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Related Collections Neoplasia Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 95 No. 3 (February 1), 2000: pp. 1066-1068 NEOPLASIA Novel SH3 protein encoded by the AF3p21 gene is fused to the mixed lineage leukemia protein in a therapy-related leukemia with t(3;11) (p21;q23) Kimihiko Sano, Akira Hayakawa, Jin-Hua Piao, Yoshiyuki Kosaka, and Hajime Nakamura From the Department of Pediatrics, Kobe University School of Medicine, Kobe, Japan.     Abstract Top Abstract Introduction Results Discussion References The mixed lineage leukemia (MLL) gene located at chromosome band 11q23 is frequently rearranged in patients with therapy-related acute monocytic leukemia who received topoisomerase II inhibitors. We have identified a novel fusion partner of MLL (FAB M5b) in a patient who developed t-AML 9 years after treatment for acute lymphoblastic leukemia (ALL). The leukemic cells had a sole karyotypic abnormality of t(3;11) (p21;q23). Screening of a genomic DNA library, prepared from leukemic cell DNA, identified rearranged clones composed of MLL and a novel gene on chromosome 3p21 (AF3p21). The AF3p21 gene encodes a protein of 722 amino acids, which contains an Src homology 3 (SH3) domain, a proline-rich domain, and a bipartite nuclear localizing signal (NLS). RNA analysis demonstrated that exon 6 of the MLL gene fused to exon 2 of the AF3p21 gene. The resulting chimeric protein consists of AT-hooks, methyltransferase, and transcription repressor domains of MLL in addition to the AF3p21 proline-rich domain and NLS but not the AF3p21 SH3 domain. (Blood. 2000;95:1066-1068) © 2000 by The American Society of Hematology.     Introduction Top Abstract Introduction Results Discussion References Therapy-related acute myelogenous leukemia (t-AML) is a complication of effective cancer chemotherapy. The mixed lineage leukemia (MLL) gene (also called the acute lymphoblastic leukemia-1 [ALL-1] gene or the human trithorax [HRX] gene) is localized on chromosome 11q231-3 and is involved in 11q23 abnormalities of t-AML.4 The MLL protein contains 2 potential DNA-binding motifs (AT-hooks and zinc fingers), a transcriptional activation domain in the -carboxyl (C) terminus, and a repression domain in the N-terminal portion. MLL possesses a highly conserved SET (Su[var]3-9, enhancer of zeste, and trithorax) domain that is found in Drosophila trithorax and Polycomb genes, which are known to regulate the expression of the homeotic genes in Drosophila. Defects in yolk sac hematopoiesis in MLL-null mouse embryos and the differentiation block in MLL-null embryonic stem (ES) cells support the notion of MLL playing a major role in hematopoietic differentiation.5,6 To date, 19 partner genes of MLL have been cloned from leukemia cells with various types of reciprocal translocations, and they form in-frame fusion products of the N-terminal portion of MLL with partner proteins.4 A mouse knock-in study showed that MLL-AF9 chimeras develop AML, whereas MLL-myc chimeras do not.7 Moreover, retrovirus-mediated gene transfer of MLL-ENL into murine hematopoietic progenitors led to their immortalization, and this activity required the presence of the ENL moiety of the fusion protein.8 These results suggest that the partner genes of MLL are critical in leukemogenesis. The detection of t(3;11)(p21;q23) in a patient with t-AML prompted us to identify the resulting molecular rearrangement. Patient and methods Patient A 14-year-old female was diagnosed as having T-cell ALL in 1988. The karyotype of the leukemic blasts was 46/XX. She was treated according to a protocol for high-risk ALL of Osaka Children's Leukemia Study Group, followed by an autologous peripheral blood stem cell transplantation. The cumulated doses of antileukemia agents used were: 300 mg/m2 daunorubicin, 2600 mg/m2 etoposide, 7500 mg/m2 cyclophosphamide, and 460 mg/m2 ranimustine. The patient had been well until she developed AML (FAB M5b) in November 1997. The karyotype of the t-AML blasts was t(3;11) (p21;q23). Complete remission was achieved with a Koseisho protocol for acute nonlymphoblastic leukemia (ANLL-91), and in September 1998, she underwent bone marrow transplantation with cells donated from a human lymphocyte antigen-identical unrelated donor. Since that time, she has been in remission with chronic graft-versus-host disease. Molecular cloning of the chimeric DNA A genomic library from the patient's leukemic cell DNA was constructed by the complete digestion of genomic DNA with BamH I and ligation into a phage vector (ZAP Express; Stratagene, La Jolla, CA). Rearranged BamH-I fragments were isolated by screening with the MLL complementary DNA (cDNA) probe. Total RNA extracted from the leukemic cells was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using MLL exon 5 (forward) and AF3p21 (reverse) primers for amplification of MLL-AF3p21 fusion transcripts. The sequences of the primers were: forward, 5'-AAGCAGCCTCCACCACCAGA, and reverse, 5'-GCTGGAGCCTGGTTCAGATG. The PCR products were subcloned into a plasmid vector (pGEM-T Easy vector; Promega, Madison, WI) and sequenced.     Results Top Abstract Introduction Results Discussion References Cloning and identification of a novel MLL fusion product To analyze the involvement of the MLL gene in this translocation, we performed Southern blot analysis of DNA prepared from the leukemic cells of the patient. Hybridization of BamH-I digested DNA with the MLL cDNA probe revealed the presence of approximately a 4-kilobase (kb) rearranged band in addition to normal-size fragments (data not shown). To clone the rearranged DNA, we constructed a genomic DNA library prepared from BamH-I digested leukemic cell DNA. Screening of the library identified 2 clones composed of a rearranged MLL segment among 13 positive clones. Sequence comparison between these clones and the 8.3-kb MLL breakpoint cluster region (bcr) revealed that MLL sequences downstream of exon 6 were substituted with the non-MLL DNA segment. Complete AF3p21 cDNA sequence A BLAST database search for the novel sequence identified a highly homologous expressed sequence tag (EST) clone (GenBank accession no. AA227 226). Since only a portion of the 5' terminus sequence of AA227 226 was deposited in the database, we determined the entire sequence of the EST clone. This clone contained a 3003-base pair (bp) insert, and a reading frame analysis suggested that this clone possibly contains an extraneous sequence in its 5' region. Therefore, we performed 5' RACE (rapid amplification of cDNA ends) to isolate the major spliced products and to determine the transcription start site of the novel gene using a cDNA of K562 leukemia cell line as a template. This analysis revealed that AA227 226 indeed contains a 29-bp intronic sequence at the exon-intron junction of exon 2 and lacks the 49-bp sequence at its 5' terminus. The corrected sequence has 3023 bp with an open reading frame of 2166 bp encoding a polypeptide of 722 amino acids with a predicted molecular mass of 80 kd. In keeping with the nomenclature for MLL fusion partner genes, we designated this gene AF3p21 (ALL-1 fused gene from chromosome 3p21). The complete sequence of AF3p21 has been deposited with the National Center for Biotechnology Information (NCBI) Data Library (accession no. AF 178432). Sequence comparisons with the public database revealed that the N-terminus of the predicted AF3p21 protein (amino acid [aa] 1-58) contains an SH3 domain. The SH3 domain of AF3p21 is mostly similar to that of Fyn proto-oncogene tyrosine kinase. A database search for other functional domains revealed the presence of a bipartite NLS (aa 171-192) and a proline-rich domain (aa 170-249) downstream of the SH3 domain. Genomic sequences at the breakpoint To determine the nucleotide sequence around the breakpoint of the chromosome 3p21 region, bacterial artificial chromosome (BAC) DNA containing the AF3p21 gene (clone 425p07; Genome Systems, St Louis, MO) was directly sequenced. A sequence comparison of the genomic segments of MLL, AF3p21, and MLL-AF3p21 at the breakpoint is shown in Figure 1A. There is an identical stretch of 3 nucleotides (CCA) in the MLL and AF3p21 genes at the rearranged site. Therefore, the breakpoint of the MLL gene is located between nucleotide positions 1587 and 1589 (numbering according to the 11q23-breakpoint sequence in GenBank accession no. HSU04 737). This is within an Alu-Sb repetitive sequence. The breakpoint on chromosome 3 is located at 61 bp upstream of the putative transcription start site of the AF3p21 gene. View larger version (35K): [in this window] [in a new window]   Fig 1. MLL is fused in-frame to the AF3p21 in the t(3,11) (A) Genomic sequences of the MLL, MLL-AF3p21 (derived from the derivative chromosome 11[der(11)]), and AF3p21 genes at the breakpoint region. (B) Sequence of the der11 MLL-AF3p21 fusion junction amplified by RT-PCR. (C) Schematic diagram of the consequences of the MLL, MLL-AF3p21, and AF3p21 proteins. RT-PCR analysis of the patient material To determine whether the translocation results in an in-frame fusion of MLL to AF3p21, RT-PCR was performed to amplify the cDNA junction of leukemic cells. Sequence analysis of the chimeric transcripts revealed an in-frame fusion between MLL exon 6 and AF3p21 exon 2 (Figure 1B). The resulting MLL-AF3p21 fusion protein consists of AT-hooks, methyltransferase, and transcription repressor domains of MLL in addition to the AF3p21 proline-rich domain and bipartite NLS but not the AF3p21 SH3 domain (Figure 1C).     Discussion Top Abstract Introduction Results Discussion References Topoisomerase II inhibitor-associated t-AML is characterized by chromosomal translocations, most of which disrupt introns within an 8.3-kb bcr between exons 5 and 11 of the MLL gene. Several translocation breakpoints in MLL and in the partner DNAs are in intronic Alu repeats, suggesting that homologous repetitive sequences may be of importance in joining MLL with its translocation partners. The breakpoint in the MLL gene of our case occurs in an Alu-Sb sequence in intron 6. Although the breakpoint region of the AF3p21 gene does not contain an Alu repetitive sequence, both MLL and AF3p21 contained a homologous CCA sequence at the breakpoint (Figure 1A). These results suggest that the base pairing of homologous DNA ends of MLL and AF3p21 may be a first step in the translocation event in this case. MLL fusion partner genes encode protein products of different types, and most of them are putative transcription factors.4 There are 2 genes, including EEN on band 19p139 and ABI-1 on 10p11.2,10 that encode SH3 domain-containing protein. EEN is a human homologue of a member of a recently described murine SH3 domain-containing protein family.11 The SH3 domain in EEN is most similar to that found in the GRB2 family proteins, suggesting that it acts as an adapter protein in signal transduction. ABI-1 is a human homologue of mouse Abi-1 (Abl-interactor-1) that suppresses v-Abl transforming activity by binding to the Abl protein.12 The SH3 domain of Abi-1 is responsible for its binding to c-Abl. Both EEN and ABI-1 have an SH3 domain in their C-terminus, and fusion proteins with MLL retain their SH3 domains. However, the SH3 domain in AF3p21 is located at the N-terminus, and the resulting MLL-AF3p21 fusion protein does not retain the SH3 domain (Figure 1C). Assuming that the MLL partner protein is required for leukemogenesis, the region downstream of the SH3 domain of AF3p21 may have functionally important domain(s). AF3p21 has a bipartite NLS downstream of the SH3 domain and a proline-rich domain. Since the N-terminus of MLL protein retains an NLS,13 the bipartite NLS of AF3p21 may not be required for nuclear localization of the MLL-AF3p21 fusion protein. Proline-rich domain is found in a number of SH3-binding proteins, and this region is responsible for physical interaction between SH3 proteins and their ligands.14 AF3p21 contains 1 class I SH3-binding motif (RXXPXXP) at aa 176-182 and 2 class II motifs (XPPXPXR) at aa 170-176 and aa 242-249. These domain structures suggest that AF3p21 might interact with other proteins in the nucleus. However, the precise intracellular localization and identification of interacting proteins (if any) of AF3p21 await further study. Chromosomal abnormalities involving 3p21 without involvement of the 11q23 region have been reported in t-AML and t-MDS.15 Moreover, the short arm of chromosome 3 is thought to contain multiple tumor suppressor genes because 1 copy of this chromosomal arm frequently is missing in carcinomas that have arisen in a variety of tissues, and region 3p21.3 is frequently deleted in lung cancers.16 It will be interesting to examine whether rearrangements of the AF3p21 gene occur in those malignancies.     Footnotes Submitted August 30, 1999; accepted September 30, 1999. Supported by grants-in-aid for Scientific Research and for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture, Japan, and by a grant for Cancer Research from the Hyogo Health Foundation, Kobe, Japan. Reprints: Kimihiko Sano, Department of Pediatrics, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan; e-mail: sanoped{at}kobe-u.ac.jp. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.     References Top Abstract Introduction Results Discussion References 1. Tkachuk DC, Kohler S, Cleary ML. Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias. Cell. 1992;71:691-700[Medline] [Order article via Infotrieve]. 2. Gu Y, Nakamura H, Alder H, et al. The t(4;11) chromosome translocation of human acute leukemias fuses the ALL-1 gene, related to Drosophila trithorax, to the AF-4 gene. Cell. 1992;71:701-708[Medline] [Order article via Infotrieve]. 3. Djabali M, Selleri L, Parry P, Bower M, Young BD, Evans GA. A trithorax-like gene is interrupted by chromosome 11q23 translocations in acute leukaemias. Nat Genet. 1992;2:113-118[Medline] [Order article via Infotrieve]. 4. Felix CA. Secondary leukemias induced by topoisomerase-targeted drugs. Biochim Biophys Acta. 1998;1400:233-255[Medline] [Order article via Infotrieve]. 5. Hess JL, Yu BD, Li B, Hanson R, Korsmeyer SJ. Defects in yolk sac hematopoiesis in Mll-null embryos. Blood. 1997;90:1799-1806[Abstract/Free Full Text]. 6. Fidanza V, Melotti P, Yano T, et al. Double knockout of the ALL-1 gene blocks hematopoietic differentiation in vitro. Cancer Res. 1996;56:1179-1183[Abstract/Free Full Text]. 7. Corral J, Lavenir I, Impey H, et al. An Mll-AF9 fusion gene made by homologous recombination causes acute leukemia in chimeric mice: a method to create fusion oncogenes. Cell. 1996;85:853-861[Medline] [Order article via Infotrieve]. 8. Lavau C, Szilvassy SJ, Slany R, Cleary ML. Immortalization and leukemic transformation of a myelomonocytic precursor by retrovirally transduced HRX-ENL. EMBO J. 1997;16:4226-4237[Medline] [Order article via Infotrieve]. 9. So CW, Cardas C, Liu M-M, et al. EEN encode for a member of a new family of proteins containing an Src homology 3 domain and is the third gene located on chromosome 19p13 that fuses to MLL in human leukemia. Proc Natl Acad Sci U S A. 1997;94:2563-2568[Abstract/Free Full Text]. 10. Taki T, Shibuya N, Taniwaki M, et al. ABI-1, a human homolog to mouse Abl-interactor 1, fuses the MLL gene in acute myeloid leukemia with t(10;11)(p11.2;q23). Blood. 1998;92:1125-1130[Abstract/Free Full Text]. 11. Sparks AB, Hoffman NG, McConnell SJ, Fowlkes DM, Kay BK. Cloning of ligand targets: systematic isolation of SH3 domain-containing proteins. Nat Biotechnol. 1996;14:741-744[Medline] [Order article via Infotrieve]. 12. Shi Y, Alin K, Goff SP. Abl-interactor-1, a novel SH3 protein binding to the carboxy-terminal portion of the Abl protein, suppresses v-abl transforming activity. Genes Dev. 1995;9:25832597. 13. Joh T, Kagami Y, Yamamoto K, et al. Identification of MLL and chimeric MLL gene products involved in 11q23 translocation and possible mechanisms of leukemogenesis by MLL truncation. Oncogene. 1996;13:1945-1953[Medline] [Order article via Infotrieve]. 14. Pawson T. Protein modules and signalling networks. Nature. 1995;373:573-580[Medline] [Order article via Infotrieve]. 15. Shi G, Weh HJ, Martensen S, Seeger D, Hossfeld DK. 3p21 is a recurrent treatment-related breakpoint in myelodysplastic syndrome and acute myeloid leukemia. Cytogenet Cell Genet. 1996;74:295-299[Medline] [Order article via Infotrieve]. 16. Daigo Y, Nishiwaki T, Kawasoe T, Tamari M, Tsuchiya E, Nakamura Y. Molecular cloning of a candidate tumor suppressor gene, DLC1, from chromosome 3p21.3. 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