- Email: [email protected]
Autophagy paradoxically regulates Sirt3 in normal and transformed hematopoietic cells
S108
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3180 - AUTOPHAGY PARADOXICALLY REGULATES SIRT3 IN NORMAL AND TRANSFORMED HEMATOPOIETIC CELLS Yixuan Fang, Gaoyue Jiang, Ni An, Ruijin Zhao, Kang Wang, Tiantian Gui, Chaorong Ge, and Jianrong Wang
3182 - COMBINING TRANSCRIPTOME, QUANTITATIVE PROTEOME AND METABOLOME APPROACHES TO IDENTIFY TARGETABLE VULNERABILITIES IN AML Ays¸eg€ ul Erdem1, Roldan Cortes2, Silvia Marin2, Marta Cascante2, and Jan Jacob Schuringa3
Soochow University, Suzhou, China (People’s Republic)
1
Experimental Hematology, UMCG Groningen, Groningen, The Netherlands; Department of Biochemistry and Molecular Biology, Universitat de Barcelona, Barcelona, Spain; 3Experimental Hematology, Groningen, The Netherlands 2
Sirtuin protein family member 3 (Sirt3) has been known as an anti-aging modulator alleviating oxidative stress primarily by acting on the mitochondrial antioxidant machinery, but its role in hematopoietic cells remains obscure. We found that progressive downregulation of autophagy activity was associated with the decline in Sirt3 expression in hematopoietic cells during the aging of mice. Loss of autophagy by deleting autophagy-essential gene atg7 with gene targeting strategy accelerated mouse aging but decelerated Sirt3 expression simultaneously, proposing an anti-aging role of Sirt3 dependent on intact autophagy machinery in normal hematopoietic cells. Paradoxically, in leukemia cells, we found that upregulation of Sirt3 was associated with mitochondrial stress, and depletion of Sirt3 decreased the generation of reactive oxygen species and lipid oxidation, but increased the ratio of reduced glutathione to oxidized glutathione, suggesting an opposite role of Sirt3 in regulating oxidative stress in transformed hematopoietic cells. In contrast to that in normal hematopoietic cells, loss of atg7 gene or pharmacological inhibition on autophagy caused a significant accumulation of Sirt3 in leukemia cells. However, induced activation of autophagy did not cause autophagic degradation of Sirt3 in leukemia cells. Furthermore, inhibiting proteasome activity accumulated Sirt3 in autophagy-intact but not autophagy-defective leukemia cells, and disrupting functional autophagy either genetically or pharmacologically caused significantly less ubiquitination of Sirt3 in leukemia cells. Therefore, unlike that in normal hematopoietic cells, in leukemia cell, basal but not enhanced autophagy activity maintains ubiquitination-proteasomal degradation of Sirt3 to limit lipid oxidative stress, representing an adaptive mechanism by which autophagy, in collaboration with the ubiquitination-proteasomal system, controls oxidative stress by controlling the level of Sirt3 protein.
3181 - A LATENT DEFECT IN LYMPHOID-BIASED HEMATOPOIETIC STEM CELLS IN UBC-GFP TRANSGENIC MICE Katerina Faltusova1, Katarina Szikszai2, Martin Molik2, Filipp Savvulidi2, and Emanuel Necas2
Depending on their cellular quiescent and active states, metabolic signatures controlling the energy production of hematopoietic stem cells (HSCs) differ. A thorough understanding of the regulation of these signatures at the molecular level is still lacking. We developed an integrative approach in order to link genetic differences with metabolomics and generated transcriptome, quantitative proteome and metabolome data in normal and leukemic human HSCs. The comparison of the proteome of human primary Acute Myeloid Leukemia (AML) CD34+ cells (n544), AML cell lines (n56) and primary healthy peripheral blood CD34+ cells (n56) demonstrated altered metabolic signatures in both glycolysis and mitochondrial oxidative phosphorylation (TCA) activity. We also performed metabolic profiling of glycolysis and TCA activity in selected primary AMLs and AML cell lines using a Seahorse assay and targeted metabolomics with LC-MS/MS (Biocrates Kit to identify w180 metabolites). Analysis of the LC-MS/MS based metabolome data is underway, but basal metabolic screening with Seahorse has already shown significantly distinct metabolic properties in a variety of AML subtypes. To functionally evaluate the correlation of proteome and metabolic changes we examined the significance of two essential enzymes Pyruvate Dehydrogenase Kinases (PDKs) and Succinate-CoA Ligase (SUCLG1/2 GDP subunits) that control glycolysis and TCA, respectively. We observed that impairment/promotion of cell proliferation varies according to the metabolic profile of the cells upon loss of PDKs with a lentiviral shRNA approach. To define the consequences of SUCLG1-2 up-regulation in AML cells we studied complex II as an upstream enzyme of SUCLGs. The treatment of AML cell lines with Complex II inhibitor impaired the growth of the cells known to be highly dependent on TCA. Spectrophotometric measurement of glycolytic flux metabolites further showed impaired glutamate production upon treatment and most drastically in the cells with higher TCA activity. Thus, we have identified a number of glycolysis and TCA intermediates that represent interesting targets for a variety of AML subtypes with distinct metabolic profiles.
3183 - THE FLCN-TFE3 AXIS REGULATES MACROPHAGE ACTIVATION THROUGH CELLULAR METABOLISM Mitsuhiro Endo1,2, Masaya Baba3, Tamie Endoh1, Terumasa Umemoto3, Michihiro Hashimoto3, Yang Chong1, and Toshio Suda1
Institute of Pathological Physiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic; 2Institute of Pathophysiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic
1 Cancer Science Institute of Singapore, University of Singapore, Singapore, Singapore; 2International Research Center for Medical Sciences, Kumamoto University, Singapore, Singapore; 3International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan
We used transgenic mice expressing GFP under the human ubiquitin promoter (UBCGFP mice; Schaefer et al, 2001) in experiments studying a spontaneous recovery of hematopoiesis damaged by submyeloablative irradiation. Results were strikingly different from corresponding experiments that used two congenic strains of mice CD45.2 and CD45.1. Analysis of this unexpected difference in results revealed that while UBC-GFP mice have normal hematopoiesis that also regenerates normally after damage, they are inferior when competing with transplanted bone marrow from wild-type mice (CD45.1 or CD45.2). Interestingly, UBC-GFP mice engrafted transplanted bone marrow of wild-type mice without conditioning, and their conditioning with a low sublethal dose of irradiation resulted in an inappropriately high level of engraftment of transplanted bone marrow of wild-type donors. Interestingly also, the wild-type mice origin hematopoietic and blood cells were consistently more represented in the peripheral blood, spleen and thymus as compared to the bone marrow. Transplanted bone marrow contributed both to myelopoiesis and lymphopoiesis but their contribution was significantly higher in the lymphoid cells. Chimeric hematopoiesis resulting from the transplantation of wild-type bone marrow to UBC-GFP recipients could be transplanted to secondary recipients confirming a defect in the lymphoid-biased hematopoietic stem cells in UBC-GFP mice. Schaefer BC, Schaefer ML, Kappler JW, Marrack P, Kedl RM: Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo. Cell Immunol. 2001 Dec 15;214(2):110-22. Research was supported by the Grant Agency od the Czech Republic (GACR 1701897S).
Cellular metabolism has been suggested to play a deterministic role in many biological processes including the regulation of stem cell properties and functions. A tumor suppressor gene, folliculin (FLCN) is responsible for Birt-Hogg-Dube (BHD) syndrome characterized by benign skin tumors, lung and kidney cysts, and kidney cancer, and has been suggested to be involved in regulation of oxidative metabolism. We previously reported that conditional depletion of Flcn in hematopoietic stem cell compartment using the Mx1-Cre system led to macrophage activation and bone marrow failure (Baba et al, 2016). In this study, we show that Mx1-Cre mediated deletion of Flcn led to induce CD11c+ CD206+ activated phagocytic macrophages which exhibited highest levels of lysosomal mass and mitochondrial membrane potential in bone marrow and spleen in mice. Flcn knockdown in a mouse monocyte/ macrophage cell line, Raw 264.7 cells, also increased both lysosomal mass and mitochondrial membrane potential and activated their phagocytic activity. Since we observed nuclear translocation of TFE3, a master regulator of lysosomal biogenesis, in Flcn-depleted cells, we next examined the effect of enforced expression of nuclear TFE3 using a TFE3-GR fusion protein system. The expression of nuclear TFE3 induced CD11c expression and augmented phagocytic activity in Raw 264.7 cells, suggesting at least a part of macrophage activation phenotypes observed in Flcn-KO mice could be caused by the activation of TFE3. Furthermore, we identified GABARAP, a member of ATG8 family proteins, as a component of FLCN/FNIP1/ AMPK complexes based on the results from our yeast two-hybrid screening using FNIP1 as a bait. We also found FLCN-TFE3 axis regulates GABARAP expression
1
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3180 - AUTOPHAGY PARADOXICALLY REGULATES SIRT3 IN NORMAL AND TRANSFORMED HEMATOPOIETIC CELLS Yixuan Fang, Gaoyue Jiang, Ni An, Ruijin Zhao, Kang Wang, Tiantian Gui, Chaorong Ge, and Jianrong Wang
3182 - COMBINING TRANSCRIPTOME, QUANTITATIVE PROTEOME AND METABOLOME APPROACHES TO IDENTIFY TARGETABLE VULNERABILITIES IN AML Ays¸eg€ ul Erdem1, Roldan Cortes2, Silvia Marin2, Marta Cascante2, and Jan Jacob Schuringa3
Soochow University, Suzhou, China (People’s Republic)
1
Experimental Hematology, UMCG Groningen, Groningen, The Netherlands; Department of Biochemistry and Molecular Biology, Universitat de Barcelona, Barcelona, Spain; 3Experimental Hematology, Groningen, The Netherlands 2
Sirtuin protein family member 3 (Sirt3) has been known as an anti-aging modulator alleviating oxidative stress primarily by acting on the mitochondrial antioxidant machinery, but its role in hematopoietic cells remains obscure. We found that progressive downregulation of autophagy activity was associated with the decline in Sirt3 expression in hematopoietic cells during the aging of mice. Loss of autophagy by deleting autophagy-essential gene atg7 with gene targeting strategy accelerated mouse aging but decelerated Sirt3 expression simultaneously, proposing an anti-aging role of Sirt3 dependent on intact autophagy machinery in normal hematopoietic cells. Paradoxically, in leukemia cells, we found that upregulation of Sirt3 was associated with mitochondrial stress, and depletion of Sirt3 decreased the generation of reactive oxygen species and lipid oxidation, but increased the ratio of reduced glutathione to oxidized glutathione, suggesting an opposite role of Sirt3 in regulating oxidative stress in transformed hematopoietic cells. In contrast to that in normal hematopoietic cells, loss of atg7 gene or pharmacological inhibition on autophagy caused a significant accumulation of Sirt3 in leukemia cells. However, induced activation of autophagy did not cause autophagic degradation of Sirt3 in leukemia cells. Furthermore, inhibiting proteasome activity accumulated Sirt3 in autophagy-intact but not autophagy-defective leukemia cells, and disrupting functional autophagy either genetically or pharmacologically caused significantly less ubiquitination of Sirt3 in leukemia cells. Therefore, unlike that in normal hematopoietic cells, in leukemia cell, basal but not enhanced autophagy activity maintains ubiquitination-proteasomal degradation of Sirt3 to limit lipid oxidative stress, representing an adaptive mechanism by which autophagy, in collaboration with the ubiquitination-proteasomal system, controls oxidative stress by controlling the level of Sirt3 protein.
3181 - A LATENT DEFECT IN LYMPHOID-BIASED HEMATOPOIETIC STEM CELLS IN UBC-GFP TRANSGENIC MICE Katerina Faltusova1, Katarina Szikszai2, Martin Molik2, Filipp Savvulidi2, and Emanuel Necas2
Depending on their cellular quiescent and active states, metabolic signatures controlling the energy production of hematopoietic stem cells (HSCs) differ. A thorough understanding of the regulation of these signatures at the molecular level is still lacking. We developed an integrative approach in order to link genetic differences with metabolomics and generated transcriptome, quantitative proteome and metabolome data in normal and leukemic human HSCs. The comparison of the proteome of human primary Acute Myeloid Leukemia (AML) CD34+ cells (n544), AML cell lines (n56) and primary healthy peripheral blood CD34+ cells (n56) demonstrated altered metabolic signatures in both glycolysis and mitochondrial oxidative phosphorylation (TCA) activity. We also performed metabolic profiling of glycolysis and TCA activity in selected primary AMLs and AML cell lines using a Seahorse assay and targeted metabolomics with LC-MS/MS (Biocrates Kit to identify w180 metabolites). Analysis of the LC-MS/MS based metabolome data is underway, but basal metabolic screening with Seahorse has already shown significantly distinct metabolic properties in a variety of AML subtypes. To functionally evaluate the correlation of proteome and metabolic changes we examined the significance of two essential enzymes Pyruvate Dehydrogenase Kinases (PDKs) and Succinate-CoA Ligase (SUCLG1/2 GDP subunits) that control glycolysis and TCA, respectively. We observed that impairment/promotion of cell proliferation varies according to the metabolic profile of the cells upon loss of PDKs with a lentiviral shRNA approach. To define the consequences of SUCLG1-2 up-regulation in AML cells we studied complex II as an upstream enzyme of SUCLGs. The treatment of AML cell lines with Complex II inhibitor impaired the growth of the cells known to be highly dependent on TCA. Spectrophotometric measurement of glycolytic flux metabolites further showed impaired glutamate production upon treatment and most drastically in the cells with higher TCA activity. Thus, we have identified a number of glycolysis and TCA intermediates that represent interesting targets for a variety of AML subtypes with distinct metabolic profiles.
3183 - THE FLCN-TFE3 AXIS REGULATES MACROPHAGE ACTIVATION THROUGH CELLULAR METABOLISM Mitsuhiro Endo1,2, Masaya Baba3, Tamie Endoh1, Terumasa Umemoto3, Michihiro Hashimoto3, Yang Chong1, and Toshio Suda1
Institute of Pathological Physiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic; 2Institute of Pathophysiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic
1 Cancer Science Institute of Singapore, University of Singapore, Singapore, Singapore; 2International Research Center for Medical Sciences, Kumamoto University, Singapore, Singapore; 3International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan
We used transgenic mice expressing GFP under the human ubiquitin promoter (UBCGFP mice; Schaefer et al, 2001) in experiments studying a spontaneous recovery of hematopoiesis damaged by submyeloablative irradiation. Results were strikingly different from corresponding experiments that used two congenic strains of mice CD45.2 and CD45.1. Analysis of this unexpected difference in results revealed that while UBC-GFP mice have normal hematopoiesis that also regenerates normally after damage, they are inferior when competing with transplanted bone marrow from wild-type mice (CD45.1 or CD45.2). Interestingly, UBC-GFP mice engrafted transplanted bone marrow of wild-type mice without conditioning, and their conditioning with a low sublethal dose of irradiation resulted in an inappropriately high level of engraftment of transplanted bone marrow of wild-type donors. Interestingly also, the wild-type mice origin hematopoietic and blood cells were consistently more represented in the peripheral blood, spleen and thymus as compared to the bone marrow. Transplanted bone marrow contributed both to myelopoiesis and lymphopoiesis but their contribution was significantly higher in the lymphoid cells. Chimeric hematopoiesis resulting from the transplantation of wild-type bone marrow to UBC-GFP recipients could be transplanted to secondary recipients confirming a defect in the lymphoid-biased hematopoietic stem cells in UBC-GFP mice. Schaefer BC, Schaefer ML, Kappler JW, Marrack P, Kedl RM: Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo. Cell Immunol. 2001 Dec 15;214(2):110-22. Research was supported by the Grant Agency od the Czech Republic (GACR 1701897S).
Cellular metabolism has been suggested to play a deterministic role in many biological processes including the regulation of stem cell properties and functions. A tumor suppressor gene, folliculin (FLCN) is responsible for Birt-Hogg-Dube (BHD) syndrome characterized by benign skin tumors, lung and kidney cysts, and kidney cancer, and has been suggested to be involved in regulation of oxidative metabolism. We previously reported that conditional depletion of Flcn in hematopoietic stem cell compartment using the Mx1-Cre system led to macrophage activation and bone marrow failure (Baba et al, 2016). In this study, we show that Mx1-Cre mediated deletion of Flcn led to induce CD11c+ CD206+ activated phagocytic macrophages which exhibited highest levels of lysosomal mass and mitochondrial membrane potential in bone marrow and spleen in mice. Flcn knockdown in a mouse monocyte/ macrophage cell line, Raw 264.7 cells, also increased both lysosomal mass and mitochondrial membrane potential and activated their phagocytic activity. Since we observed nuclear translocation of TFE3, a master regulator of lysosomal biogenesis, in Flcn-depleted cells, we next examined the effect of enforced expression of nuclear TFE3 using a TFE3-GR fusion protein system. The expression of nuclear TFE3 induced CD11c expression and augmented phagocytic activity in Raw 264.7 cells, suggesting at least a part of macrophage activation phenotypes observed in Flcn-KO mice could be caused by the activation of TFE3. Furthermore, we identified GABARAP, a member of ATG8 family proteins, as a component of FLCN/FNIP1/ AMPK complexes based on the results from our yeast two-hybrid screening using FNIP1 as a bait. We also found FLCN-TFE3 axis regulates GABARAP expression
1