Constitutive activation of FLT3 in acute myeloid ... - Wiley Online Library

British Journal of Haematology, 2000, 108, 322±330

Constitutive activation of FLT3 in acute myeloid leukaemia and its consequences for growth of 32D cells R. F E N S K I , 1 K. F L E S CH , 2 S. S E RVE , 1 M. M I Z U K I , 1 E. O E L M A N N , 1 K. K RATZ -A L B E R S , 1 J. K I E NAS T, 1 R. L E O , 1 S. S CHWA RT Z , 2 W. E. B E R DE L 1 AN D H. S E RV E 2 1University of MuÈnster, Department of Haematology/Oncology, and 2Benjamin Franklin Hospital, Department of Haematology/Oncology, Freie UniversitaÈt Berlin, Germany Received 4 June 1999; accepted for publication 26 September 1999

Summary. The receptor tyrosine kinase Flt3 is expressed on leukaemic blasts of most cases with acute myeloid leukaemia (AML). In order to evaluate the presence and signi®cance of constitutive activation of Flt3 for leukaemogenesis, we (1) analysed the expression and activation status of the receptor in AML blasts; and (2) evaluated the functional consequences of constitutively active Flt3 in a myeloid progenitor cell line. Immunoprecipitation studies revealed Flt3 expression in a high proportion of AML cases (27/32) with liganddependent Flt3 autophosphorylation in 18, constitutive autophosphorylation in three and no autophosphorylation in six cases. Only one out of three samples with constitutively active Flt3 but 3/18 samples with liganddependent autophosphorylated Flt3 contained the recently described internal tandem repeat (ITR) mutations. To test the signi®cance of Flt3 activation in myeloid cell function, we also characterized the biochemical and biological effects of

the activating mutation D838V of Flt3 (FLt3D838V) on the factor-dependent myeloid progenitor cell line 32Dcl3: cells transfected with wild-type Flt3 (32D/Flt3) grew FLt3 ligand (FL) dependent, and the receptor was ligand dependently autophosphorylated. In contrast, the receptor was constitutively autophosphorylated in 32D/Flt3D838V cells, which grew independently of FL. We conclude that, in some AML samples, Flt3 is constitutively activated and that this does not correlate with ITR mutations in the juxtamembrane domain. Furthermore, constitutively active Flt3 confers factor independence to the myeloid progenitor cell line 32D. It remains to be determined whether activation of Flt3 is leukaemogenic in vivo and whether strategies aimed at inhibition of Flt3 activation could inhibit leukaemogenesis.

The type III receptor tyrosine kinase Flt3 (Matthews et al, 1991; Rosnet et al, 1991; Small et al, 1994) and its ligand FL (Hannum et al, 1994; Lyman et al, 1993a, 1994) play an important role in the survival and self renewal of early multipotent haematopoietic progenitors, of monocytic precursors and in early lymphoid development (Mackarehtschian et al, 1995; Gotze et al, 1998; Lyman & Jacobsen, 1998). Expression of Flt3 on leukaemic cell lines and samples from patients with acute leukaemia has been studied extensively. Flt3 is expressed on most acute leukaemias of myeloid and B-lymphoid origin (Carow et al, 1996; Drexler, 1996). Incubation of leukaemic blasts with FL results in enhanced DNA synthesis in some, but not all, cases of acute myeloid leukaemia (AML; Piacibello et al, 1995; Dehmel et al, 1996; Drexler, 1996; Eder et al, 1996; McKenna et al, 1996)

and in a reduced rate of spontaneous apoptosis of AML blasts (Lisovsky et al, 1996). Interestingly, the proliferative response to FL is not necessarily predicted by surface expression of the receptor (Stacchini et al, 1996). Little is known about the functional integrity of Flt3 in leukaemic blasts. Somatic mutations involving a set of in-frame tandem repeats (ITRs) at the end of exon 11 of the Flt3 gene have been described, which result in the duplication of a stretch of several amino acids in the juxtamembrane region of the receptor (Nakao et al, 1996). These mutations are detectable in about 20% of AML samples (Yokota et al, 1997) and are associated with high leukaemic cell numbers in patients with acute promyelocytic leukaemia (Kiyoi et al, 1998). Very recently, it has been shown that the expression of ITR-containing Flt3 in COS-7 cells results in its constitutive autophosphorylation (Kiyoi et al, 1998). An activating point mutation in the kinase domain of two other receptor tyrosine kinases has been linked to neoplastic

Correspondence: Dr H. Serve, UniversitaÈtsklinik MuÈnster, Innere Medizin A, Albert-Schweitzer-Strasse 33, 48149 MuÈnster, Germany.

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Keywords: 32D cells, Flt3, acute myeloid leukaemia, receptor tyrosine kinase, leukaemogenesis.

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Flt3 Activation in AML diseases. Replacement of Asp-814 by valine (Furitsu et al, 1993) or tyrosine (Piao & Bernstein, 1996) in the structurally and functionally closely related protein c-kit (Qiu et al, 1988) has been associated with systemic mastocytosis (Longley et al, 1996). An activating mutation (Met-918!Thr) in the kinase region of the proto-oncogene ret is found in the germ line of families with multiple endocrine neoplasia type 2b and as a somatic mutation in about 30±40% of sporadic cases with medullary thyroid carcinoma (reviewed by Kolibaba & Druker, 1997). In order to analyse the functional integrity of Flt3 in AML blasts and the consequences of constitutive activation of Flt3 in myeloid cells, we therefore (1) characterized the expression and autophosphorylation pattern of Flt3 in AML blasts; and (2) analysed the functional consequences of Flt3 activation on the myeloid progenitor cell line 32Dcl3. (1) We found constitutively active Flt3 in AML blasts of a minority of patients, which did not correlate with the presence of ITR mutations. (2) Replacement of Asp-838 by valine (Flt3D838V), which is homologous to an activating mutation found in c-kit in neoplastic mast cell disorders, leads to constitutive autophosphorylation of Flt3, when expressed in 32D cells, and to their growth factor-independent proliferation. MATERIALS AND METHODS Reagents and cells Recombinant human FL was kindly provided by Dr Stuart Lyman (Immunex Corporation, Seattle, WA, USA). Rabbit polyclonal anti-mouse Flt3 antibody was purchased from UBI (Lake Placid, NY, USA) and from SantaCruz Biotechnology (Santa Cruz, CA, USA). Monoclonal rat anti-mouse Flt3 antibody was obtained from Pharmingen (San Diego, CA, USA). Monoclonal mouse antiphosphotyrosine antibody (4G10) was obtained from UBI. Goat anti-rat IgG microbeads and selection columns for magnetic cell sorting (MACS) separation were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Peripheral blood mononuclear cells were obtained by density gradient centrifugation of peripheral blood from patients with freshly diagnosed AML, which was collected after their informed consent. Blast content of the preparation was > 80% in all cases, as judged by routine morphological and immunophenotypic analysis. Reverse transcriptase±polymerase chain reaction (RT±PCR) Total RNA was isolated from 107 leukaemic blasts using RNAeasy (Qiagen, Hilden, Germany) according to the manufacturer`s instructions. First-strand synthesis was performed using oligo-dT as primers at a concentration of 4 mmol/l, a mixture of all four deoxynucleotides (125 mmol/l each) and 1 U of reverse transcriptase (Superscript II, Gibco BRL, Eggenstein, Germany) in a ®nal volume of 20 ml at 428C for 1 h. For ampli®cation, 4 ml of the RT reaction was used as templates. Ampli®cation was performed with Taq DNA polymerase at an annealing temperature of 608C at 50 mmol/l salt and 1´5 mmol/l MgCl2 concentration for 35 cycles. The sense primer was 50 -GCACATCTTGTGAGACGATCC-30 , and the antisense

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primer was 5 -CACCATAGCAACAATATTCAAAAATC-30 , yielding a 530-bp PCR product corresponding to nucleotides 1619±2149 of the published sequence of human Flt3 (Small et al, 1994). Each PCR ampli®cation was repeated at least twice with identical results. After Klenow treatment of the ends of the PCR product, it was cloned into the EcoRV site of pZero-2 (Invitrogen, The Netherlands) and ampli®ed in the bacterial strain DH5a. DNA sequencing DNA generated by RT±PCR was sequenced either directly or after cloning of the PCR product using the Dye Terminator kit (Perkin-Elmer, Weiterstadt, Germany) according to the manufacturer's instructions using 10 pmol of primer, 60±360 ng of PCR product and 1 mg of plasmid DNA. A cycle sequencing protocol with Taq polymerase was performed (2 min at 948C followed by 25 cycles of 968C for 10 s, 558C for 5 s, 608C for 4 min in a 20-ml reaction). After ethanol precipitation and a denaturing step (928C for 2 min in 25 ml of template-suppressing reagent; Perkin-Elmer), the sequencing reaction was analysed on an automated sequencing system (ABI Prism 310, Perkin-Elmer). For direct sequencing of the PCR products, the PCR primers were used (see above). The cloned PCR fragments were sequenced using the M13 forward and reverse primers. DNA constructs The cDNA of murine Flt3 was kindly provided by Dr Ihor Lemischka (Princeton, NJ, USA). The complete coding sequence was subcloned into the retroviral vector pGD (Daley et al, 1990) at the BclI site under the control of the long-terminal repeat of the myeloproliferative sarcoma virus (MPSV). Site-directed mutagenesis of Flt3 was performed on pGD/¯t3 with Quickchange (Stratagene) using the oligonucleotide primers 50 -GGACTGGCCCGAGTCATCCTGAGCGACTC-30 (sense) and 50 -GAGTCGCTCAGGATGACTCGGGCCAGTCC-30 (antisense) according to the manufacturer's instructions. Integrity of the complete Flt3 coding region was con®rmed by sequence analysis. For control experiments, a hybrid molecule of the extracellular region of c-kit and the intracellular region of Flt3 (cDNA of the intracellular domain of Flt3 kindly provided by Dr Stuart Lyman, Immunex Corporation, Seattle, WA, USA) containing the epitope of the anti-Flt3 antibodies used was cloned into pcDNA3.1 (Clontech, Heidelberg, Germany). This construct was transiently expressed in COS-1 cells. Stable transfection of 32Dcl3 cells The interleukin 3(IL-3)-dependent murine myeloid cell line 32Dcl3 (kindly provided by Dr Felicitas Rosenthal, Freiburg, Germany) was cultured in RPMI-1640 supplemented with 10% WEHI-conditioned medium as a source of IL-3, 10% fetal calf serum and antibiotics at 378C with 5% CO2. For retroviral transfer of the Flt3 constructs, 5 ´ 106 cells were co-cultivated for 2 days with E86 GP producer cells (kindly provided by Dr Arthur Banks, New York, NY, USA; Markowitz et al, 1988), which had been transfected with the plasmids previously by electroporation. For direct electroporation, 10 mg of plasmid DNA of either

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plasmid was linearized by HindIII digestion and added to cell suspensions. Samples were electroporated with a Gene Pulser (Bio-Rad) in 0´4-cm cuvettes at 300 V and 960 mF and were selected with 0´6 mg/ml G418. Fourteen days later, the geniticin-resistant cells were puri®ed twice with a MACS column (Miltenyi Biotec) using rat anti-mouse ¯t3 monoclonal antibody and goat anti-rat ferrobeads according to the manufacturer's instructions. Flow cytometry After preincubation for 15 min at 48C with mouse IgG (32D cells) or human immunoglobulin (leukaemic blasts), cells were stained for 15 min with the indicated antibody, washed twice in PBS/0´1% BSA and analysed using ¯ow cytometry (Becton Dickinson, Heidelberg, Germany). The following antibodies were used: CD34-PE (HPCA1; Becton Dickinson); CD117-PE (YB5B8; Coulter Immunotech, Hamburg, Germany); anti-mouse ¯t3 (Pharmingen). For viability testing, propidium iodide (PI) was added. Immunoprecipitation and Western blot analysis Approximately 107 32D cells or COS-1 cells transfected with the indicated cDNA constructs or 5 ´ 107 leukaemic blasts were stimulated in 1 ml of medium for 5 min at 378C with or without 100 ng/ml FL. Cells were washed once with ice-cold PBS containing 1 mmol/l sodium orthovanadate. Cells were lysed with buffer containing 50 mmol/l HEPES, pH 7´4, 10% glycerol, 150 mmol/l NaCl, 1% Triton X-100, 1 mmol/l EDTA,1 mmol/l EGTA, 50 mmol/l ZnCl, 25 mmol/l NaF, proteinase inhibitors (Complete; Boehringer Mannheim, Germany), 1 mmol/l pepstatin and 1 mmol/l sodium orthovanadate. Cell lysates were clari®ed at 20 000 g for 20 min. 32D lysates were incubated with rabbit polyclonal antibody to murine Flt3 (UBI) at 48C for 1 h, leukaemic blasts and COS-1 cells with an antibody directed against the intracellular domain of human Flt3 (SantaCruz). Protein A/GPlus-Sepharose (25 ml; SantaCruz) was added to each sample, and they were incubated for a further 1 h. The immune complex was washed three times with lysis buffer, heated in sodium dodecyl sulphate (SDS) sample buffer, separated by SDS±polyacrylamide gel electrophoresis (PAGE), immunoblotted on Immobilon P membrane (Millipore) and incubated overnight at 48C with either antiphosphotyrosine or anti¯t3 antibody in blocking reagent (Super-Block, Pierce) and detected with the ECL-Plus system (Amersham). [3H]-thymidine incorporation assay Approximately 104 32D cells that had previously been transfected with the indicated cDNA construct were starved of exogenously added growth factors for 24 h and then placed in 200 ml of medium supplemented with the indicated concentrations of FL. After 24 h incubation at 378C, 37 kBq of [3H]-thymidine was added to each well, and cells were incubated for an additional 16 h. Cells were lysed by freezing and thawing and harvested on a glass®bre ®lter, washed twice, and the b-emission of the bound DNA was analysed using a scintillation counter. Each data point represents the mean 6 standard deviation of four wells.

Cell proliferation assay Cells were placed at a density of 3 ´ 105 cells/ml in 24-well plates in duplicate and fed twice a week with RPMI and the indicated supplements. Viable cells were counted after trypan blue exclusion staining. RESULTS Flt3 is constitutively autophosphorylated in leukaemic blasts of some AML patients In order to analyse the function of Flt3 in leukaemic blasts, we immunoprecipitated Flt3 from the mononuclear cell fraction of patients with at least 80% leukaemic blasts in peripheral blood. Surprisingly, we found a rather heterogeneous distribution of expression, size and autophosphorylation pattern of the immunoprecipitated receptors (Fig 1). A total of 27/32 samples contained detectable Flt3. In the majority of the Flt3-positive samples (18/27), Flt3 displayed ligand-dependent autophosphorylation as expected (e.g. patients II, VII and VIII). On the other hand, the blasts of one patient with an acute promyelocytic leukaemia contained Flt3 with markedly reduced size without detectable autophosphorylation even after FL stimulation (patient I), whereas 5/27 Flt3-positive samples expressed Flt3 migrating as the typical double band at the expected size, but without ligand-dependent autophosphorylation

Fig 1. Heterogeneous expression and ligand-dependent autophosphorylation of Flt3 in peripheral blood from patients with AML. Mononuclear cells were separated from the peripheral blood of patients with acute leukaemia by density gradient centrifugation. Approximately 5 ´ 107 cells were incubated for 5 min with or without 100 ng/ml FL, lysed, and Flt3 was immunoprecipitated from the lysates. After SDS±PAGE (A, 10%; B, 7%), Western blot analysis was performed with antibodies (a) against the indicated antigens. For the controls, pcDNA3 alone (Neg. CTRL) or containing an Flt3 construct (Pos. CTRL) was transfected into COS-1 cells (for details, see Materials and Methods), which were treated identically to the patients' samples. PY, phosphotyrosine.


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(e.g. patient IV). Of particular interest were those samples (3/27) in which Flt3 displayed constitutive activation even in the absence of exogenously added FL (patients V, VI and IX). We also analysed the blast population in peripheral blood for the expression of CD34 or c-kit, markers of an immature phenotype, which, in normal haematopoiesis, are coregulated with Flt3 expression (Lyman & Jacobsen, 1998). No correlation between Flt3 expression and the expression of these markers on leukaemic blasts could be found (Fig 2).

Fig 2. Distribution of CD34 and c-kit positivity of ¯t3-positive AML blasts, as judged by FACS analysis. Blasts expressing ¯t3 were stained with the indicated antibodies, as described in Materials and Methods, and analysed by three-colour immuno¯uorescence. The percentage of ¯t3-positive blasts positive for CD34 and c-kit are shown.

Fig 3. Mutations in the juxtamembrane domain do not correlate with the autophosphorylation pattern of Flt3. RT±PCR was performed as speci®ed in Materials and Methods, spanning the cDNA corresponding to the juxtamembrane region of Flt3. An ethidium bromide-stained polyacrylamide gel of PCR reactions of the three samples containing constitutively active Flt3 (patients V, VI and IX) and of the four samples containing ITR mutations (patients II, VI, VII and VIII) is shown. This experiment was repeated at least twice with identical results. C, control, plasmid DNA with known wild-type Flt3 sequence; H2O, water control.

Flt3 autophosphorylation is not caused by internal tandem repeats in the juxtamembrane region As it has been reported very recently that internal tandem duplications of Flt3 result in constitutively autophosphorylated Flt3 in COS-7 cells (Kiyoi et al, 1998), we were interested in whether constitutively active Flt3 from the AML blasts contained any mutation in the juxtamembrane domain. We therefore ampli®ed the part of the mRNA corresponding to the juxtamembrane domain of Flt3 (containing the entire sequence of exon 11) by RT±PCR in all 27 AML samples expressing Flt3. Four out of 27 samples, including one of the samples displaying constitutive autophosphorylation of Flt3, displayed a PCR product that was slightly larger than expected (Fig 3, patients VI and II) or a double band (patients VII and VIII), presumably representing the two alleles of Flt3 expressed in the leukaemic blasts. We therefore analysed the DNA sequence of the PCR product. For this purpose, we puri®ed the PCR products by agarose gel electrophoresis, separating the two bands for patient VII and patient VIII and sequenced the bands separately. The sequences of the lower bands were identical to the published wild-type Flt3, whereas the sequences of the upper bands of patients VII and VIII and of all samples from patients VI and II contained the insertions indicated in translation on Fig 4. So, we found internal tandem repeat mutations in samples from four patients. Wild-type Flt3 was co-expressed with a mutated form in two samples, presumably as a result of a heterozygous mutation. In the other two samples, only the mutated form of Flt3 was detectable. As only one out of three cases showing constitutive autophosphorylation of Flt3

Fig 4. Schematic depiction of the Flt3 protein. The protein sequence in the juxtamembrane region corresponding to the part of the ¯t3 mRNA as published (Small et al, 1994) is written out in single-letter amino acid code and shaded in grey. The protein sequence deduced from the results of DNA sequencing after RT±PCR from patients V, VI and IX (constitutively active ¯t3) and patients II, VII and VIII (ligand-dependent ¯t3 activation) is written below the published sequence for comparison. q 2000 Blackwell Science Ltd, British Journal of Haematology 108: 322±330

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coincided with ITRs, other mechanisms causing constitutive receptor activation must be operative in AML blasts. On the other hand, internal tandem mutations of Flt3 do not always cause constitutive activation of Flt3. Further experiments are needed to evaluate the role of these mutations for the activation of Flt3. Stable transfection of 32D cells In order to analyse the effect of constitutively active Flt3 on the growth of myeloid cells, we introduced a single basepair exchange into the mouse Flt3 cDNA by site-directed mutagenesis. This mutation (D838V) results in a substitution of valine for Asp-838. We inserted this cDNA into a retroviral vector and transfected the construct into 32D cells (32D/Flt3D838V) with wild-type Flt3 transfectants as controls (32D/Flt3). The transfection was done ®rst by retroviral gene transfer and repeated twice by electroporation as indicated in Materials and methods. All three cultures gave similar results.

Fig 5. Expression and phosphorylation status of Flt3 and Flt3D838V in 32D cells. Approximately 5 ´ 106 32D cells transfected with the indicated cDNA constructs were incubated for 5 min with or without 100 ng/ml FL and lysed. (A) After SDS±PAGE, Western blot analysis was performed with antibodies (a) against the indicated antigens. PY, phosphotyrosine. (B) Flt3 was immunoprecipitated from the lysates. Immunoprecipitates were treated as in (A). VA, vector alone.

Flt3D838V is constitutively autophosphorylated in the myeloid progenitor cell line 32D Western blot analysis of 32D/Flt3 cells for phosphotyrosine (Fig 5A) showed a phosphoprotein in FL-stimulated cells, which co-migrates with the upper band of Flt3, presumably corresponding to the mature Flt3 protein expressed on the cell surface. On the other hand, 32D/Flt3D838V cells contained a phosphoprotein co-migrating with the lower band of Flt3, irrespective of the presence of FL. Note on the aFlt3 blots that the ratio of the mature to the immature form of Flt3D838V was dramatically changed in comparison with Flt3 and that the higher (mature) band of Flt3D838V was barely detectable in Flt3 immunoblots.

Fig 6. FACS analysis of surface expression of the different Flt3 mutants. (A) 32D cells transfected with the indicated cDNA constructs were stained with PE-conjugated a-Flt3 (dark grey) or control (light grey) antibody and analysed by ¯ow cytometry. (B) Mean ¯uorescence of a-Flt3-stained 32D cells was measured by ¯ow cytometry before and at the times indicated after the addition of 100 ng/ml FL to the medium. Mean ¯uorescence before the addition of FL was set at 100%. CTRL, control.


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Immunoprecipitation of Flt3 showed that the constitutively phosphorylated phosphoprotein in 32D/Flt3D838V was immunoprecipitated by Flt3 antibodies. Interestingly, phosphorylation of the upper band of Flt3D838V was still regulated by FL (Fig 5B). Flt3D838V is expressed at the cell surface of transfected 32D cells As the upper band of Flt3D838V was barely visible on immunoblots for Flt3, we were interested in whether this predominantly immature protein was expressed on the surface of 32D/Flt3D838V cells, as is the case for wild-type Flt3. We therefore conducted a ¯uorescence-activated cell sorting (FACS) analysis and did not see a signi®cant difference of surface expression of the two Flt3 isoforms (Fig 6A). Also, receptor downregulation after stimulation with FL was identical on both isoforms over 36 h (Fig 6B). Flt3D838V expression causes factor-independent growth of 32D cells 32Dcl3 cells need IL-3 for growth. We therefore assessed DNA synthesis in the absence of IL-3 in the transfected cells by [3H]-thymidine incorporation (Fig 7). 32D and 32D/Flt3 cells did not synthesize DNA in the absence of exogenously added growth factors. 32D/Flt3 cells responded to the addition of FL by increased [3H]-thymidine incorporation, whereas 32D cells transfected with vector alone were not in¯uenced by the addition of FL. In contrast to these two cell lines, 32D/Flt3D838V cells proliferated without exogenously added growth factors, and exogenously added FL did not in¯uence DNA synthesis in these cells. As enhanced DNA synthesis did not necessarily imply that transfection of Flt3D838V could support sustained growth of 32D cells without IL-3, we observed the culture of 32D/Flt3D838V for several weeks, the ®rst two of which are depicted in Fig 8. The culture grew exponentially during the observation period without the need for exogenously added growth factors. DISCUSSION In this report, we have shown that Flt3 is constitutively activated in leukaemic blasts of some AML patients. The constitutive activation did not correlate with the presence of ITR mutations that have been reported to cause constitutive activation of Flt3 in COS-7 cells. To obtain a model to evaluate the biological consequences of constitutive Flt3 activation, we therefore analysed a point mutation in the kinase domain of Flt3 that causes constitutive autophosphorylation and alterations in the glycosylation pattern of the protein. Expression of the mutated protein in the myeloid progenitor cell line 32D results in their factor-independent growth. Since the description of Flt3 expression on leukaemic blasts, speculation arose that it might play a role in the malignant transformation of haematopoietic progenitors. Reports about a proliferative response to FL supported that hypothesis (Drexler, 1996). Direct evidence for constitutive

Fig 7. Effects of FL on DNA synthesis of 32D cells transfected with the different Flt3 isoforms. Approximately 104 serum-starved cells transfected as indicated were incubated with increasing concentrations of FL for 40 h. For the last 16 h of the incubation period, 37 kBq of [3H]-thymidine was added. Cells were lysed, and thymidine incorporation was measured by b scintillation counting. Data are given in counts per minute. Each data point represents the mean of at least four samples 6 standard deviation. (A) Dose±response relationship of the effects of FL on thymidine incorporation. (B) Effect of 100 ng/ml IL-3 on thymidine incorporation of the different transfected 32D cell sublines.

activation of Flt3 in clinical samples has not been presented. As autophosphorylation of receptor tyrosine kinases is the main ®rst signalling event in their activation, we conclude from our data that Flt3 is constitutively active in some of the samples. The mechanism of the observed activation remains unknown. ITR mutations involving exon 11 of Flt3, which cause an insertion of several amino acids in the juxtamembrane region of the protein, have been described (Nakao et al, 1996). We showed a wild-type con®guration of the cDNA for this region in two of the three patients with autoactivated Flt3, and the presence of the mutation in samples with ligand-dependent Flt3 activation. Very recently, it has been


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Fig 8. Effect of Flt3D838V transfection on long-term growth of 32D cells. 32D cells transfected with the indicated cDNA construct were placed at a density of 3 ´ 105 cells/ml in 24-well plates in duplicate and fed twice a week with RPMI and the indicated supplements. Cell counts of viable cells are shown. (A) Growth of the culture with 10% WEHI-conditioned medium added as a source of IL-3. (B) Growth of the culture without growth factors (GF) added.

shown that ITR mutations cause constitutive Flt3 autophosphorylation in COS-7 cells, and it has been hypothesized that the ITRs represent a new mechanism of Flt3 activation (Kiyoi et al, 1998). But, in the same report, the authors could not demonstrate constitutive activation of Flt3 in clinical samples. Furthermore, constitutive autophosphorylation of Flt3 in COS-7 cells seems to be very much dependent on the experimental conditions. Wild-type Flt3 was reported to be constitutively phosphorylated in COS-7 cells with c-kit used as a control (Lyman et al, 1993b). So, the results from COS-7 cells concerning the functional consequences of the ITRs of Flt3 have to be interpreted with caution, and experiments assessing the biochemical and biological alterations caused by these mutations in haematopoietic cells are needed. As we did not see a correlation between ITRs and constitutive Flt3 autophosphorylation in our patient samples, we think that other mechanisms of activation are operational in these cases, which could be either mutations elsewhere in the protein or autocrine mechanisms. In order to gain insight into the functional relevance of Flt3 activation, we introduced a mutation into the Flt3 cDNA, which we reasoned to be the activating factor, as a homologous mutation has been described in c-kit (Nakao et al, 1996). This mutation activated the proliferation of mouse and human mast cell lines (Furitsu et al, 1993; Tsujimura et al, 1994), induced differentiation and tumorigenicity in primary mast cells (Hashimoto et al, 1996; Piao & Bernstein, 1996) and adult mouse bone marrow (Kitayama et al, 1996) and was associated with neoplastic mast cell disease in humans (Longley et al, 1996). The mechanism of activation of the type of mutation described here is still unsolved. Recently, it was shown that substitution of the homologous Asp at position 814 in c-kit by Tyr causes enhanced degradation of SHP-1, a tyrosinespeci®c phosphatase, and a change in the substrate speci®city of c-kit (Piao et al, 1996). A similar mechanism could be operative in the mutation described in Flt3. Flt3 is modi®ed post-translationally, and its characteristic two bands result from a transition from a glycosylated highmannose form to a complex carbohydrate form. Only the upper, complex carbohydrate form is present on the cell surface (Lyman et al, 1993b). We noted a consistent and

dramatic decrease in this mature form of Flt3D838V compared with Flt3. The total amount of Flt3D838V expressed in 32D/Flt3D838V cells was not higher than the amount of its wild-type counterpart (Fig 5A). On the other hand, because we also found approximately equal amounts of Flt3 and Flt3D838V on the surface of the respective cell lines (Fig 6), we concluded that the immature form of Flt3D838V was expressed on the cell surface, as the small amount of mature Flt3D838V could not explain the high level of surface expression of Flt3D838V in FACS analysis. Interestingly, the mature form of Flt3D838V was not constitutively phosphorylated, but could be phosphorylated if FL was added to the cells. So, the mechanism of constitutive activation of Flt3D838V seems to be linked to the inability of proper carbohydrate processing. It will be interesting to analyse whether constitutive phosphorylation of Flt3 inhibits its glycosylation and why the physiologically processed form of the protein retains its ligand-dependent autophosphorylation pattern. We could demonstrate that constitutively active Flt3 induced factor independence in the myeloid progenitor cell line 32D. This is the ®rst report about the effects of a constitutively active Flt3 mutation on myeloid cells. Recently, two groups have reported the effects of transfection of FL cDNA into an autonomously growing AML cell line (Braun et al, 1997) and into mouse bone marrow. FL transfection into this AML cell line induced growth enhancement and activation of MAPK, whereas introduction of FL cDNA into mouse bone marrow resulted in increased white blood cell counts and splenic ®brosis (Juan et al, 1997) and a predisposition to develop an acute leukaemic disease (Hawley et al, 1998). Further studies on the effect of activated mutations of Flt3 on tumorigenicity of myeloid cell lines and on normal mouse progenitors are needed. The system presented here could then be used to de®ne Flt3 signalling pathways involved in leukaemogenesis. ACKNOWLEDGMENTS We thank Dr Ihor Lemischka for providing the mouse ¯t3 cDNA, Dr Stuart Lyman for human Flt3 ligand and human Flt3 constructs, Dr Felicia Rosenthal for providing the 32D


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