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The spliceosomal component SF3B1 is essential for hematopoietic differentiation and splicing fidelity
Short Talk Presentations/ Experimental Hematology 44 (2016) S40–S54
2031 - OSTEOPONTIN REGULATES AND ATTENUATES AGINGASSOCIATED PHENOTYPES OF HSCS Novella Guidi1, Mehmet Sacma1, Ludger St€andker1, Karin Soller1, Gina Marka1, Johannes Weiss1, Frank Kirchhoff1, Tanja Weil1, Jose Cancelas2, Maria Carolina Florian1, and Hartmut Geiger2 1 Ulm University, Ulm, Germany; 2Cincinnati Children’s Hospital Medical Center, Cincinnati, USA
S51
discovered sf3b1 mutants were more sensitive to treatment with E7107, a small molecule inhibitor of the spliceosome, than wild type embryos indicating a potential therapeutic window for spliceosome modulators in MDS. Motivated by this finding, we have performed a large-scale chemical modulator screen and identified many new lead compounds that also show synthetic lethality in sf3b1 mutants. Together, our data demonstrate the molecular and cellular consequences of sf3b1 loss in vivo and establish sf3b1 zebrafish mutants as a novel model for discovery of therapeutics for MDS.
Hematopoietic stem cell (HSC) aging is one underlying cause of aging-associated immunosenescence as well as leukemia. Upon aging HSCs undergo changes in function and structure, including skewing to myeloid lineages, lower reconstitution potential and loss of protein polarity. While stem cell intrinsic mechanisms are known to contribute to HSC aging, little is known on whether aged-related changes in the bone marrow (BM) niche regulate HSC aging. Heterochronic transplantation of young BM cells into young or old recipients revealed that an aged environment supports agingassociated lineage skewing of HSCs (myeloid over lymphoid), a decrease in the overall level of engraftment and an increase in the frequency of long term HSCs (LTHSCs) with associated apolarity. These data imply that HSC aging is, besides intrinsic effects, also determined by aging of the niche. Osteopontin (OPN) is a secreted glycoprotein expressed by osteoblasts located close to the endosteum. Upon aging the expression of osteopontin (OPN) in the BM stroma is reduced, both in terms of expression and secretion. We then tested whether the decrease of OPN in stroma upon aging might be causatively linked to aging of HSCs. Transplantation of young BM cells into young OPN KO recipients, similarly to aged recipients, resulted in a significant decrease in stem cell engraftment with an increase of LT-HSC frequency with loss of polarity. Moreover a brief ex-vivo exposure to Thrombin-cleaved OPN functionally rejuvenated old HSCs, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity and a restored balance of lymphoid and myeloid cells in peripheral blood. Therefore our data suggest a critical causative role for reduced stroma-derived OPN in promoting HSC aging and show that addition of OPN digested by thrombin ameliorates aging of HSCs. Our data thus offer niche informed therapeutic approach for attenuating HSCs phenotypes associated with aging.
2032 - THE SPLICEOSOMAL COMPONENT SF3B1 IS ESSENTIAL FOR HEMATOPOIETIC DIFFERENTIATION AND SPLICING FIDELITY Teresa Bowman1,2,3,4, Rosannah Cameron4, Mia McKinstry4, Adriana De La Garza4, Sara Nik4, Sara Payne4, Peter Smith5, Silvia Buonamici5, and Robert Bradley6 1 Department of Developmental and Molecular Biology; 2Department of Medicine (Oncology); 3Gottesman Institute for Stem Cell Biology and Regenerative Medicine; 4Albert Einstein College of Medicine, New York City, USA; 5H3 Biomedicine, Cambridge, USA; 6Fred Hutchinson Cancer Research Center, Seattle, USA Myelodysplastic syndrome (MDS) is a disorder arising from hematopoietic stem and progenitor cell (HSPC) dysfunction resulting in ineffective hematopoiesis. Mutations in the spliceosomal component SF3B1 are prevalent in MDS, but how this leads to aberrant hematopoiesis is unclear. We elucidated the in vivo consequences of sf3b1 misregulation on hematopoiesis and splicing using a zebrafish sf3b1 (sf3b1hi3394) loss-of-function mutant. Primitive erythropoiesis initiates normally in sf3b1 mutants, as evidenced by normal expression of scl, gata1 and beta-globin, but differentiation is defective as shown by decreased hemoglobinization and an increase in immature erythroblasts at 48 hours post fertilization (hpf). Early markers of primitive myelopoiesis such as scl and pu.1 are normal, but levels of the differentiation markers l-plastin and myeloperoxidase at 24 and 28 hpf are diminished in mutants. Expression of the hemogenic endothelial and HSPC markers runx1 and gata2b at 24 hpf and formation of cmyb:gfp; kdrl:dsred double positive cells at 36 hpf were greatly diminished. In contrast, we observed normal expression of the pan-endothelial marker kdrl and aorta-specific markers notch1b and notch3 at 24 hpf. RNA-seq analysis of sf3b1 mutants revealed large-scale splicing defects and alterations, particularly affecting introns with suboptimal splice sites. Comparison of RNA-seq data from zebrafish sf3b1 mutants and MDS patients harboring SF3B1 mutations revealed a significant overlap in misspliced genes and in the corresponding affected pathways, including many pathways implicated in MDS pathology such as TGFb and immune signaling. Together, these results show conservation between zebrafish mutants and human cells with SF3B1 mutations. We next took a chemical genetic approach and
2034 - LYMPHOID AND MYELOID DIFFERENTIATION PROGRAMS ARE REGULATED BY THE COMMON GENOMIC LOCUS ENCODING FOR EVL AND MIR-342 Friederike Herbst1,2,3, Tonio Lang2,3, Elias Eckert2,3, Shayda Hemmati2,3, Peer W€unsche2,3, Oksana Zavidij2,3, Manfred Schmidt2,3, Claudia Ball2,3, and Hanno Glimm2,3 1 Department of Translational Oncology, Heidelberg, Germany; 2National Center for Tumor Diseases (NCT), Heidelberg, Germany; 3German Cancer Research Center (DKFZ), Heidelberg, Germany Extrinsic and intrinsic factors control HSC regulation to adapt the production of blood cells to the organism’s need. As retroviral integration sites (IS) are clustered nearby active genes and may lead to their upregulation, we systematically analyzed the IS repertoire of ten Wiskott-Aldrich syndrome patients enrolled in clinical gene therapy to identify novel regulators of hematopoiesis. In order to detect overrepresentation of IS in proximity to the transcription start site of gene loci, we developed a screening pipeline. By using this strategy, we identified a significant clustering of IS close to the locus encoding for Evl and its intronic miR-342. Therefore, we selected both genes encoded by one single locus to study their yet uncharacterized role in hematopoiesis. Lentiviral overexpression (OE) of Evl in murine primary LSK (lin-Sca1+ckit+) cells led to a 7.5-fold increase of preB-cell colonies as compared to mock-transduced LSK cells. By contrast, miR-342 OE led to a 2.3-fold larger number of myeloid colonies in vitro and a 3.3-fold increased number of myeloid progenitor-derived CFU-S in vivo. In line with this, global gene expression profiling revealed a profound deregulation of canonical pathways essential for the development of B-cells upon Evl, and myeloid development upon miR-342 OE. Self-renewal and multilineage reconstitution following transgene OE were assessed in serial BM transplantation experiments. Interestingly, EVL+ LSK cells led to a significantly increased donor-derived B-cell frequency within the spleens of primary recipient mice, and a 4.3-fold higher frequency of B-cells in peripheral blood after secondary transplantation. As our results point to an essential role of the Evl/miRNA-342 gene locus in lymphoid or myeloid lineage determination, we assessed its deregulation in human hematopoietic malignancies. Strikingly, the publicly available gene expression dataset of 2096 leukemia
2031 - OSTEOPONTIN REGULATES AND ATTENUATES AGINGASSOCIATED PHENOTYPES OF HSCS Novella Guidi1, Mehmet Sacma1, Ludger St€andker1, Karin Soller1, Gina Marka1, Johannes Weiss1, Frank Kirchhoff1, Tanja Weil1, Jose Cancelas2, Maria Carolina Florian1, and Hartmut Geiger2 1 Ulm University, Ulm, Germany; 2Cincinnati Children’s Hospital Medical Center, Cincinnati, USA
S51
discovered sf3b1 mutants were more sensitive to treatment with E7107, a small molecule inhibitor of the spliceosome, than wild type embryos indicating a potential therapeutic window for spliceosome modulators in MDS. Motivated by this finding, we have performed a large-scale chemical modulator screen and identified many new lead compounds that also show synthetic lethality in sf3b1 mutants. Together, our data demonstrate the molecular and cellular consequences of sf3b1 loss in vivo and establish sf3b1 zebrafish mutants as a novel model for discovery of therapeutics for MDS.
Hematopoietic stem cell (HSC) aging is one underlying cause of aging-associated immunosenescence as well as leukemia. Upon aging HSCs undergo changes in function and structure, including skewing to myeloid lineages, lower reconstitution potential and loss of protein polarity. While stem cell intrinsic mechanisms are known to contribute to HSC aging, little is known on whether aged-related changes in the bone marrow (BM) niche regulate HSC aging. Heterochronic transplantation of young BM cells into young or old recipients revealed that an aged environment supports agingassociated lineage skewing of HSCs (myeloid over lymphoid), a decrease in the overall level of engraftment and an increase in the frequency of long term HSCs (LTHSCs) with associated apolarity. These data imply that HSC aging is, besides intrinsic effects, also determined by aging of the niche. Osteopontin (OPN) is a secreted glycoprotein expressed by osteoblasts located close to the endosteum. Upon aging the expression of osteopontin (OPN) in the BM stroma is reduced, both in terms of expression and secretion. We then tested whether the decrease of OPN in stroma upon aging might be causatively linked to aging of HSCs. Transplantation of young BM cells into young OPN KO recipients, similarly to aged recipients, resulted in a significant decrease in stem cell engraftment with an increase of LT-HSC frequency with loss of polarity. Moreover a brief ex-vivo exposure to Thrombin-cleaved OPN functionally rejuvenated old HSCs, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity and a restored balance of lymphoid and myeloid cells in peripheral blood. Therefore our data suggest a critical causative role for reduced stroma-derived OPN in promoting HSC aging and show that addition of OPN digested by thrombin ameliorates aging of HSCs. Our data thus offer niche informed therapeutic approach for attenuating HSCs phenotypes associated with aging.
2032 - THE SPLICEOSOMAL COMPONENT SF3B1 IS ESSENTIAL FOR HEMATOPOIETIC DIFFERENTIATION AND SPLICING FIDELITY Teresa Bowman1,2,3,4, Rosannah Cameron4, Mia McKinstry4, Adriana De La Garza4, Sara Nik4, Sara Payne4, Peter Smith5, Silvia Buonamici5, and Robert Bradley6 1 Department of Developmental and Molecular Biology; 2Department of Medicine (Oncology); 3Gottesman Institute for Stem Cell Biology and Regenerative Medicine; 4Albert Einstein College of Medicine, New York City, USA; 5H3 Biomedicine, Cambridge, USA; 6Fred Hutchinson Cancer Research Center, Seattle, USA Myelodysplastic syndrome (MDS) is a disorder arising from hematopoietic stem and progenitor cell (HSPC) dysfunction resulting in ineffective hematopoiesis. Mutations in the spliceosomal component SF3B1 are prevalent in MDS, but how this leads to aberrant hematopoiesis is unclear. We elucidated the in vivo consequences of sf3b1 misregulation on hematopoiesis and splicing using a zebrafish sf3b1 (sf3b1hi3394) loss-of-function mutant. Primitive erythropoiesis initiates normally in sf3b1 mutants, as evidenced by normal expression of scl, gata1 and beta-globin, but differentiation is defective as shown by decreased hemoglobinization and an increase in immature erythroblasts at 48 hours post fertilization (hpf). Early markers of primitive myelopoiesis such as scl and pu.1 are normal, but levels of the differentiation markers l-plastin and myeloperoxidase at 24 and 28 hpf are diminished in mutants. Expression of the hemogenic endothelial and HSPC markers runx1 and gata2b at 24 hpf and formation of cmyb:gfp; kdrl:dsred double positive cells at 36 hpf were greatly diminished. In contrast, we observed normal expression of the pan-endothelial marker kdrl and aorta-specific markers notch1b and notch3 at 24 hpf. RNA-seq analysis of sf3b1 mutants revealed large-scale splicing defects and alterations, particularly affecting introns with suboptimal splice sites. Comparison of RNA-seq data from zebrafish sf3b1 mutants and MDS patients harboring SF3B1 mutations revealed a significant overlap in misspliced genes and in the corresponding affected pathways, including many pathways implicated in MDS pathology such as TGFb and immune signaling. Together, these results show conservation between zebrafish mutants and human cells with SF3B1 mutations. We next took a chemical genetic approach and
2034 - LYMPHOID AND MYELOID DIFFERENTIATION PROGRAMS ARE REGULATED BY THE COMMON GENOMIC LOCUS ENCODING FOR EVL AND MIR-342 Friederike Herbst1,2,3, Tonio Lang2,3, Elias Eckert2,3, Shayda Hemmati2,3, Peer W€unsche2,3, Oksana Zavidij2,3, Manfred Schmidt2,3, Claudia Ball2,3, and Hanno Glimm2,3 1 Department of Translational Oncology, Heidelberg, Germany; 2National Center for Tumor Diseases (NCT), Heidelberg, Germany; 3German Cancer Research Center (DKFZ), Heidelberg, Germany Extrinsic and intrinsic factors control HSC regulation to adapt the production of blood cells to the organism’s need. As retroviral integration sites (IS) are clustered nearby active genes and may lead to their upregulation, we systematically analyzed the IS repertoire of ten Wiskott-Aldrich syndrome patients enrolled in clinical gene therapy to identify novel regulators of hematopoiesis. In order to detect overrepresentation of IS in proximity to the transcription start site of gene loci, we developed a screening pipeline. By using this strategy, we identified a significant clustering of IS close to the locus encoding for Evl and its intronic miR-342. Therefore, we selected both genes encoded by one single locus to study their yet uncharacterized role in hematopoiesis. Lentiviral overexpression (OE) of Evl in murine primary LSK (lin-Sca1+ckit+) cells led to a 7.5-fold increase of preB-cell colonies as compared to mock-transduced LSK cells. By contrast, miR-342 OE led to a 2.3-fold larger number of myeloid colonies in vitro and a 3.3-fold increased number of myeloid progenitor-derived CFU-S in vivo. In line with this, global gene expression profiling revealed a profound deregulation of canonical pathways essential for the development of B-cells upon Evl, and myeloid development upon miR-342 OE. Self-renewal and multilineage reconstitution following transgene OE were assessed in serial BM transplantation experiments. Interestingly, EVL+ LSK cells led to a significantly increased donor-derived B-cell frequency within the spleens of primary recipient mice, and a 4.3-fold higher frequency of B-cells in peripheral blood after secondary transplantation. As our results point to an essential role of the Evl/miRNA-342 gene locus in lymphoid or myeloid lineage determination, we assessed its deregulation in human hematopoietic malignancies. Strikingly, the publicly available gene expression dataset of 2096 leukemia