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Probing IKAROS functions in B-ALL using novel mouse models
S66
Poster Presentations/ Experimental Hematology 42 (2014) S23–S68
P1172 - REGULATION OF HEMATOPOIETIC STEM CELL FATE BY SPECIAL AT-RICH SEQUENCE BINDING PROTEIN 1 Britta Will1, Tihomira Todorova1, Amit Verma1, Ulrich Steidl1, and Ari Melnick2 1 Albert Einstein College of Medicine, Bronx, New York, USA; 2Weill Cornell Medical College, New York, New York, USA Hematopoietic stem cells (HSCs) harbor extensive self-renewal capacity and the ability to commit to multilineage differentiation, thereby providing a life-long supply of mature peripheral blood cells. Cell fate decisions of self-renewal or differentiationcommitment are tightly regulated by the interplay of transcription factors instructing HSCs to function in a highly plastic manner during tissue maintenance, regeneration and repair. We have identified Special AT-rich sequence-binding protein 1 (Satb1) as a novel regulator integrating different HSC functions by promoting quiescence and repressing differentiation commitment through promoting symmetric self-renewal divisions of HSCs. In a Satb1-deficient murine model system we determined Satb1-dependent HSC function and molecular regulation utilizing congenic stem cell transplantation, cell cycle and division analyses, in vivo tracing of HSC commitment, asymmetric division assays, and clonogenic differentiation cultures. HSCs lacking Satb1 display defective self-renewal, were significantly less quiescent, and showed significantly increased and accelerated differentiation commitment caused by reduced symmetric self-renewal and increased symmetric differentiation divisions. Mechanistically, we found that Satb1 represses the expression of c-Myc and Numb, two factors known to be involved in HSC activation. Global DNA cytosine methylation analysis uncovered that Satb1 KO HSCs harbor premature differentiation-specific DNA methylation changes which closely resemble the pattern found in differentiation-committed multipotent progenitors. Our results also suggest that Satb1 can be retained at certain loci during mitosis, ensuring their appropriate transcriptional activity immediately upon cell division. Our findings uncover Satb1 as a novel transcriptional regulator in HSCs which promotes quiescence and represses differentiation commitment and thereby ensures the sustained self-renewal of HSCs.
P1174 - DELINEATING KEY MEIS1 CIS-REGULATORY ELEMENTS THROUGH CRISPR/CAS9 MEDIATED TARGETING Ping Xiang, Patricia Rosten, Wei Wei, Pamela Hoodless, and R. Keith Humphries BCCRC, Vancouver, British Columbia, Canada MEIS1 is a critical regulator of normal hematopoiesis and its overexpression is implicated in a wide range of leukemias. As demonstrated in our previous study (Xiang et al., Leukemia, 2014, 28(2):433-6), in addition to the MEIS1 promoter, multiple cisregulatory elements reside within the 140 kb MEIS1 genomic locus and are potential key components contributing to increased MEIS1 expression seen in leukemia. To further validate these elements, we are exploiting the CRISPR/Cas9 system as a powerful gene editing tool. As a first step we have targeted the Meis1 translational start site using CRISPR/Cas9 system to insert a GFP-P2A-HA tag 5’ of the MEIS1 ATG through homologous recombination. This design simplifies and enables the measurement of endogenous MEIS1 expression level by using GFP level as a reporter as well as allowing MEIS1 protein detection with an anti-HA antibody. Using P19 embryonic carcinoma cells as a model cell line, we obtained a 10% targeting efficiency. In wild type P19, Meis1 mRNA is up-regulated O 500 fold after 3 days of 1 micro M retinoic acid (RA) treatment. Similarly Clones bearing Meis1 targeted alleles showed dramatic upregulation of the GFP marker following RA induction with O95% of cells positive by FACS analysis and high level expression of HAtagged Meis1 detected by Western blot. Using these Meis1-GFP reporter lines we then used CRISPR/Cas9 editing to introduce deletions of the E1 enhancer region located 2kb upstream of the Meis1 promoter. Clones bearing sequence verified deletions of O50 bp of the E1 enhancer sequence had a significantly blunted response to retinoic induction of Meis1 expression as evidenced decreased induction of GFP expression and reduced Meis1 mRNA. These data provide important new functional evidence of the regulatory role of the E1 enhancer region for Meis1 expression and demonstrate the power of CRISPR/Cas9 mediated editing to introduce reporter genes to transcription factors and dissect their regulation. Preliminary data indicate that this system is also highly efficient for genome editing of human leukemic cell lines and experiments are now in progress to extend analysis of key MEIS1 regulatory domains to primary hematopoietic cells.
P1173 - PROBING IKAROS FUNCTIONS IN B-ALL USING NOVEL MOUSE MODELS Matthew Witkowski1, Yifang Hu1, Priyanka Sathe, Dr.1, Mark McKenzie1, Luisa Cimmino, Dr.2, Grace Liu1, Andrew Keniry, Dr.1, Gordon K. Smyth1, Nicholas Huntington, Dr.1, and Ross Dickins1 1 Walter and Eliza Hall Institute, Parkville, Victoria, Australia; 2NYU Department of Pathology, New York, New York, USA
P1175 - TRANSPLANTABLE B-1 PROGENITOR CELLS ARE PRESENT IN THE FETAL LIVER OF HEMATOPOIETIC STEM CELL DEFICIENT EMBRYOS Michihiro Kobayashi1, Williams Shelly1, Wooseok Seo2, Sasidhar Vemula1, Yan Liu1, Ichiro Taniuchi2, and Momoko Yoshimoto1 1 Indiana University School of Medicine, Indianapolis, Indiana, USA; 2RIKEN Center for Integrative Medical Sciences, IMS-RCAI, Yokohama, Japan
Loss-of-function mutations in the transcription factor IKAROS correlate with poor prognosis in human precursor B cell acute lymphoblastic leukemia (B-ALL) and are prevalent in the high-risk BCR-ABL+ and Ph-like B-ALL subtypes. Recent studies using mouse models of Ikaros-deficient B-ALL have uncovered a multitude of Ikaros-regulated genes, however the mechanisms underlying therapeutic resistance in IKAROS-mutant B-ALL remain unclear. We have generated a novel, transgenic mouse model that allows inducible, shRNA-mediated Ikaros knockdown or restoration in normal lymphocytes and leukemias in vivo. Retroviral expression of a BCR-ABL fusion oncogene co-operates with regulatable (tet-off) Ikaros knockdown to significantly accelerate B-ALL development in mice, recapitulating a common genetic interaction in high-risk pediatric B-ALL. Following leukemia transplant into multiple recipient mice, we used RNA-seq to assess the dynamic transcriptional responses to acute, Dox-induced Ikaros restoration in B-ALL in vivo. Combining this data with Ikaros ChIP-seq from normal B cell progenitors identified direct Ikarosregulated target genes in murine B-ALL. In complementary studies aimed at identifying IKAROS target genes in human B-ALL, we have virally transduced primary human CD34+ cord blood hematopoietic stem cells with vectors encoding BCRABL and the leukemia-associated dominant negative IKAROS isoform IK6. These cells display augmented proliferation in vitro and readily engraft a humanized mouse model. These mice are currently being monitored for disease progression and will be used to investigate IKAROS target genes based on transcriptional differences in BALL resulting from BCR-ABL and IK6 co-expression relative to BCR-ABL expression alone. We anticipate that this humanized system will provide new insights into the in vivo contribution of IKAROS deletion/mutation to human B-ALL genesis and therapeutic resistance.
The fetal liver is a major site containing progenitor cells to give rise to B-1 cells, especially CD5+ B-1a cells. We previously reported that hemogenic endothelial cells in the yolk sac (YS) and para-aortic splanchnopleure (P-Sp) (prior to hematopoietic stem cell [HSC] emergence) produced B-1 progenitors in vitro that engraft in immunodeficient neonates. Since the fetal liver is not a de novo site of hematopoietic cell emergence and must be seeded by various hematopoietic progenitor/stem cells derived from other hematopoietic tissues, B-1 progenitors in the fetal liver may be a mixture of YS/P-Sp derived progenies, and not only derivatives of HSCs that also seed the fetal liver at E11. Until now, it has been impossible to determine whether HSC independent B-1 progenitor cells exist in the fetal liver. We have utilized embryos genetically engineered to be devoid of HSC (CBFb-/-) but carrying a transgene expressing CBFb under the TEK promoter (CBFb-/-:TEK-CBFb/GFP mice). We have found the presence of transplantable B-1 and marginal zone B progenitors in the fetal liver despite the absence of HSCs. YS/P-Sp cells from CBFb-/-:TEK-CBFb/GFP embryos could produce B-1 progenitors in vitro. While TEK is expressed in hemogenic endothelial cells, it is not expressed in B-1 progenitors, and transplanted CBFb-/-:TEK-CBFb/GFP fetal liver cells failed to expand/ maintain B-1 cells in the host peritoneum, unless CBFb expression was rescued by retrovirus transduction. Our data provide solid evidence for the presence of the first wave of HSC independent B-1 progenitors in the fetal liver as postulated 20 years ago in the immune layered theory of B lymphopoiesis.
Poster Presentations/ Experimental Hematology 42 (2014) S23–S68
P1172 - REGULATION OF HEMATOPOIETIC STEM CELL FATE BY SPECIAL AT-RICH SEQUENCE BINDING PROTEIN 1 Britta Will1, Tihomira Todorova1, Amit Verma1, Ulrich Steidl1, and Ari Melnick2 1 Albert Einstein College of Medicine, Bronx, New York, USA; 2Weill Cornell Medical College, New York, New York, USA Hematopoietic stem cells (HSCs) harbor extensive self-renewal capacity and the ability to commit to multilineage differentiation, thereby providing a life-long supply of mature peripheral blood cells. Cell fate decisions of self-renewal or differentiationcommitment are tightly regulated by the interplay of transcription factors instructing HSCs to function in a highly plastic manner during tissue maintenance, regeneration and repair. We have identified Special AT-rich sequence-binding protein 1 (Satb1) as a novel regulator integrating different HSC functions by promoting quiescence and repressing differentiation commitment through promoting symmetric self-renewal divisions of HSCs. In a Satb1-deficient murine model system we determined Satb1-dependent HSC function and molecular regulation utilizing congenic stem cell transplantation, cell cycle and division analyses, in vivo tracing of HSC commitment, asymmetric division assays, and clonogenic differentiation cultures. HSCs lacking Satb1 display defective self-renewal, were significantly less quiescent, and showed significantly increased and accelerated differentiation commitment caused by reduced symmetric self-renewal and increased symmetric differentiation divisions. Mechanistically, we found that Satb1 represses the expression of c-Myc and Numb, two factors known to be involved in HSC activation. Global DNA cytosine methylation analysis uncovered that Satb1 KO HSCs harbor premature differentiation-specific DNA methylation changes which closely resemble the pattern found in differentiation-committed multipotent progenitors. Our results also suggest that Satb1 can be retained at certain loci during mitosis, ensuring their appropriate transcriptional activity immediately upon cell division. Our findings uncover Satb1 as a novel transcriptional regulator in HSCs which promotes quiescence and represses differentiation commitment and thereby ensures the sustained self-renewal of HSCs.
P1174 - DELINEATING KEY MEIS1 CIS-REGULATORY ELEMENTS THROUGH CRISPR/CAS9 MEDIATED TARGETING Ping Xiang, Patricia Rosten, Wei Wei, Pamela Hoodless, and R. Keith Humphries BCCRC, Vancouver, British Columbia, Canada MEIS1 is a critical regulator of normal hematopoiesis and its overexpression is implicated in a wide range of leukemias. As demonstrated in our previous study (Xiang et al., Leukemia, 2014, 28(2):433-6), in addition to the MEIS1 promoter, multiple cisregulatory elements reside within the 140 kb MEIS1 genomic locus and are potential key components contributing to increased MEIS1 expression seen in leukemia. To further validate these elements, we are exploiting the CRISPR/Cas9 system as a powerful gene editing tool. As a first step we have targeted the Meis1 translational start site using CRISPR/Cas9 system to insert a GFP-P2A-HA tag 5’ of the MEIS1 ATG through homologous recombination. This design simplifies and enables the measurement of endogenous MEIS1 expression level by using GFP level as a reporter as well as allowing MEIS1 protein detection with an anti-HA antibody. Using P19 embryonic carcinoma cells as a model cell line, we obtained a 10% targeting efficiency. In wild type P19, Meis1 mRNA is up-regulated O 500 fold after 3 days of 1 micro M retinoic acid (RA) treatment. Similarly Clones bearing Meis1 targeted alleles showed dramatic upregulation of the GFP marker following RA induction with O95% of cells positive by FACS analysis and high level expression of HAtagged Meis1 detected by Western blot. Using these Meis1-GFP reporter lines we then used CRISPR/Cas9 editing to introduce deletions of the E1 enhancer region located 2kb upstream of the Meis1 promoter. Clones bearing sequence verified deletions of O50 bp of the E1 enhancer sequence had a significantly blunted response to retinoic induction of Meis1 expression as evidenced decreased induction of GFP expression and reduced Meis1 mRNA. These data provide important new functional evidence of the regulatory role of the E1 enhancer region for Meis1 expression and demonstrate the power of CRISPR/Cas9 mediated editing to introduce reporter genes to transcription factors and dissect their regulation. Preliminary data indicate that this system is also highly efficient for genome editing of human leukemic cell lines and experiments are now in progress to extend analysis of key MEIS1 regulatory domains to primary hematopoietic cells.
P1173 - PROBING IKAROS FUNCTIONS IN B-ALL USING NOVEL MOUSE MODELS Matthew Witkowski1, Yifang Hu1, Priyanka Sathe, Dr.1, Mark McKenzie1, Luisa Cimmino, Dr.2, Grace Liu1, Andrew Keniry, Dr.1, Gordon K. Smyth1, Nicholas Huntington, Dr.1, and Ross Dickins1 1 Walter and Eliza Hall Institute, Parkville, Victoria, Australia; 2NYU Department of Pathology, New York, New York, USA
P1175 - TRANSPLANTABLE B-1 PROGENITOR CELLS ARE PRESENT IN THE FETAL LIVER OF HEMATOPOIETIC STEM CELL DEFICIENT EMBRYOS Michihiro Kobayashi1, Williams Shelly1, Wooseok Seo2, Sasidhar Vemula1, Yan Liu1, Ichiro Taniuchi2, and Momoko Yoshimoto1 1 Indiana University School of Medicine, Indianapolis, Indiana, USA; 2RIKEN Center for Integrative Medical Sciences, IMS-RCAI, Yokohama, Japan
Loss-of-function mutations in the transcription factor IKAROS correlate with poor prognosis in human precursor B cell acute lymphoblastic leukemia (B-ALL) and are prevalent in the high-risk BCR-ABL+ and Ph-like B-ALL subtypes. Recent studies using mouse models of Ikaros-deficient B-ALL have uncovered a multitude of Ikaros-regulated genes, however the mechanisms underlying therapeutic resistance in IKAROS-mutant B-ALL remain unclear. We have generated a novel, transgenic mouse model that allows inducible, shRNA-mediated Ikaros knockdown or restoration in normal lymphocytes and leukemias in vivo. Retroviral expression of a BCR-ABL fusion oncogene co-operates with regulatable (tet-off) Ikaros knockdown to significantly accelerate B-ALL development in mice, recapitulating a common genetic interaction in high-risk pediatric B-ALL. Following leukemia transplant into multiple recipient mice, we used RNA-seq to assess the dynamic transcriptional responses to acute, Dox-induced Ikaros restoration in B-ALL in vivo. Combining this data with Ikaros ChIP-seq from normal B cell progenitors identified direct Ikarosregulated target genes in murine B-ALL. In complementary studies aimed at identifying IKAROS target genes in human B-ALL, we have virally transduced primary human CD34+ cord blood hematopoietic stem cells with vectors encoding BCRABL and the leukemia-associated dominant negative IKAROS isoform IK6. These cells display augmented proliferation in vitro and readily engraft a humanized mouse model. These mice are currently being monitored for disease progression and will be used to investigate IKAROS target genes based on transcriptional differences in BALL resulting from BCR-ABL and IK6 co-expression relative to BCR-ABL expression alone. We anticipate that this humanized system will provide new insights into the in vivo contribution of IKAROS deletion/mutation to human B-ALL genesis and therapeutic resistance.
The fetal liver is a major site containing progenitor cells to give rise to B-1 cells, especially CD5+ B-1a cells. We previously reported that hemogenic endothelial cells in the yolk sac (YS) and para-aortic splanchnopleure (P-Sp) (prior to hematopoietic stem cell [HSC] emergence) produced B-1 progenitors in vitro that engraft in immunodeficient neonates. Since the fetal liver is not a de novo site of hematopoietic cell emergence and must be seeded by various hematopoietic progenitor/stem cells derived from other hematopoietic tissues, B-1 progenitors in the fetal liver may be a mixture of YS/P-Sp derived progenies, and not only derivatives of HSCs that also seed the fetal liver at E11. Until now, it has been impossible to determine whether HSC independent B-1 progenitor cells exist in the fetal liver. We have utilized embryos genetically engineered to be devoid of HSC (CBFb-/-) but carrying a transgene expressing CBFb under the TEK promoter (CBFb-/-:TEK-CBFb/GFP mice). We have found the presence of transplantable B-1 and marginal zone B progenitors in the fetal liver despite the absence of HSCs. YS/P-Sp cells from CBFb-/-:TEK-CBFb/GFP embryos could produce B-1 progenitors in vitro. While TEK is expressed in hemogenic endothelial cells, it is not expressed in B-1 progenitors, and transplanted CBFb-/-:TEK-CBFb/GFP fetal liver cells failed to expand/ maintain B-1 cells in the host peritoneum, unless CBFb expression was rescued by retrovirus transduction. Our data provide solid evidence for the presence of the first wave of HSC independent B-1 progenitors in the fetal liver as postulated 20 years ago in the immune layered theory of B lymphopoiesis.