bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abcb10 Physically Interacts with Mitoferrin1 to Enhance Its Stability for Heme Synthesis in the Erythroid Mitochondria
Authors: Chen Wen , Li Liangtao , Langer Nathaniel B. , Paradkar Prasad N. , Schultz Iman J. , Hyde Brigham B. , Shirihai Orian S. , Kaplan Jerry , Paw Barry H. 
Published: April 2020

Abstract
We previously described a zebrafish mutant, frascati (frs), with hypochromic anemia and erythroid maturation arrest. Using positional cloning, we identified mitoferrin1 (mfrn1, slc25a37) as the gene disrupted in frs mutants (Shaw GC, et al. 2006 Nature 440:96–100). Mfrn1, the principle iron-importer in the mitochondria, is essential for heme and iron-sulphur (Fe/S) cluster syntheses in erythroblasts, and is required for primitive and definitive erythropoiesis. The biochemistry for Mfrn1-mediated iron acquisition into the mitochondria of developing erythroblasts, however, is poorly understood. In order to identify Mfrn1-associated proteins involved in mitochondrial iron homeostasis, we employed the strategy of in vivo epitope-tagged affinity purification and mass spectrometry (MS). A “bait protein,” Biotag-mouse Mfrn1 (BT-Mfrn1), was engineered to affinity-purify the associated proteins. A series of control experiments were first conducted to demonstrate that the BT-Mfrn1 protein properly targeted to the mitochondria and functionally complemented the anemia in frs embryos. A two-dimensional Blue Native gel followed by SDS-PAGE and western blot showed Mfrn1 forms higher-order protein complexes with interacting proteins in the mitochondrial fraction. A stable mouse erythroleukemia (MEL) clone expressing BT-Mfrn1 protein was derived as a source for protein purification. Affinity-purified BT-Mfrn1 protein complexes were analyzed by MS in triplicate runs in comparison to control MEL cells. Abcb10, a GATA-1 induced ATP-binding cassette transporter highly expressed in hematopoietic tissues and involved in heme biosynthesis (Shirihai OS, et al. 2000EMBO J.19:2492–2502), was found as one of the proteins which physically associated with Mfrn1 during MEL differentiation. The Abcb10:Mfrn1 interaction was confirmed by immunoprecipitation (IP) and western analysis with endogenously expressed proteins in MEL cells and proteins expressed by transient transfection in heterologous cells. Mfrn1 was previously shown to have longer protein half-life in differentiated MEL cells compared to undifferentiated cells (Paradkar PN, et al. submitted). To test our hypothesis that the Abcb10:Mfrn1 interaction may enhance Mfrn1 protein stability, a pulse-chase assay using 35S-methionine labeled cells was performed, followed by IP. Abcb10 was found to stabilize Mfrn1 protein in COS7 cells co-transfected with Abcb10 and Mfrn1 compared to control cells transfected with Mfrn1 alone. Similar results were obtained when total steady-state Mfrn1 protein was evaluated by western analysis comparing lysates from Abcb10:Mfrn1 cotransfected and Mfrn1 transfected cells. Our data suggest that Abcb10 physically interacts with Mfrn1 to enhance its stability and promote Mfrn1-dependent mitochondrial heme biogenesis.
Meningeal Dissemination (MD) in Primary CNS Lymphoma (PCNSL): Multimodal Diagnosis, Presentation and Outcome in a Large Monocenter Patient Cohort
Authors: Kiewe Philipp , Korfel Agnieszka , Fischer Lars , Martus Peter , Thiel Eckhard 
Published: April 2020

Abstract
Introduction: The frequency of meningeal dissemination (MD) in primary central nervous system lymphoma (PCNSL) reported in the literature differs. The optimal management and the prognostic impact of MD have not been defined.
Methods: Data on MD from all 92 immunocompetent patients primarily diagnosed with PCNSL at our institution between January 1994 and February 2007 were retrospectively analysed for MD evaluation. All patients received cranial MRI or CT, and in 71 patients CSF was obtained by lumbar puncture. High-dose methotrexate was the intended treatment in all patients.
Results: Cerebrospinal fluid (CSF) was obtained by lumbar puncture in 71 patients (median age 62 years) of a total of 92 patients with PCNSL. Cytomorphologic CSF examination was performed in 63, immunophenotyping in 32 and PCR of the CDR3 region in 37 patients. Eight of 63 patients (13%) had positive cytomorphology, one of 32 (3%) positive immunocytology and 6 of 37 (16%) had monoclonal B-cells on PCR analysis. Evidence of MD on MRI or CT was found in three of the 71 patients (4%). Altogether, 11 (16%) patients had evidence of MD diagnosed by either of the methods used. Median CSF cell count was 5/μl (range, 0–237; n=66), and median protein concentration 76 mg/dl (range, 93–4117; n=55). An elevated cell count (&gt;5/μl) was found in 27 (41%) patients, an elevated protein (&gt;45 mg/dl) in 44 (80%). No significant correlation was found between proof of MD and CSF pleocytosis or elevated protein. Patients with MD did not differ from other patients with respect to age, gender, clinical presentation, performance status, CSF cell count and protein concentration, number of intracerebral lesions, lesion location or histology. After a median follow-up of 56 months, median EFS was 26 (95% CI, 4.3 to 47.7) months for MD patients and 34.1 (95% CI, 26.2 to 42) months for those without MD, p=0.26 (log-rank). Of 21 patients with relapse after initial treatment response, 16 had isolated cerebral relapse, one isolated meningeal relapse, two systemic relapse (one with cutaneous, and one with testicular manifestation), and one concomitant systemic (lung/thoracic wall) and cerebral relapse. Of the four MD patients with relapse after initial response, three had cerebral relapse, and one had systemic (cutaneous) relapse. The median overall survival (OAS) of patients with MD was 45.5 months (95% CI, 16.6 to 74.4), of those without MD 42.5 months (95% CI, 35.8 to 49.2) (p=0.27).
Conclusions: The frequency of MD detected in our cohort was low despite the use of highly sensitive diagnostic methods. No impact on either EFS or OAS was seen for MD.
CD3-Negative CD4+ T-Cells: A Useful Diagnostic Tool with High Specificity in Angioimmunoblastic Lymphadenopathy (AILD)-Type T-Cell Lymphoma
Authors: Singh Anju , Flörcken Anne , van Lessen Antje , Dörken Bernd , Pezzutto Antonio , Westermann Jörg 
Published: April 2020

Abstract
Angioimmunoblastic lymphadenopathy (AILD)-type T-cell lymphoma is one of the common T cell lymphomas in Western countries. Many patients present with symptoms of a systemic disease and diagnosis can often be challenging. Particularly in cases in which histological confirmation cannot be easily achieved flowcytometry of peripheral blood can give important clues for the differential diagnosis of AILD. We have previously reported that CD4-negative CD3+ T-cells in peripheral blood are a characteristic immunophenotypic finding in AILD patients. Gene scan analysis for the TCR gamma chain and intracellular CD3 expression clearly showed that these CD3-negative CD4+ cells are of T cell origin. In this study we further evaluated the phenotype of the CD4− CD3+ T cells and we compared their frequency in AILD patients with the frequency in patients with other histologically confirmed leukemic T cell lymphomas. 14 patients with AILD (clinical stage III/IV) were compared with 26 patients with other subtypes of leukemic T cell lymphomas (6 T-large granular lymphocyte leukemias (T-LGL), 4 T-chronic lymphocytic leukemias/prolymphocytic leukemias (T-CLL/PLL), 5 anaplastic large cell lymphomas (ALCL), 1 adult T-cell leukemia/lymphoma (ATLL), 1 mycosis fungoides and 9 peripheral T-cell lymphomas. Flowcytometric analysis of peripheral blood was performed in a lymphocyte gate using fluorochrome-labeled antibodies against CD3, CD2, CD4, CD5, CD7, CD8, CD16, CD56, CD57 and TCR. In 14/14 AILD patients a small but distinct population of CD3-negative CD4+ T cells was found (mean percentage of CD3-negative CD4+ T cells in the gate: 12,0 ± 17,6 %, range 0,05 – 51,8 %). In contrast, CD3-negative CD4+ T cells could be detected in only 1/26 patients with other leukemic T cell lymphomas. This patient had been diagnosed with mycosis fungoides. Further immunophenotypic analysis showed that the aberrant T cells in AILD also express pan T cell markers, such as CD2, CD5 and partially CD7, surface TCR expression could not be detected. In conclusion our comparative study shows that flowcytometric detection of CD3-negative CD4+ T cells in peripheral blood is a characteristic feature of AILD with a high specificity. Therefore, flowcytometry is particulary useful in the differential diagnosis of AILD, even if the aberrant T cell population has a very low frequency. Although AILD is mostly CD3+ on immunohistochemistry, the presence of low numbers of CD3-negative CD4+ T cells in peripheral blood appears to be a typical feature of AILD. Cytoplasmic CD3-expression is one possible explanation for this discrepancy. Since complete or partial loss of pan T cell antigens is a characteristic feature of many T cell lymphomas, the aberrant CD3-negative CD4+ T cells might also represent just a subfraction of lymphoma cells in AILD. Further biological characterization of this subpopulation should be performed.
Cholesterol Rich-Domains Regulate VEGFR-1 (FLT-1) Expression and Signaling in Acute Leukemia Cells
Authors: Casalou Cristina , Gomes Ana , Carvalho Tania , Dias Sergio 
Published: April 2020

Abstract
Vascular endothelial growth factor receptors -1 (FLT-1) and -2 (KDR) are expressed by subsets of acute and chronic leukemias, where they signal in paracrine and/or autocrine manner to induce cell survival, proliferation and migration. We have previously shown that acute lymphocytic leukemia migration in response to VEGF via FLT-1 modulates the onset of extramedullary disease, and thus has clinical predictive value (Fragoso et al, Blood 2006). Acute leukemia cell (AML) migration, induced by PlGF/VEGF activation of FLT-1 results in the formation of actin membrane protrusions with concomitant increased ERK1/2 and P38 phosphorylation and activation of Rho-GTPases (Casalou et al, 2007). Since we have found an in vitro association of FLT-1 with caveolin-1, actin and HSp90, we hypothesised that cholesterol-rich domains might regulate FLT-1 mediated survival, proliferation or migration of acute myeloid (AML) and lymphoid (ALL) leukemias. First we found by FACS and RQ-PCR that FLT-1 expression is up-regulated by increased cholesterol/HDL levels in vitro. As shown by sucrose gradient fractionation and western blotting, PlGF/VEGF stimulation of AML cells results in re-localization of FLT-1 to cholesterol-rich domains. Accordingly, FLT-1 localization within cholesterol-rich domains is abrogated by exposing leukemia cells to b-methyl-cyclodextrin (MbCD) which removes intracellular cholesterol. Additionally, FLT-1 phosphorylation is abolished by treatment of AML cells with MbCD or Nystatin, an inhibitor of lipid raft endocytosis. Functionally, AML cells exposure to high levels of total cholesterol/HDL for 24 hours exerted a protective effect from actinomycin D-induced apoptosis and promoted PlGF/VEGF-induced AML migration in transwell migration assays. Together, these results show that on subsets of acute leukemias cholesterol/HDL cellular-content regulates FLT-1 expression and signalling, resulting in decreased apoptosis and induction of cell migration. In vivo, we show that cholesterol-rich diet significantly increases bone marrow VEGF levels in mice; inoculation of FLT-1 expressing acute leukemias into mice fed with cholesterolrich diet significantly accelerated disease progression and worsened disease outcome. Taken together, our data show the molecular basis by which cellular and systemic cholesterol regulates VEGF and VEGFR-1 signalling on subsets of acute leukemias, modulating cell migration and survival and thereby regulating disease progression.
The Transmembrane Domain of PDGFR-β Plays An Important Role in ETV6-PDGFR-β Activation
Authors: Toffalini Federica , Demoulin Jean-Baptiste 
Published: April 2020

Abstract
The ETV6-PDGFRβ hybrid protein (EPβ) is found in a subset of patients with chronic myelomonocytic leukemia (CMML) associated with eosinophilia. EPβ is the archetype of a larger group of hybrid receptors that are produced by chromosomal translocations of PDGFR genes and cause atypical myeloproliferative neoplasms. In EPβ, the N-terminal portion of the ETV6 transcription factor replaces the PDGFRβ ligand-binding domain and induces the activation of the chimera through its pointed domain (PNT domain, also called helix-loop-helix or SAM domain). This domain mediates ligand-independent EPβ oligomerization, resulting in constitutive tyrosine kinase activity. In addition, we showed that EPβ and other hybrid receptor tyrosine kinases are much more stable than wild-type receptors and that the deletion of the PNT domain induced EPβ protein degradation, suggesting a link between the clustering and the stabilization of the EPβ protein. The PDGFRβ transmembrane domain (TM domain) is retained in EPβ and in most PDGFRβ hybrid proteins that have been described. We observed that the deletion of the TM domain (EPβ-ΔTM mutant) strongly impaired the ability of EPβ to sustain growth factor-independent proliferation of Ba/F3 and 32D cells. The phosphorylation of the mutant protein was also markedly reduced. We confirmed that EPβ is not inserted in membranes but resides in the cytosol, indicating that the PDGFRβ TM domain does not act as a transmembrane domain in EPβ but has a completely different function. The EPβ-ΔTM mutant retained the ability to self-associate in co-immunoprecipitation experiments, but showed a decreased level of polymerization when using cross-linking agents, suggesting that this domain is required for optimal clustering of EPβ. In line with our findings on the PNT domain, the EPβ-ΔTM protein was less stable and, as a result, was expressed at a lower level. In conclusion, we demonstrate that the TM domain plays a role in EPβ activation by promoting the clustering of the protein and by preventing its degradation in cooperation with the PNT domain.
MDM2 SNP 309 Is Not Associated with Increased Expression Levels of MDM2 Nor Clinical Significance in Childhood Acute Lymphoblastic Leukemia
Authors: He Jing , Gu Lubing , Zhou Muxiang 
Published: April 2020

Abstract
Previously, we reported that MDM2 overexpression by pediatric acute lymphoblastic leukemia (ALL) cells is linked to a poor response to chemotherapy and early relapse of disease. It was recently shown that a single nucleotide polymorphism (SNP) in the MDM2 promoter, SNP309, was related to increased MDM2 expression and poor outcomes for various malignancies. The present retrospective study investigates the association of SNP 309 with MDM2 expression levels and clinical significance in pediatric ALL. Data obtained from 81 children with ALL showed a similar genotype distribution of SNP 309 as seen in other types of cancer and healthy populations: 37 T/T (46%), 37 T/G (46%) and 7 G/G (8%). However, this SNP did not seem to control MDM2 mRNA expression in ALL. We observed that 20 of the 81 ALL cases overexpressed MDM2 mRNA (&gt; 10-fold higher than in normal bone marrow mononuclear cells), of which 8 were SNP 309 T/T, while 12 were T/G (10) or G/G (2) (p&gt;0.5). Furthermore, we found that both MDM2 mRNA and protein were undetectable in 10 of 37 T/G and 3 of the 7 G/G cases. In addition, there were no associations found between SNP 309 and the in vitro response to doxorubicin, nor and early relapse in the 81 ALL patients. Upon review of 23 ALL cell lines, we found there was a relatively higher percentage of T/G (57%) than in fresh ALL samples (46%). However, the expression level of MDM2 was not related to SNP 309, but was rather closely associated with p53 status: All 11 ALL lines with wild-type p53 overexpressed MDM2 and all 12 ALL lines with mutant p53 or with a p53-null phenotype expressed no or low levels of MDM2, regardless of their SNP 309 status. These results suggest that in pediatric ALL, the SNP 309 status affects neither the expression of MDM2 nor the clinical prognosis.
The Role of MLL in DNA Damage Checkpoint and Its Implication in Leukemogenesis
Authors: Liu Han , Westergard Todd D , Chen David Y , Cheng Emily H.-Y. , Hsieh James J.-D. 
Published: April 2020

Abstract
Cell cycle checkpoints are implemented to safeguard our genome and the deregulation of which results in human cancers. Hence, it is of great significance to discover and investigate novel key constituents of the mammalian DNA damage response network. Human chromosome band 11q23 translocation disrupting the MLL gene leads to poor prognostic leukemias. MLL is a transcription co-activator that maintains HOX gene expression. The importance of HOX gene deregulation in MLL leukemogenesis has been intensively investigated. However, physiological murine MLL leukemia knockin models have indicated that incurred HOX gene aberration alone is insufficient to initiate MLL leukemia. Thus, additional signaling pathway must be involved, which remains to be discovered. Our recent studies demonstrated an intimate relationship between MLL and the cell cycle(Takeda et al. 2006, Genes & Development, 20, 2397–2409; Liu et al. 2007, Genes & Development, 21, 2385–2398). More importantly, our studies uncovered a critical role of MLL in executing the S phase checkpoint. We showed:
Over-expression of MLL induces an S phase block. MLL accumulates in the S phase upon DNA damage. MLL deficiency results in radioresistant DNA synthesis (RDS) and chromatid-type chromosomal abnormalities, two signature characteristics of S phase checkpoint defects.
We further determined the underlying mechanisms concerning the DNA damage-induced MLL accumulation. Our data showed that MLL is phosphorylated after DNA damage, which in turn blocks its degradation by SCFSkp2 in the S phase and results in the ultimate accumulation. Our data revealed the link between MLL and the S phase checkpoint, which provides novel insights into the mammalian cell cycle checkpoint network and human leukemia pathogenesis. Future studies utilizing murine leukemia models will be performed to examine whether MLL translocation compromises the S phase checkpoint and if the resulted dysfunction contributes to MLL leukemogenesis.
Loss of Growth Arrest DNA Damage 45b (GADD45b) Enhances Oncogenicity in BCR/ABL-Induced Chronic Myelogenous Leukemia
Authors: Sha Xiaojin , Hoffman Barbara , Liebermann Dan A. 
Published: April 2020

Abstract
Growth arrest DNA damage 45b (GADD45b) is upregulated during myeloid lineage terminal differentiation. It is also involved in G2/M cell cycle arrest and apoptosis in response to exogenous stress stimuli. To investigate the effect of GADD45b in the development of leukemia, lethally irradiated mice were reconstituted with either wildtype or gadd45b null myeloid progenitors which retrovirally expressed 210-kD BCR/ABL fusion oncoprotein. We found that both wildtype and gadd45b null myeloid progenitors expressing BCR/ABL induced chronic myelogenous leukemia (CML)-like disease between 11days to 22days after bone marrow transplantation in recipients. However, gadd45b null recipients had a shorter latency (11days–19days) compared to those of wildtypes (14days–22days). This result implies that GADD45b acts as a supressor of CML. Data will be presented on further analyzing the phenotypes of these mice.
Lack of Drug-Drug Interaction Between ATI-5923, a Novel Vitamin K Epoxide Reductase Inhibitor, and Amiodarone, in Beagle Dog
Authors: Choppin Anges , Irwin Ian , Lach Leang , Davies Adrian , Shao Lei , Becker Cyrus , Palme Monica P. , Paliard Xavier , Bowersox Scott , Dennis Donn , Druzgala Paliard 
Published: April 2020

Abstract
ATI-5923 is a novel oral anticoagulant that is being developed to prevent thromboembolic events associated with atrial fibrillation, prosthetic heart valves, and deep venous thrombosis. Like warfarin, it is a vitamin K epoxide reductase inhibitor. Unlike warfarin, which is metabolized by CYP2C9 and CYP3A4, ATI-5923 is metabolized by carboxylesterases. Our hypothesis is that ATI 5923 will lead to superior INR (international normalized ratio) control compared to warfarin as it will not be subject to pharmacokinetic interactions observed with many of the drugs that affect warfarin metabolism, leading to either excessive or suboptimal anticoagulation resulting in bleeding or thrombotic complications, respectively. These studies compared the efficacy of ATI-5923 to that of warfarin, when administered either intravenously (IV) or once a day orally, to produce stable anticoagulation in beagle dogs. The drug-drug interaction potential was assessed using co-administration with amiodarone, an inhibitor of CYP3A4 and CYP2C9, with either ATI-5923 or warfarin. Effects on coagulation were assessed by measuring Factor VII and X levels and prothrombin times (PT). Continuous IV infusions (5 to 9 days per dose) and oral administration of ATI 5923 and warfarin (5 to 10 days per dose) caused a dose-dependent decrease in plasma concentrations of Factor VII and Factor X, and an associated increase in PT. On a dose basis, ATI 5923 was a more potent anticoagulant than warfarin. Intravenous fresh frozen canine plasma or subcutaneous vitamin K1 treatment reversed the anticoagulant effects of orally-administered ATI-5923 in adult beagle dogs. Co-administration of amiodarone (40mg/kg for 2 days, 20mg/kg for 6 days) significantly increased the plasma levels and anticoagulation effect of warfarin (0.25mg/kg) in beagle dogs, as measured by increase PT and decrease in factor VII and X. In contrast, amiodarone had no effect on the anticoagulation parameters and plasma levels of ATI-5923 (0.3mg/kg). We conclude that ATI-5923, via a vitamin K-dependent mechanism, causes changes in key parameters of hemostasis in beagle dogs that are consistent with effective anticoagulation but without the important drug-drug interaction commonly observed with amiodarone in warfarin-treated patients.