Oncogene (2008) 27, 2249–2256 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ONCOGENOMICS Identification of the novel AML1 fusion partner , LAF4, a fusion partner of MLL, in childhood T-cell acute lymphoblastic leukemia with t(2;21)(q11;q22) by bubble PCR method for cDNA

Y Chinen1,2, T Taki1, K Nishida2, D Shimizu2, T Okuda2, N Yoshida2, C Kobayashi3, K Koike3, M Tsuchida3, Y Hayashi4 and M Taniwaki1,2

1Department of Molecular Laboratory Medicine, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kamigyo-ku, Kyoto, Japan; 2Department of Molecular Hematology and Oncology, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kamigyo-ku, Kyoto, Japan; 3Department of Pediatrics, Ibaraki Children’s Hospital, Futabadai, Mito, Japan and 4Gunma Children’s Medical Center, Shimohakoda, Hokkitsu, Shibukawa, Gunma, Japan

The AML1 gene is frequently rearranged by chromosomal Introduction translocations in acute leukemia. We identified that the LAF4 gene on 2q11.2–12 was fused to the AML1 gene on A large number of leukemias have been found to be 21q22 in a pediatric patient having T-cell acute lympho- associated with specific chromosomal aberrations. Re- blastic leukemia (T-ALL) with t(2;21)(q11;q22) using the cent studies have demonstrated that several chromoso- bubble PCR method for cDNA. The genomic break points mal rearrangements and molecular abnormalities are were within intron 7 of AML1 and of LAF4, resulting in strongly associated with distinct clinical subgroups and the in-frame fusion of exon 7 of AML1 and exon 8 of can predict clinical features and therapeutic responses LAF4. The LAF4 gene is a member of the AF4/FMR2 (Rowley, 1999; Taki and Taniwaki, 2006). Some family and was previously identified as a fusion partner of have been associated with recurrent rearrangements and MLL in B-precursor ALL with t(2;11)(q11;q23), although have many fusion partner genes, such as MLL at 11q23, AML1-LAF4 was in T-ALL. LAF4 is the first gene fused TEL (ETV6) at 12p13 and NUP98 at 11p15; AML1 with both AML1 and MLL in acute leukemia. Almost all (RUNX1, CBFA2) at 21q22 is one of the most frequent AML1 translocations except for TEL-AML1 are asso- targets of these chromosomal rearrangements in both ciated with myeloid leukemia; however, AML1-LAF4 was acute lymphoblastic leukemia (ALL) and acute myeloid associated with T-ALL as well as AML1-FGA7 in leukemia (AML) (Miyoshi et al., 1991; Hayashi, 2000; t(4;21)(q28;q22). These findings provide new insight into Kurokawa and Hirai, 2003). To date, a number of in- the common mechanism of AML1 and MLL fusion frame fusion partners of AML1 have been cloned: in the pathogenesis of ALL. Furthermore, we YTHDF2 at 1p35 (Nguyen et al., 2006), ZNF687 at successfully applied bubble PCR to clone the novel AML1- 1q21.2 (Nguyen et al., 2006), MDS1/EVI1 at 3q26 LAF4 fusion transcript. Bubble PCR is a powerful tool for (Mitani et al., 1994), FGA7 at 4q28 (Mikhail et al., detecting unknown fusion transcripts as well as genomic 2004), SH3D19 at 4q31.3 (Nguyen et al., 2006), USP42 fusion points. at 7p22 (Paulsson et al., 2006), MTG8 (ETO, CBFA2T1) Oncogene (2008) 27, 2249–2256; doi:10.1038/sj.onc.1210857; at 8q22 (Erickson et al., 1992; Miyoshi et al., 1993), published online 29 October 2007 FOG2 at 8q23 (Chan et al., 2005), TRPS1 at 8q24 (Asou et al., 2007), TEL (ETV6) at 12p13 (Golub et al., 1995), Keywords: AML1/RUNX1; LAF4; T-cell acute lympho- MTG16 at 16q24 (Gamou et al., 1998) and PRDX4 at blastic leukemia; MLL Xp22 (Zhang et al., 2004). Most AML1 translocations, except for TEL-AML1, are associated with AML, involving the N-terminus Runt domain and lacking ONCOGENOMICS the C-terminus transactivation domain (Kurokawa and Hirai, 2003). AML1 fusion proteins are associated with leukemogenesis by dominantly interfering with normal AML1-mediated transcription and acting as a transcrip- tional repressor (Okuda et al., 1998; Wang et al., 1998). Clinically, patients with AML harboring t(8;21) in both Correspondence: Dr T Taki, Department of Molecular Laboratory children and adults show a high rate of complete Medicine, Kyoto Prefectural University of Medicine Graduate remission, and its prognosis is considered better than School of Medical Science, 465 Kajii-cho Kawaramachi-Hirokoji, that of patients with a normal karyotype or other Kamigyo-ku, Kyoto 602-8566, Japan. chromosomal aberrations (Grimwade et al., 1998). E-mail: [email protected] Received 4 May 2007; revised 13 September 2007; accepted 17 September In the present study, we analysed pediatric T-ALL 2007; published online 29 October 2007 with t(2;21)(q11;q22) and identified the LAF4 gene, AML1-LAF4 fusion gene in childhood T-ALL Y Chinen et al 2250 which is one of the fusion partners of MLL, as a novel þ der(2)t(2;21)(q11.2;q22), del(5)(p15.1), del(9)(q22), del(9) fusion partner of the AML1 gene. (p13), der(21)t(2;21)(q11.2;q22) (Supplementary Figure S1). Since AML1 is located at 21q22, we inferred that AML1 was rearranged in this case. Fluorescence in situ hybridiza- Results tion analysis using AML1-specific BAC (bacterial artificial ) clones showed split signals of Case report AML1 on two der(2)t(2;21)(q11.2;q22) and der(21)t(2;21) A 6-year-old boy with a high leukocyte count (q11.2;q22) (Figure 1a). (64 700 mlÀ1), containing 84% blasts in peripheral blood To isolate fusion transcripts of AML1, we performed the and with a mediastinal mass, was diagnosed as having bubble PCR method for cDNA (Figure 2) and obtained T-ALL. A bone marrow smear was hypercellular with various-sized products (Figure 3a). Four different-sized 69% blasts and negative for myeloperoxidase. The products were sequenced and two products contained leukemic cells, after gating of CD45-positive cells, were AML1 sequences fused to unknown sequences. Basic local positive for CD5 (90.7%), CD7 (90.7%), CD58 (69.9%) alignment search tool (BLAST) search revealed that the and cytoplasmic CD3 (92.8%), and negative for HLA- unknown sequences were part of the LAF4 gene and both DR, IgG, IgM, Igk,Igl, CD8, CD13, CD14, CD19, products had the same in-frame junctions (Figure 3b). CD20 and CD33. He was treated on the Tokyo LAF4 was located on chromosome 2q11.2–12, which was Children’s Cancer Study Group (TCCSG) L04-16 compatible with the result of spectral karyotyping analysis. extremely high-risk (HEX) protocol, including stem cell We next performed reverse transcription-PCR to confirm transplantation, because the response to initial 7-day AML1-LAF4 fusion transcripts, and obtained three prednisolone (60 mg mÀ2) monotherapy was poor. He different-sized AML1-LAF4 fusion products, including achieved complete remission after the induction phase. only one in-frame product (Figures 3c and d); however, After the early consolidation phase and two courses of reciprocal LAF4-AML1 fusion transcripts were not the consolidation phase, he received allogeneic bone generated (Figure 3c). Type 2 transcript is an out-of-frame marrow transplantation from an unrelated HLA- fusion and generated premature termination in exon 9 of matched donor 4 months after diagnosis. He has been LAF4 (Figure 3d). On the other hand, type 3 transcript is in complete remission for 17 months. an in-frame fusion of exon 7 of AML1 and exon 8 of The patient’s leukemic cells at diagnosis were LAF4, the same as the type 1 transcript; however, the type analysed after written informed consent was obtained 3 transcript contained an 85-bp intronic sequence between from his parents, and the ethics committee of Kyoto exons 9 and 10 of LAF4, which might be due to splicing Prefectural University of Medicine approved this study. error, and appeared as a premature termination codon within the intronic sequences (Figure 3d). AML1-LAF4 fusions were also confirmed by fluorescence in situ Identification of the AML1-LAF4 fusion transcript hybridization analysis (Figure 1b). Cytogenetic analysis of the leukemic cells of the patient using routine G-banding revealed 47, XY, add(1)(p36), þ der(2)t(2;21)(q13;q22), t(2;21)(q13;q22), À9, À9, þ mar1, Detection of AML1-LAF4 genomic junctions þ mar2, and spectral karyotyping (SKY) analysis revealed Southern blot analysis using a cDNA probe within exon 47, XY, der(1)t(1;17)(p36.1;q23), der(2)t(2;21)(q11.2;q22), 7ofAML1 detected a rearranged band derived from an

Figure 1 Fluorescence in situ hybridization analysis of the leukemic metaphase. (a) Both RP11-272A3 (green, 30 side of AML1) and RP11-994N6 (red, 50 side of AML1) were hybridized to normal chromosome 21 (arrowhead), RP11-272A3 to der(21)t(2;21)(q11.2;q22) (arrow, green signal) and RP11-994N6 to two der(2)t(2;21)(q11.2;q22) chromosomes (arrows, red signal). (b) Two fusion signals of RP11-994N6 (50 of AML1, red signals) and RP11-527J8 (30 of LAF4, green signals) were detected on two der(2)t(2;21)(q11.2;q22) chromosomes (arrows).

Oncogene AML1-LAF4 fusion gene in childhood T-ALL Y Chinen et al 2251

Figure 2 Outline of bubble PCR for cDNA. Bubble PCR primers (NVAMP-1 and NVAMP-2) can only anneal with one complementary sequence for bubble oligo synthesized with AML1 primer, but not bubble oligo itself; therefore, this single-stranded bubble provides the specificity of the reaction.

Figure 3 Identification of AML1-LAF4 fusion transcript. (a) Bubble PCR products by nested PCR using AML1-5S and NVAMP1 for first PCR, and AML1-E6S and NVAMP2 for second PCR (lane 1). M, size marker. (b) Sequence analysis of AML1-LAF4 fusion transcript. The single letter amino-acid sequences surrounding the fusion point are shown at the bottom of the figure. (c) Detection of AML1-LAF4 fusion transcripts by reverse transcription-PCR. Primers were AML1-PR7 and LAF4-11AS (lanes 1 and 3), AML1-PR8 and LAF4-PR5 (lanes 2 and 4), and b-actin, respectively. Lanes 1, 3 and 5, patient’s leukemic cells; lanes 2, 4 and 6, normal peripheral lymphocytes. (d) Three fusion transcripts of AML1-LAF4 are schematically depicted. Gray/dotted boxes denote predicted AML1 exons and white boxes represent predicted LAF4 exons. Type 3 contains the LAF4 intron 9 splicing donor site. AML1-PR7 and LAF4- 11AS indicate the primers used for reverse transcription-PCR. Asterisk shows the termination codon. approximately 11 kb BglII germline fragment on chro- products using primers AML1-GNM8-2S and mosome 21 (data not shown). To isolate the fusion point NVAMP2 (Figure 4a). Sequence analysis of the of chromosomes 2 and 21, we next performed bubble subcloned PCR product revealed the genomic junction PCR on genomic DNA and detected nested PCR of 50-AML1-LAF4-30 (Figures 4c and d), and the result

Oncogene AML1-LAF4 fusion gene in childhood T-ALL Y Chinen et al 2252

Figure 4 Cloning of the genomic junction of AML1 and LAF4.(a) Bubble PCR for genomic DNA. N, normal lymphocytes; P, patient’s leukemic cells. (b) Detection of the genomic fusion point of AML1-LAF4 by PCR. Primers were AML1-GNM8-4S and LAF4-GNM11-2AS (lanes 1 and 3), and LAF4-GNM11-2S and AML1-GNM8-2AS (lanes 2 and 4). Lanes 1 and 2, patient’s leukemic cells; lanes 3 and 4, normal peripheral lymphocytes. M, size marker. (c) Sequences of breakpoints in the patient’s leukemic cells. (d) Physical map of the breakpoint regions. Open vertical boxes represent defined exons in each gene. Horizontal arrows show the primers used. Restriction sites are indicated by capital letters: G, BglII; H, HindIII. AML1c1 indicates the position of the cDNA probes for Southern blot analysis. A vertical arrow shows AML1-USP42 breakpoint.

was confirmed by PCR analysis using primers AML1- Discussion GNM8-4S and LAF4-GNM11-2AS (Figure 4b); however, no 50-LAF4-AML1-30 product was generated, In this study, we identified that LAF4 was fused to suggesting interstitial deletion near genomic break AML1 in pediatric T-ALL with t(2;21)(q11;q22). Other points (Figure 4b). These sequences near the break regions with chromosomal aberrations in this patient points did not contain any lymphoid heptamer/nonamer were not considered to be associated with recurrent sequences, Alu sequences or consensus topoisomerase II cytogenetic changes involving T-ALL, except for the cleavage sites. deletion of the short arm of chromosome 9. Spectral

Oncogene AML1-LAF4 fusion gene in childhood T-ALL Y Chinen et al 2253 karyotyping analysis detected del(9)(p13), and addi- 1997; Taki et al., 1997; Chaffanet et al., 2000). tional analysis of genome array (Human Mapping 50 K Comparison of the structure and function between Hind Array, Affymetrix, Tokyo, Japan) revealed homo- AML1-LAF4 and MLL-LAF4 will facilitate our under- zygous deletion of 4.5 Mb within the 9p21 region, standing of the molecular mechanisms underlying including the CDKN2A/p16/p14 locus (data not shown), AML1- and MLL-related leukemia. which is frequently deleted in T-ALL (Ohnishi et al., The only AML1 fusion partners in T-ALL are LAF4 1995). and FGA7. It is not known how FGA7 is associated with Although the patient showed a complex chromosomal T-ALL leukemogenesis, because FGA7 does not show any abnormality, t(2;21)(q11;q22) can form regular head-to- significant to any known tail fusion transcripts of both AML1 and LAF4, because motifs and/or domains (Mikhail et al., 2004). Both patients the transcription direction of AML1 and LAF4 is with AML1-LAF4 and MLL-LAF4 fusions were diag- telomere to centromere. Furthermore, fluorescence nosed as having ALL, but they have different lymphoid in situ hybridization analysis revealed two der(2)t(2;21) lineages. MLL-LAF4 is associated with B-lineage ALL; (q11.2;q22) creating 50-AML1-LAF4-30, suggesting that however, AML1-LAF4 generatesT-ALL.Ourprevious 50-AML1-LAF4-30 is critical for leukemogenesis. study showed that LAF4 was expressed not only in B- LAF4 was previously reported to be a fusion partner of lineage ALL but also in T-lineage ALL cell lines (Hiwatari MLL in pediatric B-precursor ALL with t(2;21)(q11;q23) et al., 2003). LAF4 showed strong sequence similarity to (von Bergh et al., 2002; Bruch et al., 2003; Hiwatari et al., AF4 (Ma and Staudt, 1996), which has a role in the 2003). LAF4 is the first gene fused to both AML1 and differentiation of both B and T cells in mice (Isnard et al., MLL, and both AML1-LAF4 and MLL-LAF4 contained 2000). Furthermore, it was reported that AML1 also plays the same domains of LAF4 (Figure 5). During the an important role in T- and B-cell differentiation, because preparation of this manuscript, we found another AML1-deficient bone marrow increased defective T- and pediatric T-ALL patient with AML1-LAF4 reported in B-lymphocyte development (Ichikawa et al., 2004). These the Meeting Abstract (Abe et al., Blood (ASH Annual findings support that both AML1 and LAF4 are Meeting Abstracts) 2006; 108: 4276), suggesting that associated with T-ALL, respectively. Further functional t(2;21)(q11;q23) is a recurrent cytogenetic abnormality analysis of the AML1-LAF4 fusion gene will provide new and that the AML1-LAF4 fusion gene is associated with insights into the leukemogenesis of AML1-related T-ALL. the T-ALL phenotype. Both putative fusion proteins of Recently, it has been reported that C-terminal truncated AML1-LAF4 observed in two patients contained the AML1-related fusion proteins play critical roles in Runt domain of AML1, and the transactivation domain, leukemogenesis (Yan et al., 2004, Agerstam et al., 2007), nuclear localization sequence and C-terminal homology suggesting that the two additional types of fusion domain of LAF4, although the fused exon of LAF4 transcripts observed in our patient (types 2 and 3 in differed in the two cases. Several studies have reported Figures 3d and 5) have an additional function in that the fusion partners of MLL fused with different leukemogenesis other than that of the entire AML1- genes such as MLL-AF10 and CALM-AF10, MLL-CBP LAF4 fusion protein. and MOZ-CBP or MLL-p300 and MOZ-p300 (Ida et al., In this study, we first applied the panhandle PCR method, which is usually used for cloning the fusion partners of MLL or NUP98 (Megonigal et al., 2000; Taketani et al., 2002); however, no fusion transcripts could be obtained. Therefore, we searched for another method to clone the fusion transcripts and adapted the bubble PCR method for cDNA cloning. To date, bubble PCR has been performed for cloning unknown genomic fusion points but not fusion cDNAs (Zhang et al., 1995). Using double-stranded cDNA, we could apply the bubble PCR method for cloning fusion cDNA with fewer nonspecific products. The bubble PCR primer can only prime DNA synthesis after a first-strand cDNA has been generated by an AML1-specific primer because of the bubble-tag with an internal non-complementary region (Zhang et al., 1995). Although bubble PCR for genomic DNA generated one or two amplification products (Smith, 1992), bubble PCR for cDNA gener- ated a complex set of amplification products that appeared as a smear by SYBR green staining, suggesting Figure 5 Schematic representation of putative AML1, LAF4 and that a random hexamer generated various double- AML1-LAF4 fusion proteins. Putative MLL-LAF4 fusion protein is stranded cDNA containing the AML1 sequence. This also indicated for comparison. Arrows, break points or fusion points; means that various fusion points can be estimated, even AD, transactivation domain; AT, AT hooks; CHD, C-terminal homology domain; DNA, methyltransferase homology region; RD, if after bubble oligo ligation was generated. Further- 0 0 RUNT domain; MT, DNA methyltransferase homology region; more, bubble PCR for cDNA could amplify in both 5 –3 NLS, nuclear localization sequence. and 30–50 directions of the gene or transcript, and easily

Oncogene AML1-LAF4 fusion gene in childhood T-ALL Y Chinen et al 2254 Table 1 Comparison between bubble PCR and panhandle PCR described previously (Taniwaki et al., 1994). Fusion of AML1 and LAF4 was analysed with the patient’s leukemic cells using Characteristics Bubble PCR Panhandle RP11-994N6 (50 of AML1) and RP11-527J8 (30 of LAF4). PCR

Available orientation of fusion 50–30,30–50 Only 50–30 Bubble PCR for cDNA transcript We modified the original bubble PCR method to apply for AML1-specific random hexamera Not necessary Necessary cDNA cloning (Figure 2; Supplementary Figure S2) (Smith, Self-annealing Not necessary Necessary 1992; Zhang et al., 1995). Number of required polymerase 24Poly(A) þ RNA was extracted from the patient’s leukemic reaction Number of final products Many (smear) A few cells using a QuickPrep Micro mRNA Purification Kit (GE Nonspecific product Few Few Healthcare, Buckinghamshire, UK). Two hundred nanograms Number of extra sequences other 50–60 bp >100 bp of poly(A) þ RNA was reverse transcribed to cDNA in a total than targeted sequences in cloned volume of 33 ml with random hexanucleotide using the Ready- product To-Go You-Prime First-Strand Beads (GE Healthcare). Search for other targeted exons Easy Hardb Double-stranded cDNAs were synthesized from 10 mlof single-stranded cDNA with a phosphorylated random hex- a30-mers AML1-specific oligonucleotide with random hexamer anucleotide, blunt ended with T4 DNA polymerase, digested (AML1-N). bNecessary to use another AML1-specific random with RsaI endonuclease and ligated with bubble oligo. RsaI, a 0 hexamer if the target exons are 5 region of the initial target. 4-bp blunt-ended cutter, was chosen to shorten the bubble oligo-ligated fragments, so that almost all bubble oligo-ligated fragments would be easy to clone by standard PCR reaction. handle any exons fused to unknown partners for This suggests that poor-quality samples are also suited to this amplification. Once-ligated cDNAs are also available method, although it is unsuitable for cloning long products. for cloning any genes, other than AML1, as the target. The sequences of the primers used are listed in Supplemen- We demonstrated the efficiency and specificity of bubble tary Table S1 and their positions in the AML1 gene are shown PCR for cDNA (Table 1 and Supplementary Figure S2). in Supplementary Figure S2. Nested PCR was performed using To date, a great number of fusion genes associated primers NVAMP-1 (bubble oligo) and AML1-5S (exon 5) for with chromosomal translocations have been cloned, first round PCR, and NVAMP-2 (bubble oligo) and AML1- although these fusion genes are found as a minor part E6S (exon 6) for nested PCR. NVAMP1 and NVAMP2 can only anneal to the newly synthesized unique sequence of the of various malignancies. Recently, high frequencies of bubble oligo by AML1-5S. mutations in NOTCH1 in T-ALL (James et al., 2005), We used poly(A) þ RNA in bubble PCR for cDNA with the NPM in AML with normal karyotype (Weng et al., expectation that this approach could amplify fewer transcripts; 2004) and JAK2 in myeloproliferative disorders (poly- however, total RNA is also suitable for this method. cythemia vera, essential thrombocythemia and idiopathic myelofibrosis) (James et al., 2005) have been reported, Bubble PCR for genomic DNA and these mutations are considered to be a good target Bubble PCR for genomic DNA was performed as described for therapy. These genes were first identified as previously (Smith, 1992; Zhang et al., 1995). Primers were as associated with chromosomal translocations in a small follows: NVAMP-1 and AML1-GNM8S for first round PCR, subset of specific phenotypes of hematologic malignan- and NVAMP-2 and AML1-GNM8-2S for second round PCR cies (Ellisen et al., 1991; Morris et al., 1994; Lacronique (Supplementary Table S1). et al., 1997). These findings suggest that continuing attempts to identify genes associated with chromosomal Reverse transcription–PCR and genomic PCR analyses translocations can be expected to provide further insights Reverse transcription–PCR and genomic PCR analyses were performed as described previously. After 35 rounds of PCR into the significance of various gene alterations in cancer (30 s at 94 1C, 30 s at 55 1C, 1 min at 72 1C), 5ml of PCR product and the development of novel-targeted therapies (Taki were electrophoresed in a 3% agarose gel. Primers were and Taniwaki, 2006). The bubble PCR method for as follows: AML1-PR7 and LAF4-11AS, and AML1-PR8 cDNA will contribute to identifying numerous novel and LAF4-PR5 for reverse transcription-PCR; and AML1- translocation partners more easily and further functional GNM8-4S and LAF4-GNM11-2AS, and LAF4-GNM11-2S analysis of chimeric transcripts. and AML1-GNM8-2AS for genomic PCR (Supple- mentary Table S1).

Nucleotide sequencing Materials and methods Nucleotide sequences of PCR products and, if necessary, subcloned PCR products were analysed as described pre- Spectral karyotyping analysis viously (Hiwatari et al., 2003). Spectral karyotyping analysis was performed with a Sky- Painting kit (Applied Spectral Imaging, Migdal Ha’Emek, Israel). Signal detection was performed according to the Southern blot analysis manufacturer’s instructions. High-molecular-weight DNA was extracted from the patient’s leukemic cells by proteinase K digestion and phenol/chloroform extraction. DNA (10 mg) was digested with BglII, subjected to Fluorescence in situ hybridization analysis electrophoresis on 0.7% agarose gel and transferred to a nylon Fluorescence in situ hybridization analysis of the patient’s membrane. Blots were hybridized to probes that were labeled by leukemic cells using AML1-specific BAC clones (RP11-272A3, the Dig-labeled PCR method according to the manufacturer’s 30 of AML1 and RP11-994N6, 50 of AML1) was carried out as instructions (Roche Applied Science, Tokyo, Japan). Probes

Oncogene AML1-LAF4 fusion gene in childhood T-ALL Y Chinen et al 2255 were 112 bp AML1 cDNA fragments (AML1c1, nucleotides Acknowledgements 1233–1344; GenBank accession no. NM_001754). We express our appreciation for the outstanding technical assistance of Kozue Sugimoto, Minako Goto and Kayoko Abbreviations Kurita. This work was supported by a grant-in-aid for Scientific Research (B) from the Ministry of Education, AML, acute myeloid leukemia; ALL, acute lymphoblastic Culture, Sports, Science and Technology of Japan, and the leukemia. Takeda Science Foundation.

References

Agerstam H, Lilljebjorn H, Lassen C, Swedin A, Richter J, James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C Vandenberghe P et al. (2007). Fusion gene-mediated truncation of et al. (2005). A unique clonal JAK2 mutation leading to constitutive RUNX1 as a potential mechanism underlying disease progression in signalling causes polycythaemia vera. Nature 434: 1144–1148. the 8p11 myeloproliferative syndrome. Genes Chromosomes Cancer Kurokawa M, Hirai H. (2003). Role of AML1/Runx1 in the 46: 635–643. pathogenesis of hematological malignancies. Cancer Sci 94: 841–846. Asou N, Yanagida M, Huang L, Yamamoto M, Shigesada K, Mitsuya Lacronique V, Boureux A, Valle VD, Poirel H, Quang CT, H et al. (2007). Concurrent transcriptional deregulation of AML1/ Mauchauffe M et al. (1997). A TEL-JAK2 fusion protein RUNX1 and GATA factors by the AML1-TRPS1 chimeric gene in with constitutive kinase activity in human leukemia. Science 278: t(8;21)(q24;q22) acute myeloid leukemia. Blood 109: 4023–4027. 1309–1312. Bruch J, Wilda M, Teigler-Schlegel A, Harbott J, Borkhardt A, Ma C, Staudt LM. (1996). LAF-4 encodes a lymphoid nuclear protein Metzler M. (2003). Occurrence of an MLL/LAF4 fusion gene caused with transactivation potential that is homologous to AF-4, the gene by the insertion ins(11;2)(q23;q11.2q11.2) in an infant with acute fused to MLL in t(4;11) leukemias. Blood 87: 734–745. lymphoblastic leukemia. Genes Chromosomes Cancer 37: 106–109. Megonigal MD, Rappaport EF, Wilson RB, Jones DH, Whitlock JA, Chaffanet M, Gressin L, Preudhomme C, Soenen-Cornu V, Birnbaum Ortega JA et al. (2000). Panhandle PCR for cDNA: a rapid method D, Pebusque MJ. (2000). MOZ is fused to p300 in an acute for isolation of MLL fusion transcripts involving unknown partner monocytic leukemia with t(8;22). Genes Chromosomes Cancer 28: genes. Proc Natl Acad Sci USA 97: 9597–9602. 138–144. Mikhail FM, Coignet L, Hatem N, Mourad ZI, Farawela HM, Chan EM, Comer EM, Brown FC, Richkind KE, Holmes ML, Chong El Kaffash DM et al. (2004). FGA7, is fused to RUNX1/AML1 in BH et al. (2005). AML1-FOG2 fusion protein in myelodysplasia. a t(4;21)(q28;q22) in a patient with T-cell acute lymphoblastic Blood 105: 4523–4526. leukemia. Genes Chromosomes Cancer 39: 110–118. Ellisen LW, Bird J, West DC, Soreng AL, Reynolds TC, Smith SD Mitani K, Ogawa S, Tanaka T, Miyoshi H, Kurokawa M, Mano H et al. (1991). TAN-1, the human homolog of the Drosophila Notch et al. (1994). Generation of the AML1-EVI-1 fusion gene in the gene, is broken by chromosomal translocations in T lymphoblastic t(3;21)(q26;q22) causes blastic crisis in chronic myelocytic leukemia. neoplasms. Cell 66: 649–661. EMBO J 13: 504–510. Erickson P, Gao J, Chang KS, Look T, Whisenant E, Raimondi S Miyoshi H, Kozu T, Shimizu K, Enomoto K, Maseki N, Kaneko Y et al. (1992). Identification of breakpoints in t(8;21) acute et al. (1993). The t(8;21) translocation in acute myeloid leukemia myelogenous leukemia and isolation of a fusion transcript, AML1/ results in production of an AML1-MTG8 fusion transcript. EMBO ETO, with similarity to Drosophila segmentation gene, runt. Blood J 12: 2715–2721. 80: 1825–1831. Miyoshi H, Shimizu K, Kozu T, Maseki N, Kaneko Y, Ohki M. Gamou T, Kitamura E, Hosoda F, Shimizu K, Shinohara K, Hayashi (1991). t(8;21) breakpoints on chromosome 21 in acute myeloid Y et al. (1998). The partner gene of AML1 in t(16;21) myeloid leukemia are clustered within a limited region of a single gene, malignancies is a novel member of the MTG8 (ETO) family. Blood AML1. Proc Natl Acad Sci USA 88: 10431–10434. 91: 4028–4037. Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DN, Golub TR, Barker GF, Bohlander SK, Hiebert SW, Ward DC, Bray- Saltman DL et al. (1994). Fusion of a kinase gene, ALK,toa Ward P et al. (1995). Fusion of the TEL gene on 12p13 to the AML1 nucleolar protein gene, NPM, in non-Hodgkin’s lymphoma. Science gene on 21q22 in acute lymphoblastic leukemia. Proc Natl Acad Sci 263: 1281–1284. USA 92: 4917–4921. Nguyen TT, Ma LN, Slovak ML, Bangs CD, Cherry AM, Arber DA. Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison (2006). Identification of novel Runx1 (AML1) translocation partner G et al. (1998). The importance of diagnostic cytogenetics on genes SH3D19, YTHDf2, and ZNF687 in acute myeloid leukemia. outcome in AML: analysis of 1,612 patients entered into the MRC Genes Chromosomes Cancer 45: 918–932. AML 10 trial. The Medical Research Council Adult and Children’s Ohnishi H, Kawamura M, Ida K, Sheng XM, Hanada R, Nobori T Leukaemia Working Parties. Blood 92: 2322–2333. et al. (1995). Homozygous deletions of p16/MTS1 gene are frequent Hayashi Y. (2000). The molecular genetics of recurring chromosome but mutations are infrequent in childhood T-cell acute lympho- abnormalities in acute myeloid leukemia. Semin Hematol 37: 368–380. blastic leukemia. Blood 86: 1269–1275. Hiwatari M, Taki T, Taketani T, Taniwaki M, Sugita K, Okuya M Okuda T, Cai Z, Yang S, Lenny N, Lyu CJ, van Deursen JM et al. et al. (2003). Fusion of an AF4-related gene, LAF4,toMLL in (1998). Expression of a knocked-in AML1-ETO leukemia gene childhood acute lymphoblastic leukemia with t(2;11)(q11;q23). inhibits the establishment of normal definitive hematopoiesis and Oncogene 22: 2851–2855. directly generates dysplastic hematopoietic progenitors. Blood 91: Ichikawa M, Asai T, Saito T, Seo S, Yamazaki I, Yamagata T et al. 3134–3143. (2004). AML-1 is required for megakaryocytic maturation and Paulsson K, Bekassy AN, Olofsson T, Mitelman F, Johansson B, lymphocytic differentiation, but not for maintenance of hemato- Panagopoulos I. (2006). A novel and cytogenetically cryptic poietic stem cells in adult hematopoiesis. Nat Med 10: 299–304. t(7;21)(p22;q22) in acute myeloid leukemia results in fusion of Ida K, Kitabayashi I, Taki T, Taniwaki M, Noro K, Yamamoto M RUNX1 with the ubiquitin-specific protease gene USP42. Leukemia et al. (1997). Adenoviral E1A-associated protein p300 is involved in 20: 224–229. acute myeloid leukemia with t(11;22)(q23;q13). Blood 90: 4699–4704. Rowley JD. (1999). The role of chromosome translocations in Isnard P, Core N, Naquet P, Djabali M. (2000). Altered lymphoid leukemogenesis. Semin Hematol 36: 59–72. development in mice deficient for the mAF4 proto-oncogene. Blood Smith DR. (1992). Ligation-mediated PCR of restriction fragments 96: 705–710. from large DNA molecules. PCR Methods Appl 2: 21–27.

Oncogene AML1-LAF4 fusion gene in childhood T-ALL Y Chinen et al 2256 Taketani T, Taki T, Shibuya N, Ito E, Kitazawa J, Terui K et al. Wang J, Hoshino T, Redner RL, Kajigaya S, Liu JM. (1998). ETO, (2002). The HOXD11 gene is fused to the NUP98 gene in fusion partner in t(8;21) acute myeloid leukemia, represses acute myeloid leukemia with t(2;11)(q31;p15). Cancer Res 62: transcription by interaction with the human N-CoR/mSin3/HDAC1 33–37. complex. Proc Natl Acad Sci USA 95: 10860–10865. Taki T, Sako M, Tsuchida M, Hayashi Y. (1997). The Weng AP, Ferrando AA, Lee W, Lee W, Morris IV JP, Silverman LB t(11;16)(q23;p13) translocation in myelodysplastic syndrome fuses et al. (2004). Activating mutations of NOTCH1 in human T cell the MLL gene to the CBP gene. Blood 89: 3945–3950. acute lymphoblastic leukemia. Science 306: 269–271. Taki T, Taniwaki M. (2006). Chromosomal translocations in cancer Yan M, Burel SA, Peterson LF, Kanbe E, Iwasaki H, Boyapati A et al. and their relevance for therapy. Curr Opin Oncol 18: 62–68. (2004). Deletion of an AML1-ETO C-terminal NcoR/SMRT- Taniwaki M, Matsuda F, Jauch A, Nishida K, Takashima T, Tagawa S interacting region strongly induces leukemia development. Proc et al. (1994). Detection of 14q32 translocations in B-cell Natl Acad Sci USA 101: 17186–17191. malignancies by in situ hybridization with yeast artificial chromo- Zhang JG, Goldman JM, Cross NC. (1995). Characterization of some clones containing the human IgH gene locus. Blood 83: genomic BCR-ABL breakpoints in chronic myeloid leukemia by 2962–2969. PCR. Br J Haematol 90: 138–146. Von Bergh AR, Beverloo HB, Rombout P, van Wering ER, van Weel Zhang Y, Emmanuel N, Kamboj G, Chen J, Shurafa M, Van Dyke DL MH, Beverstock GC et al. (2002). LAF4,anAF4-related gene, is et al. (2004). PRDX4, a member of the peroxiredoxin family, is fused fused to MLL in infant acute lymphoblastic leukemia. Genes to AML1 (RUNX1) in an acute myeloid leukemia patient with a Chromosomes Cancer 37: 106–109. t(X;21)(p22;q22). Genes Chromosomes Cancer 40: 365–370.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene