scholarly journals Efficient Correction of HAX1 Mutations in Primary HSPCs of Severe Congenital Neutropenia Patients Using CRISPR/CAS9 GENE-Editing

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-22
Author(s):  
Malte U Ritter ◽  
Benjamin Secker ◽  
Masoud Nasri ◽  
Maksim Klimiankou ◽  
Benjamin Dannenmann ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Stem Cell ◽  
Protein Expression ◽  
Healthy Donor ◽  
Live Cell Imaging ◽  
Gene Editing ◽  
Cell Imaging ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia

Patients with the rare pre-leukemia bone marrow failure syndrome severe congenital neutropenia (CN) have markedly reduced numbers of neutrophils in peripheral blood (<500/μl), leading to frequent infections and requiring chronic granulocyte stimulating factor (G-CSF) treatment. Approximately 7 % of CN patients carry homozygous loss-of-function mutations in the HAX1 gene. 25 % of HAX1-CN patients develop MDS or AML. The only curative therapy for CN patients with overt MDS/AML is hematopoietic stem cell transplantation with its associated risks. A clinical need for gene therapy for CN patients is imminent. Here, we describe for the first time the application of CRISPR/Cas9 gene-editing in combination with recombinant adeno associated virus 6 (rAAV6)-based delivery of the template for homology-directed repair (HDR) for the mutated HAX1 gene in primary bone marrow mononuclear CD34+ cells (HSPCs) of HAX1-CN patients. We selected HAX1 mutation p.W44X as the most frequently described mutation in HAX1-CN. We established the delivery of the chemically modified sgRNA in combination with SpCas9 V3 in primary HSPCs using electroporation. The HDR template was generated by PCR from healthy donor HSPCs and cloned into pRC6 vector for the production of high titer rAAV6 (>12x1012 viral copies per ml). Our gene-editing protocol produced on average 79,7 % (± 8,62 %) of total editing (TE) in healthy donor HSPCs (n=6). When we transduced healthy donor HSPCs with rAAV6 containing the template at MOI 105 after electroporation with CRISPR/Cas9 RNP, we achieved 38,1 % (± 1,3 %) knock-in (KI) efficiency and 82,3 % (± 8,2 %) TE (n=2). We further applied this approach to primary HSPCs from 5 CN patients harboring the p.W44X HAX1 mutation. We achieved 84,4 % (± 4,2 %) TE and 65,8 % (± 7,12 %) KI. Too proof, that our editing reintroduced HAX1 protein expression, we performed Western Blot analysis of edited cells (n=2) and were able to detect relevant amounts of HAX1 protein. To assess the effect of HAX1 correction on the neutropenic phenotype in vitro, we performed a liquid culture differentiation assay of edited HSPCs to neutrophils. HSPCs from the same patients that were edited in the AAVS1 safe harbor were used as isogenic controls. In the AAVS1 locus the editing efficiency was 76,74 % (± 17,07 %) total indels. By morphological assessment of Wright-Giemsa stained cytospins of edited cells derived on day 14 of differentiation revealed significant (p = 0,005) increases of mature neutrophils for all five edited HAX1-CN patient samples, as compared to the respective controls. This phenotype correction was also observed in flow cytometry by a significant (p = 0,011) increase of mature CD34-CD45+ CD15+CD16+ neutrophils (n=5). To investigate if the HAX1 mutation correction and reinforced expression of HAX1 protein improved the sensitivity of HSPCs to oxidative stress as described by Klein et al. 2007, we performed live-cell imaging of caspase3/7 activation. Live-cell imaging revealed a substantial reduction of H2O2-induced apoptosis in corrected HAX1-CN patients derived HSPCs (n=3). Furthermore, the corrected differentiated cells were investigated for functional hallmarks of granulocytes. We could observe that HAX1 gene-edited HSPCs showed comparable chemotaxis, phagocytosis and no defects in ROS production to isogenic control edited cells. Taken together, we established a protocol for efficient selection-free correction of HAX1 p.W44X mutation in primary HSPCs using CRISPR/Cas9 and rAVV6 HDR repair templates. Our gene-editing reintroduced HAX1 protein expression in primary HSPCs from HAX1-CN patients. Neutrophils derived from corrected cells showed functional improvements in survival to oxidative stress and general neutrophil functions. We believe that these results are enticing to be investigated further for potential clinical translation as an autologous stem cell therapy for HAX1-CN patients. Disclosures No relevant conflicts of interest to declare.

Successful Hematopoietic Stem Cell Transplantation Using an Immunosuppressive Conditioning Regimen in Ten Patients with Severe Congenital Neutropenia: A Single-Institute Experience

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3688-3688
Author(s):  
Yoko Mizoguchi ◽  
Mizuka Miki ◽  
Aya Furue ◽  
Shiho Nishimura ◽  
Maiko Shimomura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Acute Gvhd ◽  
Conditioning Regimen ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Hematopoietic Stem ◽  
Engraftment Failure

Abstract Severe congenital neutropenia (SCN) is a rare heterogeneous genetic disorder characterized by severe chronic neutropenia, with absolute neutrophil counts below 0.5×109/L, and by recurrent bacterial infections from early infancy. Granulocyte colony-stimulating factor (G-CSF) is widely used for the treatment of neutropenia in patients with SCN. However, the long-term G-CSF therapy has a relative risk of developing myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The only curative treatment available for SCN patients is hematopoietic stem cell transplantation (HSCT). Recently, HSCTs with reduced intensity conditioning (RIC) regimens have been applied to the treatment of SCN patients without malignant transformation who have become G-CSF refractory. However, the optimal conditions of HSCT for SCN patients have not been established. In this study, we conducted bone marrow cell transplantations (BMT) in ten patients with SCN using an immunosuppressive conditioning regimen to minimize early and late transplant-related morbidity in Hiroshima University Hospital. Ten patients with a total of 11 HSCT procedures in our institution (performed from 2007 to 2015) were enrolled in this study. Four of the ten patients had experienced engraftment failure of the initial HSCT and three of them were referred to our hospital for re-transplantation. Heterozygous mutation inthe ELANE gene was identified in nine of ten patients. These nine patients received BMT less than 10 years of age. All ten patients had recurrently experienced moderate to severe bacterial or fungal infection before HSCT and received temporal or regular administration of G-CSF. Bone marrow cells (BM) were obtained from five HLA-matched related (MRD), three HLA-matched unrelated (MUD), and three HLA-mismatched unrelated (7/8) donors (MMUD), respectively. The conditioning regimen basically consisted of fludarabine (100 to 125 mg/m2), cyclophosphamide (100 to 150 mg/kg), melphalan (70 to 90 mg/m2), total body irradiation (3 to 3.6 Gy), and/or anti-thymocyte globulin (10 to 12 mg/kg). Short-term methotrexate and tacrolimus were administered for the prophylaxis of graft-versus-host disease (GVHD). Engraftment of neutrophils was successfully observed within 24 days of post-transplantation in all patients. All patients achieved complete chimerism at the time of engraftment. Two patients who underwent BMT from MRD and one patient who underwent BMT from MUD showed the gradual decrease of donor-derived cells. Donor lymphocyte infusion treatment successfully achieved the complete chimerism or stable mixed chimerism in these 3 patients. Although 3 patients experienced the acute GVHD (Grade I-II), the addition of glucocorticoids to tacrolimus prevented the extension of acute GVHD. Only one patient developed mild chronic GVHD presenting limited type of skin involvement. All patients are alive for 9 months to 9 years after HSCT with no signs of severe infections or transplantation-related morbidity. Our results demonstrate that BMT together with a sufficient immunosuppressive conditioning regimen may be a feasible and effective treatment for SCN patients, irrespective of initial engraftment failure. Although our results through the small number of cohort is limited to conclude, the BMT with the optimal donors may lead to the increased opportunity for lower risk of SCN patients especially at younger age as a curative treatment. The further analyses of accumulated cases are necessary to assess the efficacy, safety, and less late adverse effects related to HSCT including fertility. Disclosures No relevant conflicts of interest to declare.

G-CSF Receptor Mutations Found in Patients with Severe Congenital Neutropenia Confer a Strong Competitive Advantage at the Hematopoeitic Stem Cell Level That Is Dependent on Increased Systemic Levels of G-CSF.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 457-457
Author(s):  
David S. Grenda ◽  
Fulu Liu ◽  
Michael Richards ◽  
Daniel C. Link
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Competitive Advantage ◽  
B Lymphocyte ◽  
Bone Marrow Cells ◽  
Severe Congenital Neutropenia ◽  
Wild Type ◽  
Congenital Neutropenia ◽  
Gain Of Function ◽  
Donor Chimerism

Abstract Patients with severe congenital neutropenia (SCN) have a markedly increased risk of developing myelodysplasia (MDS) or acute myeloid leukemia (AML). Though the genetic basis for this increased susceptibility is unknown, gain-of-function mutations of the G-CSF receptor (G-CSFR) have been found in the great majority of patients with SCN who develop MDS/AML. These mutations are somatic and produce a truncated G-CSFR that, though remaining ligand dependent, transmits a hyperproliferative signal. We and others have shown that targeted transgenic mice expressing a representative G-CSFR mutation (termed d715) have markedly exaggerated neutrophil responses to G-CSF treatment. Based on these observations, it has been suggested that these gain-of-function G-CSFR mutations contribute to leukemogenesis. However, direct evidence supporting this hypothesis is scant. Moreover, it is unclear how hematopoietic cells expressing the mutant G-CSFR gain clonal dominance. Finally, it is not clear why these G-CSFR mutations are uniquely associated with SCN and rarely seen in de novo AML. To address these questions, we generated a series of bone marrow chimeras reconstituted with both wild type and d715 G-CSFR hematopoietic cells, thus reproducing, in part, the mixed bone marrow populations found in patients with SCN. Equal numbers of wild type or d715 G-CSFR bone marrow cells were transplanted into irradiated syngeneic hosts and donor chimerism periodically assessed by flow cytometry. At 5 weeks post-transplantation the contribution of d715 cells to the myeloid (percentage of d715 cells ± SD: 45.7 ± 12.0%, n=9), B-lymphocyte (63.5 ± 5.8%), and T-lymphocyte (46.6 ± 6.4%) lineages was near the expected level of 50%. Surprisingly, this level of chimerism was stable over the 6-month observation period, showing that the d715 G-CSFR does not confer a competitive advantage under basal conditions. In patients with SCN, systemic levels of G-CSF are elevated either due to increased endogenous production or exogenous G-CSF treatment. To simulate this condition, a cohort of chimeric mice was treated with G-CSF (10μg/kg/day) for 21 days. At the end of the treatment period, the contribution of d715 cells to the myeloid lineage in the blood increased to 97.6 ± 1.2% (n=5). Surprisingly, a marked increase in d715 donor chimerism in the B-lymphocyte lineage in the bone marrow also was observed (89.1 ± 5.7%). Remarkably, this shift in donor chimerism extended to the hematopoietic stem cell (HSC) compartment as defined by Kit+ lineage− Sca+ (KLS) cells; the contribution of d715 to the KLS cell population in G-CSF treated mice was 97.8 ± 0.8% versus 53.3 ± 11.5% in untreated mice. Transplantation of bone marrow cells from these mice into secondary recipients showed that this brief (21 day) exposure to G-CSF was sufficient to significantly expand the d715 HSC. Collectively, these data show that expression of the d715 G-CSFR results in a strong competitive advantage at the HSC level, but only in the presence of an increased concentration of G-CSF. Furthermore, they provide an explanation for the association of these mutations with SCN since SCN is one of a small number of conditions in which systemic levels of G-CSF are chronically elevated. Finally, the effect of G-CSF signals on HSC function provides further evidence for the contributions of these mutations to leukemogenesis since it is the HSC compartment in which leukemia is thought to arise.

New G-CSF-Dependent Signaling Pathway and Its Role In Patients with Severe Congenital Neutropenia and Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 385-385
Author(s):  
Julia Skokowa ◽  
Elena Chirvon ◽  
Lan Dan ◽  
Kshama Gupta ◽  
Ana Gigina ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Protein Expression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Gene Promoter ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Cd34 Cells ◽  
Acute Myeloid

Abstract Abstract 385 We demonstrated that stimulation of hematopoietic CD34+ cells with granulocyte-colony stimulating factor (G-CSF) leads to phosphorylation of hematopoietic cell-specific Lyn substrate 1 (HCLS1) protein and HCLS1 interaction with G-CSF receptor-associated tyrosine kinases Lyn and Syk. Activated HCLS1 binds to lymphoid-enhancer factor 1 (LEF-1) protein, inducing LEF-1-dependent autoregulation of the LEF-1 gene promoter. Transcription factor LEF-1 is a downstream effector of Wnt signaling and a master regulator of myeloid differentiation. In patients with severe congenital neutropenia (CN), inherited mutations in the HCLS1-associated protein × 1 (HAX1) lead to profound defects in the G-CSF-triggered expression and phosphorylation of HCLS1 protein and subsequently, to reduced expression of LEF-1 and abrogated granulopoiesis. Using immunoprecipitation assay, we identified endogenous HAX1 protein in the complex with LEF-1 and HCLS1 proteins. HAX1 is known as a mitochondrial protein, however using cell fractionation experiments of different myeloid cell lines (NB4, HL60, THP-1) and of primary CD34+ cells we identified endogenous HAX1 protein in different intracellular compartments. Thus, HAX1 protein was expressed in mitochondria, cytoplasm, nucleus, cytosol and in membranous fraction. Expression levels of HAX1 protein varied between different cell lines and between maturation stage of the cells. In vitro inhibition of HCLS1 or HAX1 by shRNA severely disrupted granulocytic differentiation of CD34+ cells, due to a lack of LEF-1 and C/EBPa protein expression and subsequently abolished activation of LEF-1 and C/EBPa gene promoters. Intriguingly, LEF-1 in turn induced HCLS1 mRNA and protein expression by direct binding to the HCLS1 gene promoter and inhibition of LEF-1 in CD34+ cells leads to severe downregulation of HCLS1 protein levels. These findings demonstrated a reciprocal feed-back regulation between LEF-1 and HCLS1 proteins. Moreover, HCLS1-/- mice are neutropenic due to a lack of LEF-1 protein expression and defective in vitro G-CSF-triggered F-actin rearrangement in bone marrow myeloid progenitor cells, as compared to WT mice. At the same time, in bone marrow biopsies of 88.46 % of tested patients (46 out of 52 patients) with acute myeloid leukemia (AML), we found significantly elevated levels of HCLS1 protein. Inhibition of HCLS1 in the AML cell lines and primary blasts of AML patients resulted in reduced proliferation and increased apoptosis. Taken together, we described a novel G-CSFR downstream signaling pathway, with dose-dependent effects on myelopoiesis: diminished expression of HCLS1 and LEF-1 resulted in a “maturation arrest” of granulopoiesis and severe neutropenia, but hyperactivation of one of these proteins led to a hyperproliferation of myeloid progenitors and AML. Disclosures: No relevant conflicts of interest to declare.

Outcomes Of Haematopoietic Stem Cell Transplantation (HSCT) for Severe Congenital Neutropenia (SCN): Preliminary Results

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3355-3355 ◽  
Author(s):  
Francesca Fioredda ◽  
Liesbeth C de Wreede ◽  
Michaela Calvillo ◽  
Bobby Gaspar ◽  
Mahmoud Aljurf ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cord Blood ◽  
Haematopoietic Stem Cell Transplantation ◽  
Conditioning Regimen ◽  
Haematopoietic Stem Cell ◽  
Unrelated Donor ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia

Abstract Introduction Haematopoietic stem cell transplantation (HSCT) is indicated in patients affected with severe congenital neutropenia (SCN) who transformed into myelodysplasia/acute myeloid leukemia or in low responders to granulocyte colony stimulating factor (G-CSF) therapy. The outcome of HSCT in SCN is not well known because the experience is based on single case report or on small series. Aim of the study describe outcomes of HSCT in SCN patients in a large cohort using EBMT data base. Methods and Patients all patients registered in the EBMT data base affected with severe congenital neutropenia (severe neutropenia diagnosed early in life with bone marrow block at promyelocytes stage in the bone marrow and G-CSF dependency) were considered eligible for the study. Data regarding HSCT and outcome were extracted from the “general” EBMT registry, while data on the history of disease before HSCT, was collected through a specific CRF sent to all the participating centers. Here we report preliminary of this survey. Results A total of 119 patients from 19 participating countries were considered eligible for the study; 66% of the eligibile patients originated from Western Europe and 34% from Eastern Europe and the Eastern Mediterranean area (Iran, Israel, Saudi Arabia,Turkey and Russia). Females were 51% of the cohort. Median age at diagnosis of neutropenia was 0.35 years (0-35.4y), while median age at first transplant was 4.8 years (range 0.2-43y). Four patients were affected with MDS at time of transplant. The cell source was bone marrow (BM) in 56%, peripheral blood (PB) in 26% and cord blood (CB) 18%. Fiftypercent of patients were engrated from a related and 50% from an unrelated donor. Conditioning regimen was myeloablative in 86% and at reduced intensity (RIC) in 14% of the cohort Engraftment was documented in 91%; 5% has no engraftment while 4% lost the engraftment for a total of 10 patients. Chimerism was assessed only in 30% of the patients. Six of the 10 patients who had graft failure died after first transplant. Four underwent a second HSCT and 2 are alive2 died (2 alive and 2 death).Overall 22 patients (18.5%) died while the remaining 97 were alive (81.5%). Causes of death were GVDH 23%, Infection 23%, organ failure 18% and “other causes” 27%. Nine percent of patients died because of relapse/progression of the disease. Transplant related mortality was assessed at 17% on the whole cohort. Acute GVDH grade 1-2 was documented in 31% grade 3-4 in 14% and no GVDH in 54%. The 5-year OS and EFS rate was respectively 77% and 70 % in the whole cohort. The 5-year OS according to HLA identical donor and matched unrelated was respectively 83% and 79% (p=0.99, log-rank). Also the OS according to source of cells (5-year OS: CB 85%, PB 79% and BM 62% was not significant (p =0.13). Likewise no significant difference was found in EFS (CB 85% BM 75% and PB 52% p =0.08). OS by age of patients (5-year OS: 82 % for patients aged 0-2y, 83 % for age 2-5 y, 83% for age 5-10 and 60% for subjects aged above 10y) (p=0.07) and by period of HSCT: (90% between 2008-2012, 75% between 2001-2007 and 64% before 2000) were again not significant. No difference was also seen in OS and EFS according to myeloablative conditioning regimen and RIC. Conclusion this preliminary analysis indicate that the 5-year survival in transplanted SCN patients is close to 80% with no difference between matched related and unrelated donor. TRM is still a not negligible and close to 17%. There is a trend towards a more favourable survival in patients younger than 5 years at time of transplant, in those transplanted with cord blood and after the year 2000. Disclosures: Marsh: Sanofi: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Dufour:Pfizer: Consultancy, Research Funding.

Assessment of bone marrow stem cell reserve and function and stromal cell function in patients with severe congenital neutropenia

2001 ◽  
Vol 67 (4) ◽  
pp. 245-251 ◽  
Author(s):  
Helen A Papadaki ◽  
Frances M Gibson ◽  
Maria Psyllaki ◽  
Edward C Gordon-Smith ◽  
Judith C.W Marsh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cell ◽  
Cell Function ◽  
Bone Marrow Stem Cell ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
And Function ◽  
Marrow Stem Cell

Umbilical Cord Mesenchymal Stem Cell Exosomes Alleviate the Progression of Kidney Failure by Modulating Inflammatory Responses and Oxidative Stress in an Ischemia-Reperfusion Mice Model

2021 ◽  
Vol 17 (9) ◽  
pp. 1874-1881
Author(s):  
Yanqiang Zhang ◽  
Chongjuan Wang ◽  
Zhuxiao Bai ◽  
Peng Li
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Renal Failure ◽  
Stem Cell ◽  
Protein Expression ◽  
Inflammatory Responses ◽  
Ischemia Reperfusion ◽  
Protein Expression Level ◽  
M1 Macrophages ◽  
And Oxidative Stress

The efficacy of stem cells for the treatment of renal failure is widely recognized; however, an excessive volume of stem cells can block the capillaries; thus, the potential risks should not be ignored. Stem cell exosomes are secretory extracellular vesicles with a size of 30–150 nm, which have similar functions to stem cells but are much smaller in size. This study aims to investigate the role of human umbilical cord mesenchymal stem cells (UCMSCs)-derived exosomes in the treatment of renal failure caused by ischemia-reperfusion. Fifty 8-week-old female C57 mice underwent bilateral renal ischemia-reperfusion surgery for 30 minutes. After 4 weeks, the treated group received UCMSCs-derived exosomes treatment, and the control group was solely injected with the same amount of PBS. At the age of 16 weeks, the kidney function, kidney damage, inflammatory responses and oxidative stress were measured. Moreover, the effect of UCMSCs-derived exosomes on the phenotype of M1 macrophages was also tested. The results showed that UCMSCsderived exosomes significantly reduced the levels of blood urea nitrogen (BUN), serum creatinine (SCR), and urinary albumin and creatinine (ACR) and 8-isoprostane. UCMSCs-derived exosomes also improved the atrophy of the kidney and glomerulus, decreased kidney pro-inflammatory factors expression (mRNA of II-1β, II-6, Tnf-α, and Mcp-1) and oxidative stress (malondialdehyde), and increased glutathione level. However, F4/80 immunohistochemistry did not show significant differences between the two groups. In systemic inflammation measurement, UCMSCs-derived exosomes decreased proinflammatory factors TNF-α, IL-6, and IL-1β levels, and increased anti-inflammatory factor IL-10 level. In vitro experiments showed that UCMSCs-derived exosomes decreased the protein expression level of TNF-α and increased the protein expression level of IL-10 in M1 macrophages. UCMSCs-derived exosomes reduce kidney inflammation and oxidative stress by improving systemic inflammation, and thus reduce kidney damage and improve kidney function. This study shows the potential application value of exosomes in the treatment of renal failure.

A Model of Severe Congenital Neutropenia Reveals Signaling Pathways Mediating Mutant Elastase-Triggered Agranulocytosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3070-3070
Author(s):  
Andrew A. Aprikyan ◽  
Tomas Vaisar ◽  
Vahagn Makaryan ◽  
Jay Heinecke
Keyword(s):  
Bone Marrow ◽  
Signaling Pathways ◽  
Myelogenous Leukemia ◽  
Severe Neutropenia ◽  
Myeloid Differentiation ◽  
Cellular Model ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Histone H2b ◽  
Maturation Arrest

Abstract Severe congenital neutropenia (SCN; Kostmann’s syndrome or infantile genetic agranulocytosis) defines an inheritable hematopoietic disorder of impaired neutrophil production due to a “maturation arrest” at the promyelocytic stage of differentiation in the bone marrow. SCN patients have recurring severe infections and often develop acute myelogenous leukemia. We and others reported accelerated apoptosis and cell cycle arrest of bone marrow-derived myeloid progenitor cells in SCN patients with autosomal dominant and autosomal recessive inheritance. Heterozygous mutations in the neutrophil elastase (NE) gene encoding a serine protease, are present in a majority of SCN patients, but not in healthy members of the family, thus indicating a key role of mutant NE in pathogenesis of this disorder. To date, there are no animal or cellular models of SCN as both the knock-in of mutant NE as well as the knock-out of normal NE failed to result in neutropenia phenotype in mice. The molecular mechanisms of mutant NE-mediated severe neutropenia remain largely unknown. We hypothesized that mutations in NE expose the protease to a new range of substrates. To explore this proposal, we established a cellular model of SCN based on tetracycline-regulated expression of mutant NE in human promyelocytic tet-off HL-60 cells that very closely recapitulated the human phenotype. Mutant NE expression resulted in a characteristic block of myeloid differentiation - the cellular hallmark of SCN. Expression of the mutant product was associated with a significant reduction in phosphatidylinosytol-3-kinase and phosphorylated PKB/Akt levels and an imbalance of anti-apoptotic Bcl-2 and pro-apoptotic Bax. These alterations contributed to observed dissipation of mitochondrial membrane potential as determined by FACS analysis, aberrant release of cytochrome C, and accelerated apoptosis. Marked changes in actin cytoskeleton that made the cells more rigid appeared to stem from a reduced level of alpha-actinin and elevated level of Rho GTPase. Immunoprecipitation of cell lysates with elastase-specific monoclonal antibodies followed by mass spectrometric analysis revealed that NE interacted with histone H2B, one of the key components of the nucleosome core of the chromatin. Interestingly, the expression level of histone H2B was substantially reduced in cells expressing mutant NE, therefore supporting the notion of altered substrate specificity of mutant NE. Thus, these observations provide the first evidence that mutant NE affects specific signaling pathways that lead to alterations in cytoskeleton and chromatin reorganization, subsequent apoptosis, and a block of myeloid differentiation in SCN. This cellular model of SCN should provide an invaluable tool for screening potential therapeutic agents capable of preventing maturation arrest and leukemogenesis in subjects suffering from severe congenital neutropenia.

Abnormal Localization of Neutrophil Elastase in Patients with Severe Congenital Neutropenia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1279-1279
Author(s):  
Takashi Sato ◽  
Masakazu Habara ◽  
Hiroki Kihara ◽  
Hiroshi Kawaguchi ◽  
Mizuka Miki ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dimensional Analysis ◽  
Protein Trafficking ◽  
Neutrophil Elastase ◽  
Plasma Membranes ◽  
Normal Subjects ◽  
Precursor Cells ◽  
Proteinase 3 ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia

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.

G-CSFSR Mutations Present in Patients with Severe Congenital Neutropenia Cooperate with PML-RARα To Induce Acute Myeloid Leukemia in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2193-2193
Author(s):  
Jill R. Woloszynek ◽  
Ghada M. Kunter ◽  
Tim Ley ◽  
Dan C. Link ◽  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Transgenic Mice ◽  
Cumulative Incidence ◽  
Myeloid Leukemia ◽  
Direct Evidence ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Acute Myeloid

Abstract Severe congenital neutropenia (SCN) is an inherited disorder of granulopoiesis that is associated with a markedly increased risk of developing acute myeloid leukemia (AML) or myelodysplasia (MDS). Somatic mutations of CSF3R, encoding the G-CSF receptor (G-CSFR), are strongly associated with the development of AML/MDS in SCN. These mutations invariably produce a truncated G-CSFR that, though remaining ligand-dependent, transmits a hyperproliferative signal. Transgenic mice carrying a targeted (knock-in) mutation of Csf3r (termed d715) reproducing a mutation found in a patient with SCN have an exaggerated neutrophil response to G-CSF treatment but do not develop AML/MDS. Moreover, we recently showed expression of the d715 G-CSFR confers a strong clonal advantage at the hematopoietic stem cell level that is dependent upon exogenous G-CSF. Collectively, these data suggest that CSF3R truncation mutations are an initiation or early progression factor for leukemic transformation. However, there is, as yet, scant direct evidence supporting this hypothesis. Previous studies have established that activating mutations of receptor tyrosine kinases, such as internal tandem duplications of FLT3, are able to cooperate with PML-RARα to induce AML. Since the CSF3R mutations in SCN also are “activating”, we asked whether the d715 G-CSFR could cooperate with PML-RARα to induce AML in mice. PML-RARα transgenic mice were intercrossed with d715 G-CSFR mice (all inbred > 10 generations onto a C57BL/6 background) to generate the cohorts listed in Table 1. A separate cohort for each genotype was treated chronically with pegylated G-CSF (1 mg/kg every 4–5 days for 6 months) to simulate the high level of serum G-CSF present in patients with SCN. Complete blood counts were performed at 3 months intervals and documented a similar increase in neutrophil counts in all mice treated with G-CSF. The cumulative incidence of AML and median follow-up for each cohort is shown in Table 1. None of the mice without the PML-RARα transgene, regardless of G-CSF treatment, developed AML, confirming that the d715 G-CSFR is not sufficient to induce AML. In mice carrying the PML-RARα transgene but not treated with G-CSF, a nonsignificant trend to increased AML was observed in mice expressing the d715 G-CSFR (P=0.12). However, in mice carrying the PML-RARα transgene and treated with G-CSF, the presence of the d715 G-CSFR significantly increased the penetrance (P=0.009) and reduced the latency of AML. In all cases, the leukemia was characterized by leukocytosis, splenomegaly, and a high percentage of blasts in the bone marrow and spleen that co-express Gr1 and c-Kit. These data provide the first direct evidence that the CSF3R mutations present in patients with SCN are leukemogenic and provide further support for the proposition that patients who acquire CSF3R mutations be considered for early stem cell transplantation. The cumulative incidence of AML and median follow-up for each cohort PML-RAR CSF3R G-CSF Rx N Median follow-up Cumulative AML% No WT No 20 454 0.0% No d715 No 45 250 0.0% No WT Yes 20 488 0.0% No d715 Yes 47 311 0.0% Yes WT No 57 286 6.4% Yes d715 No 67 267 11.9% Yes WT Yes 54 322 20.4% Yes d715 Yes 50 311 44.0%

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