bluuebunny/crossref_metadata_2025 huggingface dataset:
Scl and Notch Act Downstream of Hedgehog Signaling to Promote Embryonic Hematopoiesis From Endothelial Cells
Authors: Kim Peter G , Albacker Colleen E , Jang Il-ho , Heffner Garrett C , Lim Yoowon , Bowman Teresa V. , Lin Michelle I , Zon Leonard I. , Loewer Sabine , Daley George Q.
Published: October 2019

Abstract 1283

Adult hematopoietic cells transition through a hemogenic endothelial (HE) intermediate during development, but the signaling pathways modulating this transition are incompletely characterized. Although the Hedgehog (Hh) pathway is hypothesized to play a role in blood and endothelial cell formation, early embryonic lethality of mice lacking Hedgehog signaling precludes such analysis. To determine a role for Hh signaling in HE patterning, we assessed the effect of altered Hh signaling in differentiating mouse embryonic stem cells (mESCs), cultured embryonic day 9.5 mouse embryos, and developing zebrafish embryos. In differentiating mESCs, addition of Indian Hh ligand (IHH) increased the number of CD41+c-Kit+ hematopoietic progenitors, whereas chemical inhibition of Hh signaling led to a decrease without affecting primitive-streak mesoderm gene expression. In the setting of Hh inhibition, Notch induction rescued hemogenic VE-cadherin+ cells, demonstrating that Notch expands HE. Scl/Tal1 (stem cell leukemia/T-cell associated leukemia 1) induction rescued VE-cadherin+CD41+ cells, demonstrating that Scl/Tal1 converts endothelial cells to hematopoietic tissue. Similar experiments using cultured mouse yolk sacs demonstrated that signaling pathways are conserved in vivo. Moreover, VE-cadherin+ cells isolated from the mouse yolk sac or paraaortic splanchnopleura, when virally transduced with Notch signaling or Scl, had increased hematopoietic colony-forming activity. Finally, ectopic Notch or Scl induction in zebrafish embryos rescued the expression of the prototypical hemogenic endothelium marker Runx1 in the absence of Hh signalling. Together, our results reveal that the Hh-Notch-Scl axis promotes embryonic hematopoiesis through endothelial-to-hematopoietic transition.

Disclosures:

Zon: Fate Therapeutics: Consultancy; Stemgent: Consultancy. Daley:iPierian, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees.
Gabp Transcription Factor Is Required for Self-Renew and Proliferation of Hematopoietic Stem and Progenitor Cells
Authors: Yang Zhongfa , Drumea Karen , Cormier James , Wang Junling , Zhu Xuejun , Rosmarin Alan G.
Published: October 2019

Abstract 1284

GABP is an ets transcription factor that regulates genes which are required for normal hematopoietic development. In myeloid cells, GABP is an essential component of a retinoic acid-inducible enhanceosome that mediates granulocytic gene expression and, in lymphoid cells, GABP regulates expression of IL7-R and the essential transcription factor, Pax5. GABP is a tetrameric complex that includes GABPa, which binds DNA via its ets domain, and GABPb, which contains the transcription activation domain. Genetic disruption of mouse Gabpa caused early embryonic lethality. We created mice in which loxP recombination sites flank exons that encode the Gabpa ets domain, and bred them to mice that bear the Mx1Cre recombinase; injection with pIC induced Cre expression and efficiently deleted Gabpa in hematopoietic cells. One half of the Gabpa knock-out (KO) mice died within two weeks of pIC injection in association with widespread visceral hemorrhage. Gabpa KO mice exhibited a rapid loss of mature granulocytes, and residual myeloid cells exhibited myelodysplasia due, in part, to regulation by Gabp of the transcriptional repressor, Gfi-1. We used bone marrow transplantation to demonstrate that the defect in Gabpa null myeloid cells is cell intrinsic. Although hematopoietic progenitor cells in Gabpa KO bone marrow were decreased more than 100-fold compared to pIC treated control mice, there was not a statistically significant difference in the numbers of Lin−c-kit+Sca-1− hematopoietic stem cells (HSCs) between KO and control mice. Genetic disruption of Gfi-1 disruption in HSCs caused increased cell cycle activity – an effect that is diametrically opposite of the effect of Gabpa KO; this suggests that the effect of Gabpa on HSCs is not due to its control of Gfi-1. In contrast, Gabpa KO HSCs exhibited a marked decrease in cell cycle activity, but did not demonstrate increased apoptosis. The defects in S phase entry of Gabpa null HSCs are reminiscent of the cell cycle defects in Gabpa null fibroblasts, in which expression of Skp2 E3 ubiquitin ligase, which controls degradation of the cyclin dependent kinase inhibitors (CDKIs) p21 and p27, was markedly reduced following Gabpa disruption. We showed that Gabpa KO cells express reduced levels of Skp2. We propose that GABP controls self-renewal and proliferation of mouse bone marrow stem and progenitor cells, in part, through its regulation of Skp2. Thus, Gabpa is a key regulator of myeloid differentiation through its control of Gfi-1, but it is required for cell cycle activity of HSCs, by a distinct effect that may be due to its control of Skp2 and CDKIs.

Disclosures:

No relevant conflicts of interest to declare.
Alcam Mediated Cellular Interaction Regulates Hematopoietic Stem Cell Repopulation and Self-Renewal
Authors: Jeannet Robin , Cai Qi , Liu Hongjun , Vu Hieu , Kuo Ya-Huei
Published: October 2019

Abstract 1291

Alcam, which encodes the activated leukocyte cell adhesion molecule (CD166), is a cell surface immunoglobulin superfamily member mediating homophilic adhesion as well as heterotypic interactions with CD6. It has recently been shown that Alcam+ endosteal subset in the bone marrow contain hematopoietic niche cells able to support hematopoietic stem cell (HSC) activity. We examined Alcam mRNA levels and cell surface expression by quantitative RT-PCR and flow cytometry in various hematopoietic stem and progenitor subsets. We found that Alcam is highly expressed in long-term repopulating HSC (LT-HSC), multipotent progenitors (MPP), and granulocyte/macrophage progenitors (GMP). We use an Alcam null mouse allele to assess the function of Alcam in HSC differentiation and self-renewal. Clonogenic colony-forming progenitor serial-replating assay show that the replating potential of Alcam-deficient LT-HSCs is impaired. An in vitro single-cell differentiation assay of phenotypic LT-HSCs reveals that Alcam-deficiency leads to an enhanced granulocytic differentiation. In competitive repopulation transplantation, Alcam-deficient cells show a transient engraftment enhancement, however, the engraftment is significantly lower in secondary transplantation, suggesting that the self-renewal capacity of Alcam-deficient HSC is compromised. We performed a limiting-dilution transplantation assay and determined that the frequency of long-term repopulating cells in Alcam-deficient bone marrow is significantly reduced compared to wild type control. We further assessed the engraftment efficiency of phenotypically purified LT-HSCs. We show that the engraftment efficiency of Alcam-deleted LT-HSCs is significantly reduced compared to wild type LT-HSCs. Since Alcam-deleted HSCs are able to home efficiently to the bone marrow cavity, the engraftment defect is not due to inefficient homing upon transplantation. Collectively, These studies implicate Alcam mediated cell-cell interaction in the regulation of hematopoietic transplantation and recovery.

Disclosures:

No relevant conflicts of interest to declare.
Lack of the Transcription Factor NFAT (Nuclear Factor of Activated T cells) c2 in Hematopoietic Progenitor Cells Results in Profound Hematological Abnormalities in Mice
Authors: Arabanian Laleh S. , Haase Michael , Habermann Ivonne , von Bonin Malte , Waskow Claudia , Ordemann Rainer , Ehninger Gerhard , Kiani Alexander
Published: October 2019

Abstract 1296

Understanding the transcriptional mechanisms that control hematopoiesis and the interaction between hematopoietic stem cells and the bone marrow microenvironment in vivo is of considerable interest.

We have previously shown that aged mice lacking the transcription factor NFATc2 develop bone marrow hypoplasia, anemia, and extramedullary hematopoiesis in spleen and liver. The proliferation and differentiation of NFATc2-deficient hematopoietic progenitor cells (HPC) ex vivo, however, was found to be intact. It remained therefore unclear whether the disturbed hematopoiesis in NFATc2-deficient mice was caused by the hematopoietic or the stroma component of the bone marrow hematopoietic niche.

In the current study we dissected the relative contribution of hematopoietic and stroma cells to the phenotype of the NFATc2-deficent mice by transplanting immunomagnetically purified NFATc2-deficient (ko) HPCs to lethally irradiated wildtype (wt) mice, and vice versa. After a posttransplantation period of 6–8 months, peripheral blood, bone marrow as well as spleen and liver of the transplanted animals were analyzed and compared to wt and ko mice transplanted with control cells.

Transplantation of NFATc2-deficient HPCs into wt recipients (ko → wt) induced similar hematological abnormalities as those occurring in non-transplanted ko mice or in ko mice transplanted with ko cells (ko → ko). Compared to wt mice transplanted with wt cells (wt → wt), ko → wt mice showed evidence of anemia, thrombocytopenia and a significantly reduced number of hematopoietic cells in their bone marrow. Likewise, ko → wt mice developped clear signs of extramedullary hematopoiesis in spleen and liver, which was not the case in wt → wt control animals.

Our data demonstrate for the first time, that NFAT transcription factors directly regulate the intrinsic function of hematopoietic progenitor cells in vivo. The transcriptional targets for NFAT in these cells are yet unknown and are the focus of further investigations.

Disclosures:

No relevant conflicts of interest to declare.
Tissue-Specific HIF-1 and HIF-2 Regulatory Elements and Their Roles in Erythropoietin Gene Expression
Authors: Yoon Donghoon , Song Jihyun , Kim Hyojin , Kim Bumjun , Shin Hyejin , Arnold Gregory , Elliott Steven , Prchal Josef T.
Published: October 2019

Abstract 1303

Studies of the regulation of erythropoiesis by hypoxic induction of erythropoietin (EPO) transcription led to the discovery of oligonucleotide binding motifs, hypoxia responsive elements (HREs) essential for hypoxic upregulation of EPO. HREs bind hypoxia-inducible transcription factors (HIFs). HIFs are heterodimers of 3 α subunit isotypes; HIF-1α, HIF-2α, and HIF-3α, and a common β-subunit. EPO is expressed in diverse tissues, some regulated mainly by HIF-2 and others by HIF-1. Tissue-specific regulatory elements (i.e. Kidney Inducible Element KIE, Negative Regulatory Element NRE, and Negative Regulatory Liver specific Element NRLE) of the EPO gene spanning several kilobases were reported. Our in silico analysis of the human EPO genome found two additional canonical HIF-binding elements in the KIE and NRE regions, and comparative analysis of phylogenically conserved sequences of Epo genes refined these HIF-binding elements from several kbs to a few hundred nucleotides and further delineated them as mKIE, mNRE1, mNRE2, and mNRLE2. To further define the structure and function of these elements in vivo, we exposed mice to 8% O2 hypoxia and monitored hypoxic induction of Epo mRNA levels in the liver, kidney, brain, and spleen. Spleen, kidney, and brain had a peak induction of the Epo transcript at 3 hours of hypoxia treatment, while liver showed peak induction at 6 hours. At the end of the hypoxia treatment, the mice were sacrificed and organs were harvested, and in vivo chromatin immunoprecipitation (ChIPs) was performed with antibodies against HIF-1α and HIF-2α. The results from these studies are summarized below.

“+” denotes presence and “−” absence of binding of HIF-1 and HIF-2. “Norm” - normoxia, “Hyp” - hypoxia.

To further define the role of these elements in tissue-specific Epo expression, we cloned and mutated these elements with reporter constructs, and generated transgenic mouse strains. The reporter gene was expressed in renal tubules, the red pulp area of the spleen, and liver hepatocytes. By creating a designed point mutation to disrupt the function of KIE, we suppressed the hypoxia response in the kidney, while mutations of NRE1 and NRE2 abolished reporter expression in the liver. Studies of HREs in the brain are ongoing.

In conclusion, we have refined the previously reported HREs to a few hundred bases (particularly, KIE is a 6 nucleotide bearing motif) and now demonstrate tissue-specific differential hypoxia-induced binding of HIF-1 and HIF-2 and their functional roles in EPO induction. This provides a background to examine the molecular basis of these elements in patients with aberrant EPO expression.

Disclosures:

Elliott: Amgen, Inc: Employment.
A “Master Regulatory Cis-element” Governs the Hematopoietic Stem/Progenitor Cell Compartment, Vascular Integrity, and Cardiovascular Development
Authors: Johnson Kirby D , Ryu Myung-Jeom , Boyer Meghan E , Kelez Sunduz , Zhang Jing , Lee Youngsook , Bresnick Emery H
Published: October 2019

Abstract 1304

The expression and activities of master regulatory proteins are exquisitely controlled to ensure the proper development of complex organisms. As a master regulator can orchestrate multiple developmental processes, presumably mechanisms involving a large ensemble of cis-elements and trans-acting factors are deployed to establish this control. GATA-2, a master regulator of hematopoiesis expressed in hematopoietic, endothelial, neuronal, and fat cells is likely subject to such complex regulation. GATA-2 occupies dispersed sites of the Gata2 locus, highlighting a positive autoregulatory mechanism. As GATA-1 levels rise during erythropoiesis, GATA-1 displaces GATA-2 from the Gata2 locus, instigating repression. “GATA switches” occur at five dispersed Gata2 sites (−77, −3.9, −2.8, −1.8 and +9.5 kb). Targeted deletion of GATA factor switch sites −2.8 and −1.8 revealed that these sites individually contribute to maximal Gata2 expression, but are otherwise dispensable for embryogenesis and steady-state hematopoiesis. Herein, we describe the targeted deletion of an intronic GATA switch site (+9.5) comprised of an E-box GATA factor composite cis-element. Strikingly, whereas the Gata2 gene knockout results in lethal anemia by E10.5, the +9.5 mutation yielded embryonic lethality around E13.5. The +9.5 mutation greatly reduced Gata2 expression in fetal livers, heart and endothelial cells, but not in the brain. Consequently, by E12.5 mutant mice exhibited severely reduced numbers of hematopoietic stem/progenitor cells (HSPCs) in fetal livers as assayed by cell sorting and colony assays. However, mutant mice were not overtly anemic and Ter119+ cells were abundant in the mutant fetal livers, accounting for the delay in lethality compared to the Gata2 knockout. Additionally, +9.5 mutant embryos displayed vascular/cardiovascular malformations and severe hemorrhaging. This work reveals that, unlike the −2.8 and −1.8 GATA switch sites, the +9.5 composite element functions as a “master regulatory cis-element” required for HSPC development, vascular integrity, and cardiovascular development. Genomic analyses identified a large cohort of loci containing conserved intronic composite elements resembling the +9.5 element, with heavy representation at genes critical for HSPC and vascular development/function, as well as novel genes of unknown function.

Disclosures:

No relevant conflicts of interest to declare.
Thymoglobuline Is As Effective As Lymphoglobuline in Japanese Patients with Aplastic Anemia Possessing Increased Glycosylphosphatidylinositol-Anchored Protein (GPI-AP) Deficient Cells
Authors: Yamazaki Hirohito , Saito Chizuru , Sugimori Naomi , Hosokawa Kohei , Kiyu Yu , Mochizuki Kanako , Takamatsu Hiroyuki , Nakao Shinji
Published: October 2019

Abstract 1339

Background:

Thymoglobuline (TG), a rabbit anti-thymocyte globulin (ATG) preparation which has more potent immunosuppressive effects than horse ATG Lymphoglobuline (LG), has been shown to be as effective as LG as both first and second line immunosuppressive therapy (IST) for aplastic anemia (AA) by several retrospective studies. However, recent prospective studies from the U.S. and Europe have shown a lower effectiveness of TG compared to horse anti-thymocyte globulin (ATG). The conflicting results may be due to the heterogeneity in the pathophysiology of AA or in the ethnic backgrounds of the patients evaluated. Small populations of glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient (GPI-AP−) blood cells are detectable in approximately 60% of patients with newly-diagnosed AA, and are associated with a good response to LG (Blood, 107: 1308, 2006). TG may have comparable effects on LG if the study subjects are limited to AA patients possessing GPI-AP− blood cells. Objectives/Methods: To evaluate the effectiveness of TG in Japanese patients with AA, we retrospectively analyzed the outcomes of TG therapy in 57 patients (25 males and 32 females) with AA (40 with severe AA and 17 with non-severe AA) aged from 17 to 84 (median 58) years. Peripheral blood obtained before therapy was examined for the presence of GPI-AP− cells using flow cytometry with liquid fluorescent aerolysin, which can accurately determine the percentage of GPI-AP− granulocytes, erythrocytes, and monocytes at percentages of less than 0.01%. The cut-off values were defined as >0.003% for granulocytes, >0.005% for erythrocytes, and >0.01% for monocytes based on the analytical results of 57 healthy individuals. Results: The cumulative rate of response after 6 months of TG therapy was 59%. Significant increases in the GPI-AP− cell percentages in at least one of the three lineages were detected in 38 (67%) of the 57 patients before therapy. The response rate in patients with increased GPI-AP− cells (PNH+ patients, 69%) was significantly higher than that in patients without GPI-AP− cells (PNH− patients, 35%, P=0.016). Seventeen percent of PNH+ patients achieved complete remission (CR) within 1 year, while only 12% of PNH− patients achieved a CR. 97% of PNH+ and 84% of PNH− patients were alive at the median time of 1 year after the therapy. The response rate in PNH+ patients was comparable to that in PNH+ patients treated with LG (70%) in our recent prospective study. Conclusions: TG is therefore considered to be as effective as LG in Japanese adult patients with AA, particularly in PNH+ AA patients where immune pathophysiology is definitely involved. Alternative therapies may thus need to be considered for PNH− patients.

Disclosures:

No relevant conflicts of interest to declare.
PU.1 Is a Downstream Target of C/EBPα in Normal Hematopoiesis and Acute Myeloid Leukemia
Authors: Bach Christian , Staber Philipp B. , Ye Min , Zhang Pu , Friedman Alan D. , Delwel Ruud , Tenen Daniel G.
Published: October 2019

Abstract 1353

The transcription factors PU.1 and C/EBPα are key regulators of hematopoietic cell differentiation. Tight and coordinated regulation of these factors is essential for normal hematopoiesis and even moderate alterations can lead to acute myeloid leukemia (AML). Previous studies established that in PU.1 knockout mice myeloid differentiation is blocked at an earlier stage compared to C/EBPα knockouts, consistent with PU.1 acting upstream of C/EBPα during hematopoietic differentiation. Recently, however, we and others identified a PU.1 upstream regulatory element (URE) which contains potential C/EBP binding sites. C/EBPα binds to the PU.1 URE in vitro and in vivo. Furthermore, C/EBPα transactivated the PU.1 proximal promoter in a URE dependent manner. We, therefore, hypothesized that PU.1 is a target gene of C/EBPα in hematopoietic cells.

To assess the role of PU.1 as a downstream target of C/EBPα in normal hematopoiesis we performed gene expression analysis in immature hematopoietic cells of conditional C/EBPα knockout mice (Mx1-Cre). Of note, we observed a strong reduction of PU.1 expression in hematopoietic stem cells (HSCs: CD150+CD48-LSK) after excision of C/EBPα, corroborating that PU.1 is a target of C/EBPα in murine HSCs in vivo. Moreover, lentiviral PU.1 expression alleviated the myeloid differentiation block of C/EBPα−/− KSL cells as evidenced by the differentiation to Gr-1 and Mac1 positive myeloid cells.

Targeted deletion of the PU.1 URE reduces PU.1 expression and induces myeloid leukemia. Additionally, inactivation of C/EBPα by various mechanisms is a common observation in many AML subtypes. Therefore, we tested if dysregulation of C/EBPα is associated with decreased PU.1 expression. Gene expression studies in several human AML cell lines revealed a positive correlation between C/EBPα and PU.1 expression. Furthermore, we analyzed expression of C/EBPα and PU.1 in a well characterized cohort of 285 AML patients. Importantly, PU.1 expression was strongly reduced in cases with either C/EBPα mutations or C/EBPα promoter silencing compared to other AML subtypes.

Taken together, our data support that PU.1 is a downstream target gene of C/EBPα in normal hematopoiesis as well as human leukemia. We currently develop a mouse model containing targeted mutations of three C/EBP binding sites in the PU.1 URE. This model will help to further pinpoint the functional impact of C/EBPα mediated regulation of PU.1 in different hematopoietic populations and to determine how this regulation may contribute to leukemia development in vivo.

The first two authors contributed equally to this work.

Disclosures:

No relevant conflicts of interest to declare.
PML/RARα -Regulated MicroRNA 181b Targets the Tumor Suppressor RASSF1A in Acute Promyelocytic Leukemia
Authors: Braeuer-Hartmann Daniela , Hartmann Jens-Uwe , Gerloff Dennis , Katzerke Christiane , Falzacappa Maria Vittoria Verga , Pellicci Pier Guiseppe , Mueller-Tidow Carsten , Tenen Daniel G. , Niederwieser Dietger , Behre Gerhard
Published: October 2019

Abstract 1361

In the acute promyelocytic leukemia (APL) bearing the translocation t(15;17), all-trans-retinoic acid (ATRA) treatment induces granulocytic maturation and complete remission of leukemia. Several factors are involved in the formation of the leukemic phenotype. Latest studies identified microRNAs as critical players in this network. In a micro array based microRNA screen we could identify miR-181b as downregulated in the APL cell line NB4 by treatment with pharmacological doses of ATRA. Additionally, we showed the transcriptional induction of miR-181b by the APL-associated PML-RARα oncogene which is released after treatment with ATRA. The overexpression of miR-181b by microRNA mimics leads to an inhibition of ATRA induced granulocytic differentiation. Furthermore, the downregulation of miR-181b by locked nucleic acids (LNAs) causes the induction of granulocytic transcription factor C/EBPβ. In a PML/RARα knock in mouse model we could show the enhanced expression of miR-181b. Also, APL patient samples show a significant enhanced expression of miR-181b in comparison to AML patient samples with normal karyotype. In addition, the miR-181b expression was strongly reduced in APL patient samples after ATRA treatment. In a microRNA target search we identified the novel ATRA regulated tumor suppressor RASSF1A as a putative target of miR-181b. In functional studies we showed that enforced expression of miR-181b reduces the protein level of RASSF1A by binding to the 3'UTR of RASSF1A mRNA. Accordingly, RASSF1A protein was enriched after knock down of miR-181b. The role of RASSF1A in ATRA induced differentiation was verified by knock down of RASSF1A protein by specific siRNA: Here we could show the reduction of ATRA induced CD11b expression. In conclusion, we identified miR-181b as a new player in the PML/RARα associated APL. Moreover, we firstly described the miR-181b target RASSF1A as a crucial factor in the ATRA activated granulocytic differentiation program in APL. Our data reveal the importance of deregulated miRNA biogenesis in cancer and may provide novel biomarkers and therapeutic targets in myeloid leukemia.

Disclosures:

No relevant conflicts of interest to declare.
PDGF-RTK Inhibition Restores CD95/Fas-Mediated Apoptosis in Leukemic T-LGL Via Promoting ASM Translocation, Ceramide Production and CD95 Signal Amplification
Authors: Yang Jun , Li Yongping , Loughran Thomas P.
Published: October 2019

Abstract 1374

Human memory T cells are protected from Fas/CD95-induced apoptosis. The mechanisms involved in blocking of CD95 signal transduction revealed the presences of anti-apoptotic factors. T-LGL leukemic cells are terminal effector memory T cells (Temra [CD45RA+/CD62L−]) featuring over-expression of Fas/Fas ligand, constitutive activation of PDGF-RTK and its downstream Src family kinase (SFK), PI3K/PKB/AKT/ERK1/2 signal cascade, as well as high expression levels of c-FLIP and Mcl-1. Inhibition of the downstream targets of PDGF-RTK pathway, such as SFK and PI3K induce significant level of apoptosis in leukemic LGL. However, direct targeting of PDGF-RTK showed a limited efficacy in induction of cell death in vitro. Loss of Fas sensitivity is the key pathological aberration in leukemic LGL, and restoration of CD95/Fas-mediated apoptosis in LGL leukemic cells remains a challenge. Therefore, reprogramming LGL leukemic cells to become sensitive to the already existing over-expressed Fas/Fas L in patients can be a potential therapeutic strategy. In the current study, we observed that inhibition of PDGF-RTK activity with inhibitors, such as AG1296 or imatinib myselate (Gleevec) restored CD95/Fas sensitivity in T-LGL leukemic cells. Our data suggest that the mechanism of PDGF-RTK inhibition restores Fas sensitivity in T-LGL leukemic cells via induction of acid sphingomyelinase (ASM) cell membrane translocation and activation, promoting ceramide production and Fas receptor aggregation in the cell membrane lipid rafts of leukemic cells. We also observed that PDGF-RTK inhibition significantly downregulates Mcl-1 expression in T-LGL leukemic cells. These observations suggest a possible role for future clinical application of TKIs in clinical management of this disease.

Disclosures:

No relevant conflicts of interest to declare.
Molecular and Cellular Effects of Oncogene Cooperation in a Genetically Adequate AML Model
Authors: Reckzeh Kristian , Bereshchenko Oxana , Mead Adam J. , Rehn Matilda , Kharazi Shabnam , Jacobsen Sten Eirik W , Nerlov Claus , Cammenga Jorg
Published: October 2019

Abstract 1379

Biallelic CEBPA mutations and FLT3 length mutations are frequently identified in human acute myeloid leukemia (AML) with normal cytogenetics. However, the molecular and cellular mechanisms of oncogene cooperation remain unclear due to a lack of disease models.

We have generated an AML mouse model using knock-in mouse strains to study cooperation of FLT3 ITD with commonly observed biallelic C/EBPa mutations which in this model are expressed from the original promoter and thereby at the right dose and at the correct level of cellular differentiation.

Interestingly, Cebpa mutants with FLT3 ITD progressed to AML with shorter latency than cohorts with Cebpa mutations only. Before onset of leukemia, biallelic Cebpa mutations caused depletion of granulocyte-macrophage progenitors (GMPs), and expansion of megakaryocyte-erythroid progenitors (MkP, pMegE, pCFU-E) and HSCs. However, FLT3 ITD attenuated MegE progenitor frequencies while MPPs and myeloid progenitors (pGMP and GMP) were more frequently observed. These cellular changes are accompanied by an upregulation of hematopoietic stem cell and STAT5 target genes. Both MPP and GMP cells contain the potential to induce AML similar to corresponding cells in human AML samples showing that this AML model resembles human disease. By gene expression analysis in premalignant populations we further show a role of FLT3 ITD in activating genes involved in survival/transformation and chemoresistance.

This murine AML model is perfectly suited to test the effects of chemotherapy and tyrosine kinase inhibitors blocking FLT3 ITD function on specific cellular populations to investigate their specific role in relapse of leukemia after complete remission.

Disclosures:

No relevant conflicts of interest to declare.
Ectopic Expression of the Leukemogenic Protein E2A-PBX1 in Early Hematopoietic Progenitors Blocks B-Lymphoid Commitment
Authors: Woodcroft Mark , Thompson Patrick , Slany Robert K. , LeBrun David
Published: October 2019

Abstract 1393

The oncoprotein E2A-PBX1 is produced by t(1;19)(q23;p13) in pre-B acute lymphoblastic leukemia (ALL). Perplexingly, in vitro and in vivo models of E2A-PBX1 leukemogenesis to date generate either myeloid or T-lymphoblasts, neither of which are representative of the human disease. Furthermore, we have observed recently that lineage-depleted (lin-) murine bone marrow cells infected with an E2A-PBX1 retrovirus selectively fail to repopulate the B-lymphoid compartment on transplantation into irradiated recipients. Therefore, we have now begun to investigate the mechanism by which enforced expression of E2A-PBX1 antagonizes B-lymphopoiesis.

We show that E2A-PBX1-transduced lin- fetal liver progenitor cells (FLPs) fail to differentiate to committed CD45R+ pro-B-cells when cultured with IL-7 in the presence of OP9 stromal cells. Instead these cells manifest a relatively immature immunophenotype (CD45R-, CD11b-, CD117+), are murine stem cell factor (SCF) dependent, and undergo myeloid differentiation upon stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) or transplantation into irradiated mice. Amino acid substitutions in E2A-PBX1 that impair co-activator recruitment or DNA binding completely rescue the differentiation block, implying a critical role for target-gene regulation by E2A-PBX1. E2A-PBX1-transduced cells fail to induce the B-lymphoid commitment genes Ebf1 and Pax5, and chromatin immunoprecipitation (ChIP) analysis shows aberrant persistence of the Polycomb silencing mark H3K27me3 at these promoters. Previous studies have identified the Polycomb gene Bmi1 as a candidate E2A-PBX1 target gene. However, enforced expression of Bmi1 neither blocked induction of Ebf1 or Pax5 nor impaired B-lymphopoiesis, indicating that Bmi1 is not sufficient to mediate blocked differentiation by E2A-PBX1. Gene expression microarray analysis identified cytokines (ex., Csf2, IL1a, Il6) and transcription factors (HoxA9, Tal1) up-regulated in E2A-PBX1-transduced FLPs that are known to antagonize B-lymphoid development.

In summary, our results support the notion that enforced expression of E2A-PBX1 in early hematopoietic progenitors blocks B-lymphopoiesis, to an important degree, by inducing the transcription of genes whose products antagonize early steps of B-lymphoid commitment, including induction of Ebf1 and Pax5. HoxA9 is particularly interesting as a mediator of E2A-PBX1 effects in view of its extreme induction ratio in our system (at least 1000-fold relative to controls), its propensity to promote myeloid over lymphoid differentiation, and its well-established role in leukemogenesis. Studies to investigate a potential requirement for HoxA9 and other candidates in mediating the E2A-PBX1-driven B-lymphoid differentiation block are ongoing.

Disclosures:

No relevant conflicts of interest to declare.
IRF-4 Suppresses BCR/ABL Transformation of Myeloid Cells in a DNA-Binding Independent Manner
Authors: Jo Seung-Hee , Ren Ruibao
Published: October 2019

Abstract 1404

Interferon regulatory factor 4 (IRF-4) is essential for B and T cell development and immune response regulation, and has both nuclear and cytoplasmic functions. IRF-4 was originally identified as a proto-oncogene resulting from a t(6;14) chromosomal translocation in multiple myeloma and its expression was shown to be essential for multiple myeloma cell survival. However, we have previously shown that IRF-4 functions as a tumor suppressor in the myeloid lineage and in early stages of B cell development. The finding of IRF-4 functioning as a tumor suppressor raises caution for developing therapies aiming to downregulate IRF-4. Further studies on the mechanisms by which IRF-4 functions as an oncoprotein and as a tumor suppressor are necessary to develop improved therapies for malignancies involving IRF-4. In this study, we found that IRF-4 suppresses BCR/ABL transformation of myeloid cells. To gain insight into the molecular pathways that mediate IRF-4 tumor suppressor function, we performed a structure-function analysis of IRF-4 as a suppressor of BCR/ABL transformation. We found that the DNA binding domain deletion mutant of IRF-4, which is localized only in the cytoplasm, is still able to inhibit BCR/ABL transformation of myeloid cells. IRF-4 also functions as a tumor suppressor in bone marrow cells deficient in MyD88, an IRF-4 interacting protein found in the cytoplasm. However, IRF-4's tumor suppressor activity is lost in IRF association domain (IAD) deletion mutants. These results demonstrate that IRF-4 suppresses BCR/ABL transformation by a novel cytoplasmic function involving its IAD domain and suggest that the oncogenic and tumor suppressor functions of IRF-4 involve distinct pathways.

Disclosures:

No relevant conflicts of interest to declare.
Pre-B Cell Receptor-Mediated Activation of BCL6 Induces Pre-B Cell Quiescence Through Transcriptional Repression of MYC
Authors: Nahar Rahul , Ramezani-Rad Parham , Mossner Maximilian , Duy Cihangir , Cerchietti Leandro , Geng Huimin , Jumaa Hassan , Ye B. Hilda , Melnick Ari , Muschen Markus
Published: October 2019

Abstract 1406

Background:

Pre-B cell receptor signaling is critical to induce initial expansion of the pool of differentiating B cell precursors but also mediates subsequent cell cycle exit and quiescence. Initial cell surface expression of the pre-B cell receptor induces a series of tyrosine phosphorylation events resulting in MYC and CCND2-mediated proliferation. After 2–5 divisions, however, large pre-BII (Fraction C') cells exit cell cycle to become resting, small pre-BII cells (Fraction D). While the signaling pathway resulting in pre-B cell receptor-induced proliferation is well understood, the mechanism by which pre-BII cells exit cell cycle, however, is currently unclear.

Results:

This checkpoint and cell cycle exit at the Fraction C'-D transition is critical for immunoglobulin light chain gene recombination and to prevent malignant transformation into acute lymphoblastic leukemia. Here we demonstrate that inducible activation of pre-B cell receptor signaling recapitulates the initial proliferative burst followed by cell cycle exit, which is characterized by strong upregulation of BCL6 and subsequent loss of MYC and CCND2 expression. ChIP-on-chip analysis revealed that the transcriptional repressor BCL6 is directly recruited to the MYC promoter. Inducible activation of pre-B cell receptor signaling in pre-B cells from BCL6-deficient mice failed to induce cell cycle exit and quiescence. We conclude that activation of BCL6 downstream of the pre-B cell receptor is crucial for the induction of quiescence at the Fraction C'-D checkpoint. As expected, inducible activation of BCL6 downstream of the pre-B cell receptor results in transcriptional repression of MYC and CCND2. Overexpression of MYC prevented pre-B cell receptor/BCL6-induced cell cycle exit.

Conclusion:

Hence, pre-B cell receptor signaling induces cellular quiescence of Fraction C' pre-B cells through activation of BCL6 and BCL6-mediated transcriptional repression of MYC and CCND2.

Disclosures:

No relevant conflicts of interest to declare.
Aurora Kinases in Childhood Acute Leukemia: The Promise of Aurora Kinase B As Drugable Target
Authors: Segers Stefanie A. , Zwaan C. Michel , Exalto Carla , Luijendijk Mirjam W.J. , Calvert Valerie S. , Petricoin Emanuel F. , Evans William E. , Reinhardt Dirk , Pieters Rob , Den Boer Monique L.
Published: October 2019

Abstract 1476

AIM:

Aurora kinases (AURK) A and B are known regulators of mitosis and are overexpressed in a large number of human cancers, including leukemia. Several AURK-inhibitors have shown anti-tumor activity in vitro and in vivo. However, the efficacy of AURK inhibition in the treatment of childhood acute leukemia is unexplored. We therefore investigated the effect of targeting AURKA and AURKB in leukemic cells of children with newly diagnosed acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

Materials & Methods:

Affymetrix gene expression data of 297 ALL, 237 AML and 8 normal bone marrow (nBM) samples were analyzed for AURKA and B mRNA expression levels. Protein expression levels in 172 pediatric ALL and 10 nBM samples were determined with a reverse phase protein array. Functional studies were performed in ALL and AML cell lines, in which AURKA and B were silenced using a short hairpin RNA with a lentiviral delivery system or LNA-containing oligonucleotides. Sensitivity of leukemic cell lines to the AURKB-selective inhibitor Barasertib-hQPA (AZD1152-hQPA) was tested in vitro with an MTS assay.

Results:

AURKA and B mRNA levels were low in ALL and AML patients. In contrast, Aurora A and B proteins were expressed to a greater extent in patients (p<0.0002), especially in ALL cases with an E2A-PBX1 translocation (p<0.0001) than in nBM mononuclear cells. Silencing of AURKA by shRNA and by LNA-oligonucleotide caused no or only minor growth delay in several cell lines reflecting genetic subtypes typically found in pediatric ALL and AML. In contrast, silencing of AURKB resulted in proliferation arrest and apoptosis in these cells. Furthermore, 18 out of 20 ALL and AML cell lines tested were highly sensitive to the AURKB-selective inhibitor Barasertib-hQPA in the nanomolar range (IC50 = 19–233 nM) whereas less sensitivity was seen for other inhibitors.

Conclusion:

These data show that inhibition of AURKB but not AURKA has an anti-proliferative and pro-apoptotic effect on acute leukemic cells. Thus, targeting Aurora Kinase B may offer a new strategy to treat pediatric ALL and AML.

Disclosures:

No relevant conflicts of interest to declare.
Six Cycles of BEACOPP Tailored Based on Interim Scintigraphy Provide Favorable Outcome for Patients with Standard and Advanced Risk Hodgkin Lymphoma and Female Fertility Is Preserved: Results of a 10-Year Follow-up
Authors: Dann Eldad J , Blumenfeld Zeev , Bar-Shalom Rachel , Avivi Irit , Ben-Shachar Menachem , Goor Odelia , Libster Diana , Gaitini Diana , Rowe Jacob M. , Epelbaum Ron
Published: October 2019

Abstract 1564

Background:

Therapy of Hodgkin lymphoma (HL) is designed to prolong survival and minimize toxicity; however, an intense debate is ongoing over whether ABVD or BEACOPP should be used upfront and whether a long-term survival benefit is obtained with the BEACOPP regimen. The current study was aimed to personalize therapy based on individual risk factors and interim scintigraphy. Design and Methods: 124 patients with newly diagnosed HL and adverse prognostic factors were prospectively studied between 7/1999 and 8/2005. Patients with early unfavorable and advanced disease were eligible for the study. Study participants were assigned to therapy based on their International Prognostic Score (IPS). Those with an IPS of ≥3 were assigned to 2 cycles of escalated BEACOPP (EB). All others received 2 cycles of standard BEACOPP (SB). Subsequent therapy was prospectively allocated according to the results of early interim GA67 or PET/CT. Four cycles of EB or SB were administered following a positive or negative scan, respectively. Results: Complete remission (CR) rate, 10-year progression free (PFS) and overall survival (OS) were 97%, 87% and 88%, respectively, at a median follow-up of 89 months (5–144). Only 38% of patients, mostly with a bulky mediastinal mass, received radiation therapy. PFS and OS were similar in both groups. Predictive value of negative interim Ga67 or PET/CT was 87% and 93%, respectively. Fertility status was assessed in 38 females aged <40 years (Table). Twenty four of these patients received 6 cycles of SB, 8 patients had 2 cycles of EB and 4 cycles of SB, 3 patients were treated with 2 cycles of SB and 4 cycles of EB and one patient received 6 cycles of EB. Thirty six females did not have disease progression and 34 of them were found to preserve their cyclic ovarian function (COF). Twenty six females were co-treated with 6 cycles of GNRH agonist triptorelin (Decapeptyl 3.75 mg) for 6 months. 19 patients conceived during the follow-up (30 pregnancies), delivering 23 babies. Deliveries were reported for up to seven years from diagnosis. Notably, the female receiving 6 cycles of EB, co-administered with the GNRH agonist, was among those who conceived and delivered a healthy baby. Conclusion: Six cycles of tailored BEACOPP administered to HL patients with adverse prognostic factors provide encouraging long-term PFS and OS, and fertility is preserved in most females following reduction in the number of chemotherapy cycles and GNRH agonist co-administration.

Disclosures:

Off Label Use: GNRH agonist triptorelin was simultaneously administered for fertility preservation.
Clonal Evolution but Not Variant Chromosomal Translocations Is An Adverse Prognostic Marker for Cytogenetic Response and Survival in CML Patients Treated with Imatinib
Authors: Tantiworawit Adisak , Abou Mourad Yasser , Barnett Michael J , Broady Raewyn , Hogge Donna E. , Nantel Stephen H. , Narayanan Sujaatha , Nevill Thomas J. , Power Maryse M. , Shepherd John D. , Song Kevin W. , Sutherland Heather J. , Toze Cynthia L. , Forrest Donna L.
Published: October 2019

Abstract 1694

Background:

Clonal evolution (CE) and variant Philadelphia (Ph) chromosomal translocations (vPh) are seen in <10% of newly diagnosed CML patients. While the vPh is thought to have no prognostic significance, CE maybe associated with an inferior outcome compared to the presence of a single standard Ph for patients treated with imatinib (IM). This study aims to evaluate the prognostic significance of vPh and clonal evolution in a cohort of CML chronic phase (CP) and accelerated phase (AP) patients treated with IM.

Patients and methods:

From June 1999 to December 2008, 247 Ph+ CML CP and AP patients treated with IM (300–600 mg daily) were analyzed. Patients having had a prior allogeneic stem cell transplant were excluded. Patients were categorized into 4 groups according to karyotype at diagnosis: Group 1 (CP with CE only, n=28); Group 2 (AP, n=31); Group 3 (CP with vPh, n=20); Group 4 (CP with standard Ph, n=168). Cytogenetic response, treatment failure, event free survival (EFS) and overall survival (OS) were calculated for each group and multivariate analysis was performed to determine potential variables predictive of response and survival.

Results:

Groups 1 and 2 had lower complete cytogenetic responses (CCR) to IM and higher rates of treatment failure compared to Group 4. (Table1). At a median follow up 4.2 years, Groups 1 and 2 had lower median OS and EFS compared to Groups 3 and 4 (Table1). The 5 year probabilities of OS and EFS were 79 % and 53 % (Group 1), 77 % and 73 % (Group 2), 94 % and 87 % (Group 3), and 90 % and 83 % (Group 4). (Figures 1 and 2).

In multivariate analysis, the only significant factors predictive of CCR were Sokal score (p=.01), CE at diagnosis (p=.03) and dose of IM (p=.03). When the analysis was restricted to EFS and OS, the presence of CE at diagnosis (p<0.0001), prior treatment before IM (p=0.04), the dose of IM (p=0.01) and the response to IM (p<0.0001) were the factors associated with significant EFS and the presence of CE at diagnosis (p=0.004), the dose of IM (p=0.05) and the response to IM (p<0.0001) were predictive of OS. The presence of a vPh had no effect on EFS and OS.

Conclusion:

The presence of CE at diagnosis and/or features of AP in patients with CML treated with IM are associated with an inferior prognosis, but the presence of the vPh does not appear to have any prognostic significance. It remains to be determined whether the second generation TKI's can overcome the negative influence of CE on EFS and OS.

Disclosures:

No relevant conflicts of interest to declare.
Suppression of Murine Platelet Activation by the β Isoform of the Catalytic Subunit of Protein Phosphatase 2B
Authors: Gushiken Francisca C. , Alrehani Nawaf , Pradhan Subhashree , Kailasam Lavanya , Rumbaut Rolando , Vijayan K. Vinod
Published: October 2019

Abstract 190

Signal transduction mediated by the kinases and phosphatases are critical for platelet activation at the site of vascular injury. Compared to the kinases, a role for phosphatases in platelet activation is less understood. Our previous studies have focused on the roles of serine/threonine protein phosphatase 1 (PP1) and 2A (PP2A) in regulating integrin αIIbβ3functions. However, platelets also express protein phosphatase 2B (PP2B) and its role in platelet function is unexplored. PP2B-Aα and PP2B-Aβ constitute two ubiquitous isoforms of the PP2B catalytic subunit. Due to the general concerns regarding the specificity of the PP2B inhibitors, we have utilized mice deficient in the β isoform of the catalytic subunit of PP2B (PP2B-Aβ) to explore the role of PP2B in platelet functions. Mice lacking PP2B-Aα are short lived and are not considered in this study. Loss of PP2B-Aβ did not cause any compensatory increase in the PP2B-Aα levels in platelets. Compared to the wild type (WT) platelets, PP2B-Aβ−/− platelets displayed increased aggregation in response to low doses of protease-activated receptor 4-activating peptide (PAR4-AP), ADP, collagen and collagen related peptide (CRP). Enhanced α granule secretion in response to the low doses of PAR4-AP and CRP was noticed in PP2B-Aβ−/− platelets, relative to the WT platelets. Functions regulated by the outside-in αIIbβ3 integrin signaling like adhesion to immobilized fibrinogen and fibrin clot retraction were enhanced in the PP2B-Aβ−/− platelets. These studies indicate that PP2B-Aβ negatively regulate platelet functions in vitro. Consistent with these observations, PP2B-Aβ−/− mice exhibited a shorter tail bleeding time compared to the WT mice. In a FeCl3 induced endothelial denudation injury model, PP2B-Aβ−/− mice showed decreased time to occlusion in the carotid artery, and reduced number of emboli compared to the WT mice. These studies indicate that PP2B-Aβ suppress multiple murine platelet functions that contribute to an occlusive thrombi. Unlike a positive thrombus promoting role for the PP1cγ that was noticed in our previous study, PP2B-Aβ suppressed murine platelet activation, suggesting that different subtypes of Ser/Thr phosphatases have distinct roles in murine platelet activation.

Disclosures:

No relevant conflicts of interest to declare.