1995 85: 902-911  

F Dong, M van Paassen, C van Buitenen, LH Hoefsloot, B Lowenberg and IP Touw overexpression of a novel G-CSF-R isoform(G-CSF-R) gene in a case of acute myeloid leukemia results in the A point mutation in the granulocyte colony-stimulating factor receptor 

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RAPID COMMUNICATION

A Point Mutation in the Granulocyte Colony-Stimulating Factor Receptor (G-CSF-R) Gene in a Case of Acute Myeloid Leukemia Results in the

Overexpression of a Novel G-CSF-R Isoform By Fan Dong, Marleen van Paassen, Carin van Buitenen, Lies H. Hoefsloot, Bob Lowenberg, and Ivo P. Touw

A novel human granulocyte colony-stimulating factor (G- CSF) receptor isoform, designated SD, has been identified in which the distal C-terminal cytoplasmic region, previously shown to be essential for maturation signalling, is substi- tuted by an altered C-terminus. The SD receptor has a high affinity for G-CSF and retains the membrane-proximal cyto- plasmic region known to be sufficient for proliferative signal- ling. Nonetheless, the SD isoform lacks the ability to trans- duce growth signals in murine BAR cells and, in contrast to the wild-type G-CSF receptor, is scarcely capable of acti-

RANULOCYTE colony-stimulating factor (G-CSF) promotes the proliferation, differentiation, and sur-

vival of progenitor cells that predominantly form granulo- cytic colonies in vitro.’ G-CSF is the major growth factor responsible for the regulation of granulopoiesis and also modulates certain functions of mature granulocytes. In addi- tion, G-CSF appears to have a role in activating primitive pluripotent progenitor or stem cells into cell cycle and in inducing their pr~liferation.~.~ These biologic activities of G- CSF have led to the application of G-CSF therapy in a wide variety of clinical conditions6

The diverse activities of G-CSF are initiated after binding of G-CSF to a specific receptor (G-CSF-R) that belongs to the superfamily of cytokinehematopoietin receptors.’.* Al- though the majority of the family members, including the receptors for interleukin-2 (IL-2) to IL-7 and granulocyte- macrophage colony-stimulating factor (GM-CSF), are acti- vated through the formation of heteromeric complexes com- prising a, /3, and sometimes y sub unit^,^-'^ G-CSF-R proteins are thought to form homo(di)meric complexes upon ligand binding.l3.I4 When expressed in murine hematopoietic cells, the murine and human G-CSF-Rs are able to bind to G-CSF with high affinity and transduce G-CSF-dependent signals for proliferation and

Four different forms of the human G-CSF-R, arising from alternative RNA splicing, have been isolated from several cellular sources, one of them being a soluble receptor pro- tein.‘*.I9 The G-CSF-R protein containing a cytoplasmic do-

G

From the Department of Hematology, Dr. Daniel den Hoed Can- cer Center, Rotterdam; and the Institute of Hematology, Erasmus University, Rotterdam, The Netherlands.

Submitted August 9, 1994; accepted November 15, 1994. Supported by the Dutch Cancer Foundation. Address reprint requests to Ivo P. Touw, PhD, Dr. Daniel den

Hoed Cancer Center, PO Box 5201, 3008 AE Rotterdam, The Neth- erlands.

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 V.S.C. section 1734 solely to indicate this fact.

0 1995 by The American Society of Hematology. 0006-4971/95/8504-0032$3.00/0

902

vating JAK2 kinase. Expression of SD receptor was found to be low in normal grnnuloqtos, but was significantly in- creased in a patient with acute myeloid leukemia (AML). The leukemic cells of this patient harbour a point mutation in the SD splice donor site of the G-CSF receptor gene. Those findings provide the first evidence that mutations in the G- CSF receptor gene can occur in certain cases of clinical de novo AML. the possible contribution of defective G-CSF re- ceptor signalling to leukemogenesis is discussed. 0 1995 by The American Society of Hematology.

main of 183 amino acids shows strongest homology to the murine G-CSF-R and represents the human homologue of the wild-type (WT) G-CSF-R. Although neither type of the G-CSF-R contains consensus motifs in the cytoplasmic do- main that would indicate kinase activities, certain sequence similarities have been identified that are conserved in several members of the cytokine receptor superfamily. Two seg- ments, called box l and box 2, localized in the membrane- proximal region of the G-CSF-R, are also present in most members of cytokine receptor superfamily?o,2’ This mem- brane-proximal region has been shown to be essential for transmitting growth ~ i g n a l s . ~ ~ , ’ ~ . ~ ~ The C-terminal region of the WT G-CSF-R, which contains a segment called box 3 that is shared only with IL-6 signal transducer gp130,22.23 is involved in transduction of maturation signals and in down- modulation of proliferative signalling by the G-CSF-R.’’.16

We report here the molecular cloning of a new G-CSF-R isoform, designated SD, in which the C-terminal cytoplasmic region of the WT G-CSF-R distal to box 2 is replaced by an altered terminus. Although the SD receptor retains the membrane-proximal region including box 1 and box 2 that is essential for proliferative signalling and JAK2 activation, it fails to transduce growth signals in murine B M 3 cells and only marginally induces tyrosine phosphorylation of the JAK2 kinase. The expression of SD receptor is low in normal granulocytes, but is significantly elevated in the leukemic cells of a patient with acute myeloid leukemia (AML). The AML cells from this patient carry a point mutation in the SD splice donor site of the G-CSF-R gene. To our knowl- edge, this is the first case of de novo AML in which a mutation in the G-CSF-R gene has been identified that may result in the altered signalling properties of the encoded receptor protein.

MATERIALS AND METHODS

AML cells and normal granufocytes. Peripheral blood and bone marrow samples were obtained from normal individuals and patients with AML after informed consent. AML cells and normal granulo- cytes were isolated as described.% In brief, leukemic cells were recovered from the interface after Ficoll-Isopaque separation. Subse- quently, T cells and monocytes were removed from this fraction by E-rosette Ficoll separation and adherence to plastic, re~pectively.’~ Normal granulocytes were obtained as sedimented cell fraction after

Blood, Vol 85, No 4 (February 15), 1995: pp 902-911

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G-CSF-R MUTATION IN AML 903

Ficoll-Isopaque centrifugation and further depleted of erythrocytes by hypotonic lysis.

Cell culture. Murine pro-B BAF3 cells were provided by G. Plaetinck (Roche Research, Ghent, Belgium) and were cultured in RPM1 1640 medium supplemented with 10% fetal calf medium (FCS) and 10 ng of murine IL-3 per milliliter. Subclones of BAF3 cells expressing the different G-CSF-R forms were established as described” and were maintained in the same culture medium as that for the parental BAF3 cells.

Polymerase chain reaction (PCR) ampl$ication and cloning. TO- tal RNA was isolated using the guanidinium thiocyanate method.” RNA (1 pg) was primed with oligo (dT) and reverse transcribed into cDNA in a 20-pL reaction volume containing 200 U reverse transcriptase of moloney murine leukemia virus (MLV; GIBCO- BRL, Breda, The Netherlands). Genomic DNA was prepared as described.26 PCR primers were designed according to the published sequence of the G-CSF-R cDNAI9 and are as follows: FW2, 5’- TGTGATCATCGTGACTCCCIT-3‘ (forward, nt 1664 to 1684); FW3, 5’-CTGCTGTTGnAACCTGCCTC-3’ (nt 2070 to 2090); FW4, 5‘-CCAAGAGCAGT”ITCCACCCAG~CC-3’ (nt 2361 to 2384); FWI16, 5’-ACCCTTTGTGITCCACCAGT-3’, (nt - 125 to -105, referring to the first nucleotide of exon 17 as +l); RV1, 5’-CAAsA1CTAGTTTACAATACTGAAG-3’ (reverse, nt 2923 to 2947); RV2, 5’-GTAGATCTTAGTCATGGGCTTATGG-3’ (nt 2745 to 2769). Mismatches (underscored) were introduced to create restriction enzyme sites. One tenth of RT reaction mixtures or 1 pg of genomic DNA was used for PCR amplification in a 50-pL volume containing 0.5 pmoVL of each primer, 0.2 mmoVL of dNTPs, 1 U of Taq polymerase, and I X standard Taq buffer (Promega Corp. Madison, W). PCR amplification was performed for 30 cycles con- sisting of 1 minute at 94”C, 2 minutes at 51T , and 2 minutes at 72°C for 30 cycles. After amplification, PCR fragments were size- separated on agarose gels, purified using Geneclean I1 kit (Bio 101, Inc, La Jolla, CA), and ligated to the HincII site of pBluescript vector (Stratagene Cloning Systems, La Jolla, CA).

Plasmid construction and transfection. To reconstitute the full- length coding sequence for the SD receptor, the corresponding PCR fragment (Fig 1B) was cloned in pBluescript vector. The 3’ portion of the fragment was then cut out from the vector with BsrFI and Cla I. The resulting fragment was used to replace the 3’ sequence of the WT G-CSF-R inserted in the pLNCX expression vector.27 DNA transfection of BAF3 cells was performed by electroporation using 20 pg expression construct linearized with Pvu I. Stably transfected cells were selected by their ability to grow in culture medium containing G418 (GIBCO-BE), as previously described.”

Radioactive G-CSF binding. Binding assays with 12sI-radiola- beled G-CSF were performed as de~cribed.’~ BAF/SD cells (1.6 X lo6) were incubated with titrated concentrations of I2’I-G-CSF (20 pmol/L to 5 nmoVL) in a 100-pL volume at 37°C for 1 hour. Cells were sedimented by centrifugation through ice-cold FCS and cell- associated radioactivity was measured. Incubations with excess non- labeled G-CSF were included to determine nonspecific binding. Data were analyzed as de~cribed.’~

3H-thymidine (TdR) incorporation assay. BAF/SD cells or leu- kemic cells from an AML patient were washed and incubated in triplicate with various concentrations of indicated growth factors at lo4 celldl00 pL in 96 microtiter plates. After 24 hours (BAF/SD) or 48 hours (AML cells) of incubation, 1 pCi of 3H-TdR (2 Ci/ mmoVL; Amersham Inc, Amersham, UK) was added to each well, and the cells were incubated for further 12 hours before harvesting. ’H-TdR incorporation was measured as described.15

Immunoprecipitation and immunoblotting. Cells were deprived of growth factors for 14 hours and incubated with G-CSF or IL-3 for indicated times. Stimulation was terminated by dilution in ice- cold phosphate-buffered saline (PBS) containing 10 pmoVL

Na3V04. Cells were lysed on ice in lysis buffer (50 mmoVL Tris [pH 7.51, 150 mmoVL NaCI, 1 mmoVL EDTA, 1% [voYvol] Triton X-100, 1 mmoVL phenylmethylsulfonyl fluoride [PMSF], 1 0 0 pmoY L Na3V04, and a cocktail of proteinase inhibitors). Cell lysates were centrifuged at 10,OOOg for 30 minutes and the resultant supernatants were incubated on ice for 90 minutes with rabbit polyclonal anti- JAK2 antiserum.” Protein A-sepharose beads (Pharmacia LKB, Up- psala, Sweden) were added, incubated on ice for 30 minutes, and washed extensively in lysis buffer. Bound proteins were eluted by boiling in sample buffer (50 mmoVL Tris [pH 6.81, 1% [wt/vol] sodium dodecyl sulfate [SDS], 10% [voVvol] glycerol, 1 mmoVL dithiothreitol, and 0.008% [wtlvol] bromophenol blue) and subjected to 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Pro- teins were electrotransferred onto nitrocellulose (Schleicher & Schuell, Dassel, Germany). Filters were blocked by incubation for 30 minutes in Tris-buffered saline (TBS; 10 mmol/L Tris [pH 7.41, 150 mmol/L NaCI) containing 3% bovine serum albumin, washed extensively in TBS-T (TBS with 0.05% Tween-20), and then incu- bated with the monoclonal antiphosphotyrosine antibody 4G10 (Up- state Biotechnology Inc, Lake Placid, NY). After washing three times in TBS-T, filters were probed with peroxidase-conjugated rabbit antimouse antibodies (DAKO MS, Glostrup, Denmark). Specific signals were detected by chemiluminescence (DuPont, ’sHertogen- bosch, The Netherlands).

In vitro kinase assay. Immunoprecipitated proteins on protein A-sepharose beads were washed extensively in lysis buffer (same as above but without the proteinase inhibitor cocktail) and then in kinase buffer (50 mmol/L NaCI, 5 mmoVL MgC12, 5 mmoVL MnCI2, 0.1 mmoUL Na3V04, 10 mmol/L HEPES, pH 7.4). The immunopre- cipitates were incubated in 20 pL of kinase buffer containing 1 pmoVL ATP and 0.5 mCi of [y-32P]ATP per milliliter (Amersham) for 25 minutes at room temperature. The reaction was terminated by addition of ice-cold lysis buffer. After extensive washing, the reaction products were eluted from the beads, separated by SDS- PAGE, and visualized by autoradiography.

Northern blotting. Poly(A)+ RNA was purified on oligo(dT) col- umns (Pharmacia). Five micrograms of total RNA from normal gran- ulocytes or 4 pg of poly(A)+ RNA from leukemic cells was electro- phoresed in a 1% agarose gel containing 2.2 molk formaldehyde and then transferred to a nylon filter (Amersham). The filter was hybridized with ”P-labeled DNA probes made with the method of random priming. Final washing of filters was performed at 65°C in 0.3X SSC (1 X SSC is 0.15 moUL NaCI, 0.015 moVL sodium citrate).

RESULTS

Cloning of the G-CSF-R cDNAs from human granulo- cytes. To isolate the human G-CSF-R cDNAs, RT-PCR was performed on total RNA extracted from peripheral blood granulocytes of healthy individuals by using primers FW2 and RV1 (Fig 1A). Southern blot analysis of PCR products using the full-length G-CSF-R cDNA as a probe consistently showed 4 bands (Fig 1B). When RT-PCR was performed on RNA isolated from BAF3 cells transfected with human WT G-CSF-R cDNA or with primers FW2 and RV2 (Fig lA), only one fragment could be detected (Fig 1B and data not shown). These PCR fragments were subcloned and se- quenced. The largest fragment corresponded to the WT G- CSF-R. Unexpectedly, subclones obtained from the two mid- dle-sized fragments all had a nucleotide sequence identical to the previously described splice variant D7.I’ The reason for this phenomenon is unknown. However, similar observa- tions have been made by othersz9 and it has been suggested

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904 DONG ET AL

B 1 W

2 ( W - 1.58 - 1.38

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that some PCR products migrate abnormally on agarose gels because of heteroduplex formation."

The smallest PCR fragment had a 549-nt deletion (nt 2283-2831, Fig 1A) as compared with the WT G-CSF-R cDNA." Analysis of nucleotide sequence flanking the dele- tion boundaries showed potential RNA splicing donor and acceptor sites (Fig IC). The splice acceptor site of the SD receptor is shared with splice variant D7 (Fig IA). The pat- tern of alternative splicing for SD receptor results in a re- placement of the C-terminus of the WT G-CSF-R immedi- ately downstream of box 2 by a C-terminal tail of 34 amino acids identical to that of the D7 splice variant (Fig IA). Thus, the SD receptor cDNA predicts a truncated G-CSF-R protein lacking the C-terminal region of 130 amino acids of the WT receptor including the box 3 subdomain. The expression of the transcript of this G-CSF-R isoform was detected by RNase protection in granulocytes from 6 normal individuals (data not shown).

Function of the SD receptor in BAF3 transfectants. To study the function of the SD receptor, the full-length cDNA encoding the SD receptor was inserted into the pLNCX ret- roviral expression vecto?' and transfected into BAF3 cells.

C

+

Fig 1. RT-PCR amplification of human G-CSF receptor cDNAs. (A) Schematic representation of human G-CSF receptor cDNAs. The wide bars represent coding regions, of which the solid and stippled regions denote transmembrane domain and signal sequence, respectively. Conserved regions in the cytoplasmic domain are shown as boxes 1.2, and 3. Thin solid bars indicate noncoding regions. The sequences lacking in the SD and D7 receptors as a result of alternative splicing are indicated by thin lines. The altered coding regions in the SD and D7 receptors are hatched. Also indicated are the positions of the PCR primers used in this study. (B) Southern blot analysis of PCR prod- ucts. RT-PCR was performed on total RNA isolated from normal gran- ulocytes using primers FW2 and RV1 (lane l) or RN2 and RV2 (lane 2). Two microliters of PCR products was subjected to electrophoresis in a 0.9% agarose gel, transferred to a nitrocellulose filter, and probed with the 32P-labeled human WT G-CSF receptor cDNA. The filter was washed finally in 0 . 3 ~ SSC at 65°C and exposed t o a film for 90 minutes. (C) Sequences flanking the deletion boundaries and the corresponding translational reading frame. Numbers indicate nucleo- tide (upper) and amino acid (low) positions. The consensus nucleo- tides for RNA splicing are underlined.

After G41 8 selection, RT-PCR was performed on total RNA extracted from the BAF/SD transfectants to verify the pres- ence of SD transcript. By using primers W 2 and RV 1, the 735-bp SD product was readily detected (data not shown). The surface expression and ligand binding characteristics of the SD receptor were examined by radioactive binding assays using '2SI-labeled G-CSF. Scatchard analysis showed that BAF/SD cells expressed a single class of G-CSF binding sites with a dissociation constant (kd) of 0.34 nmoVL at a mean density of approximately 800 sites per cell (Fig 2). These binding properties were comparable to those of the WT G-CSF-R expressed on BAF3 cells as well as on other cell We next examined whether the SD receptor was able to transduce growth signals in BAF3 cells. This cell line has previously been shown to proliferate in response to G-CSF upon expression of the human G-CSF-R." In 5 independent BAF/SD clones, no increase in 'H-TdR incorpo- ration was observed after G-CSF stimulation (Fig 3A). Con- sistent with this finding, BAF/SD cells died within 2 days upon transfer from IL-3-containing medium to cultures con- taining G-CSF alone (Fig 3B).

JAK2 activation by G-CSF in BAF3 transfectants.

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G-CSF-R MUTATION IN AML 905

JAK2, a 130-kD tyrosine kinase of the Janus kinase family, has recently been implicated as a signalling molecule of several cytokine receptors, including the receptors for eryth- ropoietin (Epo), IL-3, IL-6, GM-CSF, leukemia inhibitory factor, and growth hormone.28~""' G-CSF stimulation of BAF3 cells expressing the WT G-CSF-R also resulted in the tyrosine phosphorylation of the JAK2 protein (Fig 4A, BAF/ WT). The induction of JAK2 phosphorylation was rapid but transient, occurring as early as 1 minute after G-CSF stimula- tion, peaking at 10 minutes, and declining at 60 minutes. In contrast, the SD receptor lacking the proliferative signalling capacity only marginally mediated JAK phosphorylation (Fig 4B and C). Notably, the G-CSF-R deletion mutant M1,

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Fig 2. G-CSF binding characteristics of the SD receptor expressed on BAF3 cells. (A) Equilibrium binding of "51-G-CSF to BAF/SD cells. BAFlSD cells were incubated with titrated concentrations of l"1-G- CSF at 37°C for 1 hour. Specific binding was determined after subtrac- tion of nonspecific binding. Each point represents the mean of two estimations. (B) Scatchard plot of the G-CSF binding data.

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Fig 3. Growth of BAF/SD cells in G-CSF and IL-3. (A) DNA synthe- sis of BAFlSD cells in response to G-CSF or 11-3 stimulation. Cells were washed and cultured for 24 hours in the absence or presence of 100 ng of G-CSF per milliliter or 10% of W H l cell conditioned medium as a crude source of murine 11-3. Cells were then incubated with 'H-TdR for 12 hours and 'H-TdR incorporation was measured. (B) Growth cunres of BAFlSD cdls in GCSF or IL-3. Cells were plated initially at 105/mL in medium containing 10 ng of IL-3 per milliliter 10) or 100 ng of G-CSF per milliliter W. Culture medium was re- freshed every 2 days.

which contains only 55 amino acid residues in the cyto- plasmic region that are sufficient for proliferative signal- ling,'' was still capable of inducing JAK2 phosphorylation (Fig 4C, BAFA41). Consistent with the induction of tyrosine phosphorylation, activation of the WT or the M1 receptors resulted in significantly enhanced activity of JAK2 kinase, whereas the SD receptor hardly mediated JAK2 activation (Fig 4D). Because the SD receptor contains almost the entire membrane-proximal region preserved in mutant M1, these results suggested that the SD C-terminus negatively influ- ences JAK2 activation. The data also indicate that induction

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G-CSF-R MUTATION IN AML 907

of JAK2 activation by G-CSF correlates with the growth signalling capacity of the G-CSF-R.

Expression of the SD receptor in AML. Results from RT-PCR indicated that human granulocytes predominantly express the transcript of the WT G-CSF-R, whereas tran- scripts of the D7 and SD receptors are expressed at signifi- cantly lower levels. To determine whether this expression pattern may be altered in patients with AML, RNA samples from 70 AML patients were screened for the expression of the G-CSF-R mRNA by RT-PCR using primers FW2 and RV1. In one case, the most abundant PCR fragment consis- tently ran at a position expected for the SD receptor in four independent experiments (Fig 5A). Nucleotide sequencing confirmed that this fragment is identical to the SD cDNA isolated from granulocytes (data not shown). The fragment corresponding to the WT G-CSF-R was still present, al- though less abundantly, whereas the two bands correspond- ing to the D7 isoform were not seen on ethidium bromide- stained gels. Similar results were obtained from RT-PCR using primers FW3 and RV1 (Fig lA), which amplify a fragment of 878 bp for the WT G-CSF-R and of 329 bp for the SD receptor (data not shown).

Northern blot hybridization was then performed to esti- mate the relative expression levels of the WT and SD G- CSF-R transcripts. Two different probes were used to distin- guish the WT and SD G-CSF-R transcripts. A full-length cDNA encoding the WT G-CSF-R recognizes both the WT and SD transcripts. A PCR fragment of 409 bp, defined by primers FW4 and RV2 (Fig lA), specifically recognizes the sequence of the WT G-CSF-R lacking in the SD receptor. Using the full-length cDNA as a probe, a band of 3.7 kb corresponding to the transcript for the WT G-CSF-R was seen in RNA isolated from normal granulocytes as well as from leukemia cells of the patient (Fig 5B). In addition, a smaller transcript of approximately 3.2 kb was detected in RNA from leukemia cells of the patient, but not in RNA samples from normal granulocytes. As expected, this smaller transcript could not be detected when the same blot was reprobed with the 409-bp PCR fragment (Fig 5C). As judged from Northern blot analysis, expression of the SD transcript was slightly less abundant than that of the WT transcript in the AML cells. Although this seems in contrast with the results from RT-PCR (Fig 5A), it is likely that the SD tran-

Fig 4. Tyrosine phosphorylation and activation of JAW after G- CSF and IL-3 stimulation. (A and B) Kinetics of JAW phosphorylation induced by G-CSF stimulation. BAF3 cells expressing the WT G-CSF- R (A) or the SD receptor (B) were incubated with human G-CSF 1100 ng/mL) for the indicated times. Incubation with murine IL-3 (10 ng/ mL) was also included as a positive control. Cell lysates were immu- noprecipitated with JAW antiserum. The precipitated proteins were subjected to Western blot analysis using the 4GlO antiphosphotyro- sine monoclonal antibody. IC) Tyrosine phosphorylation of JAW in BAF3 transfactants expressing the different G-CSF-R forms. Cells were incubated for 10 minutes with G-CSF (1 pg/mL), IL-3 (10 ngl mLI, or without factors. (D) Activation of in vitro kinase activity of JAW after G-CSF stimulation. Immunoprecipitates with JAW antise- rum were incubated with [p"PIATP, washed, and separated by SDS- PAGE. All lanes represent lysates from equal amounts of cells.

script was preferentially amplified during PCR because of the size difference between the WT and SD sequences.

Identijkation of a point mutation in the SD splice donor site in AML cells. RT-PCR results indicated that the en- hanced expression of the SD transcript occurred at the ex- pense of the D7 transcript, which shares the splice acceptor site with the SD transcript. This prompted us to investigate whether mutations were present in the region flanking the splice donor site of the SD receptor. The entire exon 17 together with part of the intron 16 of the G-CSF-R gene36 was amplified by PCR using primers FWI16 and RV 1 from genomic DNA of the patient's leukemia cells. The 868-bp PCR fragment was subcloned and sequenced. A G-to-A sub- stitution was identified at nt 228519 (Fig 6A), immediately downstream of the 2 consensus nucleotides at the SD splice donor site (Fig 1C). No other mutations were found in exon 17. The mutation creates a unique Kpn I site. Kpn I digestion of PCR product obtained from genomic DNA demonstrated that the AML cells carried both the normal and the mutated alleles of the G-CSF-R gene (Fig 6B). Be- cause no somatic cells were available from the patient, we were unable to determine whether the point mutation was constitutional or present only in AML cells.

To exclude that the G-to-A substitution was a polymor- phism, DNA samples from 108 control individuals were ana- lyzed either by Kpn I digestion or by nucleotide sequencing. In none of these samples could the same nucleotide change be detected. Taken together, these findings strongly argue that the G-to-A mutation resulted in the increased utility of the SD splice donor site. Notably, a minor proportion of the largest RT-PCR fragment (Fig 1B) of the patient could still be cut by Kpn I (data not shown), suggesting that not all G- CSF-R RNA precursors carrying the point mutation were spliced into the SD transcript. This result was consistent with Northern analysis, which suggested that the SD transcript was somewhat less abundant than WT transcript (Fig 5B). Because the point mutation itself does not change the amino acid (Val), the full-length mRNA transcribed from the mutant allele would encode a G-CSF-R protein identical to the WT

Growth factor responses of AML cells overexpressing the SD receptor. Because the SD receptor was incapable of transducing a mitogenic signal in BAF3 cells, it would be expected that leukemic cells from the patient were less sensi- tive to G-CSF stimulation. Indeed, the patient's leukemic cells responded poorly to G-CSF in 3H-TdR incorporation assays (Fig 7). A weak mitogenic response was observed only at extremely high concentrations of G-CSF (300 ng/ mL), most likely due to the activation of the low numbers of WT G-CSF-R homodimers that may still be formed. The poor G-CSF response of the patient's leukemia cells was in sharp contrast to the responses to IL-3 and GM-CSF, which strongly stimulated DNA synthesis in the patient's leukemia cells in a dose-dependent manner (Fig 7).

G-CSF-R.

DISCUSSION

Alternative RNA splicing resulting in the formation of different receptor isoforms has been commonly observed among the members of the hematopoietidcytokine receptor

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908 DONG ET AL

A 1 2 3 4 5 6 M

( W

4.07 3.05 2.04 1.64

B 1 2 3 c 1 2 3 -~ + - --- - - Fig 5. Expression of G-CSF-R transcripts in AMI. cells. (A) RT-PCR + 28s analysis of G-CSF-R transcripts in AMI. cells. RT-PCR was performed

with primers FW2 and RV1, which define a fragment of 1,284 bp for the WT G-CSF-R, of 865 bp for the D7 receptor, and of 735 bp for the SD receptor. The bands migrating at approximately 1 kb position may represent the heteroduplexes of the WT and D7 DNA fragments (see text). Depicted are results obtained with RNA samples from 6 AML patients (lanes 1 through 6). Note the predominance of the 735- bp fragment in lane 3. (B and C) Northern blot analysis of G-CSF-R transcripts in normal granulocytes and leukemic cells. Four micro- grams of poly(A1' RNA from leukemic cells (lane 1) and 5 p g of total RNA from granulocytes of two healthy individuals (lanes 2 and 3) was electrophoresed in a 146 formaldehyde-containing agarose gel, blotted t o a nylon filter, and probed with the 'ZP-labeled WT G-CSF- R cDNA (B). The same blot was subsequently stripped and reprobed

(C). + + 18s with a '*P-labeled PCR fragment defined by primers W 4 and RV2

~uperfamiIy.~~" In general, alternative splicing creates two types of receptor isoforms, ie, those that differ in the cyto- plasmic domain and those that lack the transmembrane do- main and therefore encode soluble receptors. The extracellu- lar domain responsible for ligand binding usually remains unchanged. Although their physiologic significance is still largely unknown, receptor isoforms may contribute to the complexity of signal transduction or modulate cellular re- sponses to growth factors.

In addition to the WT form, three other G-CSF-R isoforms have been described. We report here the molecular cloning of a novel G-CSF-R isoform SD from human granulocytes. The biologic role of the SD receptor in the regulation of normal granulopoiesis remains speculative. Because the SD receptor is incapable of growth signalling and also lacks the C-terminal region of the WT G-CSF-R essential for matura- tion induction,"."' the homodimeric form of the SD receptor could function as a "sink" for G-CSF. The SD receptor could also form heterodimers with the WT G-CSF-R, thereby interfering with the function of the WT G-CSF-R. In this respect, it is worth noting that an Epo receptor (Epo-R) isoform, also lacking the growth signalling potential, has

been shown to inhibit the activity of the WT EPO-R,~' appar- ently by heterodimerization with the WT Epo-R."'"' How- ever, in view of its low expression, at least at the RNA level, it is less likely that the SD receptor would exert a major effect on the function of the WT G-CSF-R in granulopoiesis.

We have observed that the expression of the SD receptor was markedly increased in the leukemic cells of an AML patient. Furthermore, we show this phenomenon to be associ- ated with a G-to-A point mutation at the SD splice donor site. It remains uncertain to what extent the overexpression of the SD receptor had contributed to the development of leukemia in the patient. The inability of the SD receptor to mediate a strong mitogenic signal and the poor proliferative response of the patient's leukemic cells to G-CSF might even argue against a significant role of overexpressing the SD receptor in leukemogenesis. However, overexpression of the SD receptor would conceivably lead to the increased formation of SD/SD and WT/SD receptor complexes. Nota- bly, the C-terminal cytoplasmic region of the WT G-CSF- R, lacking in the SD receptor, is responsible for maturation signalling.".'" It is plausible that heterodimerization between the WT and SD receptor molecules would result in the distur-

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G-CSF-R MUTATION IN AML

Fig 6. Identification of a point mutation in the splicing donor site of the SD receptor in leukemic cells. (A) Part of a sequencing gel showing the point mutation. Nucleotide sequencing was performed on PCR product subcloned in pBluescript. The point mutation is boxed. The arrow indicates the position of the mutation in the se- quencing gel. The Kpn I site created by the point mutation is also indicated. (B) Agarose gel electrophoresis of PCR fragments after Kpn I digestion. Using primers W116 and RV1, the entire exon 17 and part of the intron 16 was amplified from DNA isolated from normal granulocytes (lanes 1 and 2) and leukemic cells (lane 3). Twenty mi- croliters of PCR products was digested in a 40-pL volume containing 8 U of Kpn I for 2 hours and then size-separated in a 1% agarose gel. Partial digestion of PCR fragment was excluded by including a reference DNA containing a Kpn I site in the digestion mixtures (data not shown).

0.51 - 0.40 - 0.34 - 0.30 - 0.20 -

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bance of maturation signalling by the G-CSF-R. As a result, cells overexpressing SD/SD and S D N T receptor complexes would fail to mature in response to G-CSF, but accumulate under the influence of other growth factors such as IL-3 and

Our results provide the first example of aberrant splicing of hematopoietin receptors in AML. However, alternative splicing as a potential oncogenic event has its precedent. Overexpression of splice variants of W T I gene that lack growth-inhibitory activity has been suggested as a common

GM-CSF.

mechanism of W T I inactivation in Wilms tumor4' and a point mutation leading to abnormal splicing of the W T I tran- script has been detected in a patient with Wilms tumor.42

Finally, data presented here establish that JAK2 is acti- vated by the WT G-CSF-R, but hardly by the SD receptor. The fact that the MI mutant of the G-CSF-R, which contains only 55 amino acids in the cytoplasmic domain, is able to induce JAK2 phosphorylation indicates that interaction with JAK2 is mediated by the membrane-proximal region of the G-CSF-R, similar to the Epo-R, the p, chain of IL-3 and GM-

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910 DONG ET AL

N 0 7

X Y

120 - 1 00

80

60

40

20

0

Concentrations (ng/ml)

Fig 7. Responsiveness of AMI. cells overexpressing the SD recep- tor to diffsrent hematopoietic growth factors. Proliferative responses were evaluated in 'H-TdR incorporation assays, as described under Materials and Methods. (0) G-CSF; (A) GM-CSF; (0) IL-3.

CSF receptors, and the IL-6 receptor signalling molecule gp130.28,34,35 It is not clear how the C-terminus of the SD receptor interferes negatively with JAK2 activation and pro- liferative signalling. One possible explanation is that this region contains a functional subdomain that mediates growth-inhibitory signals. Alternatively, the C-terminus of the SD receptor could simply affect the steric configuration of the membrane-proximal region of the G-CSF-R in a way that hinders the interaction with cytoplasmic growth signal- ling molecules such as JAK2.

ACKNOWLEDGMENT

We thank Drs B.A. Witthuhn and J.N. Ihle for advice and for supplying JAK2 antibodies.

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