Granulocyte Colony-stimulating Factor Receptor Gene Research Articles

Structural diversity and function of the granulocyte colony-stimulating factor in medaka fish.

Diversity in the granulocyte repertoire, including neutrophils, basophils, and eosinophils, has been reported in vertebrate species. Medaka fish (Oryzias latipes) have only neutrophils; however, the storage pool of granulopoiesis tissues and the molecular mechanism of granulopoiesis in medaka fish have not been explored. Granulocyte colony-stimulating factor (G-CSF) is a cytokine responsible for neutrophil differentiation, survival, and proliferation. We performed in silico analysis to molecularly characterize the medaka G-CSF and G-CSF receptor (G-CSFR) genes. This study showed that medaka G-CSF differs considerably from human and mouse G-CSF in terms of the primary protein structure; however, the predicted tertiary structure was largely conserved. Analyses of lipopolysaccharide stimulation and G-CSF knockout and overexpression in medaka revealed that G-CSF mobilizes neutrophils into the peripheral blood. The analysis of G-CSF-deficient medaka revealed that G-CSF is involved in erythropoiesis. These findings represent an important first step toward understanding granulocyte hematopoiesis in non-mammalian species.

Human neutrophil development and functionality are enabled in a humanized mouse model.

Mice with a functional human immune system serve as an invaluable tool to study the development and function of the human immune system invivo. A major technological limitation of all current humanized mouse models is the lack of mature and functional human neutrophils in circulation and tissues. To overcome this, we generated a humanized mouse model named MISTRGGR, in which the mouse granulocyte colony-stimulating factor (G-CSF) was replaced with human G-CSF and the mouse G-CSF receptor gene was deleted in existing MISTRG mice. By targeting the G-CSF cytokine-receptor axis, we dramatically improved the reconstitution of mature circulating and tissue-infiltrating human neutrophils in MISTRGGR mice. Moreover, these functional human neutrophils in MISTRGGR are recruited upon inflammatory and infectious challenges and help reduce bacterial burden. MISTRGGR mice represent a unique mouse model that finally permits the study of human neutrophils in health and disease.

Donor CSF3R with the rs3917980A/G or G/G genotype is correlated with better leukemia-free survival after allogenic hematopoietic stem cell transplantation.

Polymorphisms in the granulocyte colony-stimulating factor receptor gene (GCSFR, CSF3R) have been reported to be associated with peripheral blood stem cell enrichment and hematological diseases. The aim of our study was to investigate the effects of donor CSF3R allelic polymorphisms on the outcomes of allogeneic stem cell transplantation. A total of 273 patients who were diagnosed with hematological diseases and treated with allogeneic hematopoietic stem cell transplantation(allo-HSCT) were enrolled in this study. Single-nucleotide polymorphisms in CSF3R were genotyped by targeted next-generation sequencing. There were six types of CSF3R genotypes with percentages over 1%. LFS and OS analyses showed that recipients receiving grafts from healthy donors with a rs3917980 G/G or A/G genotype had higher LFS rates than those receiving grafts from donors carrying a rs22754272 T/C genotype and the double-negative group (p = 0.036). Univariate cox analysis showed that donor CSF3R with the rs2275472 T/C genotype was associated with higher transplantation-related mortality (TRM) rates (HR = 2.853, 95% CI: 1.405-5.792, p = 0.00371) and lower rates of leukemia-free survival (LFS) (HR = 1.846; 95% CI: 1.018-3.347, p = 0.0435). In addition, donor CSF3R with the rs3917980G/G or A/G genotype was associated with better overall survival (OS) rates (HR = 0.560, 95% CI: 0.3162-0.9916, p = 0.047) and lower TRM rates (HR = 0.497, 95% CI: 0.2628-0.9397, p = 0.0315). Furthermore, multivariate cox analysis found that rs2275472 T/C genotype was an independent risk factors for TRM rates (HR = 3.210, 95% CI: 1.573-6.55, p = 0.001), while no statistical difference was found between rs3917980G/G or A/G genotype and clinical outcomes. Our findings demonstrate the important prognostic value of genetic variations in donor CSF3R to predict clinical outcomes in patients undergoing allo-HSCT.

Neurogenic Heterotopic Ossifications Develop Independently of Granulocyte Colony-Stimulating Factor and Neutrophils.

Neurogenic heterotopic ossifications (NHOs) are incapacitating heterotopic bones in periarticular muscles that frequently develop following traumatic brain or spinal cord injuries (SCI). Using our unique model of SCI-induced NHO, we have previously established that mononucleated phagocytes infiltrating injured muscles are required to trigger NHO via the persistent release of the pro-inflammatory cytokine oncostatin M (OSM). Because neutrophils are also a major source of OSM, we investigated whether neutrophils also play a role in NHO development after SCI. We now show that surgery transiently increased granulocyte colony-stimulating factor (G-CSF) levels in blood of operated mice, and that G-CSF receptor mRNA is expressed in the hamstrings of mice developing NHO. However, mice defective for the G-CSF receptor gene Csf3r, which are neutropenic, have unaltered NHO development after SCI compared to C57BL/6 control mice. Because the administration of recombinant human G-CSF (rhG-CSF) has been trialed after SCI to increase neuroprotection and neuronal regeneration and has been shown to suppress osteoblast function at the endosteum of skeletal bones in human and mice, we investigated the impact of a 7-day rhG-CSF treatment on NHO development. rhG-CSF treatment significantly increased neutrophils in the blood, bone marrow, and injured muscles. However, there was no change in NHO development compared to saline-treated controls. Overall, our results establish that unlike monocytes/macrophages, neutrophils are dispensable for NHO development following SCI, and rhG-CSF treatment post-SCI does not impact NHO development. Therefore, G-CSF treatment to promote neuroregeneration is unlikely to adversely promote or affect NHO development in SCI patients. © 2020 American Society for Bone and Mineral Research.

Inducible Expression of Mutant ELANE Correlates with Disruption of Differentiation Program, but Not the Unfolded Protein Response

Background. Severe congenital neutropenia (SCN) is characterized by a critical reduction in absolute neutrophil count (ANC < 500/μl), which renders the affected individual vulnerable to recurrent, life-threatening infections. A majority of SCN cases arise due to germline mutations in the neutrophil elastase gene (ELANE). Treatment with high dose granulocyte colony stimulating factor (GCSF) increases the number of neutrophils and eliminates the risk of infection. SCN patients are at a significantly high risk of developing myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML). About 70% of the MDS/AML cases report somatic mutations in the GCSF receptor gene (CSF3R), suggesting a correlation between GCSF treatment and development of MDS/AML. The current favored mechanism for ELANE mutations promoting SCN is induction of an unfolded protein response (UPR), triggering death of neutrophil precursors resulting in neutropenia. However multiple studies of UPR activation reported a requirement of treatment with chemical UPR inducers such as tunicamycin or bortezomib to observe significant changes. The mechanism of mutant ELANE-mediated neutrophil deficit thus remains elusive. In addition, the ability of G-CSF to alleviate neutropenia and its role in progression from SCN to MDS/AML is still a confounding factor in G-CSF therapy for SCN cases. Methods.We created a doxycycline inducible expression system of ELANE, consisting of wild type (wt) and point mutation (G185R) variant associated with SCN, in the murine myeloblast cell line 32D and the human promyelocytic leukemia cell line NB4. We determined the effect of mutant ELANE expression on cell proliferation (Trypan blue exclusion), survival (Trypan blue exclusion, AnnexinV-PI staining) and differentiation (Wright-Giemsa staining, CD11b expression). Underlying molecular changes were investigated using immunoblotting and qPCR based gene expression analysis. Results . Induction of wt or mutant ELANEexpression with doxycycline did not have any detrimental effects on cell proliferation and survival. However, a partial block in differentiation with an accumulation of myeloid progenitors was observed in cells expression mutant ELANE. Profiling of gene expression for markers of differentiation identified reduced expression of key transcriptional factors (Cebpa, Cebpe, Cebpb, Cebpd, Gfi1, Spi1) coupled with reduced levels of other markers of differentiation (e.g., Mpo, Ltf). Increased transcript levels were also observed in some cell cycle genes (Pola1, Ccnd1). We did not observe an increase in expression of UPR genes upon mutant ELANEinduction. Conclusion.Our data demonstrate that expression of mutant ELANE promotes an aberrant differentiation gene expression profile that leads to a partial block in differentiation. In addition, our data also suggest that mutant ELANE expression does not promote UPR. We also show a concomitant upregulation of cell proliferation genes upon mutant ELANE expression. This detour from a differentiation profile may indicate pre-leukemic underpinnings for G-CSF treatment in SCN. Disclosures No relevant conflicts of interest to declare.

Abstract B21: Epigenetic modifiers MLL1 and JMJD3 regulate neuroblastoma tumorigenicity by maintaining a cancer stem cell-like population

Abstract High-risk neuroblastoma (NB) represents a major clinical challenge in pediatric oncology. Despite significant dose escalation of intense therapies, long-term survival for NB patients remains poor (<45%), and the disease accounts for almost 15% of all pediatric cancer deaths. Relapse of metastatic, drug-resistant disease and treatment-related toxicities mandates the development of new therapeutic strategies. Recently, we discovered a highly tumorigenic, chemoresistant, and self-renewing subpopulation in NB with features similar to cancer stem cells (CSCs). This G-CSF receptor (CD114)-expressing subpopulation can escape initial therapy and cause aggressively invasive relapsed disease. Therefore, developing direct targeting strategies and understanding for the molecular mechanisms maintaining this NB CSC-like subpopulation is essential to delineate effective therapeutics for NB patients. By using siRNA screening approach and low-density pathway arrays, we found that the epigenetic regulators mixed-lineage leukemia-1 (MLL1; KMT2A; a H3K4me3 methyltransferase) and Jumonji D3 (JMJD3; KDM6B; a H3K27me3 demethylase) are important epigenetic regulators for NB proliferation and growth. Interestingly, we found that epigenetic regulators MLL1 and JMJD3 are aberrantly expressed in CD114+ subpopulation and regulate the expression of the G-CSF receptor gene (CSF3R) itself, by maintaining active histone modifications at the promoter locus. Inhibition of MLL1 and JMJD3 with specific small-molecule inhibitors reverses the histone patterns at CSF3R promoter and blocks gene expression, induces apoptosis selectively in CD114+ cells, and inhibits overall NB proliferation in vitro. Furthermore, inhibiting MLL1 and JMJD3 leads to dramatic tumor regression (p<0.001) and reduction in incidences of metastasis (p<0.001) in vivo. Most interestingly, both of these inhibitors increase the overall survival of the treated mice in vivo. As expected, reduction in tumor size was significantly correlated with the reduction in tumor CD114+ cells. Taken together, these data highlight that: a) neuroblastoma is maintained by the complex interplay of epigenetic modifiers, and b) direct targeting of these epigenetic modifiers and combining this approach with current therapy is a novel therapeutic approach to high-risk NB. Citation Format: Saurabh Agarwal, Julie Tomolonis, Sanjeev Vasudevan, Jason Shohet. Epigenetic modifiers MLL1 and JMJD3 regulate neuroblastoma tumorigenicity by maintaining a cancer stem cell-like population [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B21.

Chemo-genomic interrogation of CEBPA mutated AML reveals recurrent CSF3R mutations and subgroup sensitivity to JAK inhibitors

AbstractIn this study, we analyzed RNA-sequencing data of 14 samples characterized by biallelic CEBPA (CEBPAbi) mutations included in the Leucegene collection of 415 primary acute myeloid leukemia (AML) specimens, and describe for the first time high frequency recurrent mutations in the granulocyte colony-stimulating factor receptor gene CSF3R, which signals through JAK-STAT proteins. Chemical interrogation of these primary human specimens revealed a uniform and specific sensitivity to all JAK inhibitors tested irrespective of their CSF3R mutation status, indicating a general sensitization of JAK-STAT signaling in this leukemia subset. Altogether, these results identified the co-occurrence of mutations in CSF3R and CEBPA in a well-defined AML subset, which uniformly responds to JAK inhibitors and paves the way to personalized clinical trials for this disease.

Genetic variant of the G-CSF receptor gene is associated with lower mobilization potential and slower recovery of granulocytes after transplantation of autologous peripheral blood progenitor cells

Peripheral blood mobilized by cytokines (i.e. granulocyte colony stimulating factor, G-CSF) and chemotherapy has become a major source of hematopoietic stem and progenitor cells for transplantation (PBPCT). In this study the effect of the G-CSF receptor (CSF3R) gene polymorphism was investigated. The presence of the CSF3R variant (T allele, rs3917924) was related to CD34+ mobilization yield and the pace of granulocyte recovery after autologous PBPCT. The mobilization yield was higher in patients lacking the CSF3R variant (OR=4.756, p=0.046) and those with multiple myeloma (OR=10.534, p=0.019). The pace of granulocyte recovery was found to be associated with the CSF3R polymorphism and was significantly slower in patients carrying the CSF3R-T variant than in CC homozygotes (median of 17 vs. 13days, p<0.001). This association was confirmed (OR=4.445, p=0.014) by multiple regression analysis considering patient age and sex, the number of transplanted CD34+ cells, diagnosis and CSF3R polymorphism. These results imply that CSF3R gene polymorphism plays a significant role in PBPCT.

Hematopoietic stem cell transplantation for severe congenital neutropenia

Hematopoietic stem cell transplantation (HCT) is the only curative option for patients with severe congenital neutropenia (SCN). Transplant success is dependent on identifying at-risk patients and proceeding to transplant before the development of severe infections or malignant transformation. This review focuses on recent advancements in risk stratification of SCN patients, indications for HCT, and review of published transplant studies. Patients with poor neutrophil response despite high doses of granulocyte colony-stimulating factor (G-CSF) are at greatest risk for malignant transformation. Other studies demonstrate elevated risk with mutations in the G-CSF receptor gene and a specific mutation in the ELANE gene. These patients are at high-risk of sepsis or leukemia development and should proceed to transplant with best available donor. As recent published studies demonstrate, HCT is highly successful in patients without leukemia and, therefore, may be considered in selected low-risk patients given the life-long risk of malignancy and infection. The decision whether to proceed to HCT in healthy patients maintained on G-CSF is difficult. As transplant-related mortality continues to decrease, the role of transplant in SCN is likely to expand to more patients.

Expression of Granulocyte Colony Stimulating Factor and Its Receptor by Retinal Pigment Epithelial Cells: A Role in Maintaining Differentiation-Competent State

Purpose: Granulocyte colony stimulating factor (GCSF) is a potent hematopoietic factor that stimulates the growth of neutrophil granulocyte precursors, and also regulates the differentiation and survival of neutrophils by inhibiting apoptosis. Incidentally, GCSF is also known to act as an endogenous ligand for brain cells, counteracting acute neuronal degeneration and contributing to long-term plasticity of progenitor cells after cerebral ischemia. Since GCSF was recently reported to be present in retinal ganglions, we examined its expression in retinal pigment epithelial (RPE) cells, which, together with retinal neurons, arise from the same underlying precursor cells.Methods: We used reverse transcriptase polymerase chain reaction (PCR) to assay expression of GCSF and GCSF receptor (GCSFR) genes; immunostaining and flow cytometry to assay the presence of GCSFR on cell surfaces; bromodeoxyuridine (BrdU) incorporation measurement to monitor DNA synthesis; and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to monitor cell proliferation. The effect of GCSF on differentiation of RPE cells was assessed by immunocytochemistry to detect the presence of various marker proteins.Results: The D407 RPE cells, as well as RPE derived from cadaver eyes, were found to express both GCSF and GCSFR. Despite the presence of the GCSF receptor, exogenously added GCSF did not result in any proliferation of these cells. We found that GCSF acts like a de-differentiating factor, maintaining RPE cells in the rounded form, and in a transdifferentiation-competent state.Conclusions: The expression of GCSF and GCSFR by D407 RPE may be an important factor in RPE cell maintenance.

Granulocyte Colony-Stimulating Factor (G-CSF) Treatment of Childhood Acute Myeloid Leukemias That Overexpress the Differentiation-DefectiveG-CSFReceptor Isoform IV Is Associated With a Higher Incidence of Relapse

This prospective, multicenter Acute Myeloid Leukemia Berlin-Frankfurt-Muenster (AML-BFM) 98 study randomly tested the ability of granulocyte colony-stimulating factor (G-CSF) to reduce infectious complications and to improve outcomes in children and adolescents with acute myeloid leukemia (AML). However, a trend toward an increased incidence of relapses in the standard-risk (SR) group after G-CSF treatment was observed. Of 154 SR patients in the AML-BFM 98 cohort, 50 patients were tested for G-CSF receptor (G-CSFR) RNA isoform I and IV expression, G-CSFR cell surface expression, and acquired mutations in the G-CSFR gene. In patients randomly assigned to receive G-CSF after induction, 16 patients overexpressing the G-CSFR isoform IV showed an increased 5-year cumulative incidence of relapse (50% +/- 13%) compared with 14 patients with low-level isoform IV expression (14% +/- 10%; log-rank P = .04). The level of G-CSFR isoform IV had no significant effect in patients not receiving G-CSF (P = .19). Multivariate analyses of the G-CSF-treated subgroup, including the parameters G-CSFR isoform IV overexpression, sex, and favorable cytogenetics as covariables, revealed the prognostic relevance of G-CSFR isoform IV overexpression for 5-year event-free survival (P = .031) and the 5-year cumulative incidence of relapse (P = .049). Our results demonstrate that children and adolescents with AMLs that overexpress the differentiation-defective G-CSFR isoform IV respond to G-CSF administration after induction, but with a significantly higher incidence of relapse.

NFI-A directs the fate of hematopoietic progenitors to the erythroid or granulocytic lineage and controls β-globin and G-CSF receptor expression

AbstractIt is generally conceded that selective combinations of transcription factors determine hematopoietic lineage commitment and differentiation. Here we show that in normal human hematopoiesis the transcription factor nuclear factor I-A (NFI-A) exhibits a marked lineage-specific expression pattern: it is upmodulated in the erythroid (E) lineage while fully suppressed in the granulopoietic (G) series. In unilineage E culture of hematopoietic progenitor cells (HPCs), NFI-A overexpression or knockdown accelerates or blocks erythropoiesis, respectively: notably, NFI-A overexpression restores E differentiation in the presence of low or minimal erythropoietin stimulus. Conversely, NFI-A ectopic expression in unilineage G culture induces a sharp inhibition of granulopoiesis. Finally, in bilineage E + G culture, NFI-A overexpression or suppression drives HPCs into the E or G differentiation pathways, respectively. These NFI-A actions are mediated, at least in part, by a dual and opposite transcriptional action: direct binding and activation or repression of the promoters of the β-globin and G-CSF receptor gene, respectively. Altogether, these results indicate that, in early hematopoiesis, the NFI-A expression level acts as a novel factor channeling HPCs into either the E or G lineage.

Novel point mutations of the G‐CSF gene in a patient with neutropenia

Novel point mutations of the G‐CSF gene in a patient with neutropenia

Risk of Leukemia in Genetic Subgroups of Congenital Neutropenia (CN): Comparison of Patients with Mutations in HAX1 or ELA2

An increased risk for malignant transformation (myelodysplastic syndrome (MDS) or leukemia) is well documented in patients with congenital neutropenia (CN). However, the risk of defined genetic subgroups of CN remains unknown. We therefore assessed the incidence of leukemic transformation and potential risk factors for leukemic transformation in CN patients with ELA2 and HAX1 mutations, respectively. The overall cumulative incidence of secondary leukemias in CN, as documented by long term follow up data from the European Severe Chronic Neutropenia Registry (SCNER) and the French Neutropenia Registry (FR) is 39 out of 343 patients (11.4 %). Up to now genetic testing has been performed in 177 of the 343 CN patients revealing ELA2 mutations in 99 (56%) and HAX1 mutations in 19 (11%) of tested CN patients. In 107patients no ELA2 mutation was detectable. Out of these 107 ELA2 negative patients 59 were also negative for HAX1 mutations (33% ELA2/HAX1 negative patients). In both, patients with ELA2-CN and HAX1-CN, the clinical phenotype is characterized by a maturation arrest of myelopoiesis and severe neutropenia and cannot be discriminated morphologically. There is also no difference in the gender distribution. Both subgroups benefit from G-CSF treatment and respond well to similar G-CSF doses (median G-CSF dose in ELA2-CN is 8,5 μg/kg/day versus 6 μg/kg/day in HAX1-CN. Secondary malignancies occurred in 17 (5 MDS, .11. AML, 1 bi-phenotypic leukemia) out of 99 (17%) ELA2-CN patients, 4 (2 MDS, 1 AML, 1 bi-phenotypic leukemia) out of 19 (21%) HAX1-CN patients, and 5 (1 MDS, 3 AML, 1 ALL) out of 59 (8 %) double negative patients. Mutations in the G-CSF receptor gene have been detectable in both groups of CN patients. The frequency of mutations increases over time with a significant higher frequency of G-CSFR mutations in the CN patients who developed leukemia indicative for an involvement of G-CSFR mutations in the process of leukemic transformation. Interestingly, G-CSFR mutations are detectable in 5 of 8 ELA2-CN leukemic patients tested and 1 of 2 HAX1-CN leukemic patients tested, but also in 1 out of 4 ELA2/HAX1 negative leukemic patients. Independent of the underlying genetic defect the dose response to G-CSF treatment seems to indicate the risk for leukemia. Patients being treated with less than 5 μg/kg/day had an leukemia incidence of 14% versus 26% in patients who received 5 μg/kg/day or more (p=0.027). Within the group of patients being treated with less than 5 μg/kg/day, 14% developed leukemia, whereas in the group of patients receiving = 5 μg/kg/day 26% developed leukemia (p=0.027), suggesting that those patients requiring higher doses of GCSF may be at higher risk for malignant transformation. Leukemia outcome is dependent on the success of hematopoietic stem cell transplantation. The outcome could be improved dramatically since SCT was initiated immediately after confirmation of the leukemia diagnosis. It would be interesting to further discuss why different genetic backgrounds (ELA2 and HAX1) reveal the same clinical phenotype in terms of morphology, response to G-CSF treatment, acquisition of G-CSFR mutations and the development of leukemia.

Expression of C/EBPε Restores G-CSF-Induced Granulocytic Differentiation of Myeloid Cells Expressing the Carboxy Truncated G-CSF Receptor Associated with AML/SCN.

Mutations in the G-CSF receptor gene causing carboxy terminal truncation of the G-CSF receptor have been associated with the development of acute myelogenous leukemia (AML) in patients with severe congenital neutropenia (SCN). The truncated G-CSF receptors mediate augmented cell proliferation and survival, but are defective in inducing granulocytic differentiation in myeloid cell lines and transgenic mice. Little is known about the mechanism by which the carboxy terminus of the G-CSF receptor regulates granulocytic differentiation. It has been shown that myeloid transcription factors including C/EBPα, C/EBPε and PU.1 are critically involved in granulopoiesis. Of the three transcription factors, expression of C/EBPε was dramatically upregulated upon G-CSF treatment of myeloid 32D cells expressing the wild type G-CSF receptor (32D/WT). C/EBPε upregulation by G-CSF was abolished in 32D cells expressing the G-CSF receptor mutant D715 (32D/D715), which lacked a region of 98 amino acids in the carboxy terminus. Forced expression of C/EBPε in 32D/D715 cells at levels comparable to those in G-CSF-treated 32D/WT cells resulted in markedly attenuated cell proliferation and survival, and rapid terminal granulocytic differentiation in response to G-CSF. These data indicated that the functional defect of the D715 mutant in mediating granulocytic differentiation may arise from its inability to upregulate C/EBPε expression. Notably, forced expression of C/EBPε failed to induce terminal granulocytic differentiation of 32D/D715 cells cultured in IL-3, suggesting that the D715 receptor acted in collaboration with C/EBPε to drive granulocytic differentiation. Of the four cytoplasmic tyrosine (Tyr) residues of the G-CSF receptor, the D715 mutant contains only Tyr 704 that is involved in Stat3 activation. Because Stat3 is an important regulator of granulopoiesis, experiments are under way to address whether Tyr 704 in the D715 mutant is required for granulocytic differentiation driven by C/EBPε.

RAS and G-CSF Receptor Mutations Are Mutually Exclusive in Leukemogenesis in Severe Congenital Neutropenia.

Activating RAS mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) and were found in some patients with severe congenital neutropenia (CN) after transformation to MDS/AML (Kalra et al. Blood 1995; 86:4579). CN is a rare genetic disorder characterized by severe neutropenia leading to recurrent life-threatening bacterial infections due to the failure of myeloid cells to mature from promyelocytes and myelocytes to neutrophils. The risk for malignant transformation in CN is currently estimated at 14%. Based on earlier investigations one proposed model of the genetic events associated with progression of CN to MDS/leukemia starts with the acquirement of a somatic G-CSF Receptor gene (G-CSFR) mutation. In this model, RAS mutations and chromosomal aberrations such as monosomy 7 or trisomies represent late events in the transformation process. We have screened bone marrow specimens from 44 CN patients for mutations in the G-CSFR and in the KRAS2 and NRAS genes. Remarkably, we not only identified G-CSFR mutations in 10 out of 14 CN patients with MDS/AML, but also in 21 out of 30 patients without transformation. Only 1 out of 44 patients with CN harbored an activating RAS mutation (NRAS, c.37G>C; G13R). In this patient, who developed secondary chronic myelomonocytic leukemia at the age of 18, the NRAS mutation was not detected in a bone marrow sample obtained at age 2. Results of cytogenetic investigations were available for 10 out of the 14 CN/MDS/leukemia patients. Chromosomal abnormalities were detected in all 10 patients, and included monosomy 7 (n=3), trisomies (n=3), deletions (n=2) and translocations (n=5). In addition to the cohort of 44 patients mentioned above, we analyzed 3 of the 5 CN patients with secondary myeloid leukemia in whom a NRAS mutation, c.35G>A (G12D), was described previously (Kalra et al. Blood 1995; 86:4579). None of these patients carried a G-CSFR mutation. Combining our data with data available from the literature the frequency of RAS mutations in CN patients is now 6/55 (11%). Accordingly, RAS mutations are less common during leukemic progression in CN than suggested by earlier studies, which may reflect changes in demographics of the CN population and/or effects of early treatment with G-CSF. The sequential occurrence of G-CSFR and RAS mutations in the leukemogenesis is very rare. Instead, our genetic data suggest RAS and the G-CSFR mutations are largely mutually exclusive in CN and may deregulate common signaling pathways.

Mobilization of Human Lymphoid Progenitors after Treatment with Granulocyte Colony-Stimulating Factor

Hemopoietic stem and progenitor cells ordinarily residing within bone marrow are released into the circulation following G-CSF administration. Such mobilization has a great clinical impact on hemopoietic stem cell transplantation. Underlying mechanisms are incompletely understood, but may involve G-CSF-induced modulation of chemokines, adhesion molecules, and proteolytic enzymes. We studied G-CSF-induced mobilization of CD34+ CD10+ CD19- Lin- and CD34+ CD10+ CD19+ Lin- cells (early B and pro-B cells, respectively). These mobilized lymphoid populations could differentiate only into B/NK cells or B cells equivalent to their marrow counterparts. Mobilized lymphoid progenitors expressed lymphoid- but not myeloid-related genes including the G-CSF receptor gene, and displayed the same pattern of Ig rearrangement status as their bone marrow counterparts. Decreased expression of VLA-4 and CXCR-4 on mobilized lymphoid progenitors as well as multipotent and myeloid progenitors indicated lineage-independent involvement of these molecules in G-CSF-induced mobilization. The results suggest that by acting through multiple trans-acting signals, G-CSF can mobilize not only myeloid-committed populations but a variety of resident marrow cell populations including lymphoid progenitors.

A Novel Mutation in the Juxtamembrane Intracellular Sequence of the Granulocyte Colony-Stimulating Factor (G-CSF) Receptor Gene in a Patient with Severe Congenital Neutropenia Augments G-CSF Proliferation Activity but Not through the MAP Kinase Cascade

We analyzed the structure of the granulocyte colony-stimulating factor (G-CSF) receptor gene in a 6-year-old female patient with severe congenital neutropenia (SCN) who experienced severe recurrent infections since 1 month of age. There is no family history of any similar disease. When the patient was 4 months old, she began receiving treatment with recombinant human G-CSF that resulted in a small increase in the neutrophil count sufficient for the prevention and treatment of bacterial infection. An analysis of complementary DNA for the patient's G-CSF receptor revealed a 3-base pair deletion in the juxtamembrane intracellular sequence. This deletion at the beginning of exon 16 was thought to be caused by alternative splicing; analysis of the DNA revealed a G-to-A point mutation of the final nucleotide of intron 15. To evaluate the functional activity of the G-CSF receptor with this 3-base pair deletion of the juxtamembrane region, we transfected this G-CSF receptor mutant into an interleukin 3-dependent cell line, BAF/3. BAF/3 cells expressing the mutant G-CSF receptor showed augmented proliferation activity in response to G-CSF compared with cells having the wild-type G-CSF receptor. Although the proliferation signal of G-CSF in normal hematopoiesis is transduced through the activation of MAP kinases, this G-CSF receptor mutant showed decreased activation of ERKI/2 in response to G-CSF compared with the wild type, but the transduced sig-nal for Stat3 activation by G-CSF was of the same magnitude as that of the wild-type G-CSF receptor. This result means that the augmented proliferation activity in response to G-CSF that we observed in cells having the G-CSF receptor gene with the 3-base pair deletion is transduced through an intracellular signaling pathway other than MAP kinase. Because SCN patients with a mutation in the G-CSF receptor frequently develop leukemia, this 3-base pair deletion in the juxtamembrane sequence of the G-CSF receptor gene in this patient may be one step in the course of leukemic transformation.

A functional single-nucleotide polymorphism of the G-CSF receptor gene predisposes individuals to high-risk myelodysplastic syndrome

AbstractThe granulocyte colony-stimulating factor receptor (G-CSF-R) transmits signals for proliferation and differentiation of myeloid progenitor cells. Here we report on the identification of a rare single nucleotide polymorphism within its intracellular domain (G-CSF-R_Glu785Lys). Screening a cohort of 116 patients with primary myelodysplastic syndromes (MDS), de novo acute myeloid leukemia (AML) (84 patients), as well as 232 age- and sex-matched controls revealed a highly significant association of the G-CSF-R_785Lys allele with the development of high-risk MDS as defined by more than 5% bone marrow blasts (9.7% versus 0.9% in controls; P = .001; odds ratio [OR], 12.5; 95% confidence interval [CI], 2.4-58.9) or an International Prognostic Scoring System score of intermediate-2 or high (13.0% versus 0.9%; P < .001; OR, 14.0; 95% CI, 3.4-85.0). Functional analysis by retroviral transfer of G-CSF-R_785Lys into myeloid progenitor cells of G-CSF-R–deficient mice showed a significantly diminished colony-formation capacity after G-CSF stimulation as compared with cells transduced with the wild-type receptor. These results suggest that lifelong altered G-CSF response by the G-CSF-R_785Lys may render individuals susceptible to development of high-risk MDS.

Cellular and molecular abnormalities in severe congenital neutropenia predisposing to leukemia.

Severe congenital neutropenia (SCN) is a rare hematological disease characterized by a selective decrease in the level of circulating neutrophils in peripheral blood, maturation arrest at the promyelocyte stage of differentiation in the bone marrow, recurrent severe infections, and evolution to acute myelogenous leukemia (AML). Cellular and molecular studies of 12 SCN patients, including 5 patients that evolved to develop AML, revealed impaired proliferative characteristics and accelerated apoptosis of bone marrow progenitor cells in SCN compared with 11 healthy controls as demonstrated by flow cytometry analysis. Sequencing analysis revealed heterozygous deletion or substitution mutations in the neutrophil elastase (NE) gene in 9 of 12 patients but not in healthy controls. Expression of various NE mutants, but not normal NE, resulted in accelerated apoptosis of human promyelocytic HL-60 progenitor cells, similar to impaired survival observed in patients' cells. Bone marrow-derived primitive CD34(+) and CD33(+)/CD34(-) progenitor cells from SCN patients evolving to AML, all with mutations in the granulocyte colony-stimulating factor receptor (G-CSFR) gene, demonstrated normal cell survival, whereas more differentiated CD15(+)/CD33(-)/CD34(-) cells negative for mutant G-CSFR gene, continue to exhibit accelerated apoptosis. These data demonstrate that impaired survival of bone marrow myeloid progenitor cells, probably driven by expression of mutant NE, is the cellular mechanism responsible for neutropenia in SCN. Furthermore, our results suggest that acquired G-CSFR mutations may initiate signaling events that override the pro-apoptotic effect of mutant NE in primitive progenitor cells, resulting in an expansion of the abnormal AML clone.