Transduced Bone Marrow Research Articles

EZH2 and BCL6 Cooperate To Create The Germinal Center B-Cell Phenotype and Induce Lymphomas Through Formation and Repression Of Bivalent Chromatin Domains

The EZH2 histone methyltransferase is the enzymatic core of the Polycomb repressor 2 (PRC2 complex), is highly upregulated in germinal center (GC) B cells and is targeted by gain-of-function somatic mutations that enhance its ability to trimethylate histone 3 lysine 27 in diffuse large B cell lymphomas (DLBCLs) and follicular lymphomas (FLs). We explored the significance and mechanism of action of EZH2 in normal GC development and lymphomagenesis. We observed that EZH2-conditional knockout mice and mice exposed to the novel EZH2-specific inhibitor GSK503 both completely failed to form GCs or high affinity antibodies. Using ChIP-seq, sequential QChIP, RNA-seq and functional assays we demonstrated that EZH2 mediates the GC phenotype through de novo formation of bivalently marked chromatin domains (characterized by overlapping H3K27me3 repressive mark with the H3K4me3 activation mark) at the promoters of target genes involved in cell cycle regulation (e.g. CDKN1A) and in GC exit and terminal differentiation program (e.g. IRF4 and PRDM1). Notably, mutant EZH2 caused hyper-repression of these bivalent genes through increased H3K27me3, which we showed is causal to the mutant EZH2 phenotype. Mice engineered to conditionally express lymphoma-associated EZH2Y641F exhibited aberrant suppression of bivalent gene expression leading to increased proliferation, blockade of terminal differentiation, and massive GC hyperplasia. Transcriptional profiles of human DLBCL patients revealed that those with mutant EZH2 display a unique signature consisting of silencing of GC bivalent genes, suggesting that mutant EZH2 contributes to human lymphomagenesis through paralysis of bivalent chromatin domains.This scenario is reminiscent of the role of the transcriptional repressor BCL6, which is also required for GC formation. BCL6 also represses CDKN1A, IRF4 and PRDM1 and is required to maintain the proliferation and survival of DLBCL cells. Notably BCL6 represses its targets by associating with BCoR, which forms a variant of Polycomb repressor 1 (PRC1) complex. We hypothesized that EZH2 and BCL6 cooperate to mediate the GC B-cell phenotype and when aberrantly active may cooperate to form GC-derived B-cell lymphomas. Using ChIP-seq studies we found that the target promoters of BCL6-BCoR complex (but not promoters with BCL6 complexes lacking BCoR) significantly overlap with EZH2 bivalent promoter genes in primary human GC B cells and lymphoma cells (Hypergeometric test, p=1.5x10-26). Treatment of DLBCL cells with EZH2 or BCL6 inhibitors or siRNA partially derepressed these genes indicating that both factors cooperate and are required to mediate full repression of these crucial loci. To determine whether EZH2 and BCL6 cooperate to generate GC-derived lymphomas, we transduced bone marrow of IµHABCL6 mice (which mimic BCL6 translocations in DLBCL) with retrovirus encoding mutant EZH2Y641F or GFP alone, and transplanted them into lethally irradiated recipients. Only EZH2Y641F/BCL6 mice showed an accelerated lethal phenotype (log-rank test, p=0.007), with reduced median survival (EZH2Y641F: 309 days, empty vector: 453 days). Serial bone marrow transplantation resulted in even further increased lethality (log-rank test, p=0.004; median survival EZH2Y641F: 127 days, empty vector: 169 days). Given the oncogenic cooperation between BCL6 and EZH2, we hypothesized that rational combinatorial therapy with BCL6 and EZH2 inhibitors might synergistically kill DLBCLs. Indeed, by combining the EZH2 inhibitor GSK343 and the RI-BPI, a drug that inhibits BCL6 by abrogating its interaction with BCoR, we observed a potent synergistic effect on the inhibition of DLBCL cell lines proliferation. The combination of these two inhibitors in mice bearing DLBCL xenografts accordingly suppressed tumor growth more effectively than either agent alone. Finally, the combination also yielded further killing of primary human DLBCL cells growth in a co-culture system that we developed for testing primary human specimens.In summary we identified the first epigenetic mechanism of lymphomagenesis involving aberrant repression of GC-specific bivalent domains by EZH2 (PRC2) in cooperation with BCL6-BCoR (PRC1) complexes, as well as a rational epigenetic-based and molecular targeted therapeutic approach with the potential to eradicate lymphomas without harming normal tissues. Disclosures:Creasy:GlaxoSmithKline: Employment.

Effect Of The JAK1/2 Inhibitor GS-0387 (momelotinib) On The Anemia Associated With a Preclinical Murine Model Of Human Primary Myelofibrosis

IntroductionGS-0387 (momelotinib, formerly CYT387) is a type I JAK1/2 inhibitor. Phase 1/2 trial results in primary myelofibrosis (PMF) or post-polycythemia vera/essential thrombocythemia MF patients showed that GS-0387 was well tolerated and reduced splenomegaly and general symptoms. More interestingly, GS-0387 relieved anemia in 59% of patients, with 70% of patients requiring red blood cell transfusion becoming transfusion independent for a minimum of 12 weeks.1 Therefore, the purpose of this study was to verify if this benefit on anemia response could be transposed to a mouse model of human primary myelofibrosis (PMF). MethodsFor this purpose we used the TPOHIGH retroviral (RV) model that mimics the development of human PMF including its progressing anemia. In this model, hyperactivation of MPL, as observed in human PMF through gain-of-function mutations, is caused by high levels of circulating thrombopoietin (TPO) through an adoptive transfer of TPO retrovirally transduced bone marrow (BM) cells in C57/Bl6 recipient mice. After transplantation, mice developed a 2 stepped disease with a myeloproliferative phase characterized by high thrombocythemia, leukocytosis and anemia (hemoglobin ≈ 12 g/dL, hematocrit ≈35 %; normal levels: 16 g/dL and 45 %, respectively) and a marrow insufficiency phase characterized by thrombocytopenia, leukopenia and severe anemia (hemoglobin ≈ 8 g/dL, hematocrit level ≈22%) caused by high grade fibrosis and osteosclerosis. GS-0387 was administered as a single daily oral dose (SID) during the myeloproliferative phase in two studies with 47 and 68 days of treatment and during the marrow insufficiency phase with 101 days of treatment. ResultsGS-0387 was well tolerated at ca 80mpk with an MTD of approximately 100mpk. GS-0387 was first tested during the myeloproliferative phase, 5 weeks post transplantation of the TPO transduced BM cells. A significant improvement in anemia was observed. Starting 20 weeks after treatment, hemoglobin concentration in GS-0387-treated mice remained higher (8.5±0.4 g/dL, n=7) than in vehicle-treated mice (6.8 ±0.3 g/dL, n=7) for at least 35 days of treatment (p<0.01). Treatment with GS-0387 commencing during the marrow insufficiency phase (10 weeks post transplantation), did not significantly improve anemia, possibly due to the dramatic picture of the disease in terminal stage.Improvement of anemia during the myeloproliferative phase was associated with an increase over 35 days of treatment in:- Hematocrit levels (23±1% vs 27±1%, vehicle vs GS-0387)- Red blood cell numbers (5.5±0.2 vs 5.7±0.3 x109/mL, vehicle vs GS-0387)- Mean corpuscular volume (MCV), from microcytosis to normal levels (44.2±0.2 to 47.75 fL, vehicle vs GS-0387)An increase in erythroblast and early erythroid progenitor cell numbers was also observed in the spleen of GS-0387-treated mice compared to vehicle-treated mice. None of these increases, except for MCV, were observed during the 90 day treatment in the marrow insufficiency phase, in which no anemia response was observed.Besides improving anemia, GS-0387 prevented the development of leukocytosis and thrombocytosis and reduced splenomegaly associated with the disease without deleterious effects on hematopoiesis at efficacious doses. ConclusionGS-0387 was tested in a murine model of human PMF. It reduced the anemia associated with the development of myelofibrosis, as reported during clinical trials of patients suffering from MF. Furthermore, GS-0387, as expected from a JAK1/2 inhibitor reduced splenomegaly, thrombocytosis and leukocytosis associated with the disease. Improvement of anemia was associated with an increase in RBC, erythroid precursor and progenitor cells suggesting that GS-0387 stimulates the early stages of erythropoiesis. Further experiments are in progress to investigate whether changes in levels of cytokines known to positively or negatively effect erythropoiesis are seen. Regulation of iron metabolism, which could account for the improvement of anemia induced by GS-0387 is also being investigated.1. Pardanani A, Laborde RR, Lasho TL, et al. Safety and efficacy of CYT387, a JAK1 and JAK2 inhibitor, in myelofibrosis. Leukemia. 2013;27(6):1322-1327. Disclosures:Villeval:YMBioSciences: Research Funding. Off Label Use: JAK1/2 Inhibitor GS-0387 (momelotinib) / Human Primary Myelofibrosis. Burns:YMBioSciences: Employment; Gilead: Consultancy. Smith:YMBioSciences: Employment; Gilead: Consultancy.

Human NUP98/Iqcg Fusion Gene Induced An Acute Myelomonocytic Leukemia In Mice

NUP98 gene encoding a nucleoporin located at 11p15 has been reported to be fused with about 30 different partner genes by chromosomal translocations in hematological malignances. NUP98/IQCG was cloned in a patient of Myeloid/ T-lymphoid bi-phenotypic leukemia with 47, XX, t(3;11)(q29q13;p15)der(3)(q29), +21 karyotype. However, NUP98/IQCG's ability for leukemogenesis has not been identified yet.In this study, we established a retrovirus-mediated murine bone marrow transduction and transplantation (BMT) model of NUP98/IQCG to investigate its oncogenicity. In our model, half of the NUP98/IQCG mice developed a penetrable and transplantable acute myelomonocytic leukemia ,which was similar to the phenotype of patient with t(3;11). It suggested that NUP98/IQCG could induce the disease development.To investigate how the fusion gene promoted leukemogenesis, we transduced NUP98/IQCG into primary bone marrow cells, and found that NUP98/IQCG-expressing cells retained the ability to generate colonies in serial replating, while seldom control cells did, which indicated the increasing self-renewal ability caused by NUP98/IQCG. Meanwhile, when induced by Macrophage-Colony Stimulating Factor, NUP98/IQCG -expression bone marrow cells showed enhanced proliferation in vitro. Further molecular mechanism studies revealed that NUP98/IQCG could be involved in both NFκB and CREB pathways during leukemia development.In summary, we showed that NUP98/IQCG promoted leukemogenesis in BMT mouse model through increasing the bone marrow cells' proliferation and self-renewal capacity, which explained the fusion gene's oncogenicity in patient with t(3;11). Our mouse model will be a powerful tool both to investigate the leukemogenic mechanism of NUP98-related fusion gene, and to find the drugs for treating the disease. Disclosures:No relevant conflicts of interest to declare.

Generation of T cell receptor–retrogenic mice: improved retroviral-mediated stem cell gene transfer

The use of retrogenic mice offers a rapid and flexible approach to T cell receptor (TCR)-transgenic mice. By transducing bone marrow progenitor cells with a retrovirus that encodes a given TCR-α/β subunit, TCR-retrogenic mice can be generated in as few as 4-6 weeks, whereas conventional TCR transgenics can take 6 months or longer. In this updated protocol, we have increased the efficiency of the bone marrow transduction and bone marrow reconstitution compared with our previously published protocol. The main departure from the previous protocol is the implementation of spin transduction with the viral supernatant instead of coculture with the viral producer cell line. The changes in this protocol improve bone marrow viability, increase consistency of the bone marrow transduction and bone marrow engraftment, and they reduce the ratio of bone marrow donor mice to bone marrow recipients.

A PCR-Based Method to Genotype Mice Knocked Out for All Four CD3 Subunits, the Standard Recipient Strain for Retrogenic TCR/CD3 Bone Marrow Reconstitution Technology

The novel T-cell receptor (TCR)/CD3-retrogenic-reconstitution system represents a very useful strategy for studying TCR/CD3 signaling. Two retroviral vectors containing genes for all six subunits of the TCR/CD3 complex are used to transduce bone marrow precursors and reconstitute lethally irradiated recipient mice. Mice used in this system as bone marrow donors lack all four CD3 subunits (CD3γδɛζ−/−). These mice are generated by crossing the strains CD3ζ−/− and CD3γδɛ−/−, the latter resulting from a knockout construct targeted to CD3ɛ that additionally silences the linked genes, CD3γ and CD3δ. Lacking mature T-cell function, CD3γδɛζ−/− mice are immunocompromised animals often produced by heterozygous breeding strategies on the C57BL/6 background. As a more rapid and reliable means to identify CD3γδɛζ−/− mice than previously described Northern and Southern blots, we designed polymerase chain reactions to distinguish knockout from wild-type CD3ɛ and CD3ζ alleles, facilitating the identification of CD3γδɛζ−/− mice.

Mapping of MN1 Sequences Necessary for Myeloid Transformation

The MN1 oncogene is deregulated in human acute myeloid leukemia and its overexpression induces proliferation and represses myeloid differentiation of primitive human and mouse hematopoietic cells, leading to myeloid leukemia in mouse models. To delineate the sequences within MN1 necessary for MN1-induced leukemia, we tested the transforming capacity of in-frame deletion mutants, using retroviral transduction of mouse bone marrow. We found that integrity of the regions between amino acids 12 to 458 and 1119 to 1273 are required for MN1’s in vivo transforming activity, generating myeloid leukemia with some mutants also producing T-cell lympho-leukemia and megakaryocytic leukemia. Although both full length MN1 and a mutant that lacks the residues between 12–228 (Δ12–228 mutant) repressed myeloid differentiation and increased myeloproliferative activity in vitro, the mutant lost its transforming activity in vivo. Both MN1 and Δ12–228 increased the frequency of common myeloid progentiors (CMP) in vitro and microarray comparisons of purified MN1-CMP and Δ12–228-CMP cells showed many differentially expressed genes including Hoxa9, Meis1, Myb, Runx2, Cebpa, Cebpb and Cebpd. This collection of immediate MN1-responsive candidate genes distinguishes the leukemic activity from the in vitro myeloproliferative capacity of this oncoprotein.

YY1 controls Igκ repertoire and B-cell development, and localizes with condensin on the Igκ locus

Conditional knock-out (KO) of Polycomb Group (PcG) protein YY1 results in pro-B cell arrest and reduced immunoglobulin locus contraction needed for distal variable gene rearrangement. The mechanisms that control these crucial functions are unknown. We deleted the 25 amino-acid YY1 REPO domain necessary for YY1 PcG function, and used this mutant (YY1ΔREPO), to transduce bone marrow from YY1 conditional KO mice. While wild-type YY1 rescued B-cell development, YY1ΔREPO failed to rescue the B-cell lineage yielding reduced numbers of B lineage cells. Although the IgH rearrangement pattern was normal, there was a selective impact at the Igκ locus that showed a dramatic skewing of the expressed Igκ repertoire. We found that the REPO domain interacts with proteins from the condensin and cohesin complexes, and that YY1, EZH2 and condensin proteins co-localize at numerous sites across the Ig kappa locus. Knock-down of a condensin subunit protein or YY1 reduced rearrangement of Igκ Vκ genes suggesting a direct role for YY1-condensin complexes in Igκ locus structure and rearrangement.

Fluorescent tagged NK cell receptors expressed in vivo using retrogenic mice to study the delivery of signals in NK cells

Event Abstract Back to Event Fluorescent tagged NK cell receptors expressed in vivo using retrogenic mice to study the delivery of signals in NK cells David Tomaz1, Nadia Guerra2, Julian Dyson3 and Keith Gould1* 1 Imperial College London, Department of Immunology, United Kingdom 2 Imperial College London, Department of Life Sciences, United Kingdom 3 Imperial College London, Division of Immunology and Inflammation, United Kingdom NK cells display both activating and inhibitory receptors which recognize different ligands, and it is the integration of signals from these two classes of receptors that ultimately determines NK cell effector activity. Nevertheless, how exactly the signal integration occurs is still not well understood. Our approach is to employ retrogenic mice to generate authentic primary NK cells expressing two fluorescent tagged receptors, NKG2D (short or long isoform) and Ly49A, prototypical activating and inhibitory NK cell receptors, respectively. The aim is to use NKG2D-GFP/Ly49A-RFP Forster resonance energy transfer (FRET) to study the co-localisation of these receptors in synapses at NK-target cell-cell contact interfaces. Also, we will test the consequences of altering ligand dimensions in the synapse formation and NK cell activation. Our results show that GFP-NKG2D (short and long isoform) interacts with both adaptor molecules DAP10 and DAP12 in vitro, allowing efficient cell surface expression of fluorescent NKG2D. Also, we reconstituted irradiated C57BL/6 recipients with GFP-NKG2D transduced bone marrow cells from NKG2D-deficient C57BL/6 donor mice. Two weeks post reconstitution, we detected cell surface expression of GFP-NKG2D predominantly in NK cells. The functionality of the fluorescent receptors and their interaction with the adaptor proteins DAP10 and DAP12 are now being investigated in NK cells generated in vivo. Data on the functionality of the fluorescent receptors and the generation of NK cells in retrogenic mice will be presented. Keywords: NK cells, innate immunity, NKG2D, ly49a, retrogenic mice Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune receptors and signaling Citation: Tomaz D, Guerra N, Dyson J and Gould K (2013). Fluorescent tagged NK cell receptors expressed in vivo using retrogenic mice to study the delivery of signals in NK cells. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01194 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 22 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Keith Gould, Imperial College London, Department of Immunology, London, United Kingdom, k.gould@ic.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers David Tomaz Nadia Guerra Julian Dyson Keith Gould Google David Tomaz Nadia Guerra Julian Dyson Keith Gould Google Scholar David Tomaz Nadia Guerra Julian Dyson Keith Gould PubMed David Tomaz Nadia Guerra Julian Dyson Keith Gould Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Collaboration Between RUNX and NOTCH Pathways in T-Cell Acute Lymphoblastic Leukemia

AbstractAbstract 1279T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy characterized by the clonal outgrowth of developmentally arrested T-lymphoid blasts. Notch signaling is activated by mutation of NOTCH1 and/or FBW7 in over half of cases, and ultimately results in increased expression of target genes via the NOTCH/CSL transcriptional complex. Enforced expression of activated NOTCH1 in mouse hematopoietic progenitors leads to the development of clonal T-cell leukemias, suggesting that collaborating mutations are required for establishment and/or propagation of malignant clones. To identify candidate collaborating loci, Beverly and Capobianco performed a retroviral insertional mutagenesis screen in mice expressing a relatively weak activated Notch1 transgene and found recurrent insertions into Ikaros (Ikzf1). These insertions resulted in expression of dominant negative isoforms of Ikaros and thus potentiated Notch signaling since Ikaros and Notch/CSL compete for occupancy at target gene regulatory elements.In an attempt to identify collaborating mutations outside of the Notch pathway, we performed a similar screen, but employed instead a very potent activated NOTCH1 allele (ΔE) in hopes of saturating the Notch signaling pathway. We thus cloned out the insertion sites from 88 primary mouse leukemias generated by transduction of bone marrow with ΔE retrovirus. While recurrent insertions into Ikzf1 were again identified, we also observed frequent insertions into other regions including the Runx3 locus. The Runx3 integrations were tightly clustered in a region 40–60kb upstream of the transcriptional start site, suggesting the retroviral LTR might be inducing an increase in Runx3 expression. A single integration upstream of Runx1 was also identified in a region frequently mutated in similar screens. Of note, analysis of publically available gene expression profile data revealed that RUNX1 and RUNX3 are ubiquitously expressed in patient T-ALL samples.In order to functionally characterize the roles of RUNX1 and RUNX3 in T-ALL, we utilized lentiviral shRNAs to knock down RUNX1 and/or RUNX3 across a broad panel of 26 human T-ALL cell lines. Despite recent studies suggesting RUNX1 may act as a tumor suppressor in T-ALL, we observed the overwhelming majority of cell lines to show substantial growth defects after knock-down of RUNX1/3 as measured by competitive growth assay. These results were confirmed in a subset of cell lines and also in xenograft-expanded primary T-ALL samples by BrdU incorporation/DNA content assays which showed reduced proliferation/G1 cell cycle arrest following RUNX1/3 knock-down. Conversely, overexpression of RUNX3 induced T-ALL cells to proliferate more rapidly and to resist ABT-263-induced apoptosis.To explore potential target genes responsible for these pro-growth/survival effects, we mined available ChIP-Seq data and found NOTCH1/CSL and RUNX1 binding sites to co-localize within IGF1R and IL7R loci at intronic enhancer-like regions with associated H3K4me1>H3K4me3 marks and reduced H3K27me3 marks. Importantly, we show that NOTCH1 and RUNX factors co-regulate surface protein expression of IGF1R and IL7R in a synergistic/additive manner. As we and others have previously demonstrated important roles for both IGF1R and IL7R in T-ALL cell growth and leukemia-initiating activity, these studies reveal a novel collaborative mechanism between NOTCH1 and RUNX proteins in supporting propagation of established T-ALL disease. Disclosures:No relevant conflicts of interest to declare.

BCOR Is Involved in Myeloid Cell Growth Control by Regulating Hox Genes

AbstractAbstract 3445BCOR interacts with Polycomb group (PcG) proteins to form a PRC1-like transcriptional repressive complex. BCOR is an essential developmental regulator: hemizygous mutation of BCOR in males causes gestational lethality, and heterozygous mutation in females causes the X-linked developmental disorder Oculofaciocardiodental syndrome (OFCD). Studies in humans, cell lines and mice indicate that BCOR plays key roles in the regulation of early embryonic development, mesenchymal stem cell function and hematopoiesis. Recently, recurrent somatic BCOR mutations have been reported in retinoblastoma, medulloblastoma and acute myelogenous leukaemia (AML) suggesting BCOR functions as a tumor suppressor protein. The goal of the current study was to begin elucidating the function of BCOR in normal hematopoiesis and in hematopoietic transformation. To examine this in the mouse we transduced bone marrow (BM) from mice harboring a conditional Bcor deletion allele (BcorFl/Y) with lentivirus expressing CRE. FACS analysis showed that with CRE, 83% of Bcor-/Y (Bcor KO) BM cells kept in IL3, IL6 and stem cell factor (SCF) were CD34+ compared to 95% of non-induced BcorFl/Y (Bcor WT) BM cells. Moreover, following G-CSF treatment, Bcor KO BM cells showed a much higher incidence of cell differentiation (40.5%) compared to Bcor WT cells (5.4%) as shown by CD11b/Gr-1 FACS analysis. Surprisingly, Bcor KO BM cells also showed significantly higher proliferation rates in liquid culture and methycellulose colony assays. To identify BCOR downstream target genes, we compared the gene expression profiles between Bcor KO and WT BM cells. Real-time-PCR of individual Hox genes confirmed the upregulation of HoxA5, HoxA7 and HoxA9 in Bcor KO BM cells, which are known to increase self-renewal of immature hematopoietic cells. In addition, ChIP analysis showed specific binding of BCOR to HoxA7 promoter. In conclusion, our results from mouse experiments suggest that BCOR is involved in maintaining the CD34+ stem cell state and loss of BCOR leads to enhanced myeloid cell differentiation and proliferation possibly by regulating Hox genes. In addition, we examined BCOR expression levels in a large panel of human leukemic cell lines; high level BCOR mRNA and protein were detected in most cell lines. Notably, 2 cell lines, U937 and HL60, had low levels of BCOR mRNA and no detectable BCOR protein. Treatment of both cell lines with the differentiation inducers ATRA, Vitamin D3 or DMSO lead to a mark induction in BCOR expression both at the mRNA and protein level, suggesting that BCOR may be involved in myeloid cell differentiation. Further investigation should shed more light on the roles BCOR that plays in normal hematopoiesis and whether BCOR loss later in hematopoiesis could result in a differentiation block. Disclosures:No relevant conflicts of interest to declare.

Mutated IDH1 Has 2-Hydroxyglutarate-Independent Functions in Leukemogenesis

AbstractAbstract 770Mutations in the metabolic enzymes IDH1 and IDH2 are frequently found in glioma and AML patients. Mutant IDH produces R-2-hydroxyglutarate (2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. However, it is unknown whether R-2HG is required for transformation. To investigate the function of mutated IDH1 we established an in vivo mouse model, which allowed us to study R-2HG-dependent and –independent functions of mutated IDH1.We cloned wildtype IDH1 (IDH1wt) and mutated IDH1 (IDH1mut, R132C) from AML and MDS patients and identified a mutated splice variant that lacked exon 7 (IDH1mutΔ7) in several IDH1 mutated patients. To evaluate whether the mutated proteins produce the oncometabolite R-2HG, we expressed IDH1mut, IDH1mutΔ7, and IDH1wt in immortalized murine bone marrow cells and measured intracellular levels of 2HG by enantiomer-specific quantification. IDH1mut expressing cells produced high levels of R-2HG, but not IDH1 mutated cells that lacked exon 7 of IDH1, control-vector transduced cells (CTL), or IDH1 wildtype cells (P<.001). To functionally analyze mutated IDH1, we transduced IDH1wt, IDH1wtΔ7, IDH1mut, IDH1mutΔ7, or empty vector CTL in HoxA9-immortalized murine bone marrow cells and transplanted sorted cells in lethally irradiated mice. Mice receiving transplants with IDH1mut and IDH1mutΔ7 transduced cells had higher engraftment levels at 4, 8, and 12 weeks after transplantation (P<.001) than IDH1wt, IDH1wtΔ7, and CTL mice, and developed severe leukocytosis, anemia and thrombocytopenia, whereas IDH1wt, IDH1wtΔ7, and CTL mice had normal blood counts at 12 weeks. Mice with mutated IDH1, whether producing 2HG (IDH1mut) or not (IDH1mutΔ7), died with a median latency of 83 and 80 days after transplantation, respectively, whereas IDH1wt and CTL mice survived for a median of 167 and 210 days, respectively, and IDH1wtΔ7 mice were alive at 200 days (pooled data from 3 independently transduced cell populations that were transplanted at 3 different time points, P<.001). IDH1mut and IDH1mutΔ7 mice died with a myeloproliferative-like disease with high white blood cell counts, large spleen, anemia and thrombocytopenia. Bone marrow cells from moribund mice were readily transplantable in secondary animals, and rapidly induced disease. These data demonstrate that myeloproliferation is accelerated by an alternatively spliced mutant IDH1 independent of the metabolite R-2HG.In vivo cell cycle analysis showed a significantly higher proportion of cells in S/G2/M phase in bone marrow cells transduced with IDH1mut or IDH1mutΔ7 when compared to cells transduced with IDH1wt or CTL. Also, cyclin-dependent kinase inhibitors (Cdkn) 1A (p21), 1B (p27), 2A (p16), and 2B (p15) were markedly downregulated in IDH1mut and IDH1mutΔ7 cells when compared to IDH1wt cells. We next investigated, whether the promoters of the repressed Cdk inhibitors were hypermethylated. The CpG island in the promoter of Cdkn2a and Cdkn2b showed low levels of DNA methylation in IDH1wt, IDH1mut, IDH1mutΔ7 and CTL cells (0.6% to 4.3% average methylation), suggesting that Cdk inhibitors are repressed in cells with mutated IDH1 independently of DNA methylation. Gene set enrichment analysis from microarray data of transduced bone marrow cells showed that genes related to MAP-kinase signalling were highly enriched in IDH1mut and IDHmutΔ7 cells compared to IDH1wt or CTL transduced cells. By Western blot we found that pERK was highly upregulated in IDH1mut and IDHmutΔ7 cells compared to IDH1wt or CTL cells. Pharmacologic inhibition of transduced cells in vitro showed that IDH1mut and IDH1mutΔ7 cells were resistant to inhibition with the MEK1/2 inhibitor UO126, suggesting that MAP-kinase signalling is a relevant target in IDH1 mutated AML.In summary, we show that an alternatively spliced isoform of mutated IDH1 promotes leukemogenesis independently of R-2HG in a mouse model of transformed hematopoietic cells with the same kinetics as mutated full-length IDH1 through transcriptional repression of cyclin-dependent-kinase inhibitors p15 and p16 and activation of MAP-kinase signalling. Our mouse model of mutated IDH1 represents a powerful tool to test the efficacy of newly developed drugs targeting mutated IDH1 in a 2-HG-dependent and –independent manner. Disclosures:No relevant conflicts of interest to declare.

Rescue of Lethal Hypophosphatasia Mice by Neonatal Ex Vivo Gene Therapy Using Lentivirally Transduced Bone Marrow Cells.

Abstract 3154Hypophosphatasia (HPP) is an inherited skeletal disease caused by genetic defects of tissue-nonspecific alkaline phosphatase (TNALP). TNALP is an ectoenzyme which is attached to the outside plasma membrane via a GPI anchor and plays an essential role in bone mineralization. The major symptoms are hypomineralization of systemic bones, respiratory insufficiency and epileptic seizures. Severe HPP is often fatal. Since ALP functions on the exterior of the cells, enzyme replacement therapy (ERT) is a potential approach to treat HPP. Although previous trials of ERT using various forms of soluble ALP showed no clinical benefit, it was recently demonstrated that TNALP with deca-aspartates at the C terminus (TNALP-D10) had a high affinity for bone tissue and repeated injections of TNALP-D10 successfully rescued lethal HPP mice. HPP mice were generated by knockout the mouse TNALP gene (Akp2) and phenotypically mimic to severe infantile HPP and develop hypomineralization, growth failure and epileptic seizures after birth. The plasma ALP activity in HPP mice was less than 0.01 U/ml (approx. 0.1 U/ml in wild type (wt) mice) and the average life span of non-treated HPP mice is about 20 days. We have also shown that a single intravenous injection of either lentiviral or AAV vector expressing TNALP-D10 resulted in prolonged survival and phenotypic correction of HPP mice. In this in vivo gene therapy, bone cells were not efficiently transduced, but the plasma ALP activity derived from TNALP-D10 secreted from transduced liver or muscle cells was maintained at extremely high levels (10 to 100 folds higher than that of wt mice). As an alternative approach, we are studying the feasibility of hematopoietic stem cells (HSC) based ex vivo gene therapy for HPP. After homing of HSC to the bone marrow, local expression of TNALP in the bone should be beneficial to improve bone mineralization. Other potential advantages of this strategy compared with an in vivo systemic gene therapy include lifelong expression of TNALP, no risk of germline gene transfer, and no immunoreaction against viral vector.Lineage negative bone marrow cells (BMC) were harvested from B6.CD45.1 mice (Ly5.1) using the Mouse Hematopoietic Progenitor (Stem) Cell Enrichment Set (BD Bioscience) and incubated with lentiviral vector expressing GFP or TNALP-D10 for 20 hrs at an moi of 50 with mSCF, mIL3 and rhIL6. Transduction efficiency assessed by GFP expression was approximately 40 % under the condition used. Recipient neonatal mice (Ly5.2) were sub-lethally irradiated at 4Gy and received BMC (1 × 106′ cells) through the jugular vein on day 2. Irradiated neonatal wt mice showed a slight reduction of the growth rate but normal physical activity and healthy appearance. GFP positive donor cells migrated to the bone marrow in recipient mice. FACS analysis of the peripheral blood samples 4 to 12 weeks after transplantation demonstrated that approximately 30 % of Ly5.1 donor cells were stably detected in all lineage blood cells of recipient mice. After treatment of neonatal HPP mice with TNALP-D10 expressing BMC, the plasma ALP activity was elevated to 1 to 2 U/ml at 4 weeks of age and remained at this level during the observation period. The treated mice actively moved in the cage without epileptic seizures and the life span was prolonged over 3 months. X-ray examination of the skeleton showed that mineralization was significantly improved compared to non-treated HPP mice, but not completely normalized compared to age matched wt mice. These results indicate that lentivirally transduced BMC can serve as a reservoir for continuous supply of TNALP-D10 to rescue lethal HPP mice. However, the concentration of TNALP-D10 in the bone may not be sufficient for complete correction of skeletal abnormalities. Further optimization of gene transfer and neonatal BMT is under way to increase the plasma ALP activity. HSC mediated ex vivo gene therapy is now being applied to treat not only hematological diseases but also neurological disorders such as adreno leukodystrophy and metachromatic leukodystrophy. Hypophosphatasia, a systemic bone disease, is also an important target for ex vivo gene therapy. Disclosures:No relevant conflicts of interest to declare.

Host Thiopurine Methyltransferase Status Affects Mercaptopurine Antileukemic Effectiveness

AbstractAbstract 3560Thiopurines are routinely used to treat many hematopoietic malignancies, including acute lymphoblastic leukemia (ALL), as well as inflammatory and autoimmune diseases. Common polymorphic null alleles among humans in thiopurine methyltransferase (TPMT) render 1 in 400 patients equivalent to homozygous knock-outs (−/−), 10% of patients equivalent to hemizygous (+/−), and 90% of patients comparable to wild-type (+/+) status for TPMT enzyme activity. TPMT activity is inversely related to the accumulation of active thioguanine nucleotide metabolites among patients, resulting in a higher risk for myelosuppressive adverse effects if routine doses are given to −/− compared to +/− compared to +/+ patients. However, the impact of the host TPMT polymorphism on antileukemic efficacy in the clinic is less clear.We generated a murine model of ALL by transducing bone marrow cells of Arf-null mice on the 129X1SvJ background (Tpmt +/+) with a retroviral vector encoding p185 BCR-ABL and luciferase, and culturing them for 7–10 days. We injected 2000 BCR-ABL+ pro/pre-B cells into Tpmt-deficient (−/−), heterozygous (+/−), or wild type (+/+) unconditioned syngenic recipients and compared the antileukemic efficacy when treating with a relatively low dose of mercaptopurine (equivalent to 7–9 mg/m2—such as would be tolerated by patients homozygous for null TPMT alleles). Treatment was started one day after injection of leukemia cells and continued for 70 days or until mice were sacrificed due to moribund state (usually due to illness from leukemia). To mimic clinically relevant treatment regimens, methotrexate was injected intraperitoneally (IP) at 1 mg/kg, once a week. Mercaptopurine was administered in the drinking water at 10 mg/L, for an estimated delivered dose of 2 – 2.5 mg/kg/day of mercaptopurine.Erythrocyte TPMT activity segregated by Tpmt genotype as expected and was comparable to the differences observed in patients: 0.6 U/ml in Tpmt −/−, 6.7 U/ml in Tpmt +/−, and 11.6 U/ml in Tpmt +/+ mice (p =0.008, n=2 for each genotype). As expected, the median survival (± standard error) was similar among Tpmt genotypes in untreated mice: 16 (± 0.4) days for Tpmt +/+, 17 (± 0.7) days for Tpmt +/−, and 18 (± 0.4) days for Tpmt −/−. The difference in leukemia-free survival by Tpmt genotype was profound: at 30 days after the start of treatment, 5% (± 9%) of Tpmt +/+ mice, 47% (± 26%) of Tpmt +/− mice, and 85% (± 14%) of Tpmt −/− mice were surviving (p = 5×10−8, Figure), indicating a substantial impact of host Tpmt status on thiopurine antileukemic effectiveness. In vivo imaging of luciferase-marked ALL cells confirmed an intermediate response phenotype for the Tpmt heterozygous mice (p = 0.00001). With higher doses (closer to the doses tolerated by patients homozygous for wild-type alleles, equivalent to 50–70 mg/m2/day), leukemia-free survival improved in the Tpmt +/+ and Tpmt +/− mice, comparable to the survival of Tpmt −/− mice treated with the low dose of mercaptopurine. These findings support the notion that germline polymorphisms in Tpmt, even in the heterozygous state, affect not only host tissue toxicity, but antitumor effectiveness as well. [Display omitted] Disclosures:Evans:St. Jude Children’s Research Hospital: Dr. William Evans receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms., Dr. William Evans receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms. Patents & Royalties. Relling:St. Jude Children’s Research Hospital: Dr. Mary Relling receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms and GGH polymorphisms., Dr. Mary Relling receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms and GGH polymorphisms. Patents & Royalties; Sigma-Tau Pharmaceuticals: Research Funding.

Nfkb1 Plays a Tumor-Suppressing Role in BCR-ABL-Induced Leukemias

Abstract 1666NF-κB activation has been linked to the promotion of an assortment of malignancies. While in vitro studies have supported a tumor-promoting role for NF-κB in leukemias, in vivo evidence has not been collected. NF-κB has also been proposed as a therapeutic target for Philadelphia chromosome-positive leukemias including chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). In this study, we investigate the role the Nfkb1 gene plays in CML and B-ALL using mouse models for these leukemias induced by the BCR-ABL oncogene. Contrary to conventional thinking, we find a tumor-suppressing role for Nfkb1 in BCR-ABL leukemias. To induced CML, we transduced bone marrow cells from 5-FU-primed wild type or Nfkb1−/− mice with BCR-ABL-GFP retrovirus, followed by transplantation of the transduced cells into lethally irradiated recipient mice. We show that recipients of BCR-ABL-transduced Nfkb1−/− donor cells developed CML more rapidly than the wild type recipients, correlating to elevated percentages (82.3% vs 38.5%) and total numbers of Gr-1+GFP+ cells in peripheral blood of the mice. We next tested whether the loss of Nfkb1 also accelerated B-ALL development under the B-ALL-inducing conditions, and show that recipients of BCR-ABL-transduced Nfkb1−/− donor cells succumbed to B-ALL more quickly than the wild type recipients. These results suggest that Nfkb1 does not stimulate leukemia growth, and instead, it plays a tumor-suppressing role in CML and B-ALL. Because these findings are opposite from what people believe in the field, we confirmed our findings by conducting a rescue experiment. The Nfkb1 gene encodes the p105 protein; roughly half of p105 product is selectively proteolyzed to yield the p50 transcription factor. In this rescue experiment, we transduced Nfkb1−/− donor cells with BCR-ABL alone or BCR-ABL and p50 of Nfkb1 to induce leukemia, and we show that acceleration of leukemia development caused by the Nfkb1 loss is partially reversed by p50. To study the underlying mechanism, we focused on CARD11, because it plays a critical role in mediating NF-κB signaling in lymphocytes. We find that CARD11 expression is induced in myeloid and increased in lymphoid cell lines expressing BCR-ABL. Furthermore, we induced B-ALL using Card1−/− mice. We find that similar to the loss of Nfkb1, the loss of Card11 expression also accelerates the development of B-ALL. To increase human relevance of our findings, we overexpressed p50 in human K562 cells by retroviral transduction with p50-IRES-GFP or empty vector, and find that FACS-sorted p50/GFP K562 cells grow significantly slower than empty vector-transduced cells. These findings support an idea that Nfkb1 signaling suppresses BCR-ABL-induce leukemia at least partially through a CARD11-dependent mechanism. In summary, our results demonstrate that Nfkb1 plays a tumor-suppressing role in BCR-ABL-induced leukemias, arguing that we should be careful when targeting of Nfkb1 in leukemia treatment. Disclosures:No relevant conflicts of interest to declare.

Inducible Costimulator Gene Transduced Bone Marrow Derived Mesenchymal Stem Cells Attenuate the Severity of Acute Graft-Versus-Host Disease in a Mouse Haploidentical Model.

AbstractAbstract 2996It is reported that both block of costimulator (ICOS) or its ligand signal pathway and transfusion of mesenchymal stem cells (MSCs) can reduce the severity of acute graft-versus-host disease (GvHD). Using a MHC-haploidentical murine model of acute GvHD, we investigated the effects of MSC-ICOS-EGFP, bone marrow derived MSCs transduced with ICOS-gene using an adenoviral vector, on acute GvHD. Lethally irradiated CB6F1 mice were transplanted with bone marrow cells and spleen cells from C57BL/6 mice, and injected with MSC-ICOS-EGFP, MSCs, ICOS-Ig fusion protein or the diluent (control), respectively. Survival in recipients of MSC-ICOS-EGFP was significantly higher than that in GVHD group (74.29±7.39% vs 0, P=0.000, n=35), and also higher than in MSCs, ICOS-Ig, or MSCs+ICOS-Ig groups (74.29±7.39% vs 42.86±8.36% or 48.57±8.45% or 50.43±8.45%, P=0.0039 or 0.0323 or 0.0418, n=35). The lower mortality in recipients of MSC-ICOS-EGFP was related to lower incidence of acute GvHD. MSC-ICOS-EGFP can reduce the number of CD4+T cells in the early stage of GvHD due to the increased apoptosis, and it can produce CD4+CD25+Treg cells meanwhile. The effect on GvHD in recipient of MSC-ICOS-EGFP was also associated with higher serum levels of IL-4, IL-10 and lower levels of IFN-γ, IL-2, IL-12, IL-17A, as well as inducing expression of GATA-3, STAT6 transcription factors while inhibiting expression of T-bet, STAT4, ROR-rt in spleen. In vitro, the CD4+T cells of donor were sharply inhibited by MSC-ICOS-EGFP and the B7h blockade. In MLRs we can also detect lower levels of IFN-γ, IL-2and higher levels of IL-4, IL-10. These data indicate that MSC-ICOS-EGFP is a more prospective strategy for acute GvHD prevention, which had a synergism effect between MSCs and ICOS-Ig. The mechanisms of MSC-ICOS-EGFP on acute GvHD were closely associated with modulation of CD4+T cells, CD4+CD25+Tregs, and regulation of the Th1/Th2/Th17 balance in the early stage of GvHD. Disclosures:No relevant conflicts of interest to declare.

Dnmt3a Deletion and FLT3-ITD Cooperate in a Mouse Model of T-Lymphoblastic Leukemia (T-ALL).

Abstract 2428Aberrant DNA methylation repeatedly has been implicated in cancer development. DNA methyltransferase (DNMT) 3A, which mediates de novo DNA methylation, was found to be mutated in 20% of patients with acute myeloid leukemia and 10% of patients with myelodysplastic syndrome. Recently, mutations associated with myeloid malignancies such as DNMT3A and FLT3 have also been uncovered in patients with early T-cell precursor lymphoblastic leukemia (ETP-ALL) (Neumann et al., 2012; Van Vlierberghe et al., 2011; Zaremba et al., 2012). ETP-ALL is a type of very high-risk ALL associated with myeloid/stem cell gene expression signature and myeloid markers. We have demonstrated that Dnmt3a deletion in mouse causes increased self-renewal of hematopoietic stem cells and an impairment of differentiation (Challen et al., 2011). Dnmt3a loss also produces aberrant methylation associated with oncogenes and tumor suppressor genes. Yet, whether aberrant DNA methylation can drive leukemia remains unknown. As Dnmt3a deletion alone was insufficient for malignancy, secondary mutations are likely necessary for leukemic transformation. Because FLT3 internal tandem duplication (ITD) frequently co-exist with DNMT3A mutations in acute leukemias, we hypothesized that Dnmt3a-loss may cooperate with FLT3-ITD to promote leukemic transformation; and we established a mouse model to test this.Deletion of conditional Dnmt3a with Mx1-cre was induced by injections of pIpC. Subsequently, bone marrow from Dnmt3a-deleted (Dnmt3aKO) donor mice was transduced with MSCV-FLT3-ITD-GFP retrovirus or MSCV-GFP control and transplanted into lethally irradiated recipients. The mice were monitored monthly for development of malignancies by complete blood count and peripheral blood analysis by flow cytometry and followed for disease latency. Moribund mice were sacrificed and analyzed with peripheral blood smears, histology, and immunophenotyping.Dnmt3a deletion with overexpression of FLT3-ITD caused rapid onset T-ALL in 6/8 mice (n=6) with a median latency of 78 days compared to 121 days in WT mice (n=4) overexpressing FLT3-ITD (p<0.0001 Log-rank Mantel-Cox Test) (See figure). Mice from both groups exhibited leukocytosis, splenomegaly, and thymomegaly with high GFP expression detected by FACS. Even after we transduced bone marrow cells enriched for myeloid progenitor and stem cells, Dnmt3a deletion again accelerated T-ALL with median survival of 89 days (n=9) versus 110 days in WT-FLT3-ITD (n=10) mice. T-ALL was observed in 2/4 WT-FLT3-ITD mice and 5/6 Dnmt3aKO-FLT3-ITD mice analyzed (p<0.0001 Log-rank Mantel-Cox Test). By flow cytometry, two distinct types of T-ALL were observed in the bone marrow of Dnmt3a deleted leukemic mice: one was characterized by a double positive population (DP) of CD4+CD8+ lympoblasts (1/6) and another early immature T-cell-like type of CD4-CD8-CD44+CD25-CD11bloCD117+ lymphoblasts (4/6). Gene expression analysis by RT-PCR in the early immature T-ALL showed downregulation of Notch-pathway genes (such as Notch1, Notch 3, Deltex, Hes1) and upregulation of stem cell-associated genes Lyl1 and Scl1, suggesting an ETP-like T-ALL. The ETP-like ALL phenotype has not been seen in WT mice overexpressing FLT3-ITD. The opposite gene expression pattern was seen in the DP population with upregulation of Notch-pathway genes. Furthermore, the DP leukemia was transplantable to secondary recipients within 2 weeks. Whether ETP-like ALL can be transplanted is still under investigation. We are also currently studying the changes in global CpG methylation among the leukemias that have Dnmt3a loss, FLT3-ITD overexpression, and control and also anticipate data from transcriptome analysis by RNA-Seq.These data suggest that stem or progenitor bone marrow cells primed by early loss of Dnmt3a are transformed into DP T-ALL and ETP-like ALL fueled by the overexpression of the oncogene FLT3-ITD. The ETP-like ALL phenotype has not been seen previously in WT mice overexpressing FLT3-ITD, suggesting that Dnmt3a ablation is required. The Dnmt3a-deleted-FLT3-ITD mice with T-ALL is, to our knowledge, the first animal model of human immature T-cell leukemia. This model can enhance our understanding of the pathogenesis of ETP-like ALL with respect to aberrant DNA methylation and will serve as a powerful tool to test novel therapeutic strategies. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Negative Feedback Signaling Enables Leukemic Transformation by Oncogenic Tyrosine Kinases

AbstractAbstract 1352 Background:Oncogenic tyrosine kinases drive malignant transformation in large subsets of hematological malignancies (e.g. BCR-ABL1, FLT3ITD) and are often associated with poor clinical outcome. Current efforts to improve therapeutic options for the treatment of tyrosine kinase-driven (TKD-) leukemia are almost entirely focused on the development of more potent tyrosine kinase inhibitors (TKI) with the goal to reduce oncogenic signaling below a minimum threshold that is required for the survival of TKD-tumor cells. Here we report the surprising finding that hyperactivation of oncogenic tyrosine kinase signaling is likewise toxic when it surpasses a previously unrecognized upper threshold of maximum tolerable signaling strength. Negative feedback signaling molecules have been shown to limit tyrosine kinase signaling strength. For instance, DUSP6 and SPRY2 attenuate signaling from surface receptors and thereby function as tumor suppressors. Here we studied mechanisms of negative feedback signaling in tyrosine kinase-driven (TKD) malignancies. Results:We identified the sprouty family inhibitor of Ras signaling Spry2, the dual-specificity phosphatase Dusp6 and the Ets transcription factor Etv5 as central components of a common negative feedback signaling module that is required to calibrate signaling strength downstream of oncogenic tyrosine kinases in ALL and CML. In genetic experiments, we demonstrated that negative feedback to oncogenic tyrosine kinase signaling is required for malignant transformation and the ability of leukemia cells to form colonies. In the absence of adequate negative feedback, hyperactive tyrosine kinase signaling leads to accumulation of ROS, activation of p53/DNA damage response, cellular senescence and massive activation of the pro-apoptotic MAP kinases JNK and p38a.Studying gene expression changes by microarray analysis, RT-PCR and Western blot, we found that the DUSP6, SPRY2 and ETV5 negative feedback regulator molecules are highly expressed in a wide array of myeloid and B cell lineage leukemia. In contrast to TKD leukemia, Non-TKD leukemias lack expression of these genes and DUSP6 and SPRY2 promoters are hypermethylated. To study the function of Dusp6, Spry2 and Etv5 in a genetic mouse model of TKD-leukemia, we transduced bone marrow pre-B cells from Dusp6−/−, Spry2fl/fland Etv5−/−mice and respective wildtype controls with retroviral BCR-ABL1. Defects in one of these negative feedback mediators caused profound signaling imbalances in TKD leukemia cells. For instance, Dusp6-deficient TKD-leukemia cells rapidly underwent cellular senescence. Lack of Dusp6 and Spry2 dramatically increased cellular ROS. Owing to excessively high levels of ROS, TKD-leukemia cells lacking one of these negative feedback mediators failed to initiate colonies in semisolid agar and failed to initiate leukemia in transplant recipient mice. Inducible activation of Cre-mediated deletion of Spry2 in leukemia cells resulted in rapid cell death. Clinical relevance:To test whether negative feedback signaling molecules represent a potential target for pharmacological inhibition in the treatment of TKD-leukemia, we tested the Dusp6 small molecule inhibitor 2-benzylidene-3-(cyclohexylamino)-1-Indanone hydrochloride (BCI). At 3mmol/l, BCI induces massive accumulation of ROS and subsequent cell death in 5 patient-derived cases of Ph+ ALL including two cases with T315I mutation. To test in vivo efficacy of BCI, patient-derived Ph+ ALL cells with T315I were xenografted into NOD/SCID recipient mice. Mice were treated ten times with either vehicle or 25 mg/kg BCI (i.p.). In agreement with in vitro results, treatment with BCI resulted in significant prolongation of overall survival compared to standard TKI-treatment (BCI vs TKI Nilotinib p=0.008). Conclusion:Our studies identify negative feedback mediators including DUSP6, Spry2 and ETV5 as a novel therapeutic targets in TKD-leukemia, e.g. Ph+ ALL. Pharmacological inhibition of negative feedback signaling represents a fundamentally novel and powerful approach to increase oncogenic signaling beyond a tolerable threshold, thus causing excessive accumulation of ROS and subsequent cell death. Normal cells lacking oncogenic tyrosine kinase signaling are spared in this approach since they do not harbor an oncogenic tyrosine kinase and are not prone to excessive signaling strength. Disclosures:No relevant conflicts of interest to declare.

Mutant U2AF1(S34F) Expression Alters Hematopoiesis in Mice

Abstract 553Myelodysplastic syndromes (MDS) are stem cell disorders characterized by ineffective hematopoiesis with increased levels of hematopoietic cell apoptosis. Recent discoveries by our group and others suggest that perturbations in pre-mRNA splicing may play a role in MDS pathogenesis. Indeed, more than half of all MDS patients have a mutation in one of eight splicing factors. U2AF1 (U2AF35), a gene encoding a splicing factor involved in intronic 3’-splice site recognition, is mutated in 8.7% of MDS patients. The consequence of the highly recurrent serine to phenylalanine mutation at position 34 (S34F) of U2AF1 in hematopoiesis is unknown. Therefore, to examine the effects of mutant U2AF1(S34F) on hematopoiesis, we utilized the MSCV-IRES-GFP retroviral system to introduce mutant U2AF1(S34F) or wild type U2AF1(WT), or an empty vector control, into mouse bone marrow cells for in vitro and in vivo studies. Expression of U2AF1(S34F) results in reduced expansion of transduced bone marrow cells (marked by GFP) compared to both U2AF1(WT) or empty vector-transduced cells grown in suspension culture (2 vs 4-fold change, respectively; p<0.001, n=3). Additionally, U2AF1(S34F)-transduced cells have increased levels of apoptosis (Annexin V+/7AAD+) in culture compared to U2AF1(WT) (p=0.03) and empty vector-transduced cells (p=0.02) (n=3). We also examined the effects of the U2AF1(S34F) mutation in vivo using bone marrow transplantation. The percentage of GFP+ cells in the peripheral blood of recipient mice transplanted with MSCV-transduced bone marrow was significantly reduced at 6 months post-transplant with U2AF1(S34F) expression (average=4%) compared to U2AF1(WT) (average=44%) and empty vector (average=65%) (p<0.02, n= 6–9 mice each). Transduction efficiencies were similar within experiments. There was no consistent alteration in lineage distribution of GFP+ cells in the peripheral blood of these mice.To overcome some of the limitations of retroviral models, we created a single-copy, doxycycline-inducible U2AF1(S34F) transgenic mouse to model the effect of U2AF1(S34F) expression on hematopoiesis. As a control for U2AF1 protein overexpression, we created an additional single-copy, doxycycline-inducible U2AF1(WT) transgenic mouse with transgene integration into the same locus as the U2AF1(S34F) mouse. Induction of U2AF1(S34F) transgene expression in bone marrow cells in culture with doxycycline treatment (200 ng/ml for 5 days) resulted in reduced cell numbers when compared to uninduced U2AF1(S34F) transgenic cells (ratio of growth of induced/uninduced cells = 0.38), while cell proliferation was not altered for U2AF1(WT) transgenic cells (ratio of growth of induced/uninduced cells = 1.13) (p<0.001, n=3). In addition, doxycycline-induced U2AF1(S34F) expression results in increased apoptosis (Annexin V+) compared to uninduced U2AF1(S34F) transgenic cells (21% vs 11%, p=0.01) and induced U2AF1(WT) transgenic cells in culture (21% vs 9.3%, p=0.008) (n=4). To examine the effects of mutant U2AF1(S34F) induction in vivo, we transplanted mutant U2AF1(S34F) or U2AF1(WT) transgenic bone marrow cells into congenic wild type recipient mice and induced transgene expression 6 weeks post-transplant using 2 mg/ml doxycycline in the drinking water for 5 days. Induction of U2AF1(S34F) expression in vivo results in reduced number of WBCs in the peripheral blood of recipient mice compared to mice with uninduced U2AF1(S34F) transgenic bone marrow (3.4k vs 5.6k, p=0.01, n=3). In addition, recipient mice with induced U2AF1(S34F) bone marrow had reduced number of bone marrow cells per femur when compared to uninduced U2AF1(S34F) recipient mice (3.9M vs 13.1M, p=0.04) and induced U2AF1(WT) recipient mice (3.9M vs 12.4M, p=0.03) (n=3). The number of neutrophils in peripheral blood (p<0.001), bone marrow (p=0.04), and spleen (p=0.04) of induced U2AF1(S34F) recipient mice were all significantly lower compared to uninduced U2AF1(S34F) mice (n=3). The total numbers of c-Kit+/lineage-/Sca+ hematopoietic progenitor cells were not affected in induced U2AF1(S34F) recipient mice compared to uninduced U2AF1(S34F) (p=0.75) or induced U2AF1(WT) recipient mice (p=0.46, n=3) after 5 days of treatment. Collectively, these results suggest that the U2AF1(S34F) mutation may contribute to abnormal hematopoiesis in vivo. Longer periods of doxycycline-induction in vivo are ongoing and will be presented. Disclosures:No relevant conflicts of interest to declare.

Acetylation Is Critical for Foxo3 Protein Stability and Activity in Hematopoietic Stem and Progenitor Cells

Abstract 1198Foxo3 transcription factor is a critical regulator of hematopoietic stem cell (HSC) quiescence and hematopoietic and leukemic stem cell maintenance. In particular, loss of Foxo3 has been shown in models of both acute and chronic myeloid leukemia to prevent the maintenance of leukemogenesis, indicating that devising strategies to inhibit Foxo3 will be of critical therapeutic value. Nonetheless mechanisms that control Foxo3 activity in HSC remain unknown. FoxO are regulated by post-translational modifications including phosphorylation, acetylation and redox modulation that together determine FoxO subcellular localization and activity. In response to growth factors stimulation FoxO are phosphorylated by AKT that promote their cytosolic localization and inhibition of transcriptional activity. Interestingly, in normal bone marrow HSC and in leukemic stem cells Foxo3 is constitutively nuclear despite readily detectable phospho-AKT, strongly suggesting that negative phosphorylation may not be the sole Foxo3 regulatory mechanism in these stem cells. While acetylation of Foxo3 is linked to oxidative stress, and oxidative stress is known to activate Foxo3, whether acetylation activates or inhibits Foxo3 remains unclear and it is proposed to be context-dependent. Therefore, we sought out to determine how acetylation of Foxo3 impacts its function in HSC. To address this question we generated Flag-tagged-Foxo3 mutants mimicking (lysine to glutamine) or abrogating (lysine to arginine) acetylation of all five putative acetylation sites using a PCR-based site-directed mutagenesis strategy and then cloned these mutants into the retroviral MSCV-IRES-GFP (MIG) vector for generation of retroviral supernatant and efficient transduction of bone marrow mononuclear cells. We first evaluated subcellular distribution of Foxo3 mutants in human embryonic kidney (HEK)-293 cells. Interestingly Foxo3 is mainly cytoplasmic in HEK-293 cells but hydrogen peroxide (H2O2) treatment induces Foxo3 nuclear translocation. Ectopic expression of Foxo3 mutants mimicking acetylation where five putative acetyl-lysine residues (5KQ) are mutated in HEK-293 cells and subsequent H2O2 treatment impairs Foxo3 nuclear localization (>50% reduction of nuclear Foxo3), although to a lesser extent than some of the single mutants, indicating that distinct acetylated lysines may impact differently Foxo3 activity. Moreover, protein expression of Foxo3 acetylation-mimic mutants is increased as compared to wild type Foxo3 in HEK-293 cells suggesting that acetylation affects Foxo3 protein stability. In order to analyze the impact of Foxo3 acetylation in vivo, we transduced bone marrow mononuclear cells (BMMC) freshly isolated from 5-fluoracil (5-FU) treated mice with acetyl-lysine mimic mutant Foxo3. GFP-positive transduced BMMC were FACS-sorted, cytospun onto glass slides and analyzed by immunostaining with anti-Flag antibody, in order to discriminate exogenously expressed Foxo3-WT versus Foxo3-mutants. In agreement with HEK-293 results, overexpression of Foxo3 mutant in which five acetylation sites were mimicked (5kQ) showed a significant decrease in nuclear localization, indicating that acetylation may lead to cytoplasmic Foxo3 and ultimately abrogate its activity in BMMC. Importantly, BMMC overexpressing Foxo3-5kQ injected into lethally irradiated mice (Colony Forming Unit-Spleen – CFU-S) produced significantly less spleen colonies at day 12, indicating that the Foxo3 acetyl-lysine mimic mutant may function as a dominant-negative Foxo3. We lastly investigated Foxo3 activity in a SIRT1 conditionally knockout mouse model since SIRT1 is the main Foxo3 deacetylase. In agreement with results described here we found that in SIRT1−/−LSK cells, Foxo3 is mainly cytoplasmic. Importantly and consistent with data generated from ectopic expression of Foxo3 acetyl-lysine mutants, Foxo3 protein expression is significantly (two-fold) increased in SIRT1−/− Lin−Sca1−cKit+ myeloid progenitors. These findings strongly support the notion that acetylation of Foxo3 abrogates its function in hematopoietic stem and progenitor cells. They also suggest that acetylation of Foxo3 impacts its protein stability. Altogether these results are important for understanding the mechanism of regulation of HSC activity and are likely to have significant therapeutic value. Disclosures:No relevant conflicts of interest to declare.

Lentivirus-Mediated Platelet Gene Therapy Corrects Bleeding Diathesis and Induces Immune Tolerance in Murine Hemophilia B Mice

Abstract 1101While data from the clinical trials using AAV vector expression FIX in hemophilia B gene therapy in humans are very encouraging, for individuals with severe liver disease or neutralizing antibodies to AAV, an alternative gene therapy approach might be desired. Our previous studies have demonstrated that lentivirus-mediated platelet gene therapy can correct murine hemophilia A phenotype, but this approach has not been explored for hemophilia B. In the current study, we developed a clinical translatable approach for platelet gene therapy of hemophilia B. Platelet-FIX (2bF9) expression in hemophilia B (FIXnull) mice was introduced by transplantation of hematopoietic stem cells (HSCs) transduced with 2bF9 lentivirus (LV). The recipients were analyzed beginning at 3 weeks after bone marrow (BM) transplantation. Expression of the 2bF9 product was detected by PCR in all recipients that received 2bF9 LV-transduced BM cells, indicating viable engraftment of BM genetically modified with the 2bF9 LV transfer vector. The expression of the hFIX transgene protein in the transduced platelets was confirmed by immunofluorescent confocal microscopy. Flow cytometry showed that there were 20.8 ± 12.1% (n = 7) and 14.8 ± 10.7% (n = 6) 2bF9 LV-transduced platelets respectively in the recipients preconditioned with 1100 cGy or 660 cGy. The antigen levels of FIX (FIX:Ag) were 2.89 ± 1.75 mU/108 platelets (n = 9) in the recipients preconditioned with 1100 cGy and 1.87 ± 1.30 mU/108 platelets (n = 7) in the 660 cGy group, while the activity (FIX:C) levels were 1.67 ± 1.15 and 1.13 ± 0.85 mU/108 platelets respectively. There was a small amount of FIX detected in the 2bF9 LV-transduced recipient plasma with the average levels of 2.22 mU/ml in 1100 cGy group and 1.44 mU/ml in 660 cGy group. To analyze the distribution of the FIX between platelets and plasma, we normalized FIX levels to total whole blood FIX content. The results demonstrated that 90% to 95% of whole blood FIX was stored in platelets. The tail clip survival test demonstrated that 15 out of 16 mice that received 2bF9 LV-transduced HSCs survived the tail clip challenge, while 8 out of 10 FIXnull control mice died after tail clipping. Nine months after transplantation, sequential transplantation was performed on some of the primary recipients. Platelet-hFIX expression in the secondary recipients was sustained, leading to phenotypic correction and confirming that long-term engrafting HSCs were successfully transduced with 2bF9 LV. Notably, none of the transduced recipients developed anti-FIX antibodies after platelet gene therapy. To investigate whether immune tolerance was induced in 2bF9 LV-transduced recipients, we challenged the recipients with recombinant human FIX (rhFIX) in the presence of adjuvant. Only 1 out of 9 2bF9 LV-transduced recipients developed a low titer of inhibitory antibodies (1.6 BU/ml) as measured by a modified Bethesda assay. In contrast, all of the FIXnull controls developed inhibitory antibodies ranging from 17 – 37 BU/ml after the same challenge (n = 5). To ensure that the immune system was not defective in the 2bF9 LV-transduced recipients and that the tolerance induction is FIX antigen-specific, we further challenged the animals with ovalbumin (OVA) absorbed on Alum. Both the 2bF9 LV-transduced and FIXnull control mice developed high-titer of anti-OVA antibodies. The levels of anti-OVA IgG in the 2bF9 transduced recipients were not significantly different from FIXnull mice after the OVA immunization, confirming that tolerance induction in 2bF9 LV-transduced mice is FIX-specific. Taken together, our data suggest that lentivirus-mediated bone marrow transduction and transplantation can not only provide sustained phenotypic correction, but also induce immune tolerance in hemophilia B mice, indicating that this approach may be a promising strategy for gene therapy of hemophilia B in humans. Disclosures:No relevant conflicts of interest to declare.