bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lack of SSBP2, a Candidate Leukemia Suppressor, Promotes T Cell Lymphomagenesis in p53 Null Mice.
Authors: Wang Yang , Patel Mayur , Ma Xiaoping , Liang Hong , Brandt Brandt , Agulnick Alan , Nagarajan Lalitha 
Published: April 2020

Abstract
Our laboratory positionally cloned a candidate myeloid leukemia suppressor gene, sequence-specific single-stranded DNA binding protein 2 (SSBP2) from chromosome 5q13.3-5q14.1 interval. SSBP2 expression is lost in primary acute myelogenous leukemia (AML) and myelodysplasia (MDS) cells. AML cells induced to express SSBP2 show growth arrest and partial differentiation. SSBP2 directly binds the adaptor protein Ldb1 (LIM domain-binding protein1), which in turn binds LIM domain containing nuclear factors. Although LDB1, SSBP2 containing transcriptional complexes are ubiquitously expressed, they have been best characterized in erythroid differentiation. Stabilization of Ldb1 by its direct interaction with SSBP2 upregulated the transcriptional activity of the Ldb1-Lmo2-E2A/TAL1-containing complex on an erythroid specific gene promoter. Here, we report the generation and characterization of Ssbp2 null mice. Mice with homozygous disruption of Ssbp2 (Ssbp2−/−) are viable and fertile, with no gross hematopoietic abnormalities observed up to six months of age. In order to examine if and how loss of SSBP2 promotes transformation, we crossed Ssbp2−/− mice with p53−/− null mice. Ssbp2−/−, p53−/− mice succumbed to thymic lymphomas with a median survival of 122 days compared to a median survival of 182 days for p53−/− mice (P = 0.0002). Moreover, the spectrum of tumors observed in the Ssbp2−/−, p53−/− mice included 62.5% thymic lymphomas (20/32), whereas only 26.7% of the p53−/− mice developed thymic lymphomas (8/30). In addition, Ldb1 protein levels but not transcript levels were decreased in the thymi of Ssbp2−/−, and Ssbp2−/−, p53−/− mice. Thus, loss of Ssbp2 appears to abrogate Ldb1-Lmo2-mediated T cell differentiation by promoting Ldb1 degradation. Such a block in differentiation cooperates with the loss of cell cycle arrest and apoptosis due to loss of p53. Our findings demonstrate for the first time a functional cooperation between the candidate leukemia suppressor SSBP2 and tumor suppressor p53. Furthermore, these results suggest the Ldb1-Lmo2 complexes to be novel targets for therapeutic intervention in hematologic malignancies.
BCL6 Transcriptionally Reprograms the DLBCL Genome by Forming Distinct Biochemically Active Complexes on Genes Corresponding to Different Pathways.
Authors: Polo Jose M. , Chatzi Katerina , Dell’Oso Tania , Lev Paola , Melnick Ari 
Published: April 2020

Abstract
Aberrant gene expression is a hallmark of cancer, and so it is not surprising that the most common category of oncogenes and tumor suppressors involved in hematologic malignancies are transcription factors. These factors mediate their effects by nucleating biochemically active cofactor complexes to modify the chromatin structure of their respective target genes. BCL6 is a transcriptional repressor and the most commonly involved oncogene in diffuse large B-cell lymphomas. BCL6 represses genes by recruiting several corepressor complexes including SMRT, N-CoR, BCoR; all of which bind to BCL6 through its BTB domain. Each of these complexes has different biochemical functions (e.g. BCoR forms a polycomb complex vs. SMRT which forms an HDAC3 complex). Moreover, our preliminary data suggested that BCL6 uses different sets of corepressors to mediate distinct biological effects, possibly by using different biochemical mechanisms at specific sets of target genes. Therefore, we hypothesized that BCL6 regulates its target genes using different biochemical tools, allowing it to exquisitely fine tune gene expression and provide specific control mechanisms for different biological functions. In order to test this hypothesis we first identified the direct target genes of BCL6 SMRT, N-CoR and BCoR by ChIP-on-chip in DLBCL cells (Ly1 cells) in multiple replicates, and examined whether the overlapping sets of genes corresponded to different gene pathways. We used a 24,000 promoter microarray representing 1.5 KB of sequence for each gene. The results show reproducible binding of BCL6 at 940 promoters, While BCoR bound to 770, SMRT to 545 and N-CoR to 487 promoters respectively. BCL6 and BCoR overlapped at 400 genes, preferentially involved in involve in cell cycle, cell death chromatin structure, ubiquitin dependent process and chemotaxis. BCL6 and SMRT overlapped on 376 genes, involved in immune response, cell motility and also as BCOR cell death, while N-CoR and BCL6 overlapped on 100 genes including transcriptional control and cell death pathways. The overlap between BCoR and SMRT was at 200 genes, BCoR and N-CoR at 60 genes and SMRT and N-CoR at 85 genes. All three overlapped at 50 genes. We also examined whether these corepressors were associated with specific combinations of histone modifications including H3K9 acetylation, H3K9 methylation, H3K4 methylation, H3K27 methylation, H4K16 acetylation and H3K36 acetylation. Taken together, the data indicate that specific subsets of BCL6 target genes are dependent on distinct biochemical mechanisms, suggesting that additional layers of biochemical complexity govern formation of gene repression complexes in DLBCL cells and providing opportunities for highly specific therapeutic targeting of specific gene programs.
Activation of JAZ by Small Molecules in Human Leukemia Cells.
Authors: Yang Mingli , Jia Jinghua , Ostrov David , Qiu Jingxin , May W. Stratford 
Published: April 2020

Abstract
By screening a murine interleukin-3 (IL-3)-dependent myeloid cell cDNA library, we previously identified JAZ (Just Another Zinc Finger Protein), a novel zinc finger protein. JAZ belongs to a new class of evolutionarily conserved C2H2-type ZFPs that feature an unusually long linker sequence and preferentially bind to double-stranded RNA. JAZ localizes in the nucleus and its human gene is localized to the chromosome 5q35-qter, a specific chromosomal region at which deletions and translocations occur in leukemia and lymphoma. We have recently discovered JAZ as a novel direct, positive regulator of p53 transcriptional activity. The mechanism involves direct binding to p53’s C-terminal (negative) regulatory domain to activate “latent” p53 in response to non-genotoxic stress signals (such as interleukin-3 growth factor withdrawal). Thus, we have explored JAZ as a potentially novel molecular target in human leukemia by identifying small molecules to activate JAZ-mediated leukemic cell killing. A structure-based drug design approach was employed to screen small molecules that potentially bind and activate JAZ. Cytotoxicity assays were then carried out with a set of candidate compounds in a dose- and time-dependent manner in various human leukemia cell lines including REH, HL-60, U937, HEL and K562 cells. Several JAZ “activating” compounds were selected, which display differential abilities to induce leukemic cell killing (IC50 = &lt; 1 to 100 μM). Interestingly, biochemical analysis shows that the J-compound(s) can mediate cell death in p53 expressing leukemia cells in association with upregulation of expression of JAZ, p53 and the proapoptotic p53 target gene BAX, indicating activation of p53. Furthermore, the J-compound(s) synergizes induction of leukemic cell death when combined with other known p53 activating agents including cisplatin and nutlin-3, a recently developed small molecule MDM2 antagonist that disrupts the p53-MDM2 interaction. Therefore, the J-compound(s) may mediate leukemic cell death in a mechanism involving activation of “latent”, wild-type p53 by targeting JAZ. However, the J-compound-mediated cell death was also observed in some p53-deficient leukemia cell lines. This suggests that depending on the cell type the J-compounds may also act by a p53-independent mechanism. Since JAZ was recently reported to be a cargo protein for exportin-5, the nuclear export receptor for pre-microRNAs, this dsRNA-binding ZFP may also have an unknown p53-independent function via which the J-compound(s) may possibly act. While these possible mechanisms remain to be further investigated, the J-compound(s) points the way to develop a potentially novel therapeutic strategy targeting JAZ to treat human leukemia.
Modulation of IRP Binding Activity by Oxygen in Primary Erythroid Cells.
Authors: Schranzhofer Matthias , Schifrer Manfred , Ponka Prem , Muellner Ernst W. 
Published: April 2020

Abstract
Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are cytoplasmic RNA-binding proteins that target specific stem-loop RNA structures known as iron responsive elements (IRE). Binding of IRPs to IREs inhibits translation of ferritin mRNA and stabilizes transferrin receptor (TfR) mRNA. Various factors have been reported to regulate binding activity of IRPs, such as iron, phosphorylation, nitric oxide and hypoxia. While there is a consistent agreement on the negative effect of iron on the interaction between IRPs and IREs, reports regarding the influence of hypoxia on the IRE-binding activity of IRPs vary in a species and cell specific manner. It was the aim of this work to study the effect of hypoxic (3% oxygen) and normoxic (20% oxygen) conditions on IRP binding activity in primary erythroid cells. The cells were induced for differentiation and incubated under physiological, low (Desferrioxamine) and high (ferric ammonium citrate) iron conditions. Binding activity of IRPs and protein levels of ferritin and TfR as well as cell proliferation and differentiation parameters were determined to analyze the regulation of iron metabolism during terminal differentiation. The data show, that in developing red blood cells binding activities of IRP1 and IRP2 are reduced at 3% oxygen. This reduction correlates with increased ferritin protein levels and decreased TfR protein levels. Moreover, incubation under hypoxia strongly decreased cell expansion and reduces hemoglobinization. These results suggest that terminal erythroid differentiation in the bone marrow might occur under normoxic rather than hypoxic conditions.
Reducing Iron Stores Lowers Cancer Risk in Patients with Peripheral Arterial Disease.
Authors: Zacharski Leo R. , Chow Bruce K. , Howes Paula S. , Shamayeva Galina , Baron John A. , Dalman Ronald L. , Malenka David J. , Ozaki Keith , Lavori 6 Philip W. 
Published: April 2020

Abstract
Background: Iron accumulation in excess of requirements has been implicated in risk of malignancy through increased iron-catalyzed free radical-mediated oxidative stress. Reduction of body iron stores may lower risk of cancer.
Methods: A multi-center randomized, controlled, single-blinded clinical trial was conducted between May 1, 1999 and April 30, 2005 within the Department of Veterans Affairs Cooperative Studies Program; VA Cooperative Study #410. Its purpose was to test the hypothesis that reducing body iron stores by phlebotomy would influence all cause mortality (the primary endpoint) and combined death plus non-fatal myocardial infarction and stroke (the secondary endpoint) in a cohort of patients with symptomatic peripheral arterial disease. Patients with a diagnosis of visceral malignancy within the preceding five years were excluded. Patients were randomly assigned to control or reduction of iron stores by graded phlebotomy (avoiding iron deficiency) with stratification by hospital, age, entry ferritin, diagnosis of diabetes mellitus, smoking status and HDL/LDL ratio. Information on the occurrence of new visceral malignancies and cause-specific mortality including death due to cancer as collected prospectively.
Results: This study enrolled 1,277 patients: 641 were randomized to control and 636 to iron reduction. Ferritin levels were reduced significantly in patients randomized to iron reduction. A new visceral malignancy was diagnosed during follow-up in 60 control and 38 iron reduction patients, a 37% decrease in risk with iron reduction (p = 0.023). Reduced risk was observed for several common tumor types. Iron reduction patients had lower cancer - specific mortality and lower all - cause mortality in patients diagnosed with cancer (p = 0.003 and 0.009, respectively) than control patients. Neither cancer risk nor mortality was affected by variables used to stratify the randomization or demographic variables at entry. Mean ferritin levels at entry were comparable for all patients that subsequently acquired versus did not acquire cancer (p = 0.314) and for patients randomized to either control or iron reduction that later acquired cancer (p = 0.927). Mean ferritin levels during follow-up in patients randomized to iron reduction that acquired cancer were comparable to levels in control patients (p=0.428) while follow-up ferritin levels were significantly lower in patients not acquiring cancer (p=0.017).
Conclusions: Randomization to iron reduction was associated with a significantly lower cancer risk and mortality during follow-up. Patients randomized to iron reduction that acquired cancer were relatively non-compliant with intervention. These findings invite further study.
Oxidative Stress Contributes to Hemolysis in Hereditary Spherocytosis.
Authors: Ghoti Hussam , Amer Johnny , Shaaban Mohammad Abu , Draz Samir Abu , Issah Abd El Rahman , Fibach Eitan , Rachmilewitz Eliezer A. 
Published: April 2020

Abstract
We have previously shown that oxidative stress is generated in pathological red blood cells (RBC) in congenital (thalassemia, sickle cell anemia) and acquired (myelodysplastic syndrome and paroxysmal nocturnal hemoglobinuria) hemolytic anemias. In the present study we tested whether a similar mechanism occurs in hereditary spherocytosis (HS) which is a heterogeneous group of disorders, where RBC have short survival due to primary deficiency in spectrin, ankyrin-1, band 3 or protein 4.2 in their membranes. Secondary protein deficiencies resulting from oxidative stress are often observed and may be involved in the outcome of the disease. Seventeen patients from 7 families with clinical and laboratory diagnosis of mild to severe HS were examined. All patients were anemic, (Hb ranging from 5.3 to 10.5 g/dl) and 7 were splenectomized. Twelve patients received 5 to 58 units of blood. The diagnosis was based on spherocyte morphology, increased MCHC (33–38 g/dl), decreased MCV (&lt;80 fl in 11 patients) and increased osmotic fragility (&gt;75%) in all of them. Using flow cytometry techniques, we showed that RBC from HS patients generate high amounts (1.65-fold) reactive oxygen species (ROS), as well as membrane lipid peroxides (LP) (3.04-fold), and lower levels (2.05-fold) of the cellular anti-oxidant reduced glutathione (GSH) compared with RBC obtained from normal donors of matched gender and age (Figure). In order to determine whether oxidative stress plays a role in hemolysis in HS, we incubated HS-RBC with their autologous plasma and added antioxidants such as N-acetylcystein, vitamin C and the vitamin E derivative, tocotrinol, each at 1 mM, for 12 hours. The results showed that the antioxidants protected HS-RBC from lysis. Our results provide a direct evidence for oxidative stress in RBC from HS patients similar to findings in other hemolytic anemias which contribute to the magnitude of their lysis, and suggest that treatment with antioxidants may attenuate some of the clinical symptoms of these patients.
Figure Figure
Measurement of Urinary Hepcidin Levels by SELDI-TOF-MS in HFE-Hemochromatosis.
Authors: Bozzini Claudia , Campostrini Natascia , Trombini Paola , Nemeth Elizabetha , Castagna Annalisa , Tenuti Ilaria , Corrocher Roberto , Camaschella Clara , Ganz Tom , Olivieri Oliviero , Piperno Alberto , Girelli Domenico 
Published: April 2020

Abstract
Introduction: An insufficient production of hepcidin, the master regulator of iron metabolism, is recognized as the key pathogenetic feature of HFE-related hereditary hemochromatosis (HH). There is a growing interest in measuring the hepcidin levels, which may improve diagnosis, prognostic evaluation and clinical management of HH. Nevertheless, few investigative tools are available: an immunodot method for urinary hepcidin developed by a single centre (UCLA), not yet ready for large-scale diffusion, and mass spectrometry (MS) based assays, such as surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF-MS). The latter is well suited to small peptides like hepcidin, and can rapidly analyze crude samples with high throughput. Until now, urinary hepcidin has been measured by SELDI-TOF-MS only in small groups of C282Y homozygous patients, the majority of them under phlebotomy treatment. No data are available on C282Y/H63D compound heterozygotes, that can develop a milder clinical form of HH. This study was aimed to measure urinary hepcidin levels by SELDI-TOF-MS in a large group of HH patients.
Methods: We used a protocol based on PBSIIc mass spectromer and Normal Phase chips similar to that recently proven successful for semi-quantitative detection of urinary hepcidin. Urinary samples from 30 control subjects were compared to those obtained from 80 HH patients (57 C282Y homozygotes, 23 C282Y/H63D compound heterozygotes). Eighteen C282Y homozygotes and 11 C282Y/H63D compound heterozygotes were analyzed at diagnosis, the remainder during maintenance phlebotomy (at least 30 days from last phlebotomy).
Results: C282Y homozygotes had significantly lower urinary hepcidin levels vs. controls either at diagnosis, or after phlebotomy (P &lt; 0.05). C282Y/H63D compound heterozygotes had hepcidin levels at diagnosis similar to controls, while the hepcidin:ferritin ratio was significantly decreased (P &lt; 0.001) suggesting a relatively inappropriate hepcidin production. Moreover, also in this group means hepcidin levels after phlebotomy were significantly lower than in controls (P &lt; 0.001). Samples from 12 randomly selected control subjects were sent to UCLA for duplicate measurement by the immunodot method, yielding a good correlation (r= 0.77; P&lt;0.0001).
Conclusions: SELDI-TOF-MS assay is confirmed to be a potentially useful tool for measuring hepcidin levels in HH. The very low hepcidin levels observed in both genotypes after phlebotomy, may suggest that iron depletion as currently achieved by standard protocols may not be the best therapy from a pathophysiological standpoint.
Atiprimod Inhibits the JAK-STAT Pathway and Induces Apoptosis in Human Cells Carrying the JAK2V617F or JAK3 Mutation.
Authors: Manshouri Taghi , Golemovic Mirna , Kantarjian Hagop M.M. , Cortes Jorge E. , Zhang Ying , Priebe Waldemar , Estrov Zeev , Verstovsek Srdan 
Published: April 2020

Abstract
Janus Kinases (JAK), including JAK1, JAK2, TYKa, and JAK3, are a small family of cytoplasmic protein tyrosine kinases, which play an important role in the initiation of cytokine-triggered signaling events by signal transducer and activator of transcription (STAT) proteins, via phosphorylation. The recent reports of an activating somatic mutation in codon 617 of the gene encoding JAK2 (JAK2V617F) in patients with myeloproliferative disorders (MPDs) and 3 mutations in JAK3 (A572V, V722I, P132T) in acute megakaryoblastic leukemia patients, has opened new avenues for the development of targeted therapies for these malignancies. We report here the activity of Atiprimod, a novel compound with anti-inflammatory properties, in retrovirus-transduced JAK2 (JAK2V617F) mutant-expressing murine FDCP-EpoR cells, set-2 cells, CMK cells, and blood cells from patients with polycythemia vera (PV). We compared the growth inhibitory effect of Atiprimod against two mouse FDCP cell lines transfected with erythropoietin receptor (Epo-R), and either wild-type JAK2 (JAK2WT) or mutant JAK2 (JAK2V617F), and human megakaryoblastic leukemia cells with mutated JAK2V617F (set-2 cells) or mutated JAK3 (CMK cells). The growth inhibitory effect was assessed using 3-days MTS assay. Atiprimod was more potent against FDCP cells carrying mutant JAK2 (JAK2V617F) cells (Ic50 0.42 μM) and set-2 cells (Ic50 0.53 μM) than the CMK cells with mutant JAK3 (Ic50 0.79 μM) and FDCP wild-type JAK2 (JAK2WT) cells (Ic50 0.69 μM). Atiprimod, inhibited the phosphorylation of JAK2 and downstream STAT3, STAT5, and AKT proteins in a dose-and time-dependent manner. It exerted its inhibitory activity against cells with mutant JAK2 (JAK2V617F) by affecting the cell cycle progression and induced apoptosis, as evidenced by increase in mitochondrial membrane potential and caspase3 activity. Atiprimod also cleaved PARP protein, increased turnover of the anti-apoptotic X-linked mammalian inhibitor of apoptosis (XIAP) protein, and inhibited the apoptotic protein BCL2 in a time- and dose-dependent manner. In addition, Atiprimod inhibited the proliferation of peripheral blood hematopoietic progenitors from three patients with PV carrying the JAK2 (JAK2V617F) mutation as compared to hematopoietic progenitors from normal individuals, by 2-days MTS assay (p= 0.0013). Our data suggest that Atiprimod is active both in vitro and ex vivo against cells with activated tyrosine kinase of the JAK family.
Analysis of T-Cell Leukemia/Lymphoma (TCL1) Oncoprotein Interacting Proteins by Immunoprecipitation and Tandem Mass Spectrometry.
Authors: Elenitoba-Johnson Kojo S.J. , Basrur Venkatesha , Kronk Adam , Seiler Charles , Fermin Damian , Nesvizhskii Alexey , Lim Megan S. 
Published: April 2020

Abstract
TCL1 is a proto-oncogene whose deregulation has been implicated in the pathogenesis of T- and B-cell lymphoproliferative disorders. TCL1 expression is predominantly observed in developing B lymphocytes, whereas its overexpression in T cells is associated with mature T-cell lymphoproliferation in transgenic mice. Although recent studies indicate that dysregulated expression of TCL1 is important in mature B-cell transformation, very little is currently known about the function or interactions of TCL1, specifically in the B-cell context. In this study, we hypothesized that identification of proteins that interact with TCL1 may facilitate our understanding of the functional properties of TCL1. Proteomic analysis provides an opportunity to carry out functional studies of protein-protein interactions and characterization of functional interactomes. Using a functional proteomic approach, we determined the identity of proteins that interact with TCL1 by co-immunoprecipitation with anti-TCL1 antibody followed by liquid chromatography (LC), electrospray ionization (ESI) and tandem mass spectrometry (MS/MS). Immunoprecipitates of the TCL1 expressing SUDHL-16 B-cell lymphoma derived cell line were compared to that of hyperimmune rabbit immunoglobulin and a cell line that does not express TCL1 (SUDHL-1) by 1-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Silver-stained protein bands from both immunocomplexes were excised and analyzed by ESI-LC-MS/MS. Proteins which were unique to the TCL1 immunocomplex included transcription factors (Jun B), phosphatases (protein phosphatase 2), ubiquitin-associated proteins (ubiquitin activating enzyme), cell adhesion proteins (ADP ribosylation factor 3) and proteins associated with apoptosis regulation (peroxidoxin 1 and scaffold attachment factor). Analysis of subcellular fractions demonstrated that TCL1 and complexes were localized to both the nuclear and cytoplasmic compartments. Importantly, known TCL1 interactors such as histone 3 were uniquely identified in the TCL1 complex. Proteins identified by MS were confirmed by western blotting and reciprocal immunoprecipitation. This study reveals novel TCL1 interactors and indicates that the protein may exert diverse cellular effects impacting apoptosis, cell survival, cytoskeletal organization and cell adhesion. Our interactome analysis provides unique insights into the cellular function of TCL1, a protein whose deregulation is increasingly implicated in the pathogenesis of a variety of hematopoietic neoplasms.