bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sp1 Transcription Factor as a Novel Therapeutic Target in Multiple Myeloma (MM)
Authors: Fulciniti Mariateresa , Tassone Pierfrancesco , Hideshima Teru , Nanjappa Puru , Podar Klaus , Adamia Sophia , Amin Samir B , Prabhala Rao , Lopez Rocio , Anderson Kenneth C. , Munshi Nikhil C. 
Published: October 2019

Abstract
Sp1 transcription factor has an important role in regulating expression of cell differentiation, cell cycle and apoptosis related genes. Increased Sp1 activity has been shown to augment growth and metastatic potential of tumor cells. In multiple myeloma (MM), modulation of autocrine IL-6 secretion by Sp1 has been described. Using genome expression profiling analysis we observed high expression of Sp1 in primary myeloma cells obtained from bone marrow aspirates of MM patients. This observation was confirmed by western blot analysis. Therefore, we evaluated Sp1 as a novel therapeutic target in MM, analyzing both in vitro and in vivo anti-MM activity of a Sp1 inhibitor (Terameprocol, EM-1421). Terameprocol significantly (≥80% inhibition, P≤0.001) inhibited DNA synthesis in 12 MM cells lines tested in a dose-dependent fashion at 24 hour with an IC50 of 1–10 uM. Longer exposure of MM cells to Terameprocol resulted also in cytotoxity, as assessed by MTT assay, in all cell lines tested (IC50 of 20–40 uM at 72 hours). Terameprocol induced apoptosis in a time- and dose-dependent manner in all MM cell lines examined, as assessed by Annexin V+/PI staining, and triggered caspase-9/3 and PARP cleavage. Since Sp1 modulates transcription of survivin and survivin is an anti-apoptotic protein overexpressed in most human cancers that inhibits caspases-3 and -9 activity, we next examined effect of Terameprocol on survivin protein expression. Consistent with induction of caspase-3 and apoptosis, Terameprocol significantly reduced survivin protein in a time-dependent manner. To identify additional potential downstream targets of Sp1 inhibition in MM, we evaluated change in expression profiling in MM cells following Terameprocol treatment (10 uM for 12 hours). We observed alteration (>2-fold changes) in 68 genes. In particular, we recognized significant down-regulation of c-Jun and up-regulation of p53 and p21 following exposure to Terameprocol. Changes in protein expression of these genes were also confirmed by western blot analysis. Since bone marrow stromal cells (BMSCs) trigger growth, survival, and drug resistance of MM cells we next confirmed the ability of Terameprocol to overcome protective effects of BMSCs on MM cell growth and survival. Finally, we have confirmed in vivo activity of Terameprocol on MM cell growth in a xenograft murine model of human MM. In conclusion, our results demonstrate that Sp1 may be an attractive target for MM therapy and provide the rationale for clinical evaluation of Terameprocol in MM.
FGFR3 Associates with and Tyrosine-Phosphorylates p90RSK2, Leading to RSK2 Activation That Mediates Hematopoietic Transformation
Authors: Kang Sumin , Elf Shannon , Dong Shaozhong , Hitosugi Taro , Guo Ailan , Ruan Hong , Lonial Sagar , Khoury Hanna , Williams Ifor , Karsenty Gerard , Hanauer André , Gu Ting-Lei , Chen Jing 
Published: October 2019

Abstract
Dysregulation of receptor tyrosine kinase FGFR3 has been implicated to play a pathogenic role in a number of human hematopoietic malignancies and solid tumors. These include t(4;14) multiple myeloma associated with ectopic expression of FGFR3 and t(4;12)(p16;p13) acute myeloid leukemia associated with expression of a constitutively activated fusion tyrosine kinase TEL-FGFR3. We recently reported that FGFR3 directly tyrosine phosphorylates p90 Ribosomal S6 Kinase2(RSK2) at Y529, which consequently regulates RSK2 activation [Kang et al, Cancer Cell 2007 Sep;12(3):201–14]. Here we identified Y707 as an additional tyrosine site of RSK2 that is phosphorylated by FGFR3. Phosphorylation at Y707 contributes to RSK2 activation, through a putative disruption of the autoinhibitory αL-helix on the C-terminus of RSK2, unlike Y529 phosphorylation that facilitates ERK binding. To elucidate the role of tyrosine phosphorylation at Y707 induced by FGFR3 in RSK2 activation, we characterized the RSK2 mutants with single Y→A and Y→F substitutions at Y707. RSK2 Y707F demonstrated decreased kinase activity, suggesting substitution of Y707 attenuates activation of RSK2 induced by FGFR3. Tyrosine phosphorylation at Y529 by FGFR3 regulates RSK2 activation by facilitating inactive ERK binding, whereas substitution of Y707 in RSK2 does not similarly attenuate inactive ERK binding to RSK2. Phosphorylation at Y707 may regulate RSK2 activation by affecting the structure of the autoinhibitory C-terminal domain of RSK2 since the Y707 is localized at the C-terminal tail region which represents a conserved putative auto-inhibitory alpha helix. Since other tyrosine kinases including FGFR1 and Src also phosphorylate RSK2 at Y529 and Y707, tyrosine phosphorylation may be a general requirement for RSK2 activation through the ERK/MAPK pathway. Together, our current and previous findings represent a paradigm for tyrosine phosphorylation-dependent regulation of serine-threonine kinases. Moreover, we found that FGFR3 interacts with RSK2 through residue W332 in the linker region of RSK2, and that this association is required for FGFR3-dependent phosphorylation of RSK2 at Y529 and Y707, and subsequent RSK2 activation. Furthermore, in a murine bone marrow transplant assay, genetic deficiency in RSK2 resulted in a significantly delayed and attenuated myeloproliferative syndrome induced by TEL-FGFR3 as compared with wild type cells, suggesting a critical role of RSK2 in FGFR3-induced hematopoietic transformation.
Expression of Leukemogenic Ptpn11 Causes a Fatal Myeloproliferative Disorder by Affecting Multiple Stages of Hematopoiesis
Authors: Chan Gordon , Kalaitzidis Demetrios , Usenko Tatiana , Kutok Jeffery , Yang Wentian , Mohi Golam , Neel Benjamin 
Published: October 2019

Abstract
The Src homology-2 (SH2) domain-containing phosphatase 2 (Shp2), encoded by Ptpn11, is an ubiquitously expressed non-receptor protein tyrosine phosphatase (PTP) that positively regulates Ras/Erk activation in many receptor protein tyrosine kinases (RTK) and cytokine receptors. Mutations in PTPN11 are found in ~35% of patients with juvenile myelomonocytic leukemia (JMML) and at lower incidence in other neoplasms. To model JMML pathogenesis, we generated knock-in mice that conditionally express the leukemia-associated mutant Ptpn11D61Y. Ptpn11D61Y expression in all hematopoietic cells evokes a fatal myeloproliferative disorder (MPD), featuring leukocytosis, anemia, hepatosplenomegaly with extramedullary hematopoiesis, and factor-independent colony formation by bone marrow (BM) and spleen cells. The Lin−Sca1+cKit+ (LSK) compartment is expanded and “right-shifted”, accompanied by increased stem cell factor (SCF)-evoked colony formation and Erk and Akt activation. However, stem cell activity is decreased in diseased mice, and mice engrafted with Ptpn11D16Y stem cells fail to develop MPD. Ptpn11D16Y common myeloid (CMP) and granulocyte-monocyte (GMP) progenitors produce cytokine-independent colonies in a cell-autonomous manner, and demonstrate elevated Erk and Stat5 activation in response to granulocyte-macrophage colony-stimulating factor (GM-SCF) stimulation. Ptpn11D61Y megakaryocyte-erythrocyte progenitors (MEP) yield increased numbers of erythrocyte burst-forming units (BFU-E), but MEP and erythrocyte-committed progenitors (EP) produce fewer erythrocyte colonyforming units (CFU-E), indicating defective erythroid differentiation. Our studies provide a mouse model for Ptpn11-evoked MPD and show that this disease results from cellautonomous, and distinct lineage-specific effects of mutant Ptpn11 on multiple stages of hematopoiesis.
Bone Marrow Stromal Cells Reverse Proapoptotic Effects of JAK2 Inhibitor Atiprimod in Cells Carrying the JAK2V617F mutation
Authors: Manshouri Taghi , Estrov Zeev , Burger Jan , Livun Ana , Zhang Ying , Ma Shuhuang , Gaikwad Amos , Harris David , Kantarjian Hagop M , Verstovsek Srdan 
Published: October 2019

Abstract
Janus kinases (JAK) comprise a small family of cytoplasmic protein tyrosine kinases, which play an important role in the initiation of cytokine-triggered signaling events via signal transducer and activator of transcription (STAT) proteins. The recent reports of an activating somatic mutation in codon 617 of the JAK2 gene (JAK2V617F mutation) in patients with myeloproliferative disorders (MPDs), has opened new avenues for the development of targeted therapies for these malignancies and clinical trials with JAK2 inhibitors are underway. We report here the activity of Atiprimod (N,N-diethyl-8-dipropyl-2-azaspiro[4,5]decane-2-propanamine), a novel compound with anti-inflammatory properties, in retrovirus-transduced JAK2V617F mutant-expressing murine FDCP-EpoR cells, set-2 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 JAK2WT or mutant JAK2V617F, and human megakaryoblastic leukemia cells with mutated JAK2V617F (set-2 cells). The growth inhibitory effect was assessed using 3-days MTS assay. Atiprimod was more potent against FDCP cells carrying mutant JAK2V617F cells (IC50 0.42 μM) and set-2 cells (IC50 0.53 μM) than FDCP wildtype 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 induced apoptosis, as evidenced by increase in mitochondrial membrane potential, caspase3 activity, and cleavage of PARP protein. The anti-proliferative effect on expanded PV patient progenitor’s cells was paralleled by a decrease in JAK2V617F mutant allele frequency in BFU-E or CFU-GM clones in clonogenic assay. However, co-culturing of JAK2V617F mutant cells with three different bone marrow stromal cell lines (Hs5, ABM-MSC, NK-Tert) either directly (cell on cell) or indirectly (separated by 0.4 μm micropore membranes) for 48 hours resulted in a significant protection of mutant cells from the effect of Atiprimod. Co-culturing of bone marrow stromal cells prevented Atiprimod (0.4 and 0.8 μM) induced apoptosis, and reversed the inhibition of phosphorylation of STAT proteins. Our results suggest that cytokines secreted by stromal cells might play an important role in protecting the hematopoietic cells from a JAK2 inhibitor. Further dissection of the nature of interactions between JAK2V617F mutant cells and marrow stromal cells may lead to new therapeutic avenues for patients with MPD.
Role of Human Endogenous Retroviruses in Lymphoma Pathogenesis and a Possible Biomarker of Disease
Authors: Gitlin Scott D. , Galindo Rafael Contreras- , Kaplan Mark H. , Markovitz David M. 
Published: October 2019

Abstract
Actively replicating retroviruses entered hominid species millions of years ago and through mutations preventing replication now exist as 8% of the human genome. Active retroviral particles and antigens from the supposedly dormant human endogenous retrovirus, HERV-K (HML2), have been identified in several cancer cell lines. We have recently demonstrated very high RNA titers of HERV-K (HML2) in the plasma of HIV positive individuals by nucleic acid sequence-based amplification (NASBA) and RT-PCR. We now demonstrate very high HERV-K (HML2) RNA titers in the plasma of patients with HIV positive and HIV negative non-Hodgkin lymphoma (NHL) and in Hodgkin Disease (HD), but not in normal individuals. Different copies of HERV-K (HML-2) present throughout the human genome exist as Type 1 viruses which encode a new oncoprotein, NP9, or as Type 2 viruses which encode a functional envelope (env) and express the Rec oncoprotein. Both Types 1 and 2 viruses appear in NHLs but only Type 1 appears in the plasma of those with HD. HERV-K (HML2) Env and Gag proteins, Env and Gag RNA, and Reverse Transcriptase (RT) activity are isolated from patients with a variety of NHLs, but not in normal controls or in patients with non-malignant diseases. Viral titers dramatically decrease, up to an approximately 7.5 log drop, when patients with NHL or HD go into remission following treatment. To further establish the presence of functional viruses in NHL and HD, immuno-gold electron microscopy allowed demonstration of HERV-K (HML2) particles in the plasma of lymphoma patients. Preliminary analysis of the effect of antiretroviral agents on cell lines infected with HERV-K (HML2) demonstrate a drug class-specific reduction in viral expression at drug concentration levels that range from 0.125 – 1 mcg/mL. In conclusion, we have demonstrated evidence that human endogenous retroviruses are found in the plasma of patients with NHL and HD, suggesting that these viruses, previously presumed to be inactive, may play a role in lymphoma pathogenesis. The observation that viral expression parallels declines in disease activity with treatment of disease may allow use of HERV-K (HML2) expression as a biomarker of lymphoma activity. The role of the HERV-K (HML2)-encoded oncoproteins in disease pathogenesis is under study, as is the potential role of antiretroviral therapy for these malignancies.
Regulation of p27 by S-Phase Kinase- Associated Protein 2 Is Associated with Aggressiveness in Diffuse Large B Cell Lymphoma (DLBCL)
Authors: Seki Ritsuko , Ohshima Koichi , Fujisaki Tomoaki , Uike Naokuni , Kawano Fumio , Gondo Hisashi , Makino Shigeyoshi , Eto Tetsuya , Moriuchi Yukiyoshi , Taguchi Fumihiro , Kamimura Tomohiko , Tsuda Hiroyuki , Ogawa Ryosuke , Shimoda Kazuya , Okamura Takashi 
Published: October 2019

Abstract
The F-box protein S-phase kinase-associated protein 2 (Skp2) is one of the positive regulators of the cell cycle that promote ubiquitin-mediated proteolysis of the cyclin-dependent kinase inhibitor p27. In this study, we investigated the significance of Skp2 and p27 expression in diffuse large B cell lymphoma (DLBCL) treated with CHOP or CHOP-R. All patients (671) were diagnosed as having DLBCL at the twenty different hospitals and were treated with either CHOP (425) or CHOP-R (246) from 1996 to 2005. All specimens were histopathologically recomfirmed by one pathologist with expertise before entering into this study. Their clinical characteristics, including either the IPI or R-IPI factors, were evenly matched in both treatment groups. The median follow-up of living patients was 3.7 and 2.1 y for CHOP vs CHOP-R, respectively.
Survival analyses were performed using the Kaplan-Meier method and the Cox regression model.
There were 257 patients with high Skp2 expression (cutoff value >40% positive cells) (257/671,39.0%). High Skp2 expression was found in all IPI groups. Expression of p27 (P<.001) was associated with better overall survival (OS), whereas expression of Skp2 was associated with worse OS in CHOP treatment groups (P<.001). We also examined the prognostic impact of Skp2 according to various expression degree of Skp2, Skp2 expression remained a significant predictor of survival (<40%,40–79%,≥80%, P<.001). The multivariant analysis revealed that both the IPI score and the expression of Skp2 and p27 were independent predictors of OS and PFS in both treatment groups (P<.001).
The patients with high Skp2 expression in combination with low p27 expression were related to worst survival in CHOP group (Fig1A). Interestingly, even in CHOP-R group, both high Skp2 expression and low p27 expression were the strong biomarker of worse prognosis (Fig 1B). DLBCL patients with high Skp2 expression did not benefit from the addition of R to CHOP.
Conclusion: Addition of rituximab did not provide a beneficial outcome to DLBCL with high Skp2 and low p27 expression. Therapeutic strategies other than CHOP-R will be needed for DLBCL patients exhibiting a high Skp2 and low p27 expression at the time of diagnosis.
Figure Figure
p19Arf Cooperates with Shp2 in Lymphoid Leukemogenesis
Authors: Chen Xiaolan , Huang Yuhui , Yan Ling , Brault Marie , Wang Zhizhang , Arildsen Maryann T , Yang Elizabeth 
Published: October 2019

Abstract
Shp2 gain-of-function mutations are found in human acute lymphoblastic leukemia, as well as in juvenile myelomonocytic leukemia and acute myeloid leukemias. Genetic data from Noonan Syndrome patients bearing mutations in PTPN11/Shp2 suggest that Shp2 mutation alone is insufficient for leukemogenesis, and other genetic alterations are required. We investigated E76K Shp2-induced leukemogenesis in p19Arf+/− and p19Arf−/− backgrounds. Wild type mice reconstituted with Arf+/− or Arf−/− bone marrow cells (BMCs) transduced with E76K Shp2 developed hematologic disease with increased incidence and severity than mice reconstituted with vector-transduced Arf+/− or Arf−/− BMCs, with E76K Shp2-transduced Arf−/− BMCs causing the most aggressive disease at the highest rate. E76K Shp2 expression in Arf+/− BMCs caused myeloproliferation consisting of mainly Mac1+Gr1+ cells associated with extramedullary hematopoiesis, but E76K Shp2 expression in Arf−/− BMCs caused a shift in disease phenotype to transplantable B220+ leukemias. Consistently, E76K Shp2 transduced Arf+/− or Arf−/− BMCs from reconstituted pre-leukemic mice showed increased proB cell populations compared to vector transduced BMCs, suggesting that the E76K Shp2 expressing proB cells are the leukemia precursors. While Shp2 is well known to confer GM-CSF hypersensitivity in methylcellulose cultures, we found that E76K Shp2 transduced BMCs were also hypersensitive to the lymphoid colony-stimulating factor IL-7, and E76K Shp2 expressing lymphoid BMCs formed colonies in the absence of exogenous growth factor. These data suggest that gain-of-function Shp2 confers transformation features to B cell precursors and that Arf deletion potentiates E76K Shp2 in lymphoid leukemogenesis.
Fibrinogen Alpha and Gamma Genes and Factor VLeiden in Children with Thromboembolism: Results from Two Family-Based Association Studies
Authors: Nowak-Gottl Ulrike , Weiler Hartmut , Thedieck Sabine , Seehafer Tanja , Stoll Monika 
Published: October 2019

Abstract
Background: Fibrinogen, the precursor of fibrin, is an essential component of the hemostatic system. A previous large case-control study showed that genetic variation in the fibrinogen gamma gene (FGG) increased the risk for VT in adults. We investigated the association of haplotypes comprising the fibrinogen alpha (FGA) and gamma (FGG) genes, carriership of the Factor VLeiden-mutation and risk for VT in two large family-based study samples for pediatric thromboembolism.
Methods: We genotyped 7 single nucleotide polymorphisms in FGA and FGG, and the G1691A Factor VLeiden polymorphism in 244 pediatric VT and 268 thrombo-embolic stroke families. Association was assessed using the Transmission Disequilibrium Test (TDT) and corrected for multiple testing.
Results: Association analysis revealed that the FGA-H1 haplotype, and the FGG-H2 and -H3 haplotypes, were significantly associated with VT (FGA-H1, P=0.05; FGG: H2, P=0.032; H3, P=0.0216). In an independent study sample, FGA-H1 (P=0.0085) and FGG-H2 (P=0.05) were significantly associated with TS. When stratifying for FVLeiden carriership, the association between FGA and FGG and VT was more pronounced in FVLeiden-negative families (FGA-H1, P=0.0006; FGG-H2, P=0.0005). Homozygous carriership of the FGG-H2 risk haplotype resulted in the lowest fibrinogen γ′ content (γ′ levels: 22.7±13.7 vs. 26.8±12.0, P=0.013; % γ′: 7.63±3.05 vs. 9.46±3.17, P=2.3×10−5), with increasing concentrations of fibrinogen γ′ in heterozygote H2 carriers. Compound heterozygote carriers of one FGG-H2 risk and one FGG-H3 protective haplotype, showed significant increase in fibrinogen γ′ (P=0.000032), while fibrinogen levels remained unchanged. In contrast, homozygote carriers of the protective FGG-H3 haplotype showed the highest concentration of fibrinogen γ′ content (% γ′: 9.21±3.09, P=0.0031) with decreased total fibrinogen levels.
Conclusion: Our results support an important role of genetic variants in FGA and FGG in thromboembolism in children and adults. Our data further suggest that the genetic architecture of VT is complex and involves subtle influences through susceptibility and protective haplotypes in FGG and a genetic interaction with the FVLeiden-mutation.
Comparative Profiling of Red Blood Cells from the Beta-Adducin Knockout Mouse Model of Hemolytic Anemia Using Label Free Differential Proteomics
Authors: Wooden Jason M , Finney Greg L , Maccoss Michael J , Peters Luanne L. , Gilligan Diana M. 
Published: October 2019

Abstract
Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases. While mild to severe inherited hemolytic anemias can arise from defects in the red blood cell (RBC) membrane skeleton, fundamental questions remain unanswered surrounding the clinical variability of known red blood cell mutations. To identify candidate proteins involved in hemolytic anemia pathophysiology, we utilized a label-free comparative approach to detect differences in RBCs from normal and beta-adducin (ADD2KO) knock-out mice. For each genotype, whole blood was taken from independent biological replicates and RBCs were purified using cellulose acetate chromatography. The isolated RBCs were lysed to generate RBC ghosts whose protein complements were digested with trypsin. For each biological replicate, three replicate runs utilizing 0.5 μg digested protein were performed via microcapillary liquid chromatography coupled with tandem mass spectrometry. Using the recently developed software package CRAWDAD, we detected 7 proteins that were decreased and 31 proteins with a greater abundance in the beta-adducin knock-out RBC ghosts. Differences were detected for previously known membrane skeleton components, including the predicted absence of beta-adducin and decrease in alpha-adducin. The actin binding protein capping protein (actin filament) muscle Z-line alpha 1 was increased along with ankyrin 1, spectrin alpha, tropomyosin, and glycophorin C. Interestingly, differences were also detected for the protein oxidative stress-responsive 1 and the uncharacterized protein C10orf58 which contains a thioredoxin motif. While the protein differences span a broad range of cellular processes, some of the proteins are attractive candidates for modifiers of disease severity. This label free approach is the first demonstration of comprehensively analyzing wild type versus kockout mice that circumvents the need for metabolic or chemical labeling with isotopes. This new approach adds a valuable tool to the list of standard assays for analysis of RBCs in cases of hereditary hemolytic anemia.