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Blood, 15 January 2005, Vol. 105, No. 2, pp. 584-591. Prepublished online as a Blood First Edition Paper on September 7, 2004; DOI 10.1182/blood-2004-07-2613.
HEMATOPOIESIS Novel mechanism of G-CSF refractoriness in patients with severe congenital neutropeniaFrom the Bone Marrow Transplant Program and the Division of Hematology/Oncology, The Ohio State University, Columbus, OH; the Molecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, OH; and the Department of Hematology/Oncology, Children's Hospital, Columbus, OH.
Severe congenital neutropenia (SCN) is a rare disease diagnosed at or soon after birth, characterized by a myeloid maturation arrest in the bone marrow, ineffective neutrophil production, and recurrent infections. Most patients respond to treatment with granulocyte colony-stimulating factor (G-CSF), and the majority harbor mutations in the neutrophil elastase gene. In the subset of patients with SCN transforming to acute myeloid leukemia (AML), mutations that truncate the cytoplasmic tail of the G-CSF receptor (G-CSFR) have been detected. Here, we report a novel mutation in the extracellular portion of the G-CSFR within the WSXWS motif in a patient with SCN without AML who was refractory to G-CSF treatment. The mutation affected a single allele and introduced a premature stop codon that deletes the distal extracellular region and the entire transmembrane and cytoplasmic portions of the G-CSFR. Expression of the mutant receptor in either myeloid or lymphoid cells was shown to alter subcellular trafficking of the wild-type (WT) G-CSFR by constitutively heterodimerizing with it. WT/mutant G-CSFR heterodimers appeared to be retained in the endoplasmic reticulum and/or Golgi and accumulate intracellularly. These findings together with 2 previous case reports of extracellular mutations in the G-CSFR in patients with SCN unresponsive to G-CSF suggest a common mechanism underlying G-CSF refractoriness.
Neutrophil production is critically regulated by granulocyte colony-stimulating factor (G-CSF) and its cognate receptor (G-CSFR).1,2 The G-CSFR is a type I cytokine receptor that contains an extracellular domain with a conserved ligand binding region, a single transmembrane domain, and a cytoplasmic tail. The extracellular portion contains an N-terminal immunoglobulin (Ig)–like domain, a cytokine receptor homology (CRH) domain, and 3 fibronectin type III (FNIII) domains.3 Within the CRH region are 2 FNIII domains, 4 conserved cysteine residues, and a conserved WSXWS motif that stabilizes the CRH domain.4,5 The Ig-like and CRH domains appear to be critical for high-affinity binding.6,7 The cytoplasmic tail of the G-CSFR is not required for ligand binding but is essential for signal transduction.6,8-10 Like other members of the cytokine receptor superfamily, the cytoplasmic portion of the G-CSFR lacks intrinsic kinase activity yet activates Janus tyrosine kinases (JAKs) following ligand binding. Jak kinase activation is believed to occur by ligand-induced receptor dimerization, which brings together G-CSFR–associated JAKs permitting their trans-phosphorylation. Following phosphorylation of JAKs, the G-CSFR itself is phosphorylated on cytoplasmic tyrosine residues and recruits downstream signaling molecules. Initiation of signaling culminates in myeloid cell proliferation, neutrophilic maturation, or activation of terminally differentiated neutrophils, which are modulated by a balance of positive and negative feedback signals.11-19 A maturation arrest of myeloid progenitors in the bone marrow at the promyelocyte/myelocyte stage and low peripheral blood neutrophil counts (absolute neutrophil count [ANC] < 0.2 x 109/L [< 200/mm3]) are hallmarks of severe congenital neutropenia (SCN). Patients with SCN suffer from recurrent infections, although most respond to treatment with G-CSF with improved neutrophil counts (ANC > 1000/mm3;1 x 109/L) and decreased infections. The implementation of widespread use of G-CSF in patients with SCN in the past decade has led to dramatic improvements in the clinical course and quality of life for patients with this disease. Studies have revealed the unexpected finding of mutations in the ELA2 gene encoding neutrophil elastase (NE) in the majority of patients with SCN,20 but not in the genes for G-CSF or the G-CSFR. However, in the subset of patients with SCN developing acute myeloid leukemia (SCN/AML [acute myeloid leukemia]), nonsense mutations in the G-CSFR that are heterozygously expressed have been identified.21-23 These mutations truncate the C-terminal tail that is required for growth arrest, differentiation signaling, and down-modulation of receptor expression, and produce a dominant-negative phenotype both in vivo and in vitro that is postulated to be mediated by heterodimerization of wild-type (WT) and mutant G-CSFR forms.15,23-25 Rare patients with SCN refractory to G-CSF treatment have been reported, although the mechanisms underlying their unresponsiveness remain unclear.26,27
Here, we report a novel truncation mutation in the extracellular domain of the G-CSFR in a patient with SCN unresponsive to G-CSF therapy. The mutation disrupts the WSXWS motif after the first tryptophan and localizes to the same region of the G-CSFR where mutations were identified in 2 previous patients with SCN who were also unresponsive to G-CSF. We show that the mutant G-CSFR form we have identified (designated
Reagents and cell culture
Neutrophils were purified from peripheral blood following appropriate informed consent using Ficoll-Hypaque centrifugation (P = 1.077 g/mL) and dextran sulfate sedimentation. Contaminating erythrocytes were removed by hypotonic lysis. Neutrophils used for all subsequent studies were more than 98% pure by Wright-Giemsa staining. Buccal cells and fibroblasts were obtained following appropriate informed consent. Fibroblasts were cultured in RPMI 1640 medium supplemented with 2 mM glutamine and 10% fetal bovine serum (FBS). BaF3 cells were maintained in RPMI 1640 medium supplemented with 2 mM glutamine, 10% FBS, and 10% WEHI-3B conditioned media as a source of interleukin 3 (IL-3). Chinese hamster ovary (CHO) cells were grown in Reverse transcription–polymerase chain reaction (RT-PCR) and genomic DNA analysis
Total RNA was purified with Trizol (Invitrogen) and subsequently incubated with Superscript II reverse transcriptase (Invitrogen) to generate cDNA. Genomic DNA was purified with DNAzol (Invitrogen). Using 2 primer pairs that amplify overlapping fragments corresponding to base pair (bp) 106 to 832 and bp 772 to 1707 of the G-CSFR, the entire extracellular portion of the human G-CSFR was amplified as previously described.26 The resultant amplification products were visualized on 1% agarose/TAE (Tris [tris(hydroxymethyl)aminomethane]–acetate–EDTA [ethylenediaminetetraacetic acid]) gels and cloned into the pCR4-Blunt TOPO vector (Invitrogen) for sequencing. For detection of the Plasmid construction and transfection
The entire open reading frame of the WT G-CSFR was amplified using Advantage-HF2 DNA polymerase (Clontech, Palo Alto, CA) and cloned into the pcDNA3.1D-TOPO mammalian expression vector (Invitrogen). PCR reactions were performed with an initial 3-minute denaturation at 94°C, followed by 25 cycles of denaturation at 94°C for 30 seconds, annealing at 63°C for 30 seconds, elongation at 68°C for 90 seconds, and a final 3-minute elongation at 68°C. The forward primer used for amplification contained a Kozak consensus sequence28 (28 5'-CACCATGGCAAGGCTGGGAAACTGC-3'), and the reverse primer (5'-GAAGCTCCCCAGCGCCTCCATC-3') lacked a stop codon to allow read through to the V5 and His6 epitope tags contained within the vector sequence. The WT G-CSFR was epitope-tagged with V5 and His6 (pcDNA3.1D/WTv5h6). For generation of the Myc-tagged The tagged G-CSFR constructs were subcloned into the mammalian expression vector pcDNA6 (Invitrogen) from pcDNA3.1D, and positive clones were selected in blasticidin. The fidelity of the entire open reading frame of each expression vector was confirmed by automated DNA sequencing. Transfections of CHO cells were performed in 60-mm dishes using 1 µg DNA and Effectene (Qiagen, Valencia, CA) according to the manufacturer's instructions. Cells were either harvested at 24 to 36 hours after transfection, and transiently transfected cells were used in some experiments, or the cells were transferred to media containing G418 (75 µg/mL) to isolate positive clones stably expressing the various receptor forms. Single clones were isolated by limiting dilution and were screened for G-CSFR expression by immunoblot analysis and flow cytometry. BaF3 cells were stably transfected with the various G-CSFR forms using conditions that have previously been described.29 Single clones were isolated by limiting dilution and screened by immunoblotting. Immunoprecipitation and immunoblot analysis
Cells (2 x 107/mL) were lysed in lysis buffer (1.5% triton X-100, 0.1% sodium dodecyl sulfate [SDS], 0.1 mM sodium deoxycholate, 500 mM NaCl, 5 mM EDTA, 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES], pH 7.8) containing a cocktail of protease inhibitors (Roche, Indianapolis, IN), incubated on ice for 20 minutes, and centrifuged at 4°C at 10 000g. The supernatants were collected from each sample, and the protein concentrations were determined using the bicinchoninic acid (BCA) reagent (Pierce, Rockford, IL). A total of 150 µg protein from each sample in equivalent final volumes was mixed 1:1 with immunoprecipitation (IP) buffer (10% glycerol, 100 mM KCl, 5 mM MgCl2, 50 mM Tris, pH 8), and incubated with monoclonal antibodies (2 µg) recognizing either the V5 or Myc epitope (Invitrogen) and 40 µL protein G–agarose (Invitrogen) overnight at 4°C. Immunoprecipitates were collected by centrifugation at 2000g at 4°C, washed 3 times with a 1:1 mix of lysis and IP buffers, and resuspended in reducing lithium dodecyl sulfate (LDS) sample buffer. The samples were heated to 70°C for 10 minutes, resolved on precast 4% to 12% Bis-Tris acrylamide gels using a morpholinepropanesulfonic acid (MOPS) running buffer (Invitrogen), and transferred to nitrocellulose. For analysis of whole-cell lysates, 50 µg protein from each sample was resolved by SDS–polyacrylamide gel electrophoresis (PAGE) and subjected to immunoblot analysis. Conditioned media from cells expressing the Flow cytometric analyses Cells (5 x 105) were washed once in PSA (phosphate-buffered saline [PBS] supplemented with 1% fetal bovine serum [FBS] and 0.02% NaN3), incubated at 4°C for 1 hour with 0.5 µg biotinylated mouse anti-human G-CSFR monoclonal antibody (BD Pharmingen, San Diego, CA), washed and subsequently incubated with 2 µL Cy5-conjugated streptavidin (Caltag, San Francisco, CA). After a final wash in PSA, the cells were resuspended in 500 µL PSA, and 10 µg/mL propidium iodide was added to exclude dead cells. The cells were subsequently analyzed on a BD FACSCalibur (BD Biosciences, San Jose, CA). Fluorescent microscopy Stably transfected CHO cells (7.5 x 104) were grown on glass cover slips for 24 hours at 37°C. The adherent cells were then washed with PBS, fixed in 3% paraformaldehyde for 1 hour, and incubated with PBS containing 0.1% sodium azide and 5% BSA for 1 hour at room temperature. The cells were then incubated with a rabbit polyclonal anti–G-CSFR antibody (sc9173; Santa Cruz) overnight at 4°C, washed with PBS containing 0.1% sodium azide, and incubated for 1 hour with Alexa-633–conjugated anti-rabbit secondary antibody (Molecular Probes, Eugene, OR). Nuclei were stained with Hoechst stain (Molecular Probes) for 15 minutes at room temperature. The cover slips containing the adherent cells were mounted on glass slides using Pro-Long Antifade mounting media (Molecular Probes) and examined under a Zeiss LSM 510 multiphoton confocal fluorescence microscope (Zeiss, Jena, Germany) equipped with a c-Apochromat 63 x/1.2 Corr objective. Zeiss LSM 5 Image software was used for image processing.
Patient data A 1-month-old female infant presented with multiple abscesses, an ANC less than 200/mm3, and a normal hemoglobin and platelet count. Both parents were asymptomatic, and there was no family history of neutropenia. Autoimmune, genetic, and viral etiologies as well as paroxysmal nocturnal hemoglobinuria (PNH) and Fanconi anemia were excluded. Bone marrow analysis revealed a myeloid maturation arrest. Cytogenetics were normal. The patient was diagnosed with SCN without associated myelodysplastic syndrome (MDS) or AML. Remarkably, the patient showed no response to G-CSF even at doses of 100 µg/kg/d. Treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF) led to marked eosinophilia but no improvement in the ANC. The patient subsequently underwent an unrelated cord blood transplantation with complete resolution of the neutropenia.
Identification of the
Due to the patient's refractoriness to even high doses of G-CSF (100 µg/kg/d), we were interested in determining whether the patient might have a mutation in the extracellular region of the G-CSFR since a mutation here could affect ligand binding and explain the patient's observed G-CSF insensitivity. RT-PCR was used to amplify the entire G-CSFR extracellular region from peripheral blood neutrophils (PMNs) from the patient using 2 different primer pairs. A single-sized amplification product was obtained by RT-PCR with the 106/835 primer pair, and DNA sequencing indicated that all 30 clones examined from the patient contained the WT G-CSFR. RT-PCR with the 772/1707 primer pair is predicted to result in amplification of a single 935-bp product. However, RT-PCR using the 772/1707 primer pair and RNA from the patient's PMNs produced 2 products of approximately 935 bp (WT) and 750 bp (Figure 1A). The 750-bp product was cloned and sequenced and a 191-bp deletion identified which was present in 13 of the 30 clones examined from the patient. Numbering from the ATG codon, the deletion localizes to the region spanning bp 955 to 1145 of the G-CSFR cDNA, corresponding to a deletion in the genomic DNA spanning the region from the distal 43 bp of exon 8 to the proximal 74 bp of exon 10. The deletion produces a frame-shift immediately distal to the W318 codon, resulting in the introduction of an additional 29 missense codons followed by a premature stop codon. The mutation generates a truncated G-CSFR form containing the first 318 amino acids of the WT G-CSFR followed by the missense amino acids (Figure 1B). The truncated receptor, which we designated
Analysis of patient and parental cells
To determine whether the
Subcellular localization of the
Since the
Ligand-independent heterodimerization of the
Since we were unable to demonstrate the presence of the
To determine whether
Coexpression of the WT G-CSFR with the
To investigate the biologic consequences of coexpression of the
Increasing evidence indicates that SCN is a genetically heterogeneous disease. In most patients with SCN, mutations have been identified in the ELA2 gene encoding neutrophil elastase.20,33 Mutations in the genes encoding the Wiskott-Aldrich syndrome (WAS) protein and the transcriptional repressor oncoprotein GFI-1 have been detected in rare patients with SCN.34,35 In the approximately 15% of patients with SCN transforming to AML, acquired mutations in the cytoplasmic tail of the G-CSFR have almost universally been detected. Mutations in the extracellular region of the G-CSFR have also been reported in 2 patients with SCN without AML. Both patients had severe neutropenia that was diagnosed early in life, and both patients were unresponsive to G-CSF therapy.26,27 Despite these important observations, the mechanisms by which these mutations induce the neutropenic phenotype remain largely unknown. Although G-CSFR mutations are generally thought not to be causative of SCN, their role in SCN remains unclear. The frequent association of G-CSFR mutations with transformation to AML in patients with SCN has led to the hypothesis that these mutations contribute to leukemogenesis, although G-CSFR mutations do not invariantly occur in AML and may also appear in the absence of neoplasia.36 Notably, receptor mutations in SCN/AML localize to the cytoplasmic region of the G-CSFR.
The
Our findings imply a role for the distal region of the extracellular portion of the G-CSFR in proper targeting of the receptor to the cell membrane. We show that the
We also directly demonstrate that the truncated
Similar defects in receptor processing and assembly have been reported with truncation mutants of the erythropoietin receptor (EpoR).37 Using a series of EpoR truncation mutants terminating either just before the first tryptophan of the WSXWS motif or immediately distal to the last serine of the motif, or 9 amino acids after WSXWS motif, in which the transmembrane domain was also deleted, Miura and Ihle37 demonstrated that only the mutant terminating 9 amino acids after the WSXWS motif was secreted. Additionally, these investigators showed that all 3 truncated EpoR forms could constitutively associate with the WT receptor in a ligand-independent manner. Like the EpoR,37,38 our data also demonstrate a requirement for the WSXWS motif and/or sequences following it for correct sorting of the G-CSFR to the plasma membrane but not for constitutive receptor oligomerization. The WSXWS motif has also been shown to be important in protein folding for receptors for interleukin-2 receptor The location of any putative sorting signal/domain in the G-CSFR must lie between the WSXWS motif and the transmembrane domain, since expression of the entire extracellular portion of the G-CSFR produces a protein that is secreted.49 Furthermore, previous work by Fukunaga et al6 using deletion analysis has indicated that the 3 FNIII domains proximal to the transmembrane domain are important for correct expression of the G-CSFR. Thus, for both the EpoR and the G-CSFR, sequences in the extracellular domain appear to be critical for correct expression and sorting of the mature receptor complexes to the plasma membrane. Additionally, both receptors form oligomeric complexes, most likely dimers, during processing and transit of the receptor complexes to the plasma membrane.
Traditional dogma has held that the stimulus for cytokine receptor-induced signal transduction is ligand binding, which induces subsequent dimerization or oligomerization of receptor monomers. However, our data along with the increasing body of evidence with other cytokine receptors support a mechanism whereby ligand binding produces a conformational change in a preformed receptor dimer (or oligomer), and it is the conformational change itself that activates signal transduction.50 Both crystallographic and biochemical data indicate that, indeed, activation of the EpoR results from conformational changes in preformed receptor dimers following ligand binding.51,52 The existence of preformed receptor dimers has been reported for a number of other surface receptors.53-57 In the current paper, we show that the truncated
Notably, the 2 previously reported patients with SCN with mutations localizing to the extracellular region of the G-CSFR were both unresponsive to treatment with G-CSF. The P206H point mutation identified by Ward and coworkers26 was shown to alter ligand binding stoichiometry and drastically decreased proliferative signaling. Although the P206H mutation did not alter the apparent dissociation constant for ligand binding, an approximately 50% decrease in the number of apparent ligand binding sites per cell was observed with a concomitant decrease in the strength of the biologic response. The authors point out that Proline 206 localizes to the hinge region between the BN and BC regions of the CRH domain,32 and that crystallographic data suggest that ligand binding produces a change in the angle between the BN and BC domains. Ward et al26 suggested that the P206H mutation affected ligand-induced conformational changes in the G-CSFR, which precluded or inhibited subsequent ligand binding by prohibiting the formation of higher order ligand:receptor complexes thought to be necessary for full activation of G-CSFR–induced signaling. These data are consistent with the preformed G-CSFR dimer model, in which ligand binding induces signal transduction by initiation of conformational changes in the receptor protein rather than by formation of the actual receptor dimers. Sinhna et al27 reported a 182-bp deletion in the extracellular portion of the G-CSFR immediately distal to the codon for tryptophan 321 of the WSXWS motif (
The identification of 3 patients with SCN with mutations in the extracellular domain of the G-CSFR, all of whom were refractory to G-CSF therapy, suggests the existence of a previously unidentified subset of patients with SCN. Combination therapy with G-CSF and prednisone was found to be effective in treating the P206H patient,60 whereas GM-CSF alone or G-CSF in combination with SCF proved ineffective. Our patient, like the
Our data also provide the first direct evidence that the WT G-CSFR, indeed, heterodimerizes with truncated forms of the G-CSFR to explain the dominant-negative phenotype observed in transfected cells and in patients with SCN heterozygously expressing WT and mutant G-CSFR forms. Our results with the nonresponsive
Submitted August 14, 2004; accepted August 23, 2004.
Prepublished online as Blood First Edition Paper, September 7, 2004; DOI 10.1182/blood-2004-07-2613.
Supported by grants from the National Cancer Institute (grants CA75226, CA82859, and CA16058).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Belinda R. Avalos, The Ohio State University, Bone Marrow Transplant Program, A437A Starling-Loving Hall, 320 West Tenth Ave, Columbus, OH 43210; e-mail: avalos-1{at}medctr.osu.edu.
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Abstract
Introduction
319) heterodimerizes with the WT G-CSFR and that the formation of WT/mutant G-CSFR heterodimers occurs independent of ligand binding. We also demonstrate that the mutant G-CSFR decreases the surface expression of the WT receptor and thereby inhibits proliferative signaling by the WT G-CSFR. These findings suggest a common mechanism underlying G-CSF refractoriness in patients with SCN and also provide new insights into the basic mechanisms of G-CSFR processing and signaling. 
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chain (IL-2Rb),