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P75 interaction as a therapeutic target for mixed lineage leukemia
Poster Presentations/ Experimental Hematology 44 (2016) S56–S110
3043 - VALIDATION OF THE MLL-LEDGF/P75 INTERACTION AS A THERAPEUTIC TARGET FOR MIXED LINEAGE LEUKEMIA Sara El Ashkar1, Jan De Rijck2, Katerina Cermakova3, Petr Tesina3, Jonas Demeulemeester4, Helene Mereau5, J€urg Schwaller6, acova3, and Zeger Debyser7 V aclav Veverka3, Pavlına Rez 1 Laboratory for Molecular Virology and Drug Discovery, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven; 2Laboratory for Molecular Virology and Gene Therapy, Flanders, Belgium; 3Institute of Organic Chemistry and Biochemistry AS CR v.v.i., Prague, Czech Republic; 4The Francis Crick Institute, London, USA; 5University Hospital and Children’s Hospital Basel (UKBB) ZLF, Basel, Basel, Switzerland; 6 University Hospital Basel, Basel, Switzerland; 7Laboratory for Molecular Virology and Drug Discovery, Washington DC, USA Mixed-lineage leukemia (MLL)-rearrangements induce a genetically distinct and aggressive subset of human acute leukemia. N-terminal MLL fusions form a complex with the lens epithelium-derived growth factor (LEDGF/p75) and MENIN. This complex contributes to the association of MLL-fusion multiprotein complexes to chromatin. Knockdown and transdominant experiments have shown that both LEDGF/ p75 and MENIN are required for efficient MLL fusion-mediated transformation and for the expression of downstream MLL regulated genes like HOXA9 and MEIS1. LEDGF/p75 interacts with MLL-MENIN via its integrase binding domain (IBD). Through X-ray crystallography, NMR spectroscopy and mutational studies we and others resolved the MLL-MENIN-LEDGF/p75 complex. Colony forming assays with primary murine MLL-AF9 immortalized cells expressing MLL interactiondefective LEDGF/p75 mutants confirmed that the complex is crucial for leukemic transformation. Next to MLL, LEDGF/p75 is known to interact with several other proteins including JPO2, PogZ, and HIV-1 integrase. We analysed the IBD interaction with JPO2 and PogZ by NMR. Mutational analysis and competition experiments showed that the interaction of MLL, Jpo2 and PogZ with the IBD is mutual exclusive and maintained by an intrinsically unstructured consensus motif. The newly defined interface overlaps with the binding site of HIV-1 integrase. Overexpression of a LEDGF/p75 binding peptide, known to inhibit the LEDGF/p75-HIV-1 integrase interaction, also impaired growth of primary murine MLL-AF9 expressing leukemic blasts providing a direct rationale for the design of small molecules targeting the LEDGF/p75-MLL interaction.
3044 - SCRIBBLE IMPACTS ON THYMOCYTE NUMBERS IN VIVO Sarah Ellis1,2, Novita Novita1, Alysha Dew1, Sarah Russell3, Carl Walkley4, and Louise Purton4 1 Peter MacCallum Cancer Centre, East Melbourne, Australia, East Melbourne, Australia; 2Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Australia, East Melbourne, Australia; 3 Swinburne University of Technology, Hawthorn, Australia, Melbourne, Australia; 4St Vincent’s Institute of Medical Research, East Melbourne, Australia, Melbourne, Australia As a scaffolding protein, Scribble regulates multiple intracellular signalling pathways that control cell proliferation, migration, and apoptosis. Scribble is pivotal to the development of epithelial tissues and its deregulation has been linked to multiple epithelial cancers. Scribble’s role in haematopoiesis and haematopoietic malignancies is an exciting area of burgeoning interest. Using multiple approaches, we show that Scribble is expressed in haematopoietic stem and progenitor cells (HSPC) and their differentiated progeny, implying a putative role for Scribble in blood production. We investigated the effect of Scribble loss in haematopoiesis through two inducible knockout mouse models and an extensive phenotyping labelling regime in conjunction with multi-color flow cytometry. Surprisingly, we observed no effect of Scribble loss on either the proportion or numbers of HSPC, or cells from the B, myeloid, or erythroid lineages suggesting Scribble is dispensable in these lineages. However, mice lacking Scribble had a significantly smaller thymus compared to controls and a significant decrease in the number of DN1, DN2, DN3, single positive and double positive thymocytes. Our data suggests Scribble may regulate thymocyte development and/or the migration of the early progenitors from the bone marrow to the thymus. We are currently investigating these hypotheses and the underlying mechanism/s involved. Our research will improve the understanding of T cell development and will form the basis of future studies aimed at investigating how Scribble impacts T cell leukaemia.
S69
3045 - RADIATION FIBROSIS RESISTANT SMAD3 -/- MICE DEMONSTRATE SUPERIOR DONOR BONE MARROW STEM CELL TRANSPLANTATION CAPACITY BY COMPETITIVE REPOPULATION ASSAY Michael Epperly1, Hongmei Shen2, Xichen Zhang2, Darcy Franicola2, Donna Shields2, and Joel Greenberger2 1 University of Pittsburgh, Department of Radiation Oncology, Pittsburgh, USA; 2University of Pittsburgh, Pittsburgh, USA Smad3 -/- mice are resistant to radiation fibrosis (Flanders, Am J. Path, 163:2247-2257, 2003; Epperly, In Vivo 20:573-582, 2006) attributable to inhibition of Smad3 pathway -mediated TGF-B signal transduction. We tested the hypothesis that abrogation of TGF-B signaling in Smad3 -/-(129/Sv) marrow stem cells increased engraftment capacity into the TBF-B induced marrow microenvironment of irradiated recipient mice. The number of bone marrow stem cells in situ was determined by flow cytometry. TGF-B sensitivity was determined by measuring CFU-GEMM inhibition in vitro by TGF-B treatment of freshly explanted marrow from Smad3 -/-(129/Sv) compared to C57BL/6NTac mice. Competitive repopulation assay of Smad3 -/- and C57BL/6 bone marrow stem cells were performed by mixing bone marrow from male and female mice, injecting into TBI irradiated mice and PCR performed to detect marrow male Y chromosome to demonstrate increased stem cell repopulation capacity. Smad3 -/mice showed 95.0 stem cells per 107 bone marrow cells compared to 35.7 stem cells per 107 bone marrow cells for C57BL/6NTac mice (p 5 0.0342). CFU-GEMM formed by Smad3 -/- bone marrow was not inhibited by TGF-B or altered by the TGF-B receptor antagonist SD208 while C57BL/6NTac bone marrow CFU-GEMM was inhibited by TGF-B ( 20 and 50 ng/ml) (p 5 0.0084 and 0.0069, respectively). Competitive repopulation assay demonstrated that Smad3 -/- bone marrow had increased repopulation capacity by Y chromosome detected in all mice injected with as few as 0.1 X 106 male Smad3-/- bone marrow cells mixed with 0.9 X 106 female C57BL/6NTac bone marrow cells. In contrast, C57BL/6 male Y chromosome was not detected in mice injected with significantly higher ratios of control marrow (0.25 X 106 male C57BL/6 bone marrow cells mixed with 0.75 X 106 female Smad3 -/- bone marrow cells ) (p 5 0.0460). Smad3 -/- mice have increased baseline bone marrow stem numbers, resistance to inhibition of committed multilineage progenitor cell proliferation by TGFB in vitro, and superior in vivo engraftment capacity by competitive repopulation compared to control C57BL/6NTac mice. The data support the hypothesis that inhibition TGFB signaling may be of therapeutic benefit to marrow transplant recipients. Supported by NIAID/NIH U19-A1068021.
3046 - ELUCIDATING THE ROLE OF RNA ELONGATION DURING HEMATOPOIETIC STEM CELL SPECIFICATION Sophia Espanola1,2 and Yoonsung Lee3 1 UCSD, La Jolla; 2Biomedical Sciences, La Jolla; 3Ulsan National Institute of Science and Technology, Ulsan, South Korea Hematopoietic stem cells (HSCs) must tightly regulate the processes of quiescence, self-renewal, and differentiation to ensure proper hematopoietic homeostasis throughout the lifetime of an organism. While a general understanding of HSC emergence and migration is known, the network of signaling mechanisms that regulate these events are not well understood. Understanding the network of signaling processes occurring in the hematopoietic niche would greatly contribute to the research being done to culture and differentiate HSCs in vitro, an effort needed to advance regenerative medicine and transplantation therapeutics. To explore novel signaling pathways important in HSC differentiation, a forward genetic screening approach was used to identify zebrafish mutants defective in HSC formation. RNA-sequencing based linkage mapping was performed to identify an RNA elongation factor as a gene necessary for HSC specification. Mutants and morpholino phenocopied embryos show significantly reduced expression of the notch signaling pathway. Further experiments demonstrate the how this RNA elongation factor is required for controlling the Notch signaling pathway to stimulate HSC formation in a cell autonomous manner.
3043 - VALIDATION OF THE MLL-LEDGF/P75 INTERACTION AS A THERAPEUTIC TARGET FOR MIXED LINEAGE LEUKEMIA Sara El Ashkar1, Jan De Rijck2, Katerina Cermakova3, Petr Tesina3, Jonas Demeulemeester4, Helene Mereau5, J€urg Schwaller6, acova3, and Zeger Debyser7 V aclav Veverka3, Pavlına Rez 1 Laboratory for Molecular Virology and Drug Discovery, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven; 2Laboratory for Molecular Virology and Gene Therapy, Flanders, Belgium; 3Institute of Organic Chemistry and Biochemistry AS CR v.v.i., Prague, Czech Republic; 4The Francis Crick Institute, London, USA; 5University Hospital and Children’s Hospital Basel (UKBB) ZLF, Basel, Basel, Switzerland; 6 University Hospital Basel, Basel, Switzerland; 7Laboratory for Molecular Virology and Drug Discovery, Washington DC, USA Mixed-lineage leukemia (MLL)-rearrangements induce a genetically distinct and aggressive subset of human acute leukemia. N-terminal MLL fusions form a complex with the lens epithelium-derived growth factor (LEDGF/p75) and MENIN. This complex contributes to the association of MLL-fusion multiprotein complexes to chromatin. Knockdown and transdominant experiments have shown that both LEDGF/ p75 and MENIN are required for efficient MLL fusion-mediated transformation and for the expression of downstream MLL regulated genes like HOXA9 and MEIS1. LEDGF/p75 interacts with MLL-MENIN via its integrase binding domain (IBD). Through X-ray crystallography, NMR spectroscopy and mutational studies we and others resolved the MLL-MENIN-LEDGF/p75 complex. Colony forming assays with primary murine MLL-AF9 immortalized cells expressing MLL interactiondefective LEDGF/p75 mutants confirmed that the complex is crucial for leukemic transformation. Next to MLL, LEDGF/p75 is known to interact with several other proteins including JPO2, PogZ, and HIV-1 integrase. We analysed the IBD interaction with JPO2 and PogZ by NMR. Mutational analysis and competition experiments showed that the interaction of MLL, Jpo2 and PogZ with the IBD is mutual exclusive and maintained by an intrinsically unstructured consensus motif. The newly defined interface overlaps with the binding site of HIV-1 integrase. Overexpression of a LEDGF/p75 binding peptide, known to inhibit the LEDGF/p75-HIV-1 integrase interaction, also impaired growth of primary murine MLL-AF9 expressing leukemic blasts providing a direct rationale for the design of small molecules targeting the LEDGF/p75-MLL interaction.
3044 - SCRIBBLE IMPACTS ON THYMOCYTE NUMBERS IN VIVO Sarah Ellis1,2, Novita Novita1, Alysha Dew1, Sarah Russell3, Carl Walkley4, and Louise Purton4 1 Peter MacCallum Cancer Centre, East Melbourne, Australia, East Melbourne, Australia; 2Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Australia, East Melbourne, Australia; 3 Swinburne University of Technology, Hawthorn, Australia, Melbourne, Australia; 4St Vincent’s Institute of Medical Research, East Melbourne, Australia, Melbourne, Australia As a scaffolding protein, Scribble regulates multiple intracellular signalling pathways that control cell proliferation, migration, and apoptosis. Scribble is pivotal to the development of epithelial tissues and its deregulation has been linked to multiple epithelial cancers. Scribble’s role in haematopoiesis and haematopoietic malignancies is an exciting area of burgeoning interest. Using multiple approaches, we show that Scribble is expressed in haematopoietic stem and progenitor cells (HSPC) and their differentiated progeny, implying a putative role for Scribble in blood production. We investigated the effect of Scribble loss in haematopoiesis through two inducible knockout mouse models and an extensive phenotyping labelling regime in conjunction with multi-color flow cytometry. Surprisingly, we observed no effect of Scribble loss on either the proportion or numbers of HSPC, or cells from the B, myeloid, or erythroid lineages suggesting Scribble is dispensable in these lineages. However, mice lacking Scribble had a significantly smaller thymus compared to controls and a significant decrease in the number of DN1, DN2, DN3, single positive and double positive thymocytes. Our data suggests Scribble may regulate thymocyte development and/or the migration of the early progenitors from the bone marrow to the thymus. We are currently investigating these hypotheses and the underlying mechanism/s involved. Our research will improve the understanding of T cell development and will form the basis of future studies aimed at investigating how Scribble impacts T cell leukaemia.
S69
3045 - RADIATION FIBROSIS RESISTANT SMAD3 -/- MICE DEMONSTRATE SUPERIOR DONOR BONE MARROW STEM CELL TRANSPLANTATION CAPACITY BY COMPETITIVE REPOPULATION ASSAY Michael Epperly1, Hongmei Shen2, Xichen Zhang2, Darcy Franicola2, Donna Shields2, and Joel Greenberger2 1 University of Pittsburgh, Department of Radiation Oncology, Pittsburgh, USA; 2University of Pittsburgh, Pittsburgh, USA Smad3 -/- mice are resistant to radiation fibrosis (Flanders, Am J. Path, 163:2247-2257, 2003; Epperly, In Vivo 20:573-582, 2006) attributable to inhibition of Smad3 pathway -mediated TGF-B signal transduction. We tested the hypothesis that abrogation of TGF-B signaling in Smad3 -/-(129/Sv) marrow stem cells increased engraftment capacity into the TBF-B induced marrow microenvironment of irradiated recipient mice. The number of bone marrow stem cells in situ was determined by flow cytometry. TGF-B sensitivity was determined by measuring CFU-GEMM inhibition in vitro by TGF-B treatment of freshly explanted marrow from Smad3 -/-(129/Sv) compared to C57BL/6NTac mice. Competitive repopulation assay of Smad3 -/- and C57BL/6 bone marrow stem cells were performed by mixing bone marrow from male and female mice, injecting into TBI irradiated mice and PCR performed to detect marrow male Y chromosome to demonstrate increased stem cell repopulation capacity. Smad3 -/mice showed 95.0 stem cells per 107 bone marrow cells compared to 35.7 stem cells per 107 bone marrow cells for C57BL/6NTac mice (p 5 0.0342). CFU-GEMM formed by Smad3 -/- bone marrow was not inhibited by TGF-B or altered by the TGF-B receptor antagonist SD208 while C57BL/6NTac bone marrow CFU-GEMM was inhibited by TGF-B ( 20 and 50 ng/ml) (p 5 0.0084 and 0.0069, respectively). Competitive repopulation assay demonstrated that Smad3 -/- bone marrow had increased repopulation capacity by Y chromosome detected in all mice injected with as few as 0.1 X 106 male Smad3-/- bone marrow cells mixed with 0.9 X 106 female C57BL/6NTac bone marrow cells. In contrast, C57BL/6 male Y chromosome was not detected in mice injected with significantly higher ratios of control marrow (0.25 X 106 male C57BL/6 bone marrow cells mixed with 0.75 X 106 female Smad3 -/- bone marrow cells ) (p 5 0.0460). Smad3 -/- mice have increased baseline bone marrow stem numbers, resistance to inhibition of committed multilineage progenitor cell proliferation by TGFB in vitro, and superior in vivo engraftment capacity by competitive repopulation compared to control C57BL/6NTac mice. The data support the hypothesis that inhibition TGFB signaling may be of therapeutic benefit to marrow transplant recipients. Supported by NIAID/NIH U19-A1068021.
3046 - ELUCIDATING THE ROLE OF RNA ELONGATION DURING HEMATOPOIETIC STEM CELL SPECIFICATION Sophia Espanola1,2 and Yoonsung Lee3 1 UCSD, La Jolla; 2Biomedical Sciences, La Jolla; 3Ulsan National Institute of Science and Technology, Ulsan, South Korea Hematopoietic stem cells (HSCs) must tightly regulate the processes of quiescence, self-renewal, and differentiation to ensure proper hematopoietic homeostasis throughout the lifetime of an organism. While a general understanding of HSC emergence and migration is known, the network of signaling mechanisms that regulate these events are not well understood. Understanding the network of signaling processes occurring in the hematopoietic niche would greatly contribute to the research being done to culture and differentiate HSCs in vitro, an effort needed to advance regenerative medicine and transplantation therapeutics. To explore novel signaling pathways important in HSC differentiation, a forward genetic screening approach was used to identify zebrafish mutants defective in HSC formation. RNA-sequencing based linkage mapping was performed to identify an RNA elongation factor as a gene necessary for HSC specification. Mutants and morpholino phenocopied embryos show significantly reduced expression of the notch signaling pathway. Further experiments demonstrate the how this RNA elongation factor is required for controlling the Notch signaling pathway to stimulate HSC formation in a cell autonomous manner.