Aberrant subcellular targeting of the G185R neutrophil elastase mutant associated with severe congenital neutropenia induces premature apoptosis of differentiating promyelocytes

Abstract Mutations in the ELA2 gene encoding neutrophil elastase (NE) in patients with severe congenital neutropenia (SCN) are involved in the pathogenesis of this disorder, possibly due to the abnormal protein trafficking and accelerated apoptosis of myeloid cells. In this study we precisely examined the localization of NE in neutrophils and myeloid precursor cells in bone marrow in patients with SCN using immunofluorescence microscopy equipped with three-dimensional analysis program. Three patients with SCN were enrolled in this study. All patients with SCN showed heterozygous mutation in the ELA2 gene. In normal subjects the pattern of localization of NE in mature neutrophils was almost similar to those of myeloperoxidase (MPO), proteinase 3, lysosomal associated membrane protein 2 (LAMP2). Administration of G-CSF to normal subjects did not affect the pattern of the localization of these proteins in neutrophils. In contrast, mature neutrophils elicited by the administration of G-CSF in patients with SCN NE predominantly localized to the plasma membranes. A small part of NE was detected in the cytoplasmic compartment. The pattern of localization of NE was significantly different from those of MPO, proteinase 3, and LAMP2 in SCN patients, suggesting the abnormal traffic of NE to granules. Adaptor proteins 3 (AP3) specifically shuttles transmembrane cargo proteins from the trans-Golgi to lysosomes. AP3 of myeloid progenitor cells enriched for CD33-positive cells in normal bone marrow was localized in both cytoplasm and plasma membranes. The localization pattern of AP3 was completely consistent with those of NE, MPO, and LAMP2. The localization of AP3 of promyelocytes in patients with SCN was observed in both plasma membranes and cytoplasm. This finding was completely similar to that in normal myeloid precursor cells. However, the localization of NE of promyelocytes in SCN patients was predominantly in plasma membrane. The figures merged apparently presented the different localization of NE and AP3. This result was confirmed by the 3-dimensional analysis with histogram. The localizations of other constituents of primary granules, MPO, poteinase 3, and LAMP2, were consistent with those of AP3. These observations suggest that the mislocalization of NE in myeloid precursor cells in SCN patients does not result from a generalized impairment of protein trafficking but is specific to the mutant NE. The abnormal localization of NE may be involved in the pathogenesis of SCN associated with ELA2 mutation.

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Myelopoiesis From Induced Pluripotent Stem Cells Reveals The Role Of Elastase Activity In The Pathogenesis Of Severe Congenital Neutropenia

Blood ◽

10.1182/blood.v122.21.442.442 ◽

2013 ◽

Vol 122 (21) ◽

pp. 442-442

Author(s):

Ramesh C Nayak ◽

Lisa Trump ◽

H. Leighton Grimes ◽

Carolyn Lutzko ◽

Jose A. Cancelas

Keyword(s):

Stem Cell ◽

Neutrophil Elastase ◽

Embryoid Body ◽

Point Mutations ◽

Annexin V ◽

High Dose ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Elastase Activity ◽

Induced Pluripotent

Severe congenital neutropenia (SCN) is a hereditary neutropenia characterized by granulocytic precursor differentiation arrest in which the pathogenic mechanism is poorly understood. Over half of SCN patients associate with autosomal dominant point mutations in the gene encoding neutrophil elastase (ELANE). High-dose G-CSF therapy succeeds in increasing neutrophil counts in many SCN patients, but the molecular explanation for high dose G-CSF rescue is unknown. Mutations in the G-CSF receptor (CSF3R) associated with a dramatic risk for transformation provides a cautionary note for high dose G-CSF treatment. The pathogenic mechanism of ELANE mutations is incompletely understood due to the lack of disease recapitulation in murine models of ELANE-mutant hematopoiesis and the difficulty to obtain primary, relevant myeloid cell populations from patients. Using induced pluripotent stem cell (iPSC) derived myelopoietic cultures from peripheral blood mononuclear cells of patients with ELANE exon 3 point mutations (Q97P and I118N), we designed experiments to recapitulate G-CSF signaling in SCN myelopoiesis. The patient-derived iPSC lines were characteristically similar to human embryonic stem cell lines and normal blood-derived iPSC lines with expression of SSEA-4, Tra-1-60, Tra-1-81, and CD9 pluripotency markers >85% in all lines as determined by FACS analysis at passage 10-15. All iPSC retained a normal karyotype and ELANE locus mutations of the original specimens. Both SCN iPSC lines and control iPSC lines (normal and immune-mediated congenital neutropenia) show functional G-CSF (50 ng/mL)-induced differentiation into the myeloid lineage. However, G-CSF failed to induce terminal granulocytic differentiation of SCN iPSC derived myeloid progenitors resulting in differentiation arrest at the promyelocyte/myelocyte stage. The hematopoietic cells derived from SCN iPSC line show reduced frequency of CFU-G and CFU-GM (CFU-G+CFU-GM, 22±1.15 in 104 embryoid body cells from non SCN iPSC vs 4±2 in 104 embryoid body cells from ELANE mutated SCN iPSC). Moreover, SCN iPSC promyelocytes show significantly increased apoptosis over controls as determined by Annexin-V analysis (31% Annexin-V+ in SCN iPSC derived promyelocyte vs 3% in control iPSC). Surviving SCN promyelocytes showed decreased reactive oxygen species activity and no phagocytic activity. In contrast, suprapharmacological doses of G-CSF (1000 ng/mL) rescued SCN iPSC-derived differentiation. It has been shown that NE, expressed in promyelocytes, can cleave recombinant G-CSF and CSF3R. We evaluated the possibility that inadequate G-CSF signaling in SCN iPSC was due to abnormal elastase activity. To this end, we supplemented 50 ng/mL G-CSF cultures with a specific, cell-permeant neutrophil elastase inhibitor (Sivelastat, 230 nM) to find rescued granulocyte differentiation arrest (31% neutrophil in myeloid differentiation culture of SCN iPSC derived hematopoietic progenitors in presence of Sivelastat vs 2% mature neutrophils without Sivelastat). In conclusion, SCN modeling through patient iPSC derived myelopoiesis and G-CSF driven granulocytic recapitulation unveil a mechanism of resistance to G-CSF therapy with important translational implications for therapy. Combination of low-dose G-CSF and NE inhibitory therapy may result in an efficacious, safer approach in SCN therapy. Disclosures: No relevant conflicts of interest to declare.

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Mutations in the ELA2 gene encoding neutrophil elastase are present in most patients with sporadic severe congenital neutropenia but only in some patients with the familial form of the disease

Blood ◽

10.1182/blood.v98.9.2645 ◽

2001 ◽

Vol 98 (9) ◽

pp. 2645-2650 ◽

Cited By ~ 87

Author(s):

Phil J. Ancliff ◽

Rosemary E. Gale ◽

Ri Liesner ◽

Ian M. Hann ◽

David C. Linch

Keyword(s):

Neutrophil Elastase ◽

Autosomal Recessive ◽

Autosomal Dominant ◽

Autosomal Recessive Disorder ◽

Severe Neutropenia ◽

Base Substitution ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Gene Encoding ◽

Familial Form

Abstract Severe congenital neutropenia (SCN) was originally described as an autosomal recessive disorder. Subsequently, autosomal dominant and sporadic forms of the disease have been recognized. All forms are manifest by persistent severe neutropenia and recurrent bacterial infection. In contrast, cyclical hematopoiesis is characterized by periodic neutropenia inter-spaced with (near) normal neutrophil counts. Recently, linkage analysis on 13 affected pedigrees identified chromosome 19p13.3 as the likely position for mutations in cyclical hematopoiesis. Heterozygous mutations in the ELA2 gene encoding neutrophil elastase were detected in all families studied. Further work also demonstrated mutations in ELA2 in sporadic and autosomal dominant SCN. However, all mutations described to date are heterozygous and thus appear to act in a dominant fashion, which is inconsistent with an autosomal recessive disease. Therefore, the current study investigated whether mutations in ELA2could account for the disease phenotype in classical autosomal recessive SCN and in the sporadic and autosomal dominant types. All 5 exons of ELA2 and their flanking introns were studied in 18 patients (3 autosomal recessive, 5 autosomal dominant [from 3 kindreds], and 10 sporadic) using direct automated sequencing. No mutations were found in the autosomal recessive families. A point mutation was identified in 1 of 3 autosomal dominant families, and a base substitution was identified in 8 of 10 patients with the sporadic form, though 1 was subsequently shown to be a low-frequency polymorphism. These results suggest that mutations in ELA2are not responsible for classical autosomal recessive Kostmann syndrome but provide further evidence for the role of ELA2 in SCN.

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Severe congenital neutropenia in a multigenerational family with a novel neutrophil elastase (ELANE) mutation

Annals of Hematology ◽

10.1007/s00277-010-1056-4 ◽

2010 ◽

Vol 90 (2) ◽

pp. 151-158 ◽

Cited By ~ 5

Author(s):

Esther van de Vosse ◽

Els M. Verhard ◽

Anton J. T. Tool ◽

Adriëtte W. de Visser ◽

Taco W. Kuijpers ◽

...

Keyword(s):

Neutrophil Elastase ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Multigenerational Family

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The C-Terminal Region of the G-CSF Receptor Is Required for Induction of Neutrophil Elastase Expression in Myeloid Cells: Implication for Understanding CSF3R Mutations in Acute Myeloid Leukemia Evolving from Severe Congenital Neutropenia.

Blood ◽

10.1182/blood.v108.11.1154.1154 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1154-1154

Author(s):

Fan Dong ◽

Yaling Qiu ◽

Alan D. Friedman ◽

Qingquan Liu

Keyword(s):

Acute Myeloid Leukemia ◽

Neutrophil Elastase ◽

Myeloid Leukemia ◽

Myeloid Cells ◽

Luciferase Reporter ◽

Severe Congenital Neutropenia ◽

Wild Type ◽

Congenital Neutropenia ◽

Nonsense Mutations ◽

Acute Myeloid

Abstract Severe congenital neutropenia (SCN) is characterized by early onset of bacterial infections and maturation arrest of myeloid cells at early stages of differentiation in the bone marrow. Point mutations in ELA2 encoding neutrophil elastase (NE) have been identified in 60% to 80% of patients with SCN. SCN patients are predisposed to acute myeloid leukemia (AML), which occurs in approximately 15 % of cases. With rare exceptions, leukemic cells from these patients carry mutations in CSF3R encoding the G-CSF receptor, leading to C-terminal truncation of the receptor. Notably, the nonsense mutations in CSF3R are present only in SCN/AML patients, particularly those with ELA2 mutations, but not in other types of neutropenias and de novo AML. The mechanism for the exclusive presence of the nonsense CSF3R mutations in SCN/AML is unknown. In myeloid 32D cells transfected with the wild type (WT) G-CSF receptor (32D/WT), G-CSF treatment induced the expression of NE. However, NE expression was not upregulated by G-CSF in 32D cells expressing the truncated G-CSF receptor d715, derived from an SCN patient. It has been shown that myeloid cells from patients with SCN/AML express both the wild type and the truncated G-CSF receptors. Indeed, the d715 mutant acted in a dominant negative manner to suppress NE upregulation by the WT G-CSF receptor. In luciferase reporter assays, the WT G-CSF receptor, but not the d715 mutant, activated a 1.8-kb fragment of the mouse Ela2 promoter. Significantly, forced expression of an SCN-associated NE mutant G185R caused premature apoptosis of differentiating 32D/WT cells in response to G-CSF with no significant effect on IL-3-stimulated survival. To address whether the d715 mutant may abolish the proapoptotic effect of the G185R mutant via suppressing its expression, we transfected 32D/WT and 32D/d715 cells with an expression construct in which the expression of the G185R mutant was driven by the 1.8-kb fragment of the Ela2 promoter. G-CSF treatment induced the expression of the G185R mutant and subsequent apoptosis in 32D/WT cells. In 32D/d715 cells, however, the expression of the G185R mutant was not induced by G-CSF and accordingly its proapoptotic activity was not evident. We propose that acquisition of the nonsense mutations in CSF3R may represent a mechanism utilized by the myeloid cells harboring the ELA2 mutations to evade the proapoptotic effect of the NE mutants. However, expression of the truncated G-CSF receptors has other biological consequences: they transduce strong proliferative signals but are defective in inducing granulocytic differentiation, which may initiate the leukemogenic process.

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Small Molecule Inhibitor of Neutrophil Elastase Normalizes Myeloid Differentiation and Improves Impaired Cell Survival Triggered by Elastase Mutations In Patients with Severe Congenital Neutropenia and Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.386.386 ◽

2010 ◽

Vol 116 (21) ◽

pp. 386-386

Author(s):

Andrew A. Aprikyan ◽

Vahagn Makaryan ◽

Maxim Totrov ◽

Ruben Abagyan ◽

David C. Dale

Keyword(s):

Bone Marrow ◽

Small Molecule ◽

Neutrophil Elastase ◽

Myeloid Leukemia ◽

Small Molecule Inhibitor ◽

Myeloid Differentiation ◽

Severe Congenital Neutropenia ◽

Wild Type ◽

Congenital Neutropenia ◽

Molecule Inhibitor

Abstract Abstract 386 Heterozygous mutations in the neutrophil elastase gene ELANE have been identified as the primary cause of severe congenital neutropenia (SCN) associated with recurring severe infections and evolution to acute myeloid leukemia (AML). As of today, more than 50 substitution, truncation, insertion and deletion mutations have been identified. Animal studies based on knock-in or knockout of ELANE in mice failed to produce severe neutropenia phenotype. We and others previously reported that expression of various mutants but not wild type neutrophil elastase (NE) in human but not murine cells triggers accelerated apoptosis. We also reported that expression of mutant NE (del.145-152), identified in SCN patients one of whom evolved to develop MDS/AML, in human promyelocytic tet-off HL60 cells causes both accelerated apoptosis and characteristic block of myeloid differentiation similar to that seen in bone marrow of SCN patients. Examination of the tertiary structure of NE revealed that most of the mutations leave the active site of the mutant protease intact. We identified a small molecule inhibitor of neutrophil elastase, a derivative of L-malic acid (Merck, USA), that blocked the proteolytic activity of NE by approximately 80% and was capable of restoring impaired myeloid differentiation and normalizing production of myeloid cells expressing del145-152 NE mutant. It is important to note that block of proteolytic activity of NE with the NE-SMI had no adverse effect on control human myeloid progenitor cells expressing wild type NE, thus confirming the gain-of-function effect of NE mutants. More than 20% of SCN patients with NE mutations evolve to develop AML. Molecular modeling and analysis of the tertiary structures of NE available through the Protein Database revealed that 16 different mutations identified in AML patients affect predominantly the N95 or N144 glycosylation sites or the binding pocket of the protease suggesting that altered substrate specificity of the mutant enzyme is the cause of accelerated apoptosis and block of myeloid differentiation in SCN/AML. We sought to obtain bone marrow samples from 2 unrelated SCN/AML patients both on G-CSF treatment harboring either C122Y or insPQ94. Bone marrow purified CD34+ and/or CD34-/CD33+ myeloid progenitors from the patients showed basal level of apoptosis in a range of 20–25%, which gradually increased reaching 40–50% apoptosis by 3 days of culture. Importantly, treatment of primary bone marrow-derived cells with NE-SMI substantially reduced accelerated apoptosis to near initial rate with approximately up to 2-fold reduction of apoptosis by 3 days of culture as determined by flow cytometry. Thus, our findings demonstrate that 1) small molecule inhibitor of neutrophil elastase is effective in blocking accelerated apoptosis triggered by three different NE mutations identified in SCN patients evolved to develop MDS/AML and 2) the small molecule inhibitor of NE is a promising therapeutic agent that should be considered for testing in clinical trials in SCN/AML patients. Disclosures: Dale: Amgen: Consultancy, Research Funding; Merck: Patents & Royalties, Research Support.

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Contributions to Neutropenia from PFAAP5 (N4BP2L2), a Novel Protein Mediating Transcriptional Repressor Cooperation between Gfi1 and Neutrophil Elastase

Molecular and Cellular Biology ◽

10.1128/mcb.00596-09 ◽

2009 ◽

Vol 29 (16) ◽

pp. 4394-4405 ◽

Cited By ~ 25

Author(s):

Stephen J. Salipante ◽

Meghan E. B. Rojas ◽

Brice Korkmaz ◽

Zhijun Duan ◽

Jeremy Wechsler ◽

...

Keyword(s):

Neutrophil Elastase ◽

Target Genes ◽

Transcriptional Repressor ◽

Theoretical Models ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Cyclic Neutropenia ◽

Reporter Assays ◽

Neutrophil Differentiation ◽

Novel Protein

ABSTRACT “Neutropenia” refers to deficient numbers of neutrophils, the most abundant type of white blood cell. Two main forms of inherited neutropenia are cyclic neutropenia, in which neutrophil counts oscillate with a 21-day frequency, and severe congenital neutropenia, in which static neutropenia may evolve at times into leukemia. Mutations of ELA2, encoding the protease neutrophil elastase, can cause both disorders. Among other genes, severe congenital neutropenia can also result from mutations affecting the transcriptional repressor Gfi1, one of whose genetic targets is ELA2, suggesting that the two act through similar mechanisms. In order to identify components of a common pathway regulating neutrophil production, we conducted yeast two-hybrid screens with Gfi1 and neutrophil elastase and detected a novel protein, PFAAP5 (also known as N4BP2L2), interacting with both. Expression of PFAAP5 allows neutrophil elastase to potentiate the repression of Gfi1 target genes, as determined by reporter assays, RNA interference, chromatin immunoprecipitation, and impairment of neutrophil differentiation in HSCs with PFAAP5 depletion, thus delineating a mechanism through which neutrophil elastase could regulate its own synthesis. Our findings are consistent with theoretical models of cyclic neutropenia proposing that its periodicity can be explained through disturbance of a feedback circuit in which mature neutrophils inhibit cell proliferation, thereby homeostatically regulating progenitor populations.

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A lack of secretory leukocyte protease inhibitor (SLPI) causes defects in granulocytic differentiation

Blood ◽

10.1182/blood-2013-06-508887 ◽

2014 ◽

Vol 123 (8) ◽

pp. 1239-1249 ◽

Cited By ~ 29

Author(s):

Olga Klimenkova ◽

Wienke Ellerbeck ◽

Maksim Klimiankou ◽

Murat Ünalan ◽

Siarhei Kandabarau ◽

...

Keyword(s):

Protease Inhibitor ◽

Neutrophil Elastase ◽

Secretory Leukocyte Protease Inhibitor ◽

Myeloid Differentiation ◽

Severe Congenital Neutropenia ◽

Granulocytic Differentiation ◽

Congenital Neutropenia ◽

Key Points ◽

Natural Inhibitor

Key Points The natural inhibitor of neutrophil elastase, SLPI, is severely reduced in severe congenital neutropenia patients. SLPI controls myeloid differentiation by regulation of NFκB, ERK1/2:LEF-1, and c-myc activation.

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Mice expressing a neutrophil elastase mutation derived from patients with severe congenital neutropenia have normal granulopoiesis

Blood ◽

10.1182/blood-2002-05-1372 ◽

2002 ◽

Vol 100 (9) ◽

pp. 3221-3228 ◽

Cited By ~ 49

Author(s):

David S. Grenda ◽

Sonja E. Johnson ◽

Jill R. Mayer ◽

Morgan L. McLemore ◽

Kathleen F. Benson ◽

...

Keyword(s):

Neutrophil Elastase ◽

Blood Analysis ◽

Severe Congenital Neutropenia ◽

Normal Numbers ◽

Granulocytic Differentiation ◽

Congenital Neutropenia ◽

Gene Encoding ◽

Cyclic Neutropenia ◽

Related Disorder ◽

Increased Risk

Abstract Severe congenital neutropenia (SCN) is a syndrome characterized by an isolated block in granulocytic differentiation and an increased risk of developing acute myeloid leukemia (AML). Recent studies have demonstrated that the majority of patients with SCN and cyclic neutropenia, a related disorder characterized by periodic oscillations in the number of circulating neutrophils, have heterozygous germline mutations in the ELA2 gene encoding neutrophil elastase (NE). To test the hypothesis that these mutations are causative for SCN, we generated transgenic mice carrying a targeted mutation of theirEla2 gene (“V72M”) reproducing a mutation found in 2 unrelated patients with SCN, one of whom developed AML. Expression of mutant NE mRNA and enzymatically active protein was confirmed. Mice heterozygous and homozygous for the V72M allele have normal numbers of circulating neutrophils, and no accumulation of myeloid precursors in the bone marrow was observed. Serial blood analysis found no evidence of cycling in any of the major hematopoietic lineages. Rates of apoptosis following cytokine deprivation were similar in wild-type and mutant neutrophils, as were the frequency and cytokine responsiveness of myeloid progenitors. The stress granulopoiesis response, as measured by neutrophil recovery after cyclophosphamide-induced myelosuppression, was normal. To define the leukemogenic potential of V72M NE, a tumor watch was established. To date, no cases of leukemia have been detected. Collectively, these data suggest that expression of V72M NE is not sufficient to induce an SCN phenotype or leukemia in mice.

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