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p38a protects hematopoietic stem cells from physiological and premature aging stresses
S64
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
suggestive of an essential role for enzymatic processing in control of developmental HSPC production and migration. Taken together, our findings point to functional intersections between ECM-modifying enzymes, ECM structure and inflammatory cytokines in the onset and maintenance of vertebrate hematopoiesis.
3035 - P38A PROTECTS HEMATOPOIETIC STEM CELLS FROM PHYSIOLOGICAL AND PREMATURE AGING STRESSES Keiyo Takubo, Yukako Ootomo, and Daiki Karigane National Center for Global Health and Medicine, Tokyo, Japan Hematopoietic stem and progenitor cells (HSPCs) maintain the hematopoietic system for an organism’s entire lifetime under homeostatic or in stress settings. Along with aging, hematopoietic stem cells (HSCs) show various quantitative and qualitative changes including accumulation of phenotypic HSCs, transplantation defects, and myeloid skewing. Specific activation of p38MAPK is a candidate pathway that induces HSC aging in vivo. However, genetic dissection of p38MAPK signaling in HSC aging is still limited. Recently, we found that conditional deletion of the dominant isozyme of p38MAPK in hematopoietic cells, p38a, resulted in defective stress response of HSPCs upon acute stress loading including transplantation. Based on these findings, in this study we analyzed the hematological effects of p38a loss in physiological and premature aging models. In aged mice, loss of p38a did not ameliorate the accumulation of phenotypic HSCs in the bone marrow. In addition, transplantation capacity of aged p38a-deficient HSCs was defective than aged wild-type HSCs. Also, differentiation capacity of aged p38a-deficient HSCs was more myeloid-biased than aged wild-type HSCs. Next, we analyzed the effect of p38a loss in a premature aging model, Ataxia TelangiectasiaMutated (Atm) deficient mice. Inducible loss of Atm decreased phenotypic and functional HSCs in vivo. Co-deletion of Atm and p38a induced severe loss of HSCs and defective transplantation phenotype compared to Atm single deficiency. Neither loss of Atm nor Atm/p38a changed the apoptosis status and cell cycle quiescence of HSPCs. Also, generation of reactive oxygen species was comparable among genotypes. These findings suggest a protective role of p38a on HSC aging.
3034 - TAK1 INHIBITION DISRUPTS A VICIOUS CYCLE BETWEEN TUMOR PROGRESSION AND BONE DESTRUCTION IN MYELOMA Masahiro Abe1, Jumpei Teramachi1, Masahiro Hiasa1, Asuka Oda1, Hirofumi Tenshin1, Ryota Amachi1, Takeshi Harada1, Shingen Nakamura1, Hirokazu Miki2, Itsuro Endo2, and Toshio Matsumoto1 Tokushima University, Tokushima, Japan; 2Tokushima University Hospital, Tokushima, Japan
3036 - HIGH EFFICIENCY GENE CORRECTION IN HEMATOPOIETIC CELLS BY DONOR-TEMPLATE-FREE CRISPR/CAS9 GENOME EDITING Duran S€ ur€ un, Nina Kurrle, Hubert Serve, Harald von Melchner, and Frank Schn€utgen University Hospital Frankfurt, Frankfurt am Main, Germany
1
Multiple myeloma (MM) has a unique propensity to develop and expand almost exclusively in the bone marrow and generates destructive bone disease. MM cells constitutively overexpress Pim-2; cocultures with bone marrow stromal cells (BMSCs) or osteoclasts (OCs) were found to further upregulate Pim-2 as an anti-apoptotic mediator in MM cells and induce it also in BMSCs and OCs to progress bone destruction, indicating the critical role of Pim-2 in MM growth and bone destruction. We identified TGF-b-activated kinase-1 (TAK1) as an upstream mediator responsible for Pim-2 upregulation. In this study, we aimed to clarify the role of TAK1 in MM growth and bone destruction and therapeutic impact of TAK1 inhibition. TAK1 was constitutively overexpressed and phosphorylated in MM cells. The TAK1 inhibitor LLZ1640-2 (LLZ) abolished TNF-a-induced NF-kB, p38 and ERK activation and IL-6-induced STAT3 activation in MM cells. Importantly, LLZ suppressed Pim-2 upregulation and induced apoptosis in MM cells even in cocultures with BMSCs or OCs, while reducing VEGF production and the expression of BCMA and TACI, receptors for BAFF and APRIL. TAK1 inhibition impaired adhesive interactions between MM cells and BMSCs along with reducing VCAM-1 and RANKL expression and IL-6 production by BMSCs, which blunted protective activity of BMSCs for MM cells against anti-MM agents. The known inhibitors for osteoblastogenesis in MM, including IL-3, IL-7, TNF-a, TGF-b and activinA, as well as MM cell conditioned media all induced TAK1 activation and Pim-2 upregulation to suppress their osteoblastogenesis in MC3T3-E1 preosteoblastic cells; however, LLZ abolished the Pim-2 up-regulation and restored their osteoblastogenesis. Furthermore, although RANKL enhanced osteoclastogenesis along with activation of the TAK1-Pim-2 signaling, LLZ suppressed the osteoclastogenesis by RANKL. Finally, treatment with LLZ markedly suppressed MM growth and prevented bone destruction in mouse MM models. From these results, TAK1 appears to play a pivotal role in tumor growth and bone destruction in MM. TAK1 inhibition may become a unique anti-MM therapeutic option with bone anabolic activity.
The CRISPR/Cas9 prokaryotic adaptive immune system, and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD) - a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA guided Cas9 endonucleases (RGNs). As about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor-template free RGNs.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
suggestive of an essential role for enzymatic processing in control of developmental HSPC production and migration. Taken together, our findings point to functional intersections between ECM-modifying enzymes, ECM structure and inflammatory cytokines in the onset and maintenance of vertebrate hematopoiesis.
3035 - P38A PROTECTS HEMATOPOIETIC STEM CELLS FROM PHYSIOLOGICAL AND PREMATURE AGING STRESSES Keiyo Takubo, Yukako Ootomo, and Daiki Karigane National Center for Global Health and Medicine, Tokyo, Japan Hematopoietic stem and progenitor cells (HSPCs) maintain the hematopoietic system for an organism’s entire lifetime under homeostatic or in stress settings. Along with aging, hematopoietic stem cells (HSCs) show various quantitative and qualitative changes including accumulation of phenotypic HSCs, transplantation defects, and myeloid skewing. Specific activation of p38MAPK is a candidate pathway that induces HSC aging in vivo. However, genetic dissection of p38MAPK signaling in HSC aging is still limited. Recently, we found that conditional deletion of the dominant isozyme of p38MAPK in hematopoietic cells, p38a, resulted in defective stress response of HSPCs upon acute stress loading including transplantation. Based on these findings, in this study we analyzed the hematological effects of p38a loss in physiological and premature aging models. In aged mice, loss of p38a did not ameliorate the accumulation of phenotypic HSCs in the bone marrow. In addition, transplantation capacity of aged p38a-deficient HSCs was defective than aged wild-type HSCs. Also, differentiation capacity of aged p38a-deficient HSCs was more myeloid-biased than aged wild-type HSCs. Next, we analyzed the effect of p38a loss in a premature aging model, Ataxia TelangiectasiaMutated (Atm) deficient mice. Inducible loss of Atm decreased phenotypic and functional HSCs in vivo. Co-deletion of Atm and p38a induced severe loss of HSCs and defective transplantation phenotype compared to Atm single deficiency. Neither loss of Atm nor Atm/p38a changed the apoptosis status and cell cycle quiescence of HSPCs. Also, generation of reactive oxygen species was comparable among genotypes. These findings suggest a protective role of p38a on HSC aging.
3034 - TAK1 INHIBITION DISRUPTS A VICIOUS CYCLE BETWEEN TUMOR PROGRESSION AND BONE DESTRUCTION IN MYELOMA Masahiro Abe1, Jumpei Teramachi1, Masahiro Hiasa1, Asuka Oda1, Hirofumi Tenshin1, Ryota Amachi1, Takeshi Harada1, Shingen Nakamura1, Hirokazu Miki2, Itsuro Endo2, and Toshio Matsumoto1 Tokushima University, Tokushima, Japan; 2Tokushima University Hospital, Tokushima, Japan
3036 - HIGH EFFICIENCY GENE CORRECTION IN HEMATOPOIETIC CELLS BY DONOR-TEMPLATE-FREE CRISPR/CAS9 GENOME EDITING Duran S€ ur€ un, Nina Kurrle, Hubert Serve, Harald von Melchner, and Frank Schn€utgen University Hospital Frankfurt, Frankfurt am Main, Germany
1
Multiple myeloma (MM) has a unique propensity to develop and expand almost exclusively in the bone marrow and generates destructive bone disease. MM cells constitutively overexpress Pim-2; cocultures with bone marrow stromal cells (BMSCs) or osteoclasts (OCs) were found to further upregulate Pim-2 as an anti-apoptotic mediator in MM cells and induce it also in BMSCs and OCs to progress bone destruction, indicating the critical role of Pim-2 in MM growth and bone destruction. We identified TGF-b-activated kinase-1 (TAK1) as an upstream mediator responsible for Pim-2 upregulation. In this study, we aimed to clarify the role of TAK1 in MM growth and bone destruction and therapeutic impact of TAK1 inhibition. TAK1 was constitutively overexpressed and phosphorylated in MM cells. The TAK1 inhibitor LLZ1640-2 (LLZ) abolished TNF-a-induced NF-kB, p38 and ERK activation and IL-6-induced STAT3 activation in MM cells. Importantly, LLZ suppressed Pim-2 upregulation and induced apoptosis in MM cells even in cocultures with BMSCs or OCs, while reducing VEGF production and the expression of BCMA and TACI, receptors for BAFF and APRIL. TAK1 inhibition impaired adhesive interactions between MM cells and BMSCs along with reducing VCAM-1 and RANKL expression and IL-6 production by BMSCs, which blunted protective activity of BMSCs for MM cells against anti-MM agents. The known inhibitors for osteoblastogenesis in MM, including IL-3, IL-7, TNF-a, TGF-b and activinA, as well as MM cell conditioned media all induced TAK1 activation and Pim-2 upregulation to suppress their osteoblastogenesis in MC3T3-E1 preosteoblastic cells; however, LLZ abolished the Pim-2 up-regulation and restored their osteoblastogenesis. Furthermore, although RANKL enhanced osteoclastogenesis along with activation of the TAK1-Pim-2 signaling, LLZ suppressed the osteoclastogenesis by RANKL. Finally, treatment with LLZ markedly suppressed MM growth and prevented bone destruction in mouse MM models. From these results, TAK1 appears to play a pivotal role in tumor growth and bone destruction in MM. TAK1 inhibition may become a unique anti-MM therapeutic option with bone anabolic activity.
The CRISPR/Cas9 prokaryotic adaptive immune system, and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD) - a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA guided Cas9 endonucleases (RGNs). As about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor-template free RGNs.