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From www.bloodjournal.org by guest on August 3, 2017. For personal use only. RAPID COMMUNICATION A Serine/Proline-Rich Protein Is Fused To HRX in t ( 4 ; l l ) Acute Leukemias By Joseph Morrissey, Douglas C. Tkachuk, Athena Milatovich, Uta Francke, Michael Link, and Michael L. Cleary Translocations involving chromosome band 1 1q23 in acute leukemias have recently been shown to involve the HRX gene that codes for a protein with significant similarity to Drosophilatrithorax. HRX gene alterations are consistently observed in t(4;ll) (q21;q23)-carrying leukemias and cell lines by Southern blot analyses and are accompanied by HRXtranscriptsof anomalous size on Northern blots. HRXhomologous cDNAs were isolated from a library prepared from t(4;1l)-carrying acute leukemia cells. cDNAs representative of transcription products from the derivative 11 chromosome were shown to containHRXsequences fused to sequences derived from chromosome band 4q21. Fragments of the latter were used to clone and analyze cDNAs for wild-type4q21 transcripts that predicted a 140-Kd basic protein (named FEL) that is rich in prolines, serines, and charged amino acids. FEL contains guanosine triphosphatebinding and nuclear localization consensus sequences and uses one of two possible 5 exons encoding the first 1 2 or 5 amino acids. After t(4;ll) translocations, 913 C-terminal amino acids of FEL are fused in frame to the N-terminal portion of HRX containing its minor groove DNA binding motifs. These features are similar to predicted t ( l l ; 1 9 ) fusion proteins, suggesting that HRX consistentlycontributes a novel DNA-binding motif to at least two different chimeric proteins in acute leukemias. 0 1993 by The American Society of Hematology. T chromosomes were shown to be transcriptionally active and encode potential HRX fusion protein^.^.' The predominant t( 1 1;19) product originated from the derivative 1 1 chromosome and encoded a fusion protein with the features of a chimeric transcription factor consisting of an N-terminal portion of HRX fused to a novel serine/proline-rich protein from 1 9 ~ 1 3The . ~ predicted t(4;ll) products have not been completely characterized thus, it is unclear whether various HRX fusion partners might share significant similarities to suggest their respective contributions to the pathogenesis of leukemias carrying 1 lq23 translocations. In this report we describe the product of the chromosome 4 gene (named FEL) involved in the t(4;ll) chromosomal translocation. The FEL gene is expressed normally in both B- and T-lymphoid cell lines and encodes a predicted 140Kd protein that lacks significant homology to known proteins. Following t(4;l l), the derivative 1 1 chromosome was shown to encode a 240-Kd fusion protein containing a large C-termina1 portion of FEL fused to the N-terminal portion of HRX containing its AT hook DNA binding motifs! These features are comparable to t( 1 1;19)fusion proteins and suggest that similar HRX chimeric nuclear factors may be consistently generated by 1 lq23 translocations in acute leukemias. RANSLOCATIONS involving chromosome band 1 lq23 have been observed in several different types of hematolymphoid malignancies, including acute lymphoblastic and nonlymphocytic leukemias and non-Hodgkin’s lymphoCytogenetic observations indicate that at least 10 different chromosomal loci may participate in translocationmediated exchanges with band 11q23.’ The most common of this group is the t(4;ll) (q21;q23), which is frequently associated with biphenotypic leukemias expressing both lymphoid and myeloid surface antigens, and particularly common in leukemias of infants.’-’ The gene involved in recurring 1 lq23 translocations has recently been cloned and chara~terized~,~ following its localization within a yeast artificial chromosome that spanned several 1 lq23 translocation breakpoint^.'.^ This gene (variously called HRX, MLL, or ALLI) codes for a potential zinc finger protein of 431 Kd that shares significant but limited similarity to the Drosophila trithorax Several 1 lq23 translocation breakpoints have been localized to a cluster region within the HRX gene6,7,9.10712-14 and fusion transcripts have been isolated and characterized from t( 1 1;19) and t(4;11)-carryingcell line^.^.^ In each case, both derivative From the Laboratory of Experimental Oncology, the Departments of Pathology, Pharmacology, Pediatrics, Genetics, and the Howard Hughes Medical Institute, Stanford University School of Medicine. Stanford, CA. Submitted December 17, 1992; accepted December 18. 1992. Supported in part by National Institutes of Health research grants (CASSO29, CA496OS and HG00298) and training grants (T32 GM07149 and T32 GM08404 to J.M. and A.M., respectively), and the Howard Hughes Medical Institute of which U.F.is an investigator. D.C.T. was supported in part by a Centennial Fellowship from the Canadian Medical Research Council. M.L.C. is a Scholar of the Leukemia Society of America. Address reprint requests to Michael L . Cleary, MD, Department of Pathology, Room L-224, Stanford University School of Medicine, 300 Pasteur Dr, Stanford, CA 94305-5324. 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 solely to indicate this fact. 0 I993 by The American Society of Hematology. 0006-4971/93/81OS-OO36$3.00/0 1124 MATERIALS AND METHODS Patient materials and cell lines. Leukemia cells (ALL no. 1 and ALL no. 2) were obtained from children with acute lymphoblastic leukemia (ALL) and cryopreserved before their use in these studies. Karyotype analyses at the time of diagnosis indicated that both leukemias camed balanced t(4;ll) (q21;q23) translocations. The t(4;II)carrying cell lines RS4:ll and MV4:11 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and have been described else~here.’~.’~ Molecular cloning of t(4;l I ) fusion cDNAs. Polyadenylated RNA was prepared from leukemia cells (+I1 ALL no. 1)canying the t(4; 1 1) (42 l;q23) using commercially prepared reagents (Invitrogen, San Diego, CA). Two micrograms of poly(A+) RNA was converted to double-stranded DNA using random hexamer or oligo-dT priming and commercially prepared reagents (Superscript; BRL/GIBCO, Gaithersburg, MD). After adapter addition and size-fractionation in agarose gel, the cDNA products were cloned into XgtlO, packaged in vitro, and plated for subsequent screening using previously described methods?*” The 4;11 library was screened with a probe from the HRX cDNA that spanned the t( 1 1;19) fusion site (fragment B6). Blood, Vol81, No 5 (March 1). 1993: pp 1124-1 131 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 1125 HRX FUSION TRANSCRIPT IN t ( 4 ; l l ) LEUKEMIAS I L FEL . I BamH1 Twenty cDNAs were isolated and characterized by nucleotide sequencing using synthetic oligonucleotides homologous to HRX or to sequences determined for the fusion partner. Wild-type FEL cDNAs were isolated from cDNA librariesprepared from cell lines (HBI I19 and HAL-OI) lacking the t(4;I I)?.'' For library screening, fragments of t(4; I I ) fusion cDNAs that contained only chromosome 4 sequences were used as probes under high-stringency hybridization conditions. Southern and Northern blotting. Southern and Northern. blot analyses were performed using high-stringency conditions described previously.'*Chromosome I lq23 DNA rearrangements weredetected with a 0.9-kb BamHl fragment of the HRX cDNA that spans the I lq23 fusion region of HRY' Probes for Northern blot hybridizations consisted of portions of the HRX cDNA (fragment B6) or portions of the FEL cDNA (fragments 2 and 3, see Fig 2). Chromosomal mapping by Southern blot and J'uorescence chromosomal in situ hybridization (FISH). Chromosomal localization of the FEL locus was accomplished by using a 32PdCTPrandom primer labeled 1.6-kb probe (probe 2, see Fig 2) on Southern blots of Hindllldigested genomic DNA from normal human and Chinese hamster controls and 13 human X Chinese hamster somatic cell hybrids derived from seven independent fusion experiments'920using methods described elsewhere.2' For finer mapping of the FEL locus. FISH was performed with two plasmids with cDNA inserts as probes (probes 2 and 3, see Fig 2). The plasmids were biotin- I I d U T P labeled by nick-translation using commercially prepared reagents (Boehringer Mannheim, Indianapolis, IN). Hybridizations and washes were performed as previously described?' A biotin/avidin/fluorin isothyocyanate(FITC) detection system was used. Chromosomes were counterstained with propidium iodide ( I60 ng/mL, final concentration). Hybridization solutions consisted of 0.1 to 0.2 ng/pL of each probe DNA (either separately or together), 100 ng/pL each of human placental and salmon sperm DNA as competitors, 50% formamide, 2X sodium chloride sodium citrate (SSC), and 10%dextran sulfate. Metaphase spreads were obtained by standard cytogeneticprocedures except that the cells were synchronized by excess BrdU (200 pg/mL, final con- I T S - 28s rRNA Fig 1. (A) Southern blot analysis of t(4;1l)-canying leukemias. DNA from t(4;11)-carwing leukemias (ALL nos. 1 and 2) and cell lines (RS4:ll and M V 4 : l l ) were analyzed by Southern blot analysis using an HRX-specific probe. Germline band of 8 kb is denoted with a dash. (B) Northern blot analysis of HRX and FEL transcripts in various cell lines and leukemias. Poly(A)+ RNAs from cell lines lacking (6078 and Jurkat) and containing ( R W 1 1 and MV4: 11) t ( 4 ; l l ) translocationswere analyzed by Northern blot hybridization using FEL-specific (upper panel) or HRX-specific (lower panel) probes. Migration of HRX-FEL fusion RNAs and wild-type FEL and HRX transcripts are indicated on the right of each panel. Residual signal from prior FEL hybridization is seen in lower panel and indicated by asterisk. centration) for 17 hours and subsequently the block was released with thymidine (10 pmol/L, final concentration) for 5 hours as described.22A total of 76 metaphase spreads were scored for specific hybridization signals. Signals were considered specific only if two fluorescent signalscould be seen lying side-by-side, one on each chromatid of a particular chromosome. All other single signals were assumed to represent random hybridization. Metaphase spreads were examined in a Zeiss Axiophot microscope equipped with epifluorescence. Images were captured and enhanced using a cooled charge coupled device (CCD) camera (PM512; Photometrin, Tucson, AZ) and the Macintosh GeneJoin program developed by Tim Rand (Yale University, New Haven, CT). Pseudocolored images stored as PlCT files were converted to color slides, from which photographs were prepared for publication. Computer analyses. Compilation of nucleic acid sequences, restriction enzyme, and peptide analyses were performed using the DNA Inspector IIE (Textco, West Lebanon, NH) application. Protein similarity and consensus sequence analyses were performed using lntelligenetics (Mountain View, CA) software. Similarities were determined using Quest and FastDB algorithms against keytools-I0 and SWISSPROT file banks, respectively. RESULTS HRX DNA rearrangements in t(4;11)-carryingleukemias. DNA isolated from leukemias or cell lines carrying t(4;ll) (92 l;q23) chromosomal translocations was analyzed by Southem blot to determine the potential involvement of the previously characterized I lq23 gene HRX. Using a 0.7-kb fragment of HRX cDNA as a probe, all cases showed at least one and in most cases two nongermline bands (Fig I A) demonstrating breakpoint clustering in a limited region of the HRX gene. Detection of two rearranged HRX bands was consistent with detection of both derivative translocation products and suggested that the HRX cDNA probe used spanned the breakpoint region in the HRX gene. Northem From www.bloodjournal.org by guest on August 3, 2017. For personal use only. MORRISSEY ET AL 1126 blot analyses showed HRX RNAs with altered mobility (12 kb) slightly smaller than wild-type HRX RNAs in cell lines carrying the t(4;l l), suggesting that the translocation resulted in HRX fusion transcripts (Fig lB, lower panel). Isolation of HRX fusion transcripts from a t(4;lI ) leukemia. To further characterize potential fusion transcripts, a cDNA library was constructed from mRNA isolated from t(4;11)-carrying leukemia cells (ALL no. 1, Fig 1A). The library was screened with an HRX cDNA probe that spanned the potential fusion site (probe B6). Four types of clones were obtained (Fig 2B) representing either wild-type HRX cDNAs or three different candidate fusion cDNAs containing nonHRX sequences fused to HRX at nucleotide no. 4220.6 All clones in the latter group showed identical sequences for 426 nucleotides beyond the site of fusion with HRX,but then diverged from one another (Fig 2B and data not shown). Types la and lb differed at this point by the inclusion or exclusion of a single nucleotide and both open reading frames diverged and ended shortly thereafter. The observed variations suggested these clones were derived from RNAs that resulted from aberrant splicing at this site particularly because they were considerably shorter than the fusion RNA of 12 kb detected by Northern blotting. Type 2 clones contained an a p parently normal splicing pattern and were analyzed further as described below. Chromosomal mapping of the t(4;Il)fusion partner. To confirm that the isolated cDNAs represented 4; 1 1 fusions, the non-HRX portions were regionally mapped by Southern analysis of somatic cell hybrid lines that contained defined A regions of human chromosome 4 (Fig 3A). Three humanspecific fragments cosegregated and were concordant with human chromosome 4q2 1-q31.2. All other human chromosomes were excluded by at least two discordant hybrids (data not shown). More specific localization was obtained by fluorescence in situ hybridization to human metaphase chromosomes. Two probes (2 and 3; Fig 2, B and C) were hybridized to the chromosomes simultaneously; 11 of 31 metaphases scored had specific signal on at least one chromosome 4 homolog. Two of these 1 1 metaphases had specific signal on both chromosome 4 homologs. Overall, using the probes separately or together, in 26 of 76 metaphases examined, specific hybridization signals were seen at 4q2 1 on at least one chromosome 4 homolog. All specific signals seen were scored as located at 4q21; there were no other specific signals on any other chromosomes. These data demonstrated that the cDNAs isolated from ALL no. 1 contained sequences derived from chromosome 4q21, in addition to HRX sequences from chromosome 1 Analysis of the 4q21 fusion partner. To further characterize the 4q2 1 fusion partner, cDNA clones corresponding to the wild-type transcript were isolated from two different libraries constructed from cell lines that lacked the t(4; 1 1). Overlapping clones showed a cDNA contig of approximately 6 kb (Fig 2, A and C). When portions of these cDNAs were used as probes on Northern blot analyses, a major transcript of 11 kb was observed in all lymphoid cell lines examined along with a minor transcript of 11.5 kb (Fig 1B and data fu:t!til:b 1461 Xhol 1 .o Hindlll 2.0 Pstl 3.0 EcoRl Xbal Xhol incll 4.0 5.0 t(4;ll) Fusion Clone Type la (AM Type 1 b Type 2 Probe4 41 07 Fig 2. Physical maps of t ( 4 ; l l ) fusion clones and wild-type E L . Restriction sites in wild-type E L cDNA are indicated at the top. (A) Schematic illustration of composite wild-type E L cDNAs. Open box indicates open reading frame. Cross-hatched box indicates alternative 5 exon. Lines indicate untranslated sequences. (B) Schematic diagram of 4;ll fusion transcripts. Heavy cross-hatchindicates HRX sequences. Light cross-hatch denotes short open reading frame 3 of atypical splice site in FEL. (C) Schematic of overapping cDNA clones used to establish composite wild-type FEL map. Horizontal bars indicate positions of probes described in text. Numbers refer to nucleotide positions as shown in Fig 4. From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 1127 HRX FUSION TRANSCRIPT IN t(4;ll) LEUKEMIAS not shown). Nucleotide sequence analyses showed that the isolated portion of the transcript contained a complete open reading frame (Fig 4), suggesting that the remaining uncloned portions ofthe 1 1-kb RNA contained untranslated sequences. A a b c d 1 S( 4 + + + - Two forms of the wild-type cDNAs were isolated and found to differ at their 5’ ends, perhaps as a result of differential splicing (Figs 2A and 4). Analysis of the open reading frames for the wild-type FEL transcript showed two predicted proteins (referredto as types A and B in Fig 4) that initiated at one of two alternative methionines in highly favorable contexts for translation initiation.*’ Types A and B differed in the composition of their N terminal 12 and 5 amino acids, respectively, apparently reflecting alternative 5’ exon usage. In-frame stop codons were observed upstream of each initiating ATG, indicating that the open reading frames could not extend further in the 5’ direction. Data base searches indicated that the predicted proteins lacked significant similarity to previously reported proteins. Motif searches showed a guanosine triphosphate (GTP)-binding motif (residues 946 through 952), possible nuclear localization sequences (residues 670 through 674 and 709 through 7 13), and numerous potential phosphorylation sites possibly reflecting the high serine content of FEL. The predicted protein is quite hydrophilic with a net positive charge ($37 at normal pH) and also notable for its high serine (14.7%) and proline ( 1 1.3%)content. ChimericHRX-FELprotein in 4;ll leukemias. In all cells examined, two wild-type FEL transcripts were observed, a major RNA of approximately 11 kb and a minor, slower migrating transcript of 11.5 kb (Fig 1B). In cell lines and leukemias carrying the t(4;11) an additional transcript of 12 kb was observed (Fig 1B and data not shown). The 12-kb RNA also hybridized with an HRX probe and migrated in a position different than the wild type HRX transcripts of 13 and 15 kb (Fig IB). These data indicated that the 12-kb RNA contained both HRXand FEL sequences constitutinga fusion RNA that crossed the t(4;11) breakpoint. Hybridization with 5‘ HRX and 3’FEL probes indicated that this transcript originated from the derivative 11 translocated chromosome and corresponded to the fusion cDNAs described above. Failure to detect altered transcripts with FEL probes 5’ of the fusion site (data not shown) suggested that the der(4) was not expressed at significant levels or comigrated with the wild type FEL transcripts. < Fig 3. Mapping of FEL gene to chromosome band 4q21. (A) Gbanded chromosome 4 ideogram at the 850-band level** illustrates the mapping of the t(4;ll) clone to region q21-q31.2 by Southern blot analysis of somatic cell hybrids (SCH) and to bandq21 by FISH. The bars to the right represent specific regions of human chromosome 4 contained in somatic cell hybrids. (a) Somatic cell hybrid linesthat retained an intact human chromosome 4; (b) a hybrid that underwent a spontaneous translocation between human chromosome 4 (witha breakpointat or near the centromere) and a Chinese hamster chromosome, and retained only the long arm (cen-qter); (c) a hybrid retaining only 4q21-qter (hybrids [b] and [c] described ref 29). (d) A somatic cell hybrid line that retains the der(X)t(X:4) (p21.2;q31.22) described in.zo Hybrids (a), (b), and (c) were scored as positive for all the human-specific FEL Hindlll fragments, while (d) was scored as negative. Bracket labeled SCH represents the shortest region of overlap. Left-hand bracket represents the FISH results that place the FEL locus at 4q21. (6)Representative partial metaphase spread from the FISH experiment with specific signals on chromosome 4 at band q21 (arrowhead). From www.bloodjournal.org by guest on August 3, 2017. For personal use only. MORRISSEY ET AL 1128 a 1-trrrln.l m H p m a ~~~~~~~~~~~~~~~c~gggacag~~g~aaagcgagaagagcc~agaaaccqaagcacaqaaacq ~~~9gt~99~gt~~9~9~99cp+99~9~~CCCC~cCC9~caagaCtaCCCcaqagcaaq~~gc~~g~gtaga~gacgaac~gaccagccaccc ~ A F T E R V N S S G N cgcacrg~cc99~~cacccacagaaaqagtcucagcagcqgcaac ryP. Typr #-t*rBinal 8 mOqu.nc* cagcacaa~cgqgca99C~CCggc~ccgcggCcCC~agcgtgqgggcccgcc~ccccccccccccdq~aacc l5 ~ C 9 C C ~ 9 9 C g ~ ~ C C 9 C ~ ~ g C C C C 9 9 ~ ~ t C 9 9 d C O C ~ ~ C 9 C t C C C C O 9 9 C C C 9 C C C 9 d ~ ~ 9 U g ~ C C ~ C C g C ~ ~ I ~ C C C1C7 5~ ~ C ~ C C C ~ 9 d C ~ C C a C C d q 9 a t t ~ 9 C 9 C 9 ~ 9 C C ~ ~ ~ ~ C 9 ~ C C ~ ~ ~ C C C ~ ~ 9 c t ~ C 9 c c 9 ~ 9 9 a ~ c c c g g g g c C ~ a g a 9 ~ g g c a ~ c ~ ~ g c a ~2 7~5a c ~ g g g c g c c g c g c ~ g q a ~ ~ q g CCCCC9CC~CCC9CCda~C99t9d9C9Cg9C9CC99C~9C~999CC9C99g9C9~~99C9CtCdC99~C99~d9~CCCCC99CtCCdCdd 3 795C C 9 d ~ M A A Q S &aacaqcccaqCca S L Y N D D R N L L R I R E K E R R N Q E A H Q E K E A a ~ t C C g t a C ~ a C g ~ ~ g a ~ ~ g ~ a a c c C g c C c c g a a c C a g a g ~ g ~ a g g a a ~ g a c g c a a c c a g g ~ a g c c c a c c a a g a g a a4 7a5g a q q c a t F P E K I P L F G E P Y K T A K G D E L S S R I Q N M L G N Y E C V t c C ~ C 9 a a a ~ 9 a C C ~ ~ ~ ~ C C C t t g 9 a ~ ~ g c c c C a c a g g a c a g c d a a a g g c g a C g a g c c q C c c d g c c q a a c a c a q ~ d c ~ c g t c g g g a a a c c515 acg~~qaaqc K E F L S T K S H T H R L D A S E N R L G K P K Y P L I P D K G S qaaqqaqcccctcagtac~aaqtctcacactcatcqcc~qqacqcttccqaa~acaggccqqgaaaqccqaaacacccrttaactc~tqacaaaqq~aqc 675 S I P S S S F H T S V H H Q S I H T P A S G P L S V G N I S H N P a g C a C C ~ ~ a C C C a q C C ~ ~ C C ~ ~ a ~ a ~ C a q t q t ~ ~ ~ c ~ ~ C c ~ q C ~ ~ a C C ~ a c a ~ C c c c g ~ g c c r g g ~ c ~ c c c c c r g c c q g c1 ~ 7 5a c a c c a q c c a c a a t c c a a K M A Q P R T E P M P S L H A K S C G P P D S Q H L T Q D R L G Q E aqaCgg~g~agCcaagaaCCgaacca~CgccaagC~C~CaCg~~aaa~gCfg~ggcccacc~acagccagc~cccgacccaggatcgccctqqccaqqa 875 G F G S S H H K K G D R R A D G D H C A S V T D S A P E R E L S P qgggCCcqgcCcCaqtcaCcacaaqaaaqqcgaccgaa9a9cC9acggagacc~ccqcgcCccqgtqacagdcccgqccccagdg~ggg~gccrtccccc 975 L I S L P S P V P P L S P I H S N Q Q T L P R T Q G S S K V H G S t t a a C C C C t C C 9 C C C C C C C C d 9 t C C C C C C C C C 9 C C d C C C ~ C d C a C C C C ~ ~ C C ~ 9 C ~ d ~ C C C C C C C C C 9 9 ~ C 9 C ~ ~ g g ~ ~ g C ~ g1C0~1 5 ~ggCtC~Cqq~d~C~ S N N S K G Y C P A K S P K D L A V K V H D K E T P Q D S L V A P A gcaa~aacag~aaaggccarcgcccagccaaacccccca~ggacccagc~gcgaaagcccacgacadagagaccccccaag~caqcctgqcggcc~:tg~ 1175 Q P P S Q T F P P P S L P S K S V A ~ Q Q K P T A Y V R P M D C Q ~ ~ a ~ C C ~ ~ C t C C C C a 9 a C ~ C C t ~ C a C C C ~ ~ ~ t C ~ ~ C ~ ~ C C C C ~ a a ~ a q t g t c q c a a c q c a g c a g a d q c c c d c g g c c c d c g c c c g g c1275 ccacqgacqqtcaa D Q A P S E S P E L K P L P E D Y R Q Q T F E K T D L K V P A K A qatCa9~~~CCCagC~daCcccctgaacCgaaaccacCqccqgaggacCaccqacagcaqacctccqaaaaaac~g~cccqaaagcgcctqccaaaqcca 1375 K L T R L K ~ P S Q S V E Q T Y S N E V H C V E E I L a g c ~ c a c c a ~ a c ~ q a a q a c g c c c c c c c a g c c a g t c g a q c a q a c c c a c c c c a a c g a a g c c c a c c g c g t c g a a g a g a c c c c ~ a ~ g a ~ ~ c1 ~4 7t5c ~ ~ t q W P P P L T A I H T P S T A E P S K F P F P T K D S Q H V S S V T Q gcCgcCCcctCCg~CagcaacacaCa~ccta~acdgcCgagccacccaagtcccccccccctaca~agg~cccccagcacgccagccccqcaacccaa 1575 N Q K Q Y D T S S K T H S N S Q Q G T S S ~ L E D D L Q L S D S E aa~caaaaa~ad~a~gaca~acccccaaaaactcacccaa~tccccagcaaqgaacgccdcccacgcccgaagacg~cccccagcccagcgacagtgaqq 1675 D S D S E Q T P E K P P S S S A P P S A P Q S L P E P V A S A H S S acagCqacagtqaacaaaccccagagaagccCcccCccCcaCcCgcacccccd~gCgccccdcagccccCCccagaaccagCggc~ccagcacatcccaq 1 7 1 5 S A E S E S T S D S D S S S D S E S E S S S S D S E ~ N E P L E T ~aqtg~agagt~agaaag~accdgCg~cccagacagccccCc~qacccagdgagcgagagcagCccaagcgac~gcgaagaaa~cqagcccccaqaaacc 1875 P A P E P E P P T T N K W Q L D N Y L T K V S Q P A A P P E G P R ccagcrccggaqccCqaqccccca~caac~aac~aacggcagcCggaca~ccggccqaccaa~gccagccagccagccg~cc~ccagagqqccccaqqa 1975 S T E P P R R H P E S K G S S D S A T S Q E H S E S K D P P P K S S gcacaqagcccccdcqgcggc~ccc~gagagcaagggcagc~gcqacagcqcc~cgagccagg~gc~cCccgaacccaaag~ccccccccccaaaagccc 2075 S K A P R A P P E A P H P G R R S C Q K S P A Q Q E P P Q R Q T V caqcaaagccccccgqqccccacccga~gccccccaccccggaaagaggagccgCc~ga~gCccccggcacagcaggagcccccacaaaggcaaacc~Cc 2175 G T K Q P K K P V K A S A R A G S R T S L Q G G R E P G L L P Y G qgdaccaaacaaccc~~aaaacccgCcaaggc~cCgcccgggcaggcCucggaccagccCgcdgg999~aa~gdgcug9gccCctCccccat~~cC 2275 S R D Q T S K D K P K V K T K G R P R A A A S N E P K P A V P P S S CCCgagdCC.qaCttCC4~~gaCa~gCCC~a9gtg~dgaC9~~~gg~CggCCCCg~CCgC~9C~dgC~dC9aaCCCddgCCdgCa~C9C 2C 3 7C5C C C C C C a 9 E K K K B K S S L P A P S K A L S G P E P A K O N V E D R T P E H tqaqaagaagaaqcac~~gagccccccccccgccccccccaaggctcccccaggcccagdacccgcgaaggacaacgcggaqqacaggacccctqaqcac 2 4 7 5 F A L V P L T E S Q G P P H S G S G S R T S F C R S R G G P G G Q rccg~r~ccgcc~~~~cgaccgagagccaggqcccaccccacagcggcagcggcagcaggaccagtccccgccgaagccgtggcqqccca~qaqgaca~c 2575 P Q R Q T P I A F E R H Q A A L T A Q D T P P P Q S L M V K I T L D c g c a a a g a c ~ g a c C c c c ~ C C g c c c C C g a g a g a ~ c c a ~ g c C g c C c c c a c ~ c C c a g g a c ~ C t c c C c c C C C ~ C ~ ~ a g c C C g a C g g t g a a ~2a6C7 C 5 ~CcCt~9a L L S R I P Q P P G K G A A R G K Q K I N S R P P K K H S S ~ K R ~ ~ c g ~ c c c c t ~ g g a c a ~ ~ ~ ~ ~ g c c c c c c g g g a a g g g a g c c g c c a g ~ g g ~ ~ ~ g c a g a a g a c a ~ ~ c ~ g c ~ c c c g c c g ~ a g a a q c a2c1a1 5g c c c C q a g a a 9 a 9 9 S S D S S S K L . ~ ~ G E A E R D C D N K K I R L E K E I K S a g C C C a q ~ C d g C t C l d g C a a g t C g g C ~ ~ ~ ~ d g ~ g ~ ~ ~ ~ g C g A ~ g C ~ g ~ ~ ~ g ~ g ~ C C g C g * C ~ d C ~ ~ g ~ ~ ~2~8 C 1 5C ~ g ~ C C g g ~ g d d g 9 a ~ d C C d ~ d C C a C Q S S S S S S S H K E S S K T K P S R P S S Q S S K K E M L P P P P a q t C a t C C C C a C C C C C a C C C C C c C d C a a a g a ~ C C t t C C 4 d ~ ~ C ~ ~ ~ g C C c t C C d g ~ C C C C C ~ C ~ C ~ ~ C C C C a d A g a a g g ~ ~ a ~2915 9C~CCCCCC~C=aCC V S S S S Q K P A K P A L K R S R R E A D T C G Q D P P K V P A V C g C q t C C C C q t C C t C C C a g a d g C C d ~ C C A ~ g C ~ g C ~ C ~ C ~ ~ g ~ ~ C C ~ d g g C g g g ~ ~ g C ~ g ~ C d C C C 9 C g 9 C ~ 9 g a C3C0 7C5C C C ~ ~ ~ ~ g ~ 9 C C ~ 9 C a 9 C a P R V N H K D S S I P K Q R R V E G K G S R S S S A D K G S S G D ccaaqaqtcaaccacaaagaccccCcc~ttcccaa~cagagaagagCagaggggaagggcCccaqaagcCcct~gcagacraggggCCcCCccqqaqata 3115 T A N P F P V P S L P N G N S K P G K P Q V K F D K Q Q A D L H X R crqcaaarcctccrccagcqccccccccqccaa~~gqc~ac~craaaccagggaaqccccaagtgaagt~~gac~aacaacaagcagacc~ccacacqaq 3275 E A K K ~ K Q K A E L M T D R V G K A F K Y L E A V L S F I E C G qqaqqcaaaaaagarqaagc~gaaagcagagccaacgaerjgacagggccgg~aagqcccccaagC~ccCgga~qccgCcCCqCcctCcaCtqdqCqcq9a 3315 I A T E S E S Q S S K S A Y S V Y S E T V D L I K F X ~ S L K S F actqccacagagccrqaaagcc~gccacccaagtcagcccaccccgccc~cccagaaacc~~a~accCca~~aaaccca~aatg~ 3475 c a ~ c a a ~ aFig ~ c c ~4. ~ ~ ~ Nucleotide and preS D A T A P T Q E K I F A V L C M R C Q S I L N M A ~ F R C K K D I dicted amino acid sequences for caqacqccacagcqccaacacaagaga~~~catccgccqtcccacgcacgc~ccqccdgtccaCtccgaacaCqgcgacqccCcqccqcaaaaaaqacaC 3515 wild-type FEL. The nucleotide A I K Y S R T L N K H F E S S S K D R P G T F S M H C K K H R H T ~qcaat~aaqcacc~t~gcacccctaataaacaccccgagaqttctcccaaagarerjccca~caccttcCccat~caCC~caa~ 3675 aa~c~~a sequence ~~cd~~~~ for wild-type FEL I P S F P N A F S C Q L R R V P V K C Y Q C G E Q W G G C H Y Q H a c ~ ~ ~ ~ t ~ t c t ~ ~ ~ ~ a ~ t g c c r c ~ ~ ~ c r g ~ ~ a q c t ~ ~ g t a g g g c ~ ~ c a g c ~ a ~ g t g ~ c q g c a g t g + g q g g a q c a q c g3g1g7 5q t g g ctranscrip~ t g c c ~ c C a Care c a qshown c a c c in a 5 to 3 direction. Diierent 5 seP S H H P D M T S S Y V T I T S H V L T A F D L W E Q P R P S R G R ~ ~ a g t ~ a ~ ~ a c ~ ~ a g ~ c a t q a ~ a c c c c ~ ~ c a c g t ~ a ~ c a c c a ~ a t ~ ~ ~3 8 1a5 c g tquences c ~ t c containing a ~ ~ q ~ ~ alternative f ~ ~ g ~ ~ ~ ~ ~ ~ ~ ~ I K N S L L G S D K C V H L G P Q Q Q F G G P G A L Y T T G F S A ~ ~ c a a a q a a t ~ c ~ ~ c q c t c q q c c ~ a g a c a a a t g t g t g ~ a ~ ~ ~ t q q c c c c ~ a a ~ a g ~ a ~ c c 9 q c g g a ~ ~ c q g c g c a c t a r a c a3975 c q a c a q qATG q c ~ c ~stert c a g c codons a are denoted A T R I N Q N T L M E P Q V D S M P W E L F L H I G S L K N S P D types A and B, respectively. q ~ t a ~ a a q ~ ~ c c ~ a ~ ~ a a a a ~ ~ ~ ~ t c a a c q q ~ q c c c ~ a q g c c ~ a t t c a a t q c c c c g q g a a c r a r c r t t q c a c a c c g g a a q c c t c a a4015 a a a c a q ~~~i~~ c c a ~ a c qacids are sbwn insin- V C F I R T P N S K K E A P R D C Q D I C H L N S Q Q Q C D H ~ L D cctg~tt~at~aqqa~a~~aaacccca~aaaaqaaqcaccacqag~cqqccaggacatttqccactcaaacccccaac~acagcqcgatcaccgqcC~qa 4115 gle-leeer code. t(4;11) fusion T V V M Q K Q R . site is indicatedwith horizontal caccg~ggcratqcaqaaqcaqaqatgaqqaggccqqc~ca~agarqaccccg~ccftcc~aactraaggacaqa~qrgcaacctaqcttaaacqq~CqC 4215 acgaacqgt~taqaaa~atctctatcccccctccaaaccaqc~ggacacaacqccacctcagtaqccacqccagtccactcccagaaggaaaCC:c~~:: 4375 bracket' Underlines indicate nut t t a a ~ a a t ~ a ~ t t t t g g ~ a a a g g g c t c c q c g q a ~ q a c t t ~ t ~ c t c c c c c g ~ ~ c c t g g g a g a a a c a ~ ~ ~ g c c t a a c a a c c t c c a a c4q4g1 c 5 c a c ccleotide cqca~a (Kozak consensus23) c c a c a a q t a a t q a a g q a c t c c a c c q t g c c c c a c c c c c t g c c a a c q a a c a ~ c q q c c c g a c a ~ ~ a c c a d ~ ~ a ~ t g t ~ ~ t a a ~ r t4575 a ~ a a a a ~and t ~ a aprotein ~ ~ ~ ~ ~ ~(nuclearlocalization C C C C ~ C C c C t g C C ~ C C c c C a a C c t C C C d C C ~ C ~ ~ ~ ~ q ~ C ~ ~ C a C ~ C C ~ ~ C C C C ~ g C C C C g ~ C C C C ~ C ~ C ~ d C4615 C~C~CC~d~~gCC~qCCC~CgC~9~~ and GTP-binding' motifs gcg~tqaqqaqgagccacagcaca~gqqq~gcacc~cg~ggCctgcacaggaggactcqcgctqc~~CC~Cacccrgcca~~tccc~cccctcc~tcaqc 4115 aacagqqactctctaacaqqgcagccaccqccgaccccaCC 481s scribed in the text. de- From www.bloodjournal.org by guest on August 3, 2017. For personal use only. HRX FUSION TRANSCRIPT IN t(4;ll) LEUKEMIAS 1129 t(4;l I ) translocations.’ Expression of both derivative products is not unexpected because both wild-type HRX and FEL promoters are active in lymphoid cells. Our inability to detect or clone derivative 4 products from ALL no. 1 is consistent with detection of only one rearranged HRX band on Southern blots and may reflect deletion of chromosome 1 1 DNA 3’ (telomeric) of the breakpoint which has been observed previously.* These features underscore the pathogenetic importance of the derivative 1 I fusion product as proposed earliee and supported by the cytogenetic features of three-way transIocation~.~~ The FEL gene appears to be expressed normally in lymphoid cells because transcripts were detected in all cell lines examined (Fig I and data not shown). Characterization of cDNA clones showed two alternative amino termini for FEL that appeared to result from differential utilization of 5’ exons. It is not clear from our data whether the two wild-type FEL transcripts observed on Northern blots correlate with utilization of alternative 5’ exons or result from other differential processing of the FEL transcript. Because it is unlikely that the minor amino terminal variations in the predicted FEL proteins confer functional differences, it is possible that the 5’ FEL exons are associated with alternative promoters, but additional studies are required to address their significance and possible differential regulation. The predicted FEL protein shares several interesting features with ENL, the previously reported protein fused to HRX after t( I 1;19) translocations in ALL.6 Both proteins are unusually serine- and proline-rich; in fact, serines and prolines Nucleotide sequence analyses of 4; I I fusion transcripts showed that the junction of HRX and FEL sequences occurred at or near amino acids I406 of HRX and 362 of FEL, the precise point undetermined because of a 5-nucleotide homology between HRXand FEL at the fusion site (Fig 5A). Based on these observations, the HRX-FEL fusion protein had a predicted length of 2,319 amino acids. The fusion site in HRX is 34 amino acids upstream of that observed for the fusion protein described in the t( I 1;19)-carrying cell line HBl I 19.6 The fusion site in FEL is 15 amino acids downstream to that observed in a partial 4; I I clone reported prev i o ~ s l yThese .~ observations suggested that translocation-associated fusion sites in HRX and FEL are not invariant but are restricted in their distribution. DISCUSSION The studies presented here establish a complete structure for the product of the chromosome 4 gene involved in t(4; 1 1) translocations in acute leukemias. Fusion of FEL to HRX appears to be a consistent feature of t(4; I 1) (q21;q23) translocations. Both t(4;l I)-carrying cell lines in this study contained FEL-HRX fusion transcripts of comparable size detectable with an FEL cDNA probe isolated from a t(4;l l) leukemia. In addition, partial sequence analyses of fusion cDNAs reported earlier further support the involvement of FEL (AF-4) in the RS4;l I cell line.7 Although we detected and characterized the derivative 1 1 translocation product, other studies have reported the presence of both derivative 4 and 1 I fusion products following A Fusion site n ATC AGA GTG GAC TTT AAG GAG GAT TGT GAA GCA GAA AAT GTG I R V D F K E D C E A E N V HRX 1401 I I I I 1414 I HRX-FEL ATC AGA GTG GAC TTT AAG GAA ATG ACC CAT TCA TGG CCG CCT I R V D F K E M T H S W P P FEL GTT GAA GAG ATT CTG AAG GAA ATG ACC CAT TCA TGG CCG CCT V E E I L K E M T H S W P P 1 1 1 1 1 1 1 I I 356 B A-T hooks HRX I Ill A-T hodts 369 1O00 2060 3OoO I I I zinc finaen, I SP-rich HRX-FEL Fig 5. Structure of H R X 4 1 1 fusion products. (A) Nucleotide and predictedpmtein sequences at fusion sites betweenHRX and E L RNAs. (B)Schematic representation of wild-type and fusion proteins involved in the t ( 4 ; l l ) and t ( l l ; l 9 ) . 411 der(l1) product SP-rich FEL A.T honks SP-rich 11;19 der(l1) product From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 1130 MORRISSEY ET AL constitute fully 26% of the residues in each protein. ENL and FEL are both positively charged, hydrophilic proteins with many potential phosphorylation sites. Both ENL and FEL may be nuclear proteins as each contains sequences homologous to consensus nuclear localization sequences present in known nuclear proteins.25In addition, both proteins contain sequences that conform to a consensus GTP-binding site (GXXXXGK) suggesting possible energy requiring functions for ENL and E L . Despite these similarities, ENL and FEL are not significantly homologous to one another beyond their overall amino acid composition nor are they significantly similar to previously reported proteins preventing further functional comparisons. The data suggest that ENL and FEL may be widely expressed, nuclear proteins without obvious DNA binding motifs. Because serine/proline-rich domains have been associated with transcriptional activator proteins, ENL and FEL may play an accessory role in some aspects of transcription regulation. Interestingly, as a result of translocations with the 1 lq23 HRYgene, large C-terminal portions of ENL and FEL are fused to the amino terminal portion of HRX that previous studies have demonstrated contains motifs (AT hooks and 'SPKK') implicated in minor groove DNA binding.6326,27 Therefore, a consistent feature of two 1 lq23 translocations is the fusion of HRX DNA binding motifs with different serine/proline-rich partners resulting in proteins that could bind DNA inappropriately and function as chimeric transcription factors. Comparison of the chimeric products resulting from t( 1 1;19) and t(4; 1 1) translocations supports the previous hypothesis6 that an important contribution of HRX to 1 lq23 translocation-associatedfusion proteins is its amino terminal DNA binding domain. A common HRX DNA binding motif in both the HRX-ENL and HRX-FEL fusion proteins suggests they may deregulate a similar set of subordinate genes, although the sequence-specific DNA binding properties of the HRX motifs remain to be determined. The importance and specific contributions of each fusion partner in the t(4; 1 l), t( 1 1;19), and other 1 lq23 translocations to different leukemia phenotypes are not resolved by our studies. The compositional similarities of FEL and ENL may imply a similar leukemogenic role; however, their respective contributions and potential transcriptional properties need to be addressed in appropriate model systems. ACKNOWLEDGMENT We thank Cita Nicolas and Roxane Brown for technical assistance and Phil Verzola and Judith Quenvold for photography. REFERENCES 1. Abe R, Sandberg AA: Significance of abnormalities involving chromosomal segment 1lq22-25 in acute leukemia. Cancer Genet Cytogenet 13: 121, 1984 2. Mitelman F: Catalog of Chromosome Aberrations in Cancer (ed 3). New York, NY, Lis, 1988 3. Kaneko Y, Shikano T, Maseki N, Sakurai M, Sakurai M, Takeda T, Hiyoshi Y, Miyama J-I, Fujimoto T: Clinical characteristics of infant acute leukemia with or without 1 lq23 translocations.Leukemia 2:672, 1988 4. Raimondi SC, Peiper SC, Kitchingman GR, Behm FG, Williams DL, Hancock ML, Mirro J: Childhood acute lymphoblastic leukemia with chromosomal breakpoints at 1 lq23. Blood 73:1627, 1989 5. Shimizu H, Culbert SJ, Cork A, Iacuone JI: A lineage switch in acute monocytic leukemia. Am J Pediatr Hematol Oncol 11: 162, 1989 6. Tkachuk DC,Kohler S, Cleary M L Involvement ofa homolog of Drosophila trithoraxby 1lq23 chromosomal translocations in acute leukemias. Cell 7 1:69 1, 1992 7. Gu Y, Nakamura T, Alder H, Prasad R, Canaani 0, Cimino G, Croce CM, Canaani E: The t(4;ll) chromosome translocation of human acute leukemias fuses the ALL-I gene, related to Drosophila trithorax, to the AF-4 gene. Cell 71:701, 1992 8. Rowley JD, Diaz MO, Espinosa R, Patel YD, van Melle E, Ziemin S,Taillon-Miller P, Lichter P, Evans GA, Kersey JH, Ward DC, Domer PH, LeBeau MM: Mapping chromosome band I lq23 in human acute leukemia with biotinylated probes: Identification of 1 lq23 translocation breakpoints with a yeast artificial chromosome. Proc Natl Acad Sci USA 87:9358, 1990 9. Ziemin-van der Poel S, McCabe NR,Gill HJ, Espinosa R, Patel Y, Harden A, Rubinelli P, Smith SD, LeBeau MM, Rowley JD, Diaz M O Identification of a gene, MLL, that spans the breakpoint in 1 lq23 translocations associated with human leukemias. Proc Natl Acad Sci USA 86:10735, 1991 10. Djabali M, Selleri L, Parry P, Bower M, Young BD, Evans G: A trithorax-like gene is interrupted by chromosome 1lq23 translocations in acute leukemias. Nature Genetics 2:113, 1992 1 1. Mazo AM, Huang D-H, Mozer BA, Dawid IB: The tnthorax gene, a trans-acting regulator of the bithorax complex in Drosophila, encodes a protein with zinc-binding domains. Proc Natl Acad Sci USA 87:2112, 1990 12. Gu Y, Cimino G, Alder H, Nakamura T, Prasad R, Canaani 0,Moir DT, Jones C, Nowell PC, Croce CM, Canaani E: The t(4;ll) (92 1;q23) chromosome translocations in acute leukemias involve the VDJ recombinase. Proc Natl Acad Sci USA 89:10464, 1992 13. Cimino G, Moir DT, Canaani 0, Williams K, Crist WM, Katzav S, Cannizzaro L, Lange B, Nowell PC, Croce CM, Canaani E Cloning of ALL-I ,the locus involved in leukemias with the t(4; 1 1) (q21;q23), t(9;ll) (p22;q23), and t(l1;19) (q23;p13) chromosome translocations. Cancer Res 5 1:67 12, 199I 14. Cimino G, Nakamura T, Gu Y, Canaani 0, Prasad R, Crist WM, Carroll AJ, Baer M, Bloomfield CD, Nowell PC, Croce CM, Canaani E An altered 1 I-kilobase transcript in leukemic cell lines with the t(4;ll) (q21;q23) chromosome translocation. Cancer Res 52:3811, 1992 15. Stong RC, Kersey JH: In vitro culture of leukemic cells in t(4;I 1) acute leukemia. Blood 66:439, 1985 16. Lange B, Valtieri M, Santoli D, Caracciolo D, Mavilio F, Gemperlein I, Griffin C, Emanuel B, Finan J, Nowell P, Rovera G: Growth factor requirements of childhood acute leukemia: Establishment of GM-CSF dependent cell lines. Blood 70192, 1987 17. Hunger SP, Ohyashiki K, Toyama K, Cleary M L HLF, a novel hepatic bZIP protein, shows altered DNA-binding properties following fusion to E2A in t( 17;19)-ALL. Genes Dev 6: 1608, 1992 18. Cleary ML, Smith SD, Sklar J: Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t( 1438) translocation. Cell 47:19, 1986 19. Francke U, Yang-Feng TL, Brissenden JE, Ullrich A: Chromosomal mapping of genes involved in growth control. Cold Spring Harbor Symp Quant Biol51:855, 1986 20. Giacalone JP, Francke U: Common sequence motifs at the rearrangement sites of a constitutional X/autosome translocation and associated deletion. Am J Hum Genet 50725, 1992 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. HRX FUSION TRANSCRIPT IN t(4;ll) LEUKEMIAS 2 1. Milatovich A, Travis A, Grosschedl R, Francke U: Gene for lymphoid enhancer-bindingfactor (LEFl) mapped to human chromosome 4 (q23-q25) and mouse chromosome 3 near Egf. Genomics 11:1040, 1991 22. Zakl BU, Naylor SL, Sakaguchi AY, Bell GI, Shows TB: High resolution chromosomal localization of human genes for amylase, proopiomelanocortin, somatostatin,and a DNA fragment (D3S1) by in situ hybridization. Proc Natl Acad Aci USA 806932, 1983 23. K O AM: Point mutationsdefine a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44283, 1986 24. Rowley JD. The der(l1) chromosome contains the critical breakpoint junction in the 4;11, 9;11, and 11;19 translocations in acute leukemia. Genes Chrom Cancer 5:264, 1992 1131 25. Silver PA: How proteins enter the nucleus. Cell 64489, 1991 26. Reeves R, Nissen MS: The AT-DNA-binding domain of mammalian high mobility group I chromosomal proteins: A novel peptide motif for recognizing DNA structure.J Biol Chem 265:8573, 1990 27. Solomon MJ, Straws F, Varshavsky A: A mammalian high mobility group protein recognizes any stretch of six A-T base pairs in duplex DNA. Roc Natl Acad Sci USA 83:1276, 1986 28. Francke U: High resolution ideograms of trypsin-Giemsa banded human chromosomes. Cytogenet Cell Genet 3 1:24, 1981 29. Brissenden JE, Ullrich A, Francke U Human chromosomal mapping of genes for insulin-like growth factors I and I1 and epidermal growth factor. Nature 310781, 1984 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 1993 81: 1124-1131 A serine/proline-rich protein is fused to HRX in t(4;11) acute leukemias J Morrissey, DC Tkachuk, A Milatovich, U Francke, M Link and ML Cleary Updated information and services can be found at: http://www.bloodjournal.org/content/81/5/1124.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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