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Takuji Suzuki - One of the best experts on this subject based on the ideXlab platform.
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statin as a novel pharmacotherapy of pulmonary alveolar proteinosis
Nature Communications, 2018Co-Authors: Cormac Mccarthy, Takuji Suzuki, Claudia Chalk, Tisha Wang, Elinor Lee, James P Bridges, Anthony Sallese, Jason C Woods, Brian J Bartholmai, Brenna CareyAbstract:Pulmonary alveolar proteinosis (PAP) is a syndrome of reduced GM-CSF-dependent, macrophage-mediated surfactant clearance, dysfunctional foamy alveolar macrophages, alveolar surfactant accumulation, and hypoxemic respiratory failure for which the pathogenetic mechanism is unknown. Here, we examine the lipids accumulating in alveolar macrophages and surfactant to define the pathogenesis of PAP and evaluate a novel pharmacotherapeutic approach. In PAP patients, alveolar macrophages have a marked increase in cholesterol but only a minor increase in phospholipids, and pulmonary surfactant has an increase in the ratio of cholesterol to phospholipids. Oral statin therapy is associated with clinical, physiological, and radiological improvement in autoimmune PAP patients, and ex vivo statin treatment reduces cholesterol levels in explanted alveolar macrophages. In CSF2RB-/- mice, statin therapy reduces cholesterol accumulation in alveolar macrophages and ameliorates PAP, and ex vivo statin treatment increases cholesterol efflux from macrophages. These results support the feasibility of statin as a novel pathogenesis-based pharmacotherapy of PAP.
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iPSC-Derived Macrophages Effectively Treat Pulmonary Alveolar Proteinosis in CSF2RB-Deficient Mice
Elsevier, 2018Co-Authors: Adele Mucci, Takuji Suzuki, Mania Ackermann, Christine Happle, Miriam Hetzel, Elena Lopez-rodriguez, Serena Liu, Henning Kempf, Roman Hillje, Jessica KunkielAbstract:Summary: Induced pluripotent stem cell (iPSC)-derived hematopoietic cells represent a highly attractive source for cell and gene therapy. Given the longevity, plasticity, and self-renewal potential of distinct macrophage subpopulations, iPSC-derived macrophages (iPSC-Mφ) appear of particular interest in this context. We here evaluated the airway residence, plasticity, and therapeutic efficacy of iPSC-Mφ in a murine model of hereditary pulmonary alveolar proteinosis (herPAP). We demonstrate that single pulmonary macrophage transplantation (PMT) of 2.5–4 × 106 iPSC-Mφ yields efficient airway residence with conversion of iPSC-Mφ to an alveolar macrophage (AMφ) phenotype characterized by a distinct surface marker and gene expression profile within 2 months. Moreover, PMT significantly improves alveolar protein deposition and other critical herPAP disease parameters. Thus, our data indicate iPSC-Mφ as a source of functional macrophages displaying substantial plasticity and therapeutic potential that upon pulmonary transplantation will integrate into the lung microenvironment, adopt an AMφ phenotype and gene expression pattern, and profoundly ameliorate pulmonary disease phenotypes. : Mucci and colleagues demonstrate marked plasticity of iPSC-derived macrophages and rapid adoption of an alveolar macrophage phenotype upon pulmonary transplantation, as well as profound therapeutic efficacy of iPSC-derived macrophages in the context of the severe lung disease pulmonary alveolar proteinosis. Key words: iPSC, hematopoiesis, macrophages, lung, cell therap
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murine ipsc derived macrophages as a tool for disease modeling of hereditary pulmonary alveolar proteinosis due to CSF2RB deficiency
Stem cell reports, 2016Co-Authors: Adele Mucci, Takuji Suzuki, Alexandra Kuhn, Mania Ackermann, Christine Happle, Jessica Kunkiel, Sebastian Brennig, Silke Glage, Mark Philipp Kuhnel, Axel SchambachAbstract:Induced pluripotent stem cells (iPSCs) represent an innovative source for the standardized in vitro generation of macrophages (Mφ). We here describe a robust and efficient protocol to obtain mature and functional Mφ from healthy as well as disease-specific murine iPSCs. With regard to morphology, surface phenotype, and function, our iPSC-derived Mφ (iPSC-Mφ) closely resemble their counterparts generated in vitro from bone marrow cells. Moreover, when we investigated the feasibility of our differentiation system to serve as a model for rare congenital diseases associated with Mφ malfunction, we were able to faithfully recapitulate the pathognomonic defects in GM-CSF signaling and Mφ function present in hereditary pulmonary alveolar proteinosis (herPAP). Thus, our studies may help to overcome the limitations placed on research into certain rare disease entities by the lack of an adequate supply of disease-specific primary cells, and may aid the development of novel therapeutic approaches for herPAP patients.
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use of induced pluripotent stem cells to recapitulate pulmonary alveolar proteinosis pathogenesis
American Journal of Respiratory and Critical Care Medicine, 2013Co-Authors: Takuji Suzuki, Brenna Carey, Robert E Wood, Claudia Chalk, Anthony Sallese, Christopher N Mayhew, Punam Malik, Bruce C TrapnellAbstract:Rationale: In patients with pulmonary alveolar proteinosis (PAP) syndrome, disruption of granulocyte/macrophage colony–stimulating factor (GM-CSF) signaling is associated with pathogenic surfactant accumulation from impaired clearance in alveolar macrophages.Objectives: The aim of this study was to overcome these barriers by using monocyte-derived induced pluripotent stem (iPS) cells to recapitulate disease-specific and normal macrophages.Methods: We created iPS cells from two children with hereditary PAP (hPAP) caused by recessive CSF2RAR217X mutations and three normal people, differentiated them into macrophages (hPAP-iPS-Mφs and NL-iPS-Mφs, respectively), and evaluated macrophage functions with and without gene-correction to restore GM-CSF signaling in hPAP-iPS-Mφs.Measurements and Main Results: Both hPAP and normal iPS cells had human embryonic stem cell–like morphology, expressed pluripotency markers, formed teratomas in vivo, had a normal karyotype, retained and expressed mutant or normal CSF2RA gen...
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hereditary pulmonary alveolar proteinosis caused by recessive CSF2RB mutations
European Respiratory Journal, 2011Co-Authors: Takuji Suzuki, Takuro Sakagami, Brenna Carey, Claudia Chalk, Bruno Maranda, P Catellier, C Y Couture, Bruce C TrapnellAbstract:To the Editors: Pulmonary alveolar proteinosis (PAP) is a syndrome characterised by accumulation of surfactant in alveoli resulting in respiratory insufficiency 1. Surfactant homeostasis is critical for lung function and is tightly regulated, in part, by pulmonary granulocyte-macrophage colony-stimulating factor (GM-CSF), which is required for surfactant clearance by alveolar macrophages 2 and alveolar macrophage maturation 1. The effects of GM-CSF are mediated by cell-surface receptors composed of GM-CSF-binding α-chains and affinity-enhancing β-chains (encoded by CSF2RA and CSF2RB , respectively) 3. Ligand binding activates signalling via multiple pathways including the signal transducer and activator of phosphorylation (STAT)5 4. Disruption of GM-CSF signalling causes PAP by impairing surfactant catabolism in alveolar macrophages 1. In 90% of patients, PAP is caused by neutralising GM-CSF auto-antibodies 5, 6. Through the Rare Lung Diseases Network global PAP detection programme, we identified PAP caused by recessive CSF2RA mutations and developed novel diagnostic methods to identify patients with PAP caused by GM-CSF receptor dysfunction 4, 7. Herein, we report a case of hereditary PAP caused by disruption of GM-CSF receptor β-chain function. A previously healthy 9-yr-old female presented with bilateral pneumonia, followed 3 months later by progressive dyspnoea of insidious onset. The diagnosis of PAP was suggested by chest radiograph findings, high-resolution computed tomography and bronchoalveolar cytology, and was confirmed by surgical lung biopsy. Pulmonary histopathology was typical of primary PAP (fig. 1) and she was successfully treated by serial whole lung lavage therapy. Details of the case history are included in the online supplement. A GM-CSF auto-antibody test was negative and the serum GM-CSF level was increased (25.9 pg·mL−1) suggesting GM-CSF receptor dysfunction as the molecular basis of PAP 4, 7. A molecular evaluation was undertaken and included GM-CSF receptor detection, STAT-5 phosphorylation, CSF2RA …
Katherine R Hyde - One of the best experts on this subject based on the ideXlab platform.
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runx1 and cbfb myh11 are required for the maintenance of inv 16 aml
Blood, 2016Co-Authors: Yiqian Wang, Lemlem Alemu, Kira Hannon, Michelle Becker, Lisa Garett, Cecilia Rivas, Lisa Richter, Ling Zhao, Katherine R HydeAbstract:The inversion of chromosome 16 (inv(16)) is found in 5-12% of human AML cases. Although considered a marker of favorable prognosis, approximately half of inv(16) AML patients eventually relapse. Inv(16) generates a fusion gene between the transcription factor gene CBFB and the MYH11 gene. Expression of the CBFB-MYH11 fusion gene, which encodes CBFβ-SMMHC, is the initiating event, but cooperating mutations are required for transformation to a frank leukemia. In previous work, we showed that CBFβ and CBFβ-SMMHC binding partner RUNX1 is required for efficient leukemia development. Small molecule inhibitors of the CBFβ-SMMHC: RUNX1 complex decrease leukemic burden and increase survival in mouse models, indicating that both proteins also play a role in leukemia maintenance. However, it is not currently known whether inhibition of this complex alone is sufficient to cure inv(16) AML. To test the requirement for CBFB-MYH11 after leukemic transformation, we generated knockin mice that have a Cbfb-MYH11 allele flanked by loxP sites ( Cbfb flMYH11 ), which allows for deletion of Cbfb-MYH11 by Cre recombinase (Cre). Chimeric founder mice were treated with N-ethyl-N-nitrosourea (ENU) to induce cooperating mutations and leukemia. Leukemia cells from three different founder mice had a similar histological appearance and immunophenotype as leukemia cells derived from previous Cbfb-MYH11 knockin models. Importantly, the leukemia was transplantable, with similar latency as the previous knockin models. These findings indicate that the Cbfb flMYH11 allele causes frank leukemia, similar to other Cbfb-MYH11 alleles. To induce excision of the fusion gene, Cbfb +/flMYH11 leukemia cells were transduced with a lentivirus expressing Cre and GFP. Excision of the Cbfb-MYH11 allele was verified by PCR and showed an average excision frequency of 72.3%, +/- 1.8. To test if deletion of Cbfb-MYH11 affected cell survival, leukemia cells were infected with Cre and control viruses, and stained for Annexin V. At 48 hours post-transduction, total Cre-infected Cbfb +/flMYH11 leukemia cells showed a statistically significant increase in Annexin V staining, as compared to cells infected with the control virus. Importantly, increased Annexin V staining was seen in CSF2RB - cells, a population we previously showed to be enriched for leukemia stem cells (LSCs). To test if loss of Cbfb-MYH11 induced differentiation of leukemia cells, we stained Cre and control transduced Cbfb flMYH11 leukemia cells for the myeloid differentiation markers Gr-1 and Mac-1. We found no difference in the expression of either marker with Cbfb-MYH11 excision. These findings indicate that Cbfb-MYH11 is required for the survival of leukemic cells, including the LSC population, and its loss does not cause differentiation. To test if Runx1 is required for Cbfb-MYH11 activities during leukemia maintenance, we utilized a lentiviral vector expressing an shRNA against Runx1 and infected Cbfb-MYH11 expressing mouse leukemia cells. Runx1 knockdown was verified by quantitative RT-PCR and by western blot. To test if Runx1 knockdown induced apoptosis, leukemia cells infected with Runx1 knockdown or a scrambled shRNA control virus were stained for Annexin V. With an average decrease in Runx1 of 60.0% +/- 0.17, we observed an overall increase in Annexin V staining, but not in the CSF2RB-, LSC enriched population. This implies that LSCs may be less sensitive to decreased RUNX1 activity, than non-LSCs. To examine the effect of Runx1 knockdown on LSC activity in vitro, we performed colony forming cell (CFC) assays. We found that cells with decreased expression of Runx1 produced significantly fewer colonies as compared to the scrambled shRNA-infected cells. While some of the observed colonies may have been due to rare cells that lost or silenced the shRNA vector, our preliminary data indicates some colonies retained Runx1 knockdown (70.0% decrease, as compared to control infected cells) after growth in culture. These findings indicate that Runx1 is required for leukemia maintenance, but that LSCs may be less sensitive to decreased RUNX1 activity than non-LSCs. Taken together, our results imply that both Cbfb-MYH11 and Runx1 are important for the maintenance of inv(16) AML, and that inhibition of CBFβ-SMMHCand RUNX1 have potential as a cure for inv(16) AML. Disclosures No relevant conflicts of interest to declare.
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runx1 and cbfb myh11 are required for the maintenance of inv 16 aml
Blood, 2016Co-Authors: Yiqian Wang, Lemlem Alemu, Kira Hannon, Michelle Becker, Lisa Garett, Cecilia Rivas, Lisa Richter, Ling Zhao, Katherine R HydeAbstract:The inversion of chromosome 16 (inv(16)) is found in 5-12% of human AML cases. Although considered a marker of favorable prognosis, approximately half of inv(16) AML patients eventually relapse. Inv(16) generates a fusion gene between the transcription factor gene CBFB and the MYH11 gene. Expression of the CBFB-MYH11 fusion gene, which encodes CBFβ-SMMHC, is the initiating event, but cooperating mutations are required for transformation to a frank leukemia. In previous work, we showed that CBFβ and CBFβ-SMMHC binding partner RUNX1 is required for efficient leukemia development. Small molecule inhibitors of the CBFβ-SMMHC: RUNX1 complex decrease leukemic burden and increase survival in mouse models, indicating that both proteins also play a role in leukemia maintenance. However, it is not currently known whether inhibition of this complex alone is sufficient to cure inv(16) AML. To test the requirement for CBFB-MYH11 after leukemic transformation, we generated knockin mice that have a Cbfb-MYH11 allele flanked by loxP sites ( Cbfb flMYH11 ), which allows for deletion of Cbfb-MYH11 by Cre recombinase (Cre). Chimeric founder mice were treated with N-ethyl-N-nitrosourea (ENU) to induce cooperating mutations and leukemia. Leukemia cells from three different founder mice had a similar histological appearance and immunophenotype as leukemia cells derived from previous Cbfb-MYH11 knockin models. Importantly, the leukemia was transplantable, with similar latency as the previous knockin models. These findings indicate that the Cbfb flMYH11 allele causes frank leukemia, similar to other Cbfb-MYH11 alleles. To induce excision of the fusion gene, Cbfb +/flMYH11 leukemia cells were transduced with a lentivirus expressing Cre and GFP. Excision of the Cbfb-MYH11 allele was verified by PCR and showed an average excision frequency of 72.3%, +/- 1.8. To test if deletion of Cbfb-MYH11 affected cell survival, leukemia cells were infected with Cre and control viruses, and stained for Annexin V. At 48 hours post-transduction, total Cre-infected Cbfb +/flMYH11 leukemia cells showed a statistically significant increase in Annexin V staining, as compared to cells infected with the control virus. Importantly, increased Annexin V staining was seen in CSF2RB - cells, a population we previously showed to be enriched for leukemia stem cells (LSCs). To test if loss of Cbfb-MYH11 induced differentiation of leukemia cells, we stained Cre and control transduced Cbfb flMYH11 leukemia cells for the myeloid differentiation markers Gr-1 and Mac-1. We found no difference in the expression of either marker with Cbfb-MYH11 excision. These findings indicate that Cbfb-MYH11 is required for the survival of leukemic cells, including the LSC population, and its loss does not cause differentiation. To test if Runx1 is required for Cbfb-MYH11 activities during leukemia maintenance, we utilized a lentiviral vector expressing an shRNA against Runx1 and infected Cbfb-MYH11 expressing mouse leukemia cells. Runx1 knockdown was verified by quantitative RT-PCR and by western blot. To test if Runx1 knockdown induced apoptosis, leukemia cells infected with Runx1 knockdown or a scrambled shRNA control virus were stained for Annexin V. With an average decrease in Runx1 of 60.0% +/- 0.17, we observed an overall increase in Annexin V staining, but not in the CSF2RB-, LSC enriched population. This implies that LSCs may be less sensitive to decreased RUNX1 activity, than non-LSCs. To examine the effect of Runx1 knockdown on LSC activity in vitro, we performed colony forming cell (CFC) assays. We found that cells with decreased expression of Runx1 produced significantly fewer colonies as compared to the scrambled shRNA-infected cells. While some of the observed colonies may have been due to rare cells that lost or silenced the shRNA vector, our preliminary data indicates some colonies retained Runx1 knockdown (70.0% decrease, as compared to control infected cells) after growth in culture. These findings indicate that Runx1 is required for leukemia maintenance, but that LSCs may be less sensitive to decreased RUNX1 activity than non-LSCs. Taken together, our results imply that both Cbfb-MYH11 and Runx1 are important for the maintenance of inv(16) AML, and that inhibition of CBFβ-SMMHCand RUNX1 have potential as a cure for inv(16) AML. Disclosures No relevant conflicts of interest to declare.
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cbfb runx1 repression independent blockage of differentiation and accumulation of CSF2RB expressing cells by cbfb myh11
Blood, 2010Co-Authors: Katherine R Hyde, Yasuhiko Kamikubo, Martha Kirby, Lemlem Alemu, Stacie M. Anderson, Ling ZhaoAbstract:It is known that CBFB-MYH11, the fusion gene generated by inversion of chromosome 16 in human acute myeloid leukemia, is causative for oncogenic transformation. However, the mechanism by which CBFB-MYH11 initiates leukemogenesis is not clear. Previously published reports showed that CBFB-MYH11 dominantly inhibits RUNX1 and CBFB, and such inhibition has been suggested as the mechanism for leukemogenesis. Here we show that Cbfb-MYH11 caused Cbfb/Runx1 repression–independent defects in both primitive and definitive hematopoiesis. During primitive hematopoiesis, Cbfb-MYH11 delayed differentiation characterized by sustained expression of Gata2, Il1rl1, and CSF2RB, a phenotype not found in Cbfb and Runx1 knockout mice. Expression of Cbfb-MYH11 in the bone marrow induced the accumulation of abnormal progenitor-like cells expressing CSF2RB in preleukemic mice. The expression of all 3 genes was detected in most human and murine CBFB-MYH11+ leukemia samples. Interestingly, Cbfb-MYH11+ preleukemic progenitors and leukemia-initiating cells did not express CSF2RB, although the majority of leukemia cells in our Cbfb-MYH11 knockin mice were CSF2RB+. Therefore CSF2RB can be used as a negative selection marker to enrich preleukemic progenitor cells and leukemia-initiating cells from Cbfb-MYH11 mice. These results suggest that Cbfb/Runx1 repression–independent activities contribute to leukemogenesis by Cbfb-MYH11.
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cbfb runx1 repression independent blockage of differentiation and accumulation of CSF2RB expressing cells by cbfb myh11
Blood, 2010Co-Authors: Katherine R Hyde, Yasuhiko Kamikubo, Martha Kirby, Lemlem Alemu, Stacie M. Anderson, Ling ZhaoAbstract:It is known that CBFB-MYH11, the fusion gene generated by inversion of chromosome 16 in human acute myeloid leukemia, is causative for oncogenic transformation. However, the mechanism by which CBFB-MYH11 initiates leukemogenesis is not clear. Previously published reports showed that CBFB-MYH11 dominantly inhibits RUNX1 and CBFB, and such inhibition has been suggested as the mechanism for leukemogenesis. Here we show that Cbfb-MYH11 caused Cbfb/Runx1 repression–independent defects in both primitive and definitive hematopoiesis. During primitive hematopoiesis, Cbfb-MYH11 delayed differentiation characterized by sustained expression of Gata2, Il1rl1, and CSF2RB, a phenotype not found in Cbfb and Runx1 knockout mice. Expression of Cbfb-MYH11 in the bone marrow induced the accumulation of abnormal progenitor-like cells expressing CSF2RB in preleukemic mice. The expression of all 3 genes was detected in most human and murine CBFB-MYH11+ leukemia samples. Interestingly, Cbfb-MYH11+ preleukemic progenitors and leukemia-initiating cells did not express CSF2RB, although the majority of leukemia cells in our Cbfb-MYH11 knockin mice were CSF2RB+. Therefore CSF2RB can be used as a negative selection marker to enrich preleukemic progenitor cells and leukemia-initiating cells from Cbfb-MYH11 mice. These results suggest that Cbfb/Runx1 repression–independent activities contribute to leukemogenesis by Cbfb-MYH11.
Bruce C Trapnell - One of the best experts on this subject based on the ideXlab platform.
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pulmonary alveolar proteinosis
Nature Reviews Disease Primers, 2019Co-Authors: Bruce C Trapnell, Cliff Morgan, Koh Nakata, Francesco Bonella, Ilaria Campo, Matthias Griese, John A Hamilton, Tisha Wang, Vincent Cottin, Cormac MccarthyAbstract:Pulmonary alveolar proteinosis (PAP) is a syndrome characterized by the accumulation of alveolar surfactant and dysfunction of alveolar macrophages. PAP results in progressive dyspnoea of insidious onset, hypoxaemic respiratory failure, secondary infections and pulmonary fibrosis. PAP can be classified into different types on the basis of the pathogenetic mechanism: primary PAP is characterized by the disruption of granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling and can be autoimmune (caused by elevated levels of GM-CSF autoantibodies) or hereditary (due to mutations in CSF2RA or CSF2RB, encoding GM-CSF receptor subunits); secondary PAP results from various underlying conditions; and congenital PAP is caused by mutations in genes involved in surfactant production. In most patients, pathogenesis is driven by reduced GM-CSF-dependent cholesterol clearance in alveolar macrophages, which impairs alveolar surfactant clearance. PAP has a prevalence of at least 7 cases per million individuals in large population studies and affects men, women and children of all ages, ethnicities and geographical locations irrespective of socioeconomic status, although it is more-prevalent in smokers. Autoimmune PAP accounts for >90% of all cases. Management aims at improving symptoms and quality of life; whole-lung lavage effectively removes excessive surfactant. Novel pathogenesis-based therapies are in development, targeting GM-CSF signalling, immune modulation and cholesterol homeostasis.
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use of induced pluripotent stem cells to recapitulate pulmonary alveolar proteinosis pathogenesis
American Journal of Respiratory and Critical Care Medicine, 2013Co-Authors: Takuji Suzuki, Brenna Carey, Robert E Wood, Claudia Chalk, Anthony Sallese, Christopher N Mayhew, Punam Malik, Bruce C TrapnellAbstract:Rationale: In patients with pulmonary alveolar proteinosis (PAP) syndrome, disruption of granulocyte/macrophage colony–stimulating factor (GM-CSF) signaling is associated with pathogenic surfactant accumulation from impaired clearance in alveolar macrophages.Objectives: The aim of this study was to overcome these barriers by using monocyte-derived induced pluripotent stem (iPS) cells to recapitulate disease-specific and normal macrophages.Methods: We created iPS cells from two children with hereditary PAP (hPAP) caused by recessive CSF2RAR217X mutations and three normal people, differentiated them into macrophages (hPAP-iPS-Mφs and NL-iPS-Mφs, respectively), and evaluated macrophage functions with and without gene-correction to restore GM-CSF signaling in hPAP-iPS-Mφs.Measurements and Main Results: Both hPAP and normal iPS cells had human embryonic stem cell–like morphology, expressed pluripotency markers, formed teratomas in vivo, had a normal karyotype, retained and expressed mutant or normal CSF2RA gen...
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hereditary pulmonary alveolar proteinosis caused by recessive CSF2RB mutations
European Respiratory Journal, 2011Co-Authors: Takuji Suzuki, Takuro Sakagami, Brenna Carey, Claudia Chalk, Bruno Maranda, P Catellier, C Y Couture, Bruce C TrapnellAbstract:To the Editors: Pulmonary alveolar proteinosis (PAP) is a syndrome characterised by accumulation of surfactant in alveoli resulting in respiratory insufficiency 1. Surfactant homeostasis is critical for lung function and is tightly regulated, in part, by pulmonary granulocyte-macrophage colony-stimulating factor (GM-CSF), which is required for surfactant clearance by alveolar macrophages 2 and alveolar macrophage maturation 1. The effects of GM-CSF are mediated by cell-surface receptors composed of GM-CSF-binding α-chains and affinity-enhancing β-chains (encoded by CSF2RA and CSF2RB , respectively) 3. Ligand binding activates signalling via multiple pathways including the signal transducer and activator of phosphorylation (STAT)5 4. Disruption of GM-CSF signalling causes PAP by impairing surfactant catabolism in alveolar macrophages 1. In 90% of patients, PAP is caused by neutralising GM-CSF auto-antibodies 5, 6. Through the Rare Lung Diseases Network global PAP detection programme, we identified PAP caused by recessive CSF2RA mutations and developed novel diagnostic methods to identify patients with PAP caused by GM-CSF receptor dysfunction 4, 7. Herein, we report a case of hereditary PAP caused by disruption of GM-CSF receptor β-chain function. A previously healthy 9-yr-old female presented with bilateral pneumonia, followed 3 months later by progressive dyspnoea of insidious onset. The diagnosis of PAP was suggested by chest radiograph findings, high-resolution computed tomography and bronchoalveolar cytology, and was confirmed by surgical lung biopsy. Pulmonary histopathology was typical of primary PAP (fig. 1) and she was successfully treated by serial whole lung lavage therapy. Details of the case history are included in the online supplement. A GM-CSF auto-antibody test was negative and the serum GM-CSF level was increased (25.9 pg·mL−1) suggesting GM-CSF receptor dysfunction as the molecular basis of PAP 4, 7. A molecular evaluation was undertaken and included GM-CSF receptor detection, STAT-5 phosphorylation, CSF2RA …
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pulmonary alveolar proteinosis a primary immunodeficiency of impaired gm csf stimulation of macrophages
Current Opinion in Immunology, 2009Co-Authors: Bruce C Trapnell, Brenna Carey, Kanji Uchida, Takuji SuzukiAbstract:Pulmonary alveolar proteinosis (PAP) is a rare syndrome characterized by accumulation of pulmonary surfactant, respiratory insufficiency, and increased infections. It occurs in various clinical settings that disrupt surfactant catabolism in alveolar macrophages, including a relatively more common autoimmune disease caused by GM-CSF autoantibodies and a rare congenital disease caused by CSF2RA mutations. Recent results demonstrate that GM-CSF is crucial for alveolar macrophage terminal differentiation and immune functions, pulmonary surfactant homeostasis, and lung host defense. GM-CSF is also required to determine the basal functional capacity of circulating neutrophils, including adhesion, phagocytosis, and microbial killing. PAP research has illuminated the crucial role of GM-CSF in innate immunity and led to novel therapy for PAP and the potential use of anti-GM-CSF therapy in other common disorders.
Ling Zhao - One of the best experts on this subject based on the ideXlab platform.
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runx1 and cbfb myh11 are required for the maintenance of inv 16 aml
Blood, 2016Co-Authors: Yiqian Wang, Lemlem Alemu, Kira Hannon, Michelle Becker, Lisa Garett, Cecilia Rivas, Lisa Richter, Ling Zhao, Katherine R HydeAbstract:The inversion of chromosome 16 (inv(16)) is found in 5-12% of human AML cases. Although considered a marker of favorable prognosis, approximately half of inv(16) AML patients eventually relapse. Inv(16) generates a fusion gene between the transcription factor gene CBFB and the MYH11 gene. Expression of the CBFB-MYH11 fusion gene, which encodes CBFβ-SMMHC, is the initiating event, but cooperating mutations are required for transformation to a frank leukemia. In previous work, we showed that CBFβ and CBFβ-SMMHC binding partner RUNX1 is required for efficient leukemia development. Small molecule inhibitors of the CBFβ-SMMHC: RUNX1 complex decrease leukemic burden and increase survival in mouse models, indicating that both proteins also play a role in leukemia maintenance. However, it is not currently known whether inhibition of this complex alone is sufficient to cure inv(16) AML. To test the requirement for CBFB-MYH11 after leukemic transformation, we generated knockin mice that have a Cbfb-MYH11 allele flanked by loxP sites ( Cbfb flMYH11 ), which allows for deletion of Cbfb-MYH11 by Cre recombinase (Cre). Chimeric founder mice were treated with N-ethyl-N-nitrosourea (ENU) to induce cooperating mutations and leukemia. Leukemia cells from three different founder mice had a similar histological appearance and immunophenotype as leukemia cells derived from previous Cbfb-MYH11 knockin models. Importantly, the leukemia was transplantable, with similar latency as the previous knockin models. These findings indicate that the Cbfb flMYH11 allele causes frank leukemia, similar to other Cbfb-MYH11 alleles. To induce excision of the fusion gene, Cbfb +/flMYH11 leukemia cells were transduced with a lentivirus expressing Cre and GFP. Excision of the Cbfb-MYH11 allele was verified by PCR and showed an average excision frequency of 72.3%, +/- 1.8. To test if deletion of Cbfb-MYH11 affected cell survival, leukemia cells were infected with Cre and control viruses, and stained for Annexin V. At 48 hours post-transduction, total Cre-infected Cbfb +/flMYH11 leukemia cells showed a statistically significant increase in Annexin V staining, as compared to cells infected with the control virus. Importantly, increased Annexin V staining was seen in CSF2RB - cells, a population we previously showed to be enriched for leukemia stem cells (LSCs). To test if loss of Cbfb-MYH11 induced differentiation of leukemia cells, we stained Cre and control transduced Cbfb flMYH11 leukemia cells for the myeloid differentiation markers Gr-1 and Mac-1. We found no difference in the expression of either marker with Cbfb-MYH11 excision. These findings indicate that Cbfb-MYH11 is required for the survival of leukemic cells, including the LSC population, and its loss does not cause differentiation. To test if Runx1 is required for Cbfb-MYH11 activities during leukemia maintenance, we utilized a lentiviral vector expressing an shRNA against Runx1 and infected Cbfb-MYH11 expressing mouse leukemia cells. Runx1 knockdown was verified by quantitative RT-PCR and by western blot. To test if Runx1 knockdown induced apoptosis, leukemia cells infected with Runx1 knockdown or a scrambled shRNA control virus were stained for Annexin V. With an average decrease in Runx1 of 60.0% +/- 0.17, we observed an overall increase in Annexin V staining, but not in the CSF2RB-, LSC enriched population. This implies that LSCs may be less sensitive to decreased RUNX1 activity, than non-LSCs. To examine the effect of Runx1 knockdown on LSC activity in vitro, we performed colony forming cell (CFC) assays. We found that cells with decreased expression of Runx1 produced significantly fewer colonies as compared to the scrambled shRNA-infected cells. While some of the observed colonies may have been due to rare cells that lost or silenced the shRNA vector, our preliminary data indicates some colonies retained Runx1 knockdown (70.0% decrease, as compared to control infected cells) after growth in culture. These findings indicate that Runx1 is required for leukemia maintenance, but that LSCs may be less sensitive to decreased RUNX1 activity than non-LSCs. Taken together, our results imply that both Cbfb-MYH11 and Runx1 are important for the maintenance of inv(16) AML, and that inhibition of CBFβ-SMMHCand RUNX1 have potential as a cure for inv(16) AML. Disclosures No relevant conflicts of interest to declare.
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runx1 and cbfb myh11 are required for the maintenance of inv 16 aml
Blood, 2016Co-Authors: Yiqian Wang, Lemlem Alemu, Kira Hannon, Michelle Becker, Lisa Garett, Cecilia Rivas, Lisa Richter, Ling Zhao, Katherine R HydeAbstract:The inversion of chromosome 16 (inv(16)) is found in 5-12% of human AML cases. Although considered a marker of favorable prognosis, approximately half of inv(16) AML patients eventually relapse. Inv(16) generates a fusion gene between the transcription factor gene CBFB and the MYH11 gene. Expression of the CBFB-MYH11 fusion gene, which encodes CBFβ-SMMHC, is the initiating event, but cooperating mutations are required for transformation to a frank leukemia. In previous work, we showed that CBFβ and CBFβ-SMMHC binding partner RUNX1 is required for efficient leukemia development. Small molecule inhibitors of the CBFβ-SMMHC: RUNX1 complex decrease leukemic burden and increase survival in mouse models, indicating that both proteins also play a role in leukemia maintenance. However, it is not currently known whether inhibition of this complex alone is sufficient to cure inv(16) AML. To test the requirement for CBFB-MYH11 after leukemic transformation, we generated knockin mice that have a Cbfb-MYH11 allele flanked by loxP sites ( Cbfb flMYH11 ), which allows for deletion of Cbfb-MYH11 by Cre recombinase (Cre). Chimeric founder mice were treated with N-ethyl-N-nitrosourea (ENU) to induce cooperating mutations and leukemia. Leukemia cells from three different founder mice had a similar histological appearance and immunophenotype as leukemia cells derived from previous Cbfb-MYH11 knockin models. Importantly, the leukemia was transplantable, with similar latency as the previous knockin models. These findings indicate that the Cbfb flMYH11 allele causes frank leukemia, similar to other Cbfb-MYH11 alleles. To induce excision of the fusion gene, Cbfb +/flMYH11 leukemia cells were transduced with a lentivirus expressing Cre and GFP. Excision of the Cbfb-MYH11 allele was verified by PCR and showed an average excision frequency of 72.3%, +/- 1.8. To test if deletion of Cbfb-MYH11 affected cell survival, leukemia cells were infected with Cre and control viruses, and stained for Annexin V. At 48 hours post-transduction, total Cre-infected Cbfb +/flMYH11 leukemia cells showed a statistically significant increase in Annexin V staining, as compared to cells infected with the control virus. Importantly, increased Annexin V staining was seen in CSF2RB - cells, a population we previously showed to be enriched for leukemia stem cells (LSCs). To test if loss of Cbfb-MYH11 induced differentiation of leukemia cells, we stained Cre and control transduced Cbfb flMYH11 leukemia cells for the myeloid differentiation markers Gr-1 and Mac-1. We found no difference in the expression of either marker with Cbfb-MYH11 excision. These findings indicate that Cbfb-MYH11 is required for the survival of leukemic cells, including the LSC population, and its loss does not cause differentiation. To test if Runx1 is required for Cbfb-MYH11 activities during leukemia maintenance, we utilized a lentiviral vector expressing an shRNA against Runx1 and infected Cbfb-MYH11 expressing mouse leukemia cells. Runx1 knockdown was verified by quantitative RT-PCR and by western blot. To test if Runx1 knockdown induced apoptosis, leukemia cells infected with Runx1 knockdown or a scrambled shRNA control virus were stained for Annexin V. With an average decrease in Runx1 of 60.0% +/- 0.17, we observed an overall increase in Annexin V staining, but not in the CSF2RB-, LSC enriched population. This implies that LSCs may be less sensitive to decreased RUNX1 activity, than non-LSCs. To examine the effect of Runx1 knockdown on LSC activity in vitro, we performed colony forming cell (CFC) assays. We found that cells with decreased expression of Runx1 produced significantly fewer colonies as compared to the scrambled shRNA-infected cells. While some of the observed colonies may have been due to rare cells that lost or silenced the shRNA vector, our preliminary data indicates some colonies retained Runx1 knockdown (70.0% decrease, as compared to control infected cells) after growth in culture. These findings indicate that Runx1 is required for leukemia maintenance, but that LSCs may be less sensitive to decreased RUNX1 activity than non-LSCs. Taken together, our results imply that both Cbfb-MYH11 and Runx1 are important for the maintenance of inv(16) AML, and that inhibition of CBFβ-SMMHCand RUNX1 have potential as a cure for inv(16) AML. Disclosures No relevant conflicts of interest to declare.
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cbfb runx1 repression independent blockage of differentiation and accumulation of CSF2RB expressing cells by cbfb myh11
Blood, 2010Co-Authors: Katherine R Hyde, Yasuhiko Kamikubo, Martha Kirby, Lemlem Alemu, Stacie M. Anderson, Ling ZhaoAbstract:It is known that CBFB-MYH11, the fusion gene generated by inversion of chromosome 16 in human acute myeloid leukemia, is causative for oncogenic transformation. However, the mechanism by which CBFB-MYH11 initiates leukemogenesis is not clear. Previously published reports showed that CBFB-MYH11 dominantly inhibits RUNX1 and CBFB, and such inhibition has been suggested as the mechanism for leukemogenesis. Here we show that Cbfb-MYH11 caused Cbfb/Runx1 repression–independent defects in both primitive and definitive hematopoiesis. During primitive hematopoiesis, Cbfb-MYH11 delayed differentiation characterized by sustained expression of Gata2, Il1rl1, and CSF2RB, a phenotype not found in Cbfb and Runx1 knockout mice. Expression of Cbfb-MYH11 in the bone marrow induced the accumulation of abnormal progenitor-like cells expressing CSF2RB in preleukemic mice. The expression of all 3 genes was detected in most human and murine CBFB-MYH11+ leukemia samples. Interestingly, Cbfb-MYH11+ preleukemic progenitors and leukemia-initiating cells did not express CSF2RB, although the majority of leukemia cells in our Cbfb-MYH11 knockin mice were CSF2RB+. Therefore CSF2RB can be used as a negative selection marker to enrich preleukemic progenitor cells and leukemia-initiating cells from Cbfb-MYH11 mice. These results suggest that Cbfb/Runx1 repression–independent activities contribute to leukemogenesis by Cbfb-MYH11.
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cbfb runx1 repression independent blockage of differentiation and accumulation of CSF2RB expressing cells by cbfb myh11
Blood, 2010Co-Authors: Katherine R Hyde, Yasuhiko Kamikubo, Martha Kirby, Lemlem Alemu, Stacie M. Anderson, Ling ZhaoAbstract:It is known that CBFB-MYH11, the fusion gene generated by inversion of chromosome 16 in human acute myeloid leukemia, is causative for oncogenic transformation. However, the mechanism by which CBFB-MYH11 initiates leukemogenesis is not clear. Previously published reports showed that CBFB-MYH11 dominantly inhibits RUNX1 and CBFB, and such inhibition has been suggested as the mechanism for leukemogenesis. Here we show that Cbfb-MYH11 caused Cbfb/Runx1 repression–independent defects in both primitive and definitive hematopoiesis. During primitive hematopoiesis, Cbfb-MYH11 delayed differentiation characterized by sustained expression of Gata2, Il1rl1, and CSF2RB, a phenotype not found in Cbfb and Runx1 knockout mice. Expression of Cbfb-MYH11 in the bone marrow induced the accumulation of abnormal progenitor-like cells expressing CSF2RB in preleukemic mice. The expression of all 3 genes was detected in most human and murine CBFB-MYH11+ leukemia samples. Interestingly, Cbfb-MYH11+ preleukemic progenitors and leukemia-initiating cells did not express CSF2RB, although the majority of leukemia cells in our Cbfb-MYH11 knockin mice were CSF2RB+. Therefore CSF2RB can be used as a negative selection marker to enrich preleukemic progenitor cells and leukemia-initiating cells from Cbfb-MYH11 mice. These results suggest that Cbfb/Runx1 repression–independent activities contribute to leukemogenesis by Cbfb-MYH11.
Brenna Carey - One of the best experts on this subject based on the ideXlab platform.
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statin as a novel pharmacotherapy of pulmonary alveolar proteinosis
Nature Communications, 2018Co-Authors: Cormac Mccarthy, Takuji Suzuki, Claudia Chalk, Tisha Wang, Elinor Lee, James P Bridges, Anthony Sallese, Jason C Woods, Brian J Bartholmai, Brenna CareyAbstract:Pulmonary alveolar proteinosis (PAP) is a syndrome of reduced GM-CSF-dependent, macrophage-mediated surfactant clearance, dysfunctional foamy alveolar macrophages, alveolar surfactant accumulation, and hypoxemic respiratory failure for which the pathogenetic mechanism is unknown. Here, we examine the lipids accumulating in alveolar macrophages and surfactant to define the pathogenesis of PAP and evaluate a novel pharmacotherapeutic approach. In PAP patients, alveolar macrophages have a marked increase in cholesterol but only a minor increase in phospholipids, and pulmonary surfactant has an increase in the ratio of cholesterol to phospholipids. Oral statin therapy is associated with clinical, physiological, and radiological improvement in autoimmune PAP patients, and ex vivo statin treatment reduces cholesterol levels in explanted alveolar macrophages. In CSF2RB-/- mice, statin therapy reduces cholesterol accumulation in alveolar macrophages and ameliorates PAP, and ex vivo statin treatment increases cholesterol efflux from macrophages. These results support the feasibility of statin as a novel pathogenesis-based pharmacotherapy of PAP.
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use of induced pluripotent stem cells to recapitulate pulmonary alveolar proteinosis pathogenesis
American Journal of Respiratory and Critical Care Medicine, 2013Co-Authors: Takuji Suzuki, Brenna Carey, Robert E Wood, Claudia Chalk, Anthony Sallese, Christopher N Mayhew, Punam Malik, Bruce C TrapnellAbstract:Rationale: In patients with pulmonary alveolar proteinosis (PAP) syndrome, disruption of granulocyte/macrophage colony–stimulating factor (GM-CSF) signaling is associated with pathogenic surfactant accumulation from impaired clearance in alveolar macrophages.Objectives: The aim of this study was to overcome these barriers by using monocyte-derived induced pluripotent stem (iPS) cells to recapitulate disease-specific and normal macrophages.Methods: We created iPS cells from two children with hereditary PAP (hPAP) caused by recessive CSF2RAR217X mutations and three normal people, differentiated them into macrophages (hPAP-iPS-Mφs and NL-iPS-Mφs, respectively), and evaluated macrophage functions with and without gene-correction to restore GM-CSF signaling in hPAP-iPS-Mφs.Measurements and Main Results: Both hPAP and normal iPS cells had human embryonic stem cell–like morphology, expressed pluripotency markers, formed teratomas in vivo, had a normal karyotype, retained and expressed mutant or normal CSF2RA gen...
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hereditary pulmonary alveolar proteinosis caused by recessive CSF2RB mutations
European Respiratory Journal, 2011Co-Authors: Takuji Suzuki, Takuro Sakagami, Brenna Carey, Claudia Chalk, Bruno Maranda, P Catellier, C Y Couture, Bruce C TrapnellAbstract:To the Editors: Pulmonary alveolar proteinosis (PAP) is a syndrome characterised by accumulation of surfactant in alveoli resulting in respiratory insufficiency 1. Surfactant homeostasis is critical for lung function and is tightly regulated, in part, by pulmonary granulocyte-macrophage colony-stimulating factor (GM-CSF), which is required for surfactant clearance by alveolar macrophages 2 and alveolar macrophage maturation 1. The effects of GM-CSF are mediated by cell-surface receptors composed of GM-CSF-binding α-chains and affinity-enhancing β-chains (encoded by CSF2RA and CSF2RB , respectively) 3. Ligand binding activates signalling via multiple pathways including the signal transducer and activator of phosphorylation (STAT)5 4. Disruption of GM-CSF signalling causes PAP by impairing surfactant catabolism in alveolar macrophages 1. In 90% of patients, PAP is caused by neutralising GM-CSF auto-antibodies 5, 6. Through the Rare Lung Diseases Network global PAP detection programme, we identified PAP caused by recessive CSF2RA mutations and developed novel diagnostic methods to identify patients with PAP caused by GM-CSF receptor dysfunction 4, 7. Herein, we report a case of hereditary PAP caused by disruption of GM-CSF receptor β-chain function. A previously healthy 9-yr-old female presented with bilateral pneumonia, followed 3 months later by progressive dyspnoea of insidious onset. The diagnosis of PAP was suggested by chest radiograph findings, high-resolution computed tomography and bronchoalveolar cytology, and was confirmed by surgical lung biopsy. Pulmonary histopathology was typical of primary PAP (fig. 1) and she was successfully treated by serial whole lung lavage therapy. Details of the case history are included in the online supplement. A GM-CSF auto-antibody test was negative and the serum GM-CSF level was increased (25.9 pg·mL−1) suggesting GM-CSF receptor dysfunction as the molecular basis of PAP 4, 7. A molecular evaluation was undertaken and included GM-CSF receptor detection, STAT-5 phosphorylation, CSF2RA …
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hereditary pulmonary alveolar proteinosis pathogenesis presentation diagnosis and therapy
American Journal of Respiratory and Critical Care Medicine, 2010Co-Authors: Takuji Suzuki, Maurizio Luisetti, Takuro Sakagami, Lisa R Young, Brenna Carey, Robert E Wood, Susan E Wert, Bruce K Rubin, Katharine Kevill, Claudia ChalkAbstract:Rationale: We identified a 6-year-old girl with pulmonary alveolar proteinosis (PAP), impaired granulocyte-macrophage colony–stimulating factor (GM-CSF) receptor function, and increased GM-CSF. Objectives: Increased serum GM-CSF may be useful to identify individuals with PAP caused by GM-CSF receptor dysfunction. Methods: We screened 187 patients referred to us for measurement of GM-CSF autoantibodies to diagnose autoimmune PAP. Five were children with PAP and increased serum GM-CSF but without GM-CSF autoantibodies or any disease causing secondary PAP; all were studied with family members, subsequently identified patients, and controls. Measurement and Main Results: Eight children (seven female, one male) were identified with PAP caused by recessive CSF2RA mutations. Six presented with progressive dyspnea of insidious onset at 4.8 ± 1.6 years and two were asymptomatic at ages 5 and 8 years. Radiologic and histopathologic manifestations were similar to those of autoimmune PAP. Molecular analysis demonstrated that GM-CSF signaling was absent in six and severely reduced in two patients. The GM-CSF receptor β chain was detected in all patients, whereas the α chain was absent in six and abnormal in two, paralleling the GM-CSF signaling defects. Genetic analysis revealed multiple distinct CSF2RA abnormalities, including missense, duplication, frameshift, and nonsense mutations; exon and gene deletion; and cryptic alternative splicing. All symptomatic patients responded well to whole-lung lavage therapy. Conclusions: CSF2RA mutations cause a genetic form of PAP presenting as insidious, progressive dyspnea in children that can be diagnosed by a combination of characteristic radiologic findings and blood tests and treated successfully by whole-lung lavage.
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pulmonary alveolar proteinosis a primary immunodeficiency of impaired gm csf stimulation of macrophages
Current Opinion in Immunology, 2009Co-Authors: Bruce C Trapnell, Brenna Carey, Kanji Uchida, Takuji SuzukiAbstract:Pulmonary alveolar proteinosis (PAP) is a rare syndrome characterized by accumulation of pulmonary surfactant, respiratory insufficiency, and increased infections. It occurs in various clinical settings that disrupt surfactant catabolism in alveolar macrophages, including a relatively more common autoimmune disease caused by GM-CSF autoantibodies and a rare congenital disease caused by CSF2RA mutations. Recent results demonstrate that GM-CSF is crucial for alveolar macrophage terminal differentiation and immune functions, pulmonary surfactant homeostasis, and lung host defense. GM-CSF is also required to determine the basal functional capacity of circulating neutrophils, including adhesion, phagocytosis, and microbial killing. PAP research has illuminated the crucial role of GM-CSF in innate immunity and led to novel therapy for PAP and the potential use of anti-GM-CSF therapy in other common disorders.