a novel human homeobox gene lies at the chromosome 10

A Novel Human Homeobox Gene Lies at the Chromosome 10 Breakpoint in Lymphoid Neoplasias With Chromosomal Translocation t (10;14)

By Ian D. Dube, Suzanne Kamel-Reid, Chiu Chin Yuan, Ming Lu, Xin Wu, George Corpus, Susana C. Raimondi, William M. Crist, Andrew J. Carroll, J u n Minowada, and Jason B. Baker

The translocation t(10; 14)(q24;qll) is an acquired change seen in 4% to 7% of T-cell acute lymphoblastic leukemias IT-ALL). We previously demonstrated that the translocation juxtaposes the T-cell receptor (TCR) &chain gene in chromosome 14q11 with a novel region in chromosome 10q24 and is likely catalyzed by recombinases normally involved in the generation of immunoglobulin and TCR diversity. We now present the sequence of a gene on chromosome 10 that lies immediately telomeric of the breakpoints in nine new ALL patients with acquired rearrangements in 10q24. The gene is

ANY ACQUIRED chromosome changes in human M leukemia are highly specific and result in the activation or mutation of genes involved in cell proliferation and its regulation. Well characterized examples include the deregulation of MYC expression by juxtaposition with immunoglobulin (Ig) or T-cell receptor (TCR) genes in lymphoid neoplasia’ and the formation of a fusion BCR- ABL gene in chronic myeloid leukemia.’ Emerging dogma in cancer cytogenetics describes primary chromosome translocations as morphologically appreciable manifestations of specific genetic recombinational events. In these recombinations, two genes are juxtaposed in such a manner that one gene deregulates or mutates the other. One gene plays the role of an oncogene, while the other is often one normally expressed in the cell compartment from which the tumor arises. Inappropriate expression of the putative oncogene, or its expression in a mutated form, plays a key role in the transformation to malignant growth. This model predicts that genes important in the pathogenesis of particular cancers exist close to translocation breakpoints nonran- domly associated with those cancers.

We first observed a translocation between the long arms

From the University of Toronto Hospitals’ Cancer Cytogenetics Program, Toronto, Ontario, Canada; the Oncology Research Program and the Department of Pathology, Toronto General Hospital; St Jude Children’s Research Hospital and the University of Tennessee, Mem- phis College of Medicine, Memphis, TN; the University of Alabama Medical Center, Birmingham, AL; the Pediatric Oncology Group, St Louis, MO; and the Fujisaki Cell Center, Hyashibara Biochemical Laboratories, Inc, Fujisakr, Japan.

Submitted June 14, 1991; acceptedAugust 6, 1991. Supported by research grants from the Medical Research Council of

Canada (MT10528) and the Hospital for Sick Children Foundation (XG88-61), and by grants from the National Cancer Institute, including CA21765, CA30969, CA05587, and by the Lebanese Syrian Associated Charities (ALSAC).

Address reprint requests to Ian D. Dub&!, PhD, Department of Pathology, Toronto General Hospital, Room 77, 100 College St, Toronto, Ontario, Canada M5G 1L5.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 sole& to indicate this fact.

0 1991 by The American Society of Hematology. OOO6-4971I91 I7811-oO12$3.oO/O

a novel human homeobox gene and is expressed in leukemic cells from ALL patients with rearrangements in a defined chromosome 10 breakpoint cluster region, but not in other adult tissues or cell lines. This new gene has been designated HOXll. Our results strongly support a role for homeobox genes in oncogenesis and may represent the first example of a human cancer in which deregulated expression of an unaltered homeobox gene is involved in tumorigenesis. o 1991 by The American Society of Hematology.

of chromosomes 10 and 14, t(10;14)(q24;qll), in malignant cells from patients with T-cell acute lymphoblastic leukemia (T-ALL).334 Our studies, and data from other centers, indicate that the t(10;14) translocation is one of the most frequently observed nonrandom cytogenetic changes in T-cell neoplasia, occurring in approximately 4% to 7% of T-ALL.3-6 We initially hypothesized that this translocation deregulates a novel oncogene in chromosome 10 via illegitimate recombination with a TCR gene in 14q11.’ Kagan et a1 subsequently demonstrated that the breakpoint in chromosome 14 did indeed split the TCR a-chain locus in one patient and termed the putative gene on chromosome 10 TCL3.7 In a subsequent study,’ the translocation was shown to actually interrupt the &chain gene, located within the a-chain locus^ in three patients. The breakpoints in chromosome 10q24 in these and other patients were found to be tightly In one study,” a chromosome 10 breakpoint flanking sequence detected a homologous transcript exclusively in leukemic cells from t( 10;14) T-ALL, but no corresponding gene has yet been identified.

We recently presented sequence analysis of both translocation breakpoints from a t( 10;14) translocation and our results indicated that the mechanism of the translocation involved illegitimate recombination catalyzed by Ig/TCR recombinases and recombination motifs on both chromosomes 10 and 14.” This latter finding supported the concept that translocations appearing to involve TCR gene loci at the cytogenetic level did indeed involve these genes at the molecular level and occurred via aberrant physiological recombinational events.13

In this report, we describe the loqlization of the chromosome 10 breakpoints in nine additional patients with acute leukemia and clonal rearrangements of 10q24. In all nine cases, and in the cases previously reported,s”0.” breakpoints were clustered within a 15-kb region. We report here that immediately telomeric of this breakpoint cluster region is the coding sequence of a novel human homeobox gene. This gene has been designated HOXll and is expressed in cells from patients with acute leukemia and rearrangements in 10q24, but not in other adult tissues or hematopoietic cell lines. Our results suggest that HOXll is deregulated in the course of acquired translocations in lymphoid neoplasia involving 10q24 and support the notion that abnormal expression of homeobox containing genes can contribute to a malignant phenotype.

2996 Blood, Vol78, No 11 (December 1). 1991: pp 2996-3003

/-/OX77 IN LYMPHOID NEOPLASIAS WITH t(10;14) 2997

MATERIALS AND METHODS

Leukemic cells from patients with ALL were obtained from the Pediatric Oncology Group Tumor Bank at the St Jude Children’s Research Hospital, the Fujisaki Cell Center in Japan, and from patients at the Hospital for Sick Children in Toronto. Approval for biologic studies was obtained from the appropriate institutional review boards. The diagnosis of ALL was based on standard criteria. Cytogenetic studies were performed on leukemic cells obtained at diagnosis and on the cell line K3P according to standard cytogenetic techniques.g” The cell line K3P was obtained from a patient with t(10;14) TALL by modifications of established technique^.'^ Briefly, malignant cells were incubated in methylcellulose with 10% human serum for 21 days. Arising colonies were individually removed and suspended in complete media containing growth factors. Continuous cell growth in RPMI 1640 with 5% fetal calf serum without growth factor supplement was attained after 4 to 6 weeks.

Genomic DNA was prepared according to standard protocols by cell lysis with sodium dodecyl sulfate (SDS), digestion with proteinase K, and phenol-chloroform extraction.” DNA was transferred onto nylon membraned6 and the membranes prehybridized for 3 hours at 65°C with 0.1 mg/mL salmon sperm DNA in 6 x SSC ( l x SSC is 0.15 mol/L NaCl plus 0.015 mol/L sodium citrate), 5x Denhardts, and 0.5% SDS. Hybridization was also performed at 65°C in 6x SSC, 5x Denhardts, and 0.5% SDS. The filters were washed twice in 2x SSC and 0.1% SDS, first at room temperature for 15 minutes and then at 52°C for 30 minutes, and then once with 0 . 1 ~ SSC and 0.1% SDS at 52°C. Subsequently, the filters were exposed at -70°C to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) with intensifying screens.

Total cellular RNA was extracted using a modified guanidinium thiocyanate method” and RNA samples

Patient material.

Southern blotting.

Northern analysis.

were electrophoresed on a 3% formaldehyde/ 1.4% agarose gel. After ethidium bromide staining, the gel was photographed under UV illumination to check the amounts and integrity of the mRNA. The RNA was then transferred onto nylon membranes (Amersham Hybond-N, Arlington Heights, IL) and the filters hybridized overnight with ”P-labeled DNA probes. The Northern blots were hybridized under the same conditions as for the Southern hybridizations. Filters were washed in 2x SSC, 0.1% SDS at room temperature for 30 minutes and then in 0.1X SSC, 0.1% SDS at 65°C for 30 minutes. The quantity and integrity of the RNA transferred onto the filters was subsequently checked by hybridization with a p-actin probe.

An oligo(dT)cDNA library was constructed in pBluescript (Stratagene, La Jolla, CA) from K3P poly(A)+RNA. Total RNA was extracted from the cells and subjected to oligo(dT)-cellulose chromatography. cDNA synthesis was accomplished using M-MuLV reverse transcriptase after denaturation with methyl mercuric hydroxide. Second-strand synthesis was carried out using DNA polymerase I, RNAase H, and Escherichia coli DNA ligase. The 3’ overhangs were blunted using T4 DNA polymerase and EcoRI linkers were ligated to the DNA. The cDNA was electrophoresed and fragments larger than 5 kb were selected for ligation into pBluescript. Recombinant clones, 6 x lo5, were screened with the probe CH104.8 (Fig 1). Four positive clones were obtained and the one with the largest insert (2.1 kb) was selected for further analysis.

Sequencing was performed using Sequenase according to the manufacturer’s instructions (US Biochemical, Cleveland, OH) directly from double-stranded DNA by the dideoxynucleotide chain-termination method as described.18 The composite cDNA sequence was obtained by sequencing both strands.

cDNA cloning and sequence analysis.

H t Kb

Centromere

Chromosome IO

Fig 1. Restriction map of chromosome 10 illustrating the breakpoints (arrows) in ALL patients with reprrangements in 10q24, and the repeat-free probes used in our studies (stippled rectangles). Horizontal lines at the arrow heads indicate restriction fragments t o which breakpoints have been localized. For chromosome walking, repeat-free probes were used to screen normal placental and fibroblast phage and cosmid libraries. Breakpoint locations were determined by digesting DNA from bone marrow cells (or a marrow-derived cell line in the case of K3P) with EcoRI. BamHI, or Hindlll and probing with MLlOl.la. ML101.2a. MLlO1.SH. and CH104.8. The probe CH104.8 was used to successfully screen the cDNA library from the cell line K3P bearing the der(l4)t(lO;l4)(q24;qll). Restriction sites are: H, Hindlll; B, BamHI; E, EcoRI. Numbers indicate distances in kb between restriction sites. Rt, Ry, En, Rr. were all patients with t(10;14) ALL. K3P is a cell line derived from a patient with t(10; 14)T-ALL. LI was a patient with t(7;lO) T-ALL. Rearrangements in Ne and Er were detected in the course of screening 50 unselected cases of T-ALL. Rearrangements in On and Em were detected in the course of screening 56 unselected cases of pre-B-ALL. The breakpoint in En has been previously reported in greater detail.” The most likely genomic organization of HOXl I is indicated with shaded rectangles.

2998

RESULTS

Clustering of breakpoints in IOq24. To characterize the breakpoint region in chromosome 10, breakpoint-flanking probes (Fig 1) obtained in the course of our previous studies" were used to screen human genomic DNA libraries. Overlapping clones covering 55 kb of chromosome 10 were obtaincd and repeat-free probes were used in South- ern analyses of leukemic cell DNA from four patients with t(10;14) T-ALL and one with t(7;lO) T-ALL to determine the locations of breakpoints. All breakpoints were clustered within a IO-kb region of chromosome 10 (Fig 1).

To determine the frequency with which rearrangements occur in this chromosomal region in pediatric ALL, we studied malignant cells from 106 unselected cases by Southern analysis, using our chromosome 1Oq24 probcs MLlOl.la, ML101.2a, MLlOl.9H, and CH104.8 (Fig 1). Rearrangements were detected in the chromosome 10 breakpoint cluster region in two of 56 patients with pre-B (CALLA+) ALL and in two of 50 patients with T-ALL (Fig 1). Cancer cytogenetic studies were not informative (ie, failcd or normal) in these latter four cases. Of the remaining 102 cases, none had cytogenetic evidence for rearrangements involving 10q24. The results for T-ALL are consis- tent with the cytogenetic data suggesting a 4% to 7% frequency of acquired t( 10;14) translocations in T-ALL."' The finding of rearrangements in a similar proportion of pre-B-ALL suggests that this region may also be involved in rearrangements in B-lineage neoplasias.

We reasoned that candidate coding sequences of the chromosome 10 gene postu- lated to be involved in this translocation may lie close to the breakpoint cluster region and may be evolutionarily conserved. The CH104.8 probe (Fig 1) detected conserved sequences in Southern blots of DNA from dog, cow, mouse, chicken, and snake (data not shown). This probe was then used to screen human cell lines and adult tissues for expression of a homologous transcript (Table 1). Only the cell line K3P expressed a 2.1-kb message (Table 1, Fig 2). K3P is a cell line derived from the leukemic cells of a patient with t( 10;14) T-ALL and established according to published methods.'& Cytogenetic analysis in our routine diagnostic laboratory (University of Toronto Hospitals' Cancer Cytogenetics Program) indicated that the cell line had lost the derivative chromosome 10 and the normal chromosome 10, but retained the derivative chromosome 14 bearing the short and proximal long arms of 14 and the translocated chromosome 10 material distal to the breakpoint in 10q24.

To characterize the transcript detected in the cell line U P , a cDNA library was made from K3P poly(A)' RNA. The library was screened with CH104.8 and the largest clone, containing a 2.1-kb insert, was selected for further analysis. The insert cDNA hybridized only to chromosome 10 genomic probes CH104.8 and ML101.9H (Fig 1). It detected a 2.1-kb transcript expressed in K3P (Fig 2) and in bone marrow cells from two of two tested T-ALL patients with 10q24 rearrangements; one ascertained by cytogenetic demonstration of a t(10;14) and the other by Southern analysis using 10q24 probes, as described above (Table 1).

Isolation of a candidate cDNA.

DUB€ ET AL

Table 1. Results of Northern Analyses

TissuelCell Line

Probe 2.1 cDNA Probe CH104.8

K3P DND41 SKW3 JURKAT YT Nalm 6 Hoon Cordick DAUDI HL60 u937 Tonsil Thymus

t(10:14) cells + (early T) - (early T) - (early T) - (NK-like) - (early B) - (early B) - (mature B) - (Burkitt's) -

(promyelocyte) - (monocyte) -

K3P t(10:14) cells + Rt t(10;14) marrow + Ne t(10;14) marrow + Kidney -

Melanoma - Pericardium -

Spleen - Colon Testis Marrow Lymphoblasts -

- -

-

Results of Northern analyses of human cell lines, tissues, and bone marrow cells from patients with T-ALL and rearrangements in 10q24 demonstrated cytogenetically by the presence of the t(10;14) (K3P and Rt) or demonstrated by Southern analysis using chromosome 10 breakpoint cluster region probes (Ne). The probes used are CH104.8 (Fig 1) and the 2.1-kb cDNA insert. In cases where hybridizations were observed (+),the hybridizing transcripts were 2.1 kb in size.

These latter two patients were the only cases of those described above from which lcukcmic cells were available for Northern analyses.

The hybridization results and the fact that breakpoints in the nine patients studied here and in eight previously reported casesX."'.'' were all clustered in the genomic region corresponding to the 5' end of the 2.1-kb transcript,

A B C D E F 9.94 b 7.46 b

4.40 +

2.37 b

I .35 b

b 0.24

Fig 2. Expression of the 2.1-kb transcript. Northern analysis of total RNA from human kidney, melanoma, pericardium, spleen, the cell line K3P. and colon (lanes A-F, respectively) probing with the 2.1-kb cDNA probe (see text). The cell line K3P shows expression of the 2.1-kb message.


strongly suggested that the 2.1-kb cDNA contained coding sequences of a gene transcribed from chromosome 10 sequences immediately telomeric of the breakpoint on the derivative 14. That the cell line K3P contained the derivative chromosome 14, but not the derivative 10 or the normal 10, further supported this interpretation, since the only allele of the putative chromosome 10 gene present in K3P was the one involved in the translocation.

Sequencing of the 2.1-kb cDNA showed a 1,218 nucleotide long open-reading frame between nucleotides 192 and 1,409 (Fig 3). There was an in-frame stop codon (TAG) at position -66 and the sequences flanking the ATG at position 1 were found to share several features common to translation initiation sites of vertebrate mRNAs.” There were 3‘ untranslated sequences after the stop codon (TGA) at 1,407 and a polyadenylation signal at 2,026. These data suggested that the 2.1-kb cDNA contained the entire coding sequence of a gene including 5’ and 3’ untranslated regions.

The coding region predicted a 43-kD protein of 405 amino acids (Fig 3). The predicted protein was compared with those in the MBIR/PIR protein database using the search algo- rithm FASTA (Baylor College of Medicine, Houston, TX). The results showed strong homologies with the homeopro- tein family. A 61 amino acid sequence between residues 199 and 259 showed striking homology with the evolutionarily conserved DNA-binding homeobox domain (Fig 4). Com- parison with murine HOX-2.6, for example, showed identity or conservative changes at 33 of 61 amino acids. The strict conservation of tryptophan, phenylalanine, asparagine, and arginine residues at positions 49, 50, 52, and 54, in the consensus 61 amino acid sequence that occurs in all homeoproteins” occurred in our protein at positions 247, 248, 250, and 252. Furthermore, there was identity at 13 of the 17 amino acids that are less strictly conserved in this 61 amino acid region among homeodomains of 83 eukaryotes (Fig 4).” The sequence Pro-Trp-Met found upstream of the homeodomain of several homeobox genes21,zz occurred in our peptide at amino acid 174 (-22 from the homeodomain). The new gene did not belong to any of the known classes of homeobox genes.“ In addition, no homeobox gene has been mapped to human chromosome 10 or the syntenic regions in the mouse genome. These results indicated that the 2.1-kb cDNA was derived from a novel human homeobox-containing gene. The nomenclature as- signed to this gene is HOXll (P.J. McALpine, Chair, HGM Nomenclature Committee, University of Manitoba, Win- nipeg).

Analysis of the chromosome 14 breakpoint in K3P (data not shown) indicated that a potential fusion gene created by the translocation would contain sequences derived from the joining subunits of the TCR 8-chain gene. Consequently, we analyzed the 2.1 cDNA for TCR &chain gene sequences. None were found and thus there was no support at this level of analysis for the notion that the 2.1-kb cDNA transcript was derived from a TCRG/HOXll fusion gene.

cDNA sequence anafysis.

The DNA sequence predicts a novel homeobox gene.

1 192

17 240

33 288

49 336

65 304

81 432

97 4 8 0

113 520

129 576

145 624

161 612

111 720

193 160

209 816

225 864

241 912

251 960

273 1008

209 1056

305 1104

321 1152

337 1200

353 1240

369 1296

mt GLY nis m u G ~ Y PTO Hi8 Hi. ~ e u BID pro ~i~ nis ~ i a GI” pro ATG GAG U C CTG GGT CCG CAC U C CTC U C CCG GGT U C GU GAG CCC

11. Sex Phe Gly 11s ASP Gln 11. Leu Asn Ser 9x0 Asp Gln Gly Gly ATT AGC TTC GGC ATC GAC U G hTC CTC M C AGC CCG GAC U G GGT GGC

CyS Met G1y PI0 Ala Ssr A r g Idu Gln Anp Gly Glu Tyl Gly Leu Gly TGC ATG GGA CCC GCC TCG CGC CTC U G GAC GGA GM TAC GGC CTT GGC

Cy8 Leu V a l GlY Gly A l a Tyr Thr Tyr Gly Fly Gly Gly 5-1 Ala A l a TGC TTG GTC GGA GGC GCC TIC ACT TIC GGC GGC Gv. GGC TCC GCG GCC

Ala Thr Gly Ala Gly Gly Ala Gly l l la Tyr G ~ Y Thr Gly Gly PIO Gly GCG ACG GGG GCT GGA GGA GCG GGG GCC TAT GGT ACT GGA GGT CCC GGC

Gly Pro Gly Gly Pro A l a G1Y Gly Gly Gly A l a Cys SOT Met Gly Pro GGC CCC GGA GGC CCG GU GGC GGC GGC GGC GCC TGC AGC ATG GGT CCT

Leu Thr Gly SBr Tyr &on Val AS“ Met Ala Leu Ala Gly Gly Pro Gly CTG ACC GGC TCC TIC M C GTG M C ATG GCC TTG G U GGC GGC CCC GGT

Len Ala Ala Ala Ala Ala Ala Acg Gly Ala Gly Ala Leu Ser ala Ala CTG GCG GCG GCG GCG GU GCA CGC GGT GCC GGG G U CTC AGC GCT GCG

Gly V a l Ile Arg V a l Pro Ala Hi3 Arg Pro Leu Ala Gly Ala Val Ala GGG GTA ATC CGG GTG CCG GCA U C AGG CCG CTC GCC GGA GCC GTG GCC

Hi3 Pro Gln Pro Leu Ala Thr Gly Leu Pzo Thr V a l Pro Ser V a l Pro CAC CCC CAG CCC CTG GCC ACC GGC TTG CCC ACC GTG CCC TCT GTG CCT

Ala Met PI0 Gly Val As” Asn leu Thr Gly Leu Thr Phe Pro Trp Met GCC ATG CCG GGC GTC AAC M C CTC ACT GGC CTC ACC TTC CCC TGG RTG

Glu Ser Rsn A r g A r g Tyr Thr Lya Asp Arg PhB Thr Gly His Pro Tyr GIG AGT AAC CGC AGI TIC A U M G GAC AGG TTC ACA GGT U C CCC TAT

Gln Asn Azg Thr pro Pro CAG M C CGG ACG CCC CCC AAG M G M G M G CCG CGC ACG TCC TTC ACA

CGC CTG CAG ATC TGC GAG CTG GAG AAG CGC TTC U C CGC CAG AAG TAC

CTG GCC TCG GCC GAG CGC GCC GCC CTG GCC M G GCG CTC &AA ATG ACC

GAT GCG CAG GTC AAA ACC TGG TTC CAG M C CGG CGG liCA M G TGG AGA

Arg Arg A S n Gly Fly Arg Alp Arg G l n A111 Thr Rrg Ser CGC AGI CTG CGG AGG M C GGA GGC CGA GAC AGG CM GCA ACC C W TCC

Ssr Ser Ser Cy* Ser Arg A r q Pro Ser Arg Arg A l a TXT HIS SeI Acg TCC TCG AGT TGC AOC AGG AGG CCT TOC AG4 A W GCC TGG U C Am CGC

Cyn Pro Leu Thr Leu Cya Ala Cys Thr Thr Arg Arg Ser SBr Pro Cy5 TGC CCG CTG ACC CTC TGT GCG TGC ACA ACT CGT CGC TClP TCG CCC TGC

Arg Ile Cy. Ser Arg Gly Leu Thi Thr ATg Pro Ly3 Ser Leu A l a Ser A m ATC TGC AGC CGT GGT CTG ACG ACT CGA CCA huL T U CTA GCG T U

Arg Irg Trp A r g Arg Pro Ala S e x Flu Pro Ala Hi3 Ser Ala Leu Trp CGT CGG TGG CGT COG CCT GCG AGT GAG CCT GCC CAT TCT GCC CTG TGG

Asp Pro A19 PI0 Thr Gln Gly Ser Leu Arg PI0 G1.u Thr Gln ASP SCr GAC CCC AGG CCC ACT U G GGG T U CTG AGG CCT GAG ACC U G GAC TCC

SBT PTO pro Sei ~ r p PTO Gln Thr A l a A r g A r 4 Arg Gly Thr Leu P r o TCC C U CCC TCC TGG CCT U G ACT GU CGC AGG aGG GGA ACA CTG CCC

Ser Hi5 Gly Pro Lys Gly Pro PTo HI3 Isu Cy3 Arg Hi0 Cy3 Ser Pro TCG U C GGC CCC AAA GGG CCC C U CAT TTG TGC CGA U C TGT TCT CCC

385 1341

Phe Gly Gly Arg Ala Gln G1Y Thr Arg Thr Ax9 Ala Pro Leu Pro Glu TTC GGT GGA AGA GCT CAA GGG A U AGG ACl CGC GCC CCC CTC CCA GAG

4 0 1 AT9 Pro Ala Pro V a l *** 1392 CGT CCC GCA CCT GTC TGA ACT GTT AAG A M TCT GTT TTT GTT TAT TTC

ITTTTATTTTMTTTTTMCGTGGGATTUGAGMAGGCAAGGGAGGTMGGGAGGAGGAGCTTCTGGG GTCCCUGGGCTGTWTCTGMTTTGCCCTGOGMICCCCTTCTCTGTGACCUCTTCTUTUUUC ATGGIMCCUTAGGTCUUUUGGTGGTGTUCTGTCCCTCCTGGTGTUCCCUGAGCUUUT GGGCATCTIITGGGAGAGTGTCMCUGAUG~GGGTUUGTGTTTACACTTTGGACCTTACWTUGG U U G G T U G O F G T G A U U G A C T U T C C T G M U G U T W C T C C T F O C U U C T G T G A U U C T A U C ~ U U ~ U G C - C A G C T A ~ ~ G C C T C A C T T ~ T C T G C U

CTCUDMCCUTTTGAGGGGTGGGGGOGTGTTMTTTATGUCTTATMGGTGTTTTCTGTGTMCU G O C C ~ C U U U ~ C ~ ~ C ~ T C U ~ T A C G I U W U G G T T T T U U T ~ T G U G C C U T T T

T T T T A T A M G T G C T T G T G T M T T T A T G T ~ T A A M G C C T C C G A U G ’ = M C G G A A T T C

Fig 3. Nucleotide sequence of the 2.1-kb cDNA showing 5’ and 3’ untranslated regions and the predicted amino acid sequence corresponding to nucleotides 192 to 1.409. The nucleotide sequence is numbered on the left below the amino acid sequence. The amino acid sequence corresponding to the homeodomain and used in the analysis illustrated in Fig 4 is underlined. The 3’ polyadenylation signal and the in frame stop codon (TAG) in the 5’ untranslated region are also underlined.

DISCUSSION

Molecular dissection of translocation t(10;14) (q24;qI 1). In this report, we present the localization of the breakpoints in chromosome 10 in four previously unreported patients with T-ALL and leukemic clones bearing the standard t(10;14) translocation and in one patient with the variant translocation, t(7;lO). Rearrangements in 10q24 in

3000 DUB€ ET AL

TURN n

HELIX 1 HELIX 2 HELIX 3

Fig 4. Amino acid identities of selected homeodomains with

m ..'-l 7... HOXll K K R K P R T S F T R L Q I C e L E K R F H R P K Y L A S A G R A A G A R A L I Human

1 10 20 30 40 5 0 60 No of the corresponding region of Identical matches'61

HOXl1. Identical matches are de- noted by dashes. The number of

. . . . . . . **;*;* ; ; A1 1 R Y Q L F Y R A L L QKIWFQNPAKK

. . . . . . .

NK-3 NK-2 H40 Hox2.6 NK- 1 HOX H55 HOXl H17 HOXl . 3 HOXl .3 ZF-25 NK-4 H90 H15 ~ 0 ~ 3 . 3 Ceh-7 Hb3 Ceh-5 Hox6. 1 nox5.4 Hoxl HOXZ .I Ubxlb nox5.2

30 30

B e e 28 Mouse 28 FlY 29 Z.danio 27 Bee 30 Human 28 Bee 30 Human 28 Mouse 28 z.danio 28

30

Bee 27 Mouse 26 C.slegans 25 Sea urchn 26 C.elegans 27 Mouse 2 5 Human 25 East Newt 2 5 Mouse 24 F l Y 26 Human 2 5

identical matches over the region shown is indicated on the right. The four invariant amino acids in all homeoproteins are indicated by asterisks. Identical matches or conservative changes at 14 of 17 less strictly conserved amino acids are marked by co- lons. The consensus amino acids for 83 higher eukaryote homeodomains (A//) is indicated.m The three predicted crheliciesand the turn are indicated. The number- ing of the amino acids in HOXl1 corresponds to that convention- ally used for homeodomains. The dotted lines indicate areas of un- certainty with respect to the ex- tent of the helix region.

four additional patients were detected only by Southern analyses of 56 unselected cases of B-lineage ALL and 50 cases of T-ALL. In all cases, the breaks in chromosome 10 occurred in the 15-kb region to which breakpoints have been identified in other Our finding of rearrangements in chromosome 10 in pre-B-ALL is the first indication that this region of chromosome 10 may be involved in rearrangements in other cancers. Furthermore, the demonstration of chromosome 10 breaks in patients without cytogenetic evidence for rearrangements raises the possibility that mechanisms other than chromosomal translocation may be involved. There is precedence for this in T-ALL as the SCL gene is mutated by the chromosomal translocation t(1;14) in only 3% of patients, but is rearranged at the molecular level in 25% of T-ALL.23324

To identify the putative gene on chromosome 10 involved in this translocation, we used several repeat-free probes derived from a 55-kb region of chromosome 10 that encompasses the breakpoint cluster region. Only one of these probes, ML104.8 (Fig 1) detected evolutionarily conserved sequences. When this probe was used in the Northern analysis of a t(10;14) leukemic cell line (K3P), expression of a 2.1-kb mRNA species was detected. The cDNA corresponding to this message was cloned and its chromosome 10 origin confirmed by Southern analyses using genomic probes. When used as a probe in Northern analyses, the 2.1-kb cDNA hybridized to a 2.1-kb mRNA species in K3P and in leukemic cells from two of two patients with T-ALL on whom material was available for study. One of these latter patients had a cytogenetically demonstrable t( 10;14) translocation, while a chromosome 10 rearrangement in the other was ascertained by Southern screening of 50 unselected cases of T-ALL using our 10q24 probes (Fig 1). Cytogenetic studies in the latter case failed. Unfortunately, no bone marrow cells were available on the remaining patients for Northern analyses.

The 2.1-kb cDNA corresponding to the mRNA cloned

from the cell line K3P was sequenced. Comparative sequence analysis indicated that we had cloned 5' and 3' untranslated regions and the entire coding region of a novel gene. The gene, HOXII, is quite clearly a homeobox- containing gene, as there is greater than 50% amino acid conservation with the homeodomains of other homeobox genes.20 Moreover, HOXll shares several key features of homeoproteins, including conservation of the sequence Pro-Trp-Met 22 amino acids upstream of the homeodomain.z'~zz No amino acid insertions are required for correct alignment of the homeodomain ofHOXll with that of other homeoproteins. Despite these similarities, HOXIl does not belong to any of the known classes of homeobox genes."

The t(10;14) translocation of T-ALL has been the subject of several molecular dissection^.^^^^^^^'^ These data supported our original hypothesis3 in that all studied breakpoints involved the TCR &chain gene on chromosome 14 and a defined region in 10q24. In an additional case with a related t(7;lO) translocation, the breakpoints involved the TCR P-chain gene in chromosome 7 and the breakpoint cluster region in 10q24.'0~25 Actual sequencing of translocation breakpoints8s'n~'' strongly supported the hypothesis that these translocations may arise as a result of aberrant physiological recombinations. The tight clustering of breakpoints in chromosome 10 strongly supports the notion that a gene in this region is deregulated as a result of the translocation with chromosome 14. HOXII, a new human homeobox gene, is the most likely candidate for this gene. Its normal expression pattern appears to be developmen- tally restricted, but it is expressed in t(10;14) T-ALL cells. Furthermore, the 2.1-kb HOXll transcript is derived from a genomic region that lies immediately downstream of the chromosome 10 breakpoints in all eight informative patients (Ne, On, Em, Er, Rt, RY, K3P, and L1 in Fig l).

Interestingly, in one study," a 2.9-kb mRNA transcript was detected in t(10;14) T-ALL cells using sequences 3' of

HOX77 IN LYMPHOID NEOPLASIAS WITH t(10;14) 3001

the chromosome 10 breakpoint cluster region to probe Northern blots. In the latter case, the chromosome 10 breakpoint is more telomeric than other cases reported to date, and it is thus possible that a fusion gene was created and that this accounts for the difference between the transcript observed by us and that described by Zutter et a1 (2.1 kb v 2.9 kb).” Nevertheless, our cDNA sequence analysis (Fig 4) strongly supports our message size of 2.1 kb as being correct.

The homeobox is a conserved structural domain encoding three short a-helical segments containing a DNA binding helix-turn-helix motif.” Homeobox domains were first described as highly conserved features of homeotic proteins important in controlling the developmental fate of embryonic cells in Drosophila.26 In recent years, homeobox-containing genes have been cloned from a variety of vertebrate genomes and it appears that their function as regulators of transcription, with temporally and spatially restricted expression patterns, is evolutionarily We speculate that homeobox-containing genes could also play important roles in differentiation in the adult, particularly in cell renewal systems such as the hematopoietic system where decisions concerning proliferation, commitment, and differentiation are made continually. The observation of expression of homeobox-containing genes in a variety of hematopoietic cell type^^^"^ supports this notion.

The relationship between the oncogenic process and the control of cellular growth has long been recogni~ed.~’ The discovery that many cellular oncogenes were in fact genes involved in growth regulation was not surprising to those who study cancer biology. Likewise, it should come as no surprise that genes involved in the control of cellular differentiation would have tumorigenic potential under appropriate conditions. There is some support for this concept. For example, in the murine myelomonocytic leukemia cell line WEHI3B, integration of an intracisternal A particle upstream of the Hox-2.4 gene results in constitutive expression of H O X - ~ . ~ . ’ ~ . ~ An upregulation of undefined homeobox gene transcripts has also been detected in human carcinomas by in situ hybridization and RNAse pr~tection.~‘

Transcription factors and acquired chromosome translocations. Potential transcription factors have been identified at the sites of certain chromosome translocations nonran- domly associated with human leukemia, particularly T-cell neoplasias. A helix-loop-helix protein, lyl-1, has been associated with a t(7;19) translocation seen rarely in T-cell neoplasia. The lyl-1 locus on chromosome 19 is structurally altered as a result of a head-to-head juxtaposition with the TCR P-chain gene on chromosome 7.4’

The SCL gene on chromosome 1 encodes a helix-loop- helix protein that is rearranged in approximately 25% of

Homeobox genes in cancer.

patients with T-ALL.23,43 This gene was first identified in the course of the molecular dissection of the t( 1;14) translocation observed in approximately 3% of In the t(ll;l4)(pl5;qll) translocation associated with T-ALL, expression of a zinc finger DNA binding protein in 1 1 ~ 1 5 is deregulated as a result of aberrant recombination with the TCR &-chain gene in 14q11.”-48

Recently, molecular dissection of a primary chromosome translocation in human B-lineage leukemia provided evidence that transcription factors with homeodomain-like regions could play important roles in the pathogenesis of certain human hematological malignancies. The translocation t(1;19) of pre-B-ALL results in the production of a novel fusion gene:935o The fusion mRNA is translated into a chimeric protein that consists of the transcriptional- activating domain of the common transcription factor, E2A, fused to the DNA binding domain of a homeobox-like gene termed PBXl. The PBXl homeodomain is quite divergent from those described previously, with less than 36% amino acid identity with known homeodomains and a requirement for a 3 amino acid insertion for optimal alignment.49 In vitro assessment of the oncogenic potential of E2A-PBXl suggested that the chimeric protein has transforming ability.”

This report presents several key points. First, it repre- sents the elucidation of the molecular basis of the t(10;14) translocation of T-ALL. Second, our data indicate that the gene on chromosome 10 involved in this translocation, and most likely in the t(7;lO) variant, is a novel human homeobox gene, HOXll . Third, our report is the first indication that deregulation of intact homeobox genes as a result of acquired chromosome changes may play a role in the pathogenesis of human cancer. Fourth, this may be the first example of deregulated expression of a true human homeobox gene. Fifth, our finding of breakpoints in the 10q24 breakpoint cluster region in patients with pre-B-ALL suggests that aberrant expression of HOXll may not be restricted to T-ALL. Future studies will be directed at studying the role of this gene in normal cells and determin- ing how its deregulation contributes to pathogenesis in human leukemia.

NOTE ADDED IN PROOF

reported by Hatano et alsz and Kennedy et aLS3 Since this manuscript was submitted similar results have been

ACKNOWLEDGMENT

We thank Dr Mike Link of Stanford University for T-cell immunophenotyping and Drs Ronald Worton, Robert Phillips, Gordon Mills, and Alex Joyner for their comments regarding the manuscript and Kent Williams for excellent specimen handling. The expert assistance of the staff of the University of Toronto Hospitals’ Cancer Cytogenetics Program is greatfully acknowl- edged.

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