The aberrant IRF-1 transcript generated an abnormal protein without a transcriptional activation domain. No mutations were found within exons or splice junctions. Although the exact mechanism that causes this aberrant splicing remains to be identified, it was proposed to be a mechanism to inactivate tumor-suppressor genes.40 Taken together, our findings provide direct evidence that leukemic cells from children with ALL express high levels of non–DNA-binding isoforms or aberrant DNA-binding isoforms of Ikaros. This study establishes a previously unknown link between specific molecular defects that involve the Ikaros gene and the most common form of childhood cancer. Because the vast majority of the patients in the present study had high-risk ALL, future studies of much larger patient populations are required to determine the true incidence of Ikaros abnormalities in standard-risk ALL as well as to elucidate the potential prognostic significance of Ikaros abnormalities. Finally, our results motivate a comprehensive analysis of other hematopoietic as well as nonhematopoietic malignancies for similar changes in Ikaros expression.
Supported in part by Children's Cancer Group Chairman's Grant no. CA-13539 from the National Cancer Institute, National Institutes of Health (F.M.U.).
F.M.U. is a Stohlman Scholar of the Leukemia Society of America, New York, NY. Presented in part during the Plenary Session at the Fortieth Annual Meeting of the American Society of Hematology, Miami Beach, FL, December 4-8, 1998. The first three authors have contributed equally to this work.
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ABSTRACT
INTRODUCTION
/ß genes coexpressing the B-lineage surface antigen CD19 as well as the T-lineage surface antigen CD2) and FL8.2- (a CD2-CD19+CD10+CD34+ Cµ-sIg- pro-B cell line with germline IgH genes). The establishment and characterization of these normal lymphocyte precursor cell lines were previously reported in detail.
KSSMPQKFLG, due to a 30-bp deletion at the 3' end of exon 6 (
