cloning of the all.1 fusion partner, the af-6 gene ...primers contained cloning sites at their...

[CANCER RESEARCH 53, 5624-5628, December 1, 1993 I

Advances in Brief

Cloning of the ALL.1 Fusion Partner, the AF-6 Gene, Involved in Acute Myeloid

Leukemias with the t(6;ll) Chromosome Transiocation 1

17, Prasad , Y. Gu, H. Alder, T. N a k a m u r a , O. Canaani , H. Saito, K. Huebner , R. P. Gale, P. C. NoweU, K. K u r i y a m a , Y. Miyazaki , C. M. Croce, and E. Canaan i 2

Jefferson Cancer Institute, Jefferson Cancer Center and Department of Microbiology and Immunology, Jefferson Medical College of Thomas Jefferson University, Philadelphia~ Pennsylvania 19107lR. R, Y G., H. A., T. N., O. C., H. S., K. H., C. M. C., E. C.]; University of California Medical Center, Los Angeles, California 90024 [R. P. G.]; Un&ersity of Pennsyh, ania, Philadelphia, Pennsylvania 19104 [P. C. N.]; and Nagasaki University School of Medicine, Nagasaki 852, Japan [K. K., Y M.]

Abstract

Reciprocal chromosome translocations involving 11q23 are frequently associated with acute leukemias, with the t(4;ll) translocation predomi- nating among acute lymphoblastic leukemias, and the t(9;ll), t(ll;19) and t(6;ll) translocations most common among acute myeloid leukemias. In each of these translocations the ALL-1 gene, located at 11q23 and consti- tuting the human homologue of Drosophila trithorax, fuses to a specific gene on the partner chromosome to produce a chimeric protein. Here we report the cloning and the characterization of the partner gene from chromosome 6 (AF.6). AF-6 is expressed in a variety of cell types and encodes a protein of 1612 amino acids. The protein contains short stretches rich in prolines, charged amino acids, serines, or glutamines. In addition, the AF-6 protein contains the GLGF motif shared with several proteins of vertebrates and invertebrates thought to be involved in signal transduc. tion at special cell-cell junctions.

Introduction

The majority of infant acute leukemias and at least 5% of ALLs 3 and AMLs of older children and adults show abnormalities of chromosome band 11q23 (1, 2). In addition, 11q23 aberrations occur at very high frequency in secondary acute leukemias induced by treat- ment of malignancies with inhibitors of topoisomerase II (3, 4). Leu- kemias involving 11q23 abnormalities share unique clinical and bio- logical features such as massive cell burden, frequent mixed lineage with markers of both lymphoid and myeloid blasts, and a bad prog- nosis.

Recently we cloned (5) a DNA segment from chromosome 11 which is rearranged in most or all 11q23 abnormalities (6). We and others have subsequently cloned the gene spanning the breakpoint cluster region (7, 8). This gene was designated ALL-1 (5-7), M L L (9), or HRX (8, 10). ALL-1 shows strong homology to three regions with Drosophi la tr i thorax and therefore is thought to constitute the human homologue of the latter. ALL-l-encoded protein has two types of DNA binding motifs, zinc fingers and AT hooks; in addition it contains a domain shared with DNA methyltransferase and is presumably involved in recognition of hemimethylated and unmethylated DNA (11, 12).

11q23 chromosome translocations sever theALL-1 gene in a region containing exons 5-11 and result in fusion of the open reading frames of ALL-1 and the partner genes in phase. One protein fusion product will contain the NH2-terminal --1400 amino acids of ALL-1 including the AT hook motifs and the domain shared with DNA methyltrans-

ferase; the reciprocal protein product will include most or all of the zinc fingers. Cytogenetic analysis of complex 11q23 translocations indicated that the chromosome der( l l ) , but not the reciprocal deriva- tive, is the common feature of these abnormalities (13). This sug- gested that the fusion protein product containing the NH2 terminus of ALL-1 is the oncogene.

The partner genes in the t(4;l l) , t(9;l l) , and t ( l l ;19) were cloned and characterized by us or others (7, 8, 12, 14, 15) and were designated AF-4, AF-9, and ENL, respectively. AF-9 and ENL encode highly homologous proteins that vary completely from the polypeptide coded by the A F - 4 gene. The three proteins, however, share a nuclear targeting sequence and serine/proline-rich domains (14). In the present communication we describe the cloning and characterization of the partner gene involved in a fourth common translocation involving l lq23, the t(6; ll)(q27;q23).

Materials and Methods

Patients and Cells. The patient 01 was a 47-year-old female, diagnosed as AML(M4). Her karyotype was 46,XX, t(6;ll)(q27;q23) in 20 of 20 of bone marrow ceils analyzed. Patient Ed was a male diagnosed as AML(M5) with a karyotype of 46,XY, del(llq23). The celI lines used for RNA analysis included K562 and KC122 (erythroid and myeloid acute phase of chronic myeloid ~eukemia) (16, 17), B-t and MV4:ll [ALL with the t(4;11) abnormality (18, 19)], SKDHL (B-cell lymphoma) (20), T98G (glioblastoma) (21), and the 293 cell line derived from kidney (22).

Molecular Cloning. The rearranged genomic fragments of ALL-1 from patients 01 and Ed were cloned into the EMBL-3 phage vector (Promega) after partial digestion of the DNAs with the MboI enzyme and size selection. Phage libraries were screened using a 0.86-kilobase BamHI fragment derived from ALL-I cDNA and spanning exons 5-11. A normal genomic library was constructed in a similar way from normal WBC DNA. The cDNA library was constructed utilizing a kit from Pharmacia. Cytoplasmic polyadenylate-selected RNA was prepared from KC122 cells. For RT-PCR reactions, aliquots of 2 ~g of patients' RNAs were reverse transcribed utilizing the AF-6 oligo- nucleotide 5' ATC TGAAT-F CTC CGC TGA CAT GCA CTI" CAT AG 3'. The cDNA was amplified using the same AF-6 primer together with the ALL.1 primer 5' ATC TGA ATI" CTC CGC TGA CAT GCA c T r CAT AG 3'. (Both primers contained cloning sites at their 5' termini.) The amplified products were cloned into the SK plasmid vector and sequenced.

Sequencing. cDNAs and genomic DNAs were excised from the phage vectors and recloned into the SK plasmid vector. Sequencing was performed using the ABI automatic sequencer. The sequence was analyzed using the FASTA, TFASTA, and motifs programs.

Received 10/4/93; accepted 10/28/93. The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by grants from the National Cancer Institute (CA39860) and from the Falk Medical Research Trust; Genbank accession number is U02478.

z To whom requests for reprints should be addressed. 3 The abbreviations used are: ALL, acute lymphoblastic leukemia; AML, acute my-

eloid leukemia; cDNA, complementary DNA; RT-PCR, reverse transcription-polymerase chain reaction.

Results

A rearranged ALL-1 segment was cloned from the genomic DNA of leukemic cells of patient 01. Mapping of this segment indicated that it originated from the der(6) chromosome (Fig. 1A). Sequencing of the junction region (Fig. 1C) showed neither extra nucleotides nor hep- tamer-like signal at the junction point. Therefore, unlike two t (4; l l ) and one (9;11) translocation breakpoints that we previously studied

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A

CLONING OF AF-6 GENE

2kb

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Cen

B R G X R XXBH

Lql f f 5 6 7 8 91011

Tel

Chr.llq23 - - 1 2 k b

R H

II RVP0.5

H X R G X R XXBH

III I I I IIII

R H H X RXR H X HB X HX HR B

XR0.5 Xinf0.6

der (6)

Chr . 6q27

+el

a b

C

~T~TGTTTCTCTGCCATTTU~AC~TGTATTCTATTTTGT~TTATCcTTGAcTTCTATGTAGA~G~TTTCTTAAAATTAAGAAA Chr. i lq2 3 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I i i i i i i i i i i i i i i i i i i i i i i i i i i

TTCC TCATAGGAAATAAAATCTITTAAATTAGCTTGTTTAG CTTATCC~TTCTATGTAGATGGCAGTGGAATTTCTTAAAATTAAGAAA der (6) l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l i l l l l l l l l i l l l l TTC~TCA~ACK~'AAA~AAAATCTTTTAAATT~~L-~-A-~C~AAAACCCAACAAAACCATTG~A~TTTTAGTTAC~~~A~~ Chr. 6q2 7

Fig. 1. Oenomic analysis of the t(6;ll)(q27;q23) chromosome translocation. (A) Physical map of the t(6;ll) junction in patient 01, as well as of the corresponding regions from chromosomes 11 and 6. The RVP0.5 probe was used to isolate the corresponding normal DNA of 6q27. (B) Chromosome 6+specific probe XR0.5 detects DNA rearrangement in the bone marrow ceils from the patient (Ed), whose karyotype showed 11q23 deletion; high moleculer weight DNAs were digested with BamHI. (C) Sequence of the t(6;ll) breakpoint region in patient 01. Cen and Tel denote the direction of the centromeres and telomeres of the two chromosomes. Open vertical boxes rcpresent defined exons. Restriction sites: B, BamHI; 11, HindlII; G, BgllI; R, EcoRI; X, Xba]. kb, kilobase.

(23, 24), here the VDJ recombinase was probably not involved in the recombination process.

We next used a repeat-free EcoRV-PstI 0.5-kilobase fragment (RVP 0.5) as a probe to clone the corresponding region from normal DNA (Fig. IA, bottom). To examine whether this region of chromosome 6 constitutes a breakpoint cluster region, we probed genomic blots of some selected patients' DNAs with the 0.5-kilobase XbaI-EcoRI (XRO.5) radiolabeled fragment. While the DNA of another patient with AML and t(6;l l) showed only germ line configuration of this region, the DNA of the patient Ed with AML and the del(llq23) aberration contained a rearranged BamHl fragment of 12 kilobases (Fig. 1B). This indicated that the cloned DNA spanned a breakpoint cluster region and that a cytogenetic pattern of del(llq23) could correspond to a t(6;11) translocation.

The entire area of 30 kilobases cloned from 6q27 was searched for segments reacting with clones from a normal cDNA library. A 0.6- kilobase HinfI DNA reacted with the K12 cDNA clone (Fig. 14). By subsequent "walking" it was possible to clone overlapping cDNA clones which spanned the complete coding region of the gene. We named the latter AF-6 for ALL-1 fused gene from chromosome 6. AF-6 encodes a protein of 1612 amino acids. In cDNA clone K10 we find two additional amino acids, glutamic acid at position 101 and a lysine in position 139; both are probably due to alterations in splicing similar to those which we previously detected in ALL-1 (11, 14). To

directly demonstrate a fused transcript we performed RT-PCR reactions on RNAs from patients 01 and Ed using ALL-1 and AF-6 primers flanking the expected junction region. Products of the reactions were cloned, screened for hybridization to ALL-1 and AF-6 probes, and sequenced. The RT-PCR products of both patients showed identical chimeric ALL-1/AF-6 RNAs transcribed from the der(ll) chromosome (Fig. 2C). The two open reading frames were linked in phase.

The nucleotide and the amino acid sequences of AF-6 were exam- ined for motifs and homology to other genes. Throughout the protein, but in particular towards the COOH terminus of AF-6 there exist small domains rich in prolines, serines, acidic amino acids, or glutamines. AF-6 protein within residues 745-925 shows 23.2% identity over 181 amino acids with the COOH terminus of yeast myosin-1 isoform (25). The protein also shows high similarity, although low identity, (66% similarity plus identity) over amino acids 1000-1594 to amino acids 1400-1980 of the myosin heavy chain from Dictyostelium discoideum (26). In the latter protein this region is part of the tail domain which assumes, due to a high a-helical potential, a rod structuce. A striking homology was detected in the polypeptide spanning amino acids 997-1080. A series of amino acids in this domain are conserved (Fig. 3) in three other proteins: in the human tight junction protein ZO-1 (27); in the rat PSD-95 protein present in brain synapses (28); and in a tumor suppressor gene of Drosophila (dig) located at septate junctions (which are thought to be the invertebrate equivalent of tight

5625



AF-6 cDNA

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1 MSAGGRDEERRKLADI I HHWNANRLDLFE ISQPTEDLEFHGVMRFYFQDKAAGNFATKCI RVSS TATTQDVIETLAEKFRP DMRMLSSP KYSLYEVHVSG

i01 ERRLD I D EKP LVVQLNWNKDDREGRFVLKNENDAI PPKAQSNGP EKQEKEGVI QNFKRTLSKKE KKEKKKREKEALRQASDKDDRPFQGEDVENS RLAAE

201 VYKDMPETSFTRT I SNPEVVMKRRRQQKLEKRMQEFRSSDGRPDSGGTLRIYADSLKPNI PYKT I LLSTTDPADFAVAEALEKYGLEKENPKDYC IARVM

301 LPPGAQHSDEKGAKE I I LDDDECP LQIFREWP SDKG I LVFQLKRRP PDHIPKKTKKHLEGKTPKGKERADGSVYGSTLP PEKLPYLVELSPDGSDSRDKP

401 KLYRLQLSVTEVGTEKLDDNSI QLFGPG IQPHHCDLTNMDGVVTVTPRSMDAETYVEGQR I SETTMLQSGMKVQFGASHVFKFVDPSQDHAIAKRSVDGG

501 LMVKGPRHKPG IVQETTFDLGGDI HSGTALPTSKSTTRLDSDRVSSASSTAERGMVKPMI RVEQQp DYRRQESRTQDASGP EL I LPASI EFRESSEDSFL

601 SAI INYTNSSTVHFKLSPTYVLYMACRYVLSNQYRPDI SP TERTH_KVIAVVNKMVS~9~EGVI QKQKNIAGALAYWMANASE LLNF IKQDRDLSRI TLDAQ

701 DVLAHLVQMAFKYLVHCLQSELNNYMPAFLDDPEENSLQRPK I DDVLHTLTGAMSLLRRCRVNAALT I QLFSQLFHF INMWLFNRLVTDPDSGLCSHYWG

801 AI I RQQLGHIEAWAEKQGLE LAADCHLSRIVQATTLLTMDKYAP DD I PNINSTCFKLNSLQLQALLQNYHCAP DEPF Ip TDLI ENVVTVAENTADELARS

901 DGREVQLEEDPDLQLPFLLPEDGYSCDVVRNIPNGLQEFLDP LCQRGFCRLIPHTRSPGTWTIYFEGADYESHLLRENTELAQPLRKEPEI ITVTLKKQN

I001 GMGLS IVAAKGAGQDKLG IYVKSVVKGGAADVDGRLAAGDQL LSVDGRSLVG LSQE RAAE LMTRTS SVVT LEVAKQGAI YHGLAT LLNQP SPMMQRI SDR

1101 RG•GKPRPKsEGFELYNNSTQNG•PESPQLPWAEYSEPKKLPGDDRLMKNRADHR•SPNVANQPP•PGGKSAYASGTTAKIT•VsTGNLCTEEQTPPPRP

1201 EAYPI PTQTYTREYFTFPASKSQDRMAPPQNQWPNYEEKPHMHTDSNHSS IAI QRVTRSQEELREDKAYQLERHRIEAA RKSDSDMWINQSSSLDSS T

1301 SSQEHLNHSSKSVTPASTLTKSGPGRWKTPAAI PATPVAVSQP I RTDLPP pp pp PPVHYAGDFDGMSMDLP Lppp PSANQI GLPSAQVAAAERRKREEHQ

1401 RWYEKEKAP LEEERERKRREQERKLGQMRTQSLNPAPFSP LTAQQMKPEKPSTLQRPQETVI RE LQpQQQPRTIERRDLQY ITVSKEELSSGDSLSPDPW

1501 KRDAKEKLEKQQQMHIVDMLsKEIQELQ•KPDRSAEEsDRLRKLMLEWQFQKRLQE•KQKDEDDEEEEDDDVDTMLIMQRLEAERRARVKGGVLWLCP•V

1601 VPILASACFPWG* 1612

GTCCAGAGCAGAGCAAACAGAAAAAAGT GGC TCCCCGCCCAAG TATCCC TG TAAAACAAA P E Q S K Q K K V A P R P S I P V K Q K

ALL-1 exon6 ~ _ AF-6 exon

AAC CAAAAGAAAAGGATT TGGAGTTCCATGGAGTGAT GAGATT TTAT TT TCAAGATAAAG P K E K D L E F H G V M R F Y F Q D K A

C TGCTGGAAACT TTGCAACAAAATGTAT TCGGGTC TC TAGT AC TGCCACCACTCAAGAT G A G N F A T K C I R V S S T A T T Q D V

TAATCGAAACC-C TCGCGG~F~T TTCGACCTGATATGCGAATGC TGTCCTCTCCCAAGT I E T L A E K F R P D M R M L S S P K Y

AT TCAC TCTATGAAGTGCATGTCAGCGGAG S L Y E V H V S G

Fig. 2. Cloning and sequencing of AF-6 eDNA and of ALL-1/AF-6 fusion transcript. (,4)AF-6 cDNA clones. - - - - , different sequences possibly representing alternative non-coding exons. Restriction sites: ,4, Apal; B, BamHI; tt, H/ndlII; S, SacI. (B) Predicted amino acid sequence o lAF-6 cDNA coding region. Arrow, RNA fusion point. (C) Fusion transcript of ALL-1 and AF-6 cloned from the RNAs of patients 01 and Ed. Kb, kilobase.

5626




AF- 6 KKQNGMGL S IVAAKGAGQ.. DKLGXYVKSWKGGAADVDGRIdkAGDQLLSVDGRSLVGLS Q~t~AE LM.. TRTS SV%'TLEVAKQGAIY ZO-I (3) RKGD SVGLRL ..... AGG.. NDVGXF%rAGVLED SPAAKEG. LEEGDQ ILRVNNVDF TN I IRZEAVLFLLDLPKGEEVTI LAQKKKDVY psd95 (2) KGPKGL~SIAGGVGNQHIPGDNSXYVTKIIEGGA~KDGR~IGDKIZdkVNSVGLEDVMHI~AVAAL.. KNTYDVVYLKVAKPSNAY dlg (3) KGPQGL&'FNIVG .... GE. . DGQGIrY%'SFI LAGGPJ~3LGSEId~(~DQLLS~qNVNLTHATHZEAAQAL.. KTSGGVVTLLAQYRPEEM

Fig. 3. Comparison of the GLGF repeat within the AF-6 protein to GLGF repeats of other proleins. GLGF repeats are the third GLGF in human ZO-1 (ZO-1 3), the second GLGF in rat PSD95 (PSD95 2), and the third GLGF in Drosophila large disc tumor suppressor gene (dtg3). Bold amino acids are consensus amino acids conserved among the four proteins.

junctions) (29). In this domain, termed the GLGF repeat (28), AF-6 shows identity of 28, 36, and 42% and similarity of 57, 59, and 67% t6 the human, rat, and Drosophila proteins, respectively.

To examine the expression of AF-6 in different cell types, we performed a Northern analysis on RNAs extracted from several cell lines (Fig. 4). An 8-kilobase transcript was detected in cell lines of myeloid (Fig. 4, Lane a), erythroid (Fig. 4, Lane b), lymphoid (Fig. 4, Lanes c-e), glial (Fig. 4, Lane f) and epithelial (Fig. 4, Lane g) origin. Thus, it appears that AF-6 is expressed in a variety of hematopoietic and nonhematopoietic cells.

Discussion

The t(6;11)(q27;q23) translocation is one of the most frequent translocations involving 11q23. Cloning of the AF-6 gene involved in this abnormality should enable now the use of Southern blotting and the RT-PCR technique to identify relevant patients whose karyotype was different, complex, or not clear. In addition it is possible now to examine residual disease in patients in remission. The analysis re- ported here of the patient Ed illustrates the first point. This patient showed a typical del(llq23) abnormality. Using the molecular ap- proaches we found here that he had the ALL-1/AF-6 fusion product. Presumably, del(l lq23) and t(6;l l) are difficult to distinguish cyto- genetically. Using chromosome 6-specific probes and fluorescence in situ hybridization analysis, others have recently concluded that some patients with del(llq23) in fact carry the t(6;l l) chromosome translocation (30).

One of the main reasons for cloning AF-6 was to see if it is related to the partner genes AF-4, AF-9, and ENL. Among these, AF-9 and ENL are highly reiated. However, AF-6 showed no sequence homology to any of the three partner genes. Short domains rich in prolines, serines, and charged amino acids were the only motifs shared by the four genes. The COOH terminus of AF-6 showed homology to the tail domain of myosin-1 isoform from yeast and myosin heavy chain from

5 k b -

2 k b -

D. discoideum; this domain presumably confers the rod structure on the myosin protein. Within this region AF-6 displays a remarkable homology to the GLGF repeat found in the ZO-1, PSD-95, and dig proteins from human, rat, and Drosophila, respectively. The first and the third proteins are thought to play a role in signal transduction on the cytoplasmic surface of intercellular junctions (27, 29). The second protein localizes to synaptic junctions and is thought to be involved in synaptic signaling or organization (28). The three proteins are associated with the cytoskeleton. Therefore, the presence of the GLGF domain in AF-6 raises the possibility that AF-6 is not a nuclear protein.

Acknowledgments

We thank Jean Letofsky, Christine Beatty, and Kate Wildauer for their

technical assistance.

a b c d e f g Fig. 4. Northern analysis of AF-6 RNA in human cell lines. From 5 to 10 /zg of

polyadenylated RNA were analyzed on agarose gel containing formaldehyde. RNAs were obtained from lines KCL22, K562, B-l, MV4;ll, SKDHL, T98G, and 293 (Lanes a-g, respectively).

5627

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1993;53:5624-5628. Cancer Res R. Prasad, Y. Gu, H. Alder, et al. Translocationin Acute Myeloid Leukemias with the t(6;11) Chromosome

Gene, InvolvedAF-6 Fusion Partner, the ALL-1Cloning of the

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