bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of Phospholipids in the Activation of Factor X by Tissue Factor/VIIa.
Authors: Hathcock James J. , Rusinova Elena , Väänänen Heikki , Nemerson Yale 
Published: October 2019

Abstract
The activation of coagulation factors IX and X by tissue factor(TF)/VIIa is the key step in initiating coagulation. The mechanisms by which FX binds to phospholipid and interacts with TF/VIIa remains disputed; and the reported dissociation constants between FX and FXa to well-defined phospholipid vary by orders of magnitude. In accord with other investigators we have found that at low lipid to TF ratios, adding additional lipid vesicles lacking TF, can accelerate the activation of FX. This acceleration has previously been attributed to the physically unrealistic notion that bound FX, regardless of whether it be bound to vesicles containing TF or naked vesicles lacking TF, is the preferred substrate for TF/VIIa. We have explored an alternative approach, namely that this acceleration is due to relief of Xa-mediated inhibition. Whereas it is well known that FXa can bind to TF/VIIa thus inhibiting further activation of FX, the presence of additional lipid appears to segregate FXa away from the enzymatic complex and localize it to lipid far from the reactive site. Using classical initial velocity measurements of product formation (typically 30 pM TF), we found that as we increased the lipid to TF ratio from 1.1 to 15 to 110 (x103), the level of inhibition achieved by 40 nM Phe-Phe-Arg-chloromethylketone-inactivated Xa (Xai), dropped from 67% to 40% to 10%. This concept is supported further by our new observation, evaluated by ellipsometry, that at saturation 30% PS/PC surfaces (phosphotidylserine/phosphatidylcholine) bind exactly twice as much FXa as FX; The measured dissociation constants (Kd) for FX and FXa binding to our lipid were 34.5 ± 2.9 nM and 53.9 ± 2.7 nM respectively and have been verified using independent techniques. We also consider herein the likely possibility that lipid-bound FXa exists on the surface as an opposing dimer. At very high lipid to TF ratios (>2x106) we observed reaction velocities decreasing with increasing lipid concentration. This decrease in velocity was associated with an increase in the apparent Km, which we show is due to loss of available substrate to phospholipid surfaces far from the reactive site. The interactions between TF/VIIa, FX, FXa and phospholipid surfaces, although extensively studied, have not been well understood nor well-agreed upon. The true parameters describing these interactions are needed to correctly interpret typical TF/VIIa experimental results as well as to model and understand the roles of these proteins in coagulation. Results of a computational kinetic model addressing the interplay of these reaction components will be presented.
Three-Drug Induction and BFM-Type Intensive Consolidation Chemotherapy for Treatment of Standard Risk Childhood Acute Lymphoblastic Leukemia Defined Also by DNA index. The Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) Study ALL-9501.
Authors: Arico’ Maurizio , Conter Valentino , Valsecchi Maria Grazia , Boccalatte Marie France Pinta , Barisone Elena , Messina Chiara , De Rossi Giulio , Nigro Luca Lo , Pession Andrea , Locatelli Franco , Micalizzi Concetta , Basso Giuseppe , Masera Giuseppe 
Published: October 2019

Abstract
In this study, we tried to select a very small subset of children with acute lymphoblastic leukemia (ALL) at minimal risk of treatment failure - identified to not only by early response in vivo, one of the strongest predictors in the I-BFM-SG experience, but also by age, blood count and in particular high DNA content - which we treated with a reduced-intensity BFM schedule. The AIEOP-ALL 9501 study enrolled patients with standard-risk (SR) ALL, defined as: <20,000 WBC/mm3, age 1 to <6 years, non-T immunophenotype, DNA index between 1.16 and 1.6, non t(9;22), no extramedullary leukemia, good response to prednisone (PGR, defined as <1,000/mmc blasts in the peripheral blood after 7 days of prednisone and one injection of IT-MTX), CR at the end of induction therapy. Follow-up was updated at December,31st 2003 and median follow-up was 5.9 years. Treatment consisted of a modified BFM schedule: 3-drug (VCR + PDN + Erwinia ASP), 43-day induction, no phase IB, 4x2 g/m2 MTX, reinduction with protocol II followed by 6MP+MTX continuation therapy up to 2 years; CNS directed therapy consisted of IT-MTXx18. Between May 95 and August 2000, 137 patients were identified as SR (7.8% of the ALL-95 population), of whom 102 received the SR protocol and are here reported.Of them, 1 died in remission of septicemia, and 1 had developed a second malignant neoplasm (T-ALL after initial B-lineage ALL); 11 patients relapsed (bone marrow, n=8; central nervous system, n=1; marrow + testis, n=1; eye, n=1) and their re-treatment included chemotherapy only (n=3, 1 dead) or plus bone marrow transplant (n=8) either autologous (n=2) or allogeneic from matched (n=4, 1 dead) or partially matched (n=2) unrelated donor. The remaining 89 are in first CR; the probabilities (and related standard errors) of survival and event-free survival (EFS) were 97.0% (1.7) and 86.7% (3.5) at 5 years, 95.3% (2.4) and 86.7% (3.5) at 7 years, respectively. There was no difference in the outcome between the 56 females [7 events, 7-yrs EFS, 87.3 (4.5)] and the 46 males [6 events, 85.9 (5.4)]. Although most of the relapsed patients were rescued, the long-term EFS in this small, very selected group of patients remains inferior to expectance. Thus, alternative selection criteria, for instance related to minimal residual disease as in current AIEOP-ALL 2000, should be considered in order to address the issue of treatment reduction.
Genetic Polymorphisms and the Toxicity of Antileukemic Agents in Children with Acute Lymphoblastic Leukemia.
Authors: Kishi Shinji , Cheng Cheng , Pei Deqing , Das Soma , Cook Edwin H. , Hijiya Nobuko , Rizzari Carmelo , Rosner Gary , Pui Ching-Hon , Evans William E. , Relling Mary V. 
Published: October 2019

Abstract
Regimen-related toxicity, one of the main reasons for discontinuation of chemotherapy in acute lymphoblastic leukemia (ALL), can not only be life-threatening but may affect the risk of relapse. In 246 children with newly-diagnosed ALL who were treated using a single protocol (St. Jude Total XIIIB), we used a candidate-gene approach to determine whether acute toxicities [gastrointestinal (stomatitis or diarrhea), hyperbilirubinemia, infection, or neurotoxicity] were related to 16 common polymorphisms in genes plausibly linked to the pharmacodynamics of the drug therapy. During the high-dose methotrexate consolidation therapy, only genotypes related to methotrexate disposition were associated with grade 3–4 gastrointestinal toxicity, adjusting for demographic factors and ALL risk group. Patients having the RFC AA/AG (p = 0.025) genotypes or those having the MTHFR 677 TT (p = 0.048) genotypes had a higher risk of the toxicity (OR = 10.4 and 3.2 respectively), than those with RFC GG or MTHFR CT/CC. In addition, the risk of hyperbilirubinemia during consolidation was higher among those with the lower activity UGT1A1 7/7 genotype than those with other UGT1A1 genotypes (OR = 12.2, p < 0.0001). Adjusting for time at risk over the entire therapeutic period, RFC AA/AG was again prognostic for gastrointestinal toxicity (p < 0.0001); the GSTP1 AA/AG genotypes (p = 0.021) and TPMT heterozygous genotypes (p = 0.047) were also risk factors. The RFC AA/AG genotype was also a modest risk factor for infection (p = 0.05). There were only weak predictors of neurotoxicity, with the MDR1 exon 26 TT genotype a risk factor (p = 0.025) for presumed methotrexate-related neurotoxicity over the entire therapeutic period. We conclude that germline polymorphisms influence the toxicity of antileukemic therapy and their identification may provide a tool for tailoring therapy in childhood ALL.
Identical Novel C-Kit Transcripts in Two Patients with Mast Cell Leukemia.
Authors: Ozer Ozden , Anastasi John , Vardiman James W. , Godley Lucy A. 
Published: October 2019

Abstract
Background: Mutations of c-kit have been well documented in mast cell neoplasms. The most common alterations found in human cutaneous and systemic mastocytosis are V560G and D816V. Mast cell leukemia is the most aggressive mast cell neoplasm, characterized by diffuse bone marrow and peripheral blood involvement. It has not been well studied due to its rarity. To date, no C-KIT mutational analysis of primary mast cell leukemia cells has been reported, although a mast cell leukemia cell line expresses the V560G and D816V mutations. Here we report on C-KIT cDNA analysis on two patients diagnosed with mast cell leukemia at the University of Chicago Hospitals. Both patients had peripheral blood involvement. Their diagnosis was confirmed on bone marrow biopsies with tryptase stains. Patient 1, diagnosed in 1989, died shortly after diagnosis. Patient 2 achieved a complete remission with allogeneic stem cell transplant. Upon relapse, treatment with Gleevec, resulted in an incomplete but significant response. This patient eventually died of sepsis following refractory cytopenias.
Methodology: We retrieved liquid nitrogen frozen, pre-treatment bone marrow aspirate samples from both patients. We performed RT-PCR analysis of the entire coding region of C-KIT by amplifying 21 exons in smaller fragments. Each PCR product was cloned and sequenced. A thorough sequencing analysis was performed by screening several clones of each amplification product, with the purpose of being able to identify low abundance transcripts.
Results: In both patients, the most abundant transcript was alternatively spliced and lacked exons 12 and 13. In addition, coexisting on the same allele, there were novel but not identical single amino acid deletions: deletion of L521 in patient 1 and of S715 in patient 2. A second novel alteration detected in both patients was the duplication of Q252. Patient 2 also demonstrated two independent, low abundance transcripts, one encoding the point mutation V559G and the other containing a deletion of exon 12.
Discussion: Here we describe two novel and identical C-KIT alterations in two patients with mast cell leukemia (deletion of exons 12 and 13 and duplication of Q252). We hypothesize that both are key molecular changes underlying mast cell leukemia. Exons 12 and 13 encode the N-terminus of the kinase domain, which is thought to have modulatory effect on the kinase active site. Amino acid 252 is located in the fourth immunoglobulin-like domain of the extracellular region, which is thought to be critical for receptor dimerization and phosphorylation. We predict that these mutations result in the superactivation of C-KIT, based on the aggressive behaviour of mast cell leukemia. The response to Gleeevec seen in patient 2 indicates that at least some of these transcripts are Gleevec sensitive. Functional assays to test if the encoded C-KIT proteins are constitutively activated and whether Gleevec inhibits the novel C-KIT proteins are in progress.
Prognostic Impact of C-Kit Mutations in Core Binding Factor-Leukemia.
Authors: Cairoli Roberto , Beghini Alessandro , Nadali Gianpaolo , Elice Francesca , Lunghi Maria , Cuneo Antonio , Grillo Giovanni , Nichelatti Michele , Ripamonti Carla B. , Lazzarino Mario , Rodeghiero Francesco , Pizzolo Giovanni , Larizza Lidia , Morra Enrica 
Published: October 2019

Abstract
C-KIT gene mutations are not a rare event in Core Binding Factor Leukemia (CBFL). To investigate their prognostic impact in this setting , we attempted at correlating the KIT mutational status with the WBC count, the level of the WBC-index, the presence of extramedullary disease (EMD) and the outcome, in t(8;21)(n=30) and inv(16) (n=20) AML patients. On the basis of genomic DNA analysis of c-KIT gene exons 2, 8, 10, 11, 17 for mutation detection, we categorized 21 patients (42%) in the “KIT mutated group” (KIT+) and 29 patients (58%) in the “KIT unmutated group” (KIT−). The median age at diagnosis was 46.4 and 45.6 years for the KIT(+) and KIT(−) groups, respectively (P=0.872). Among the KIT(+) patients, we found mutations in exon 8 (n = 4), exon 10 (n =1), exon 11 (n = 1) and exon 17 (n = 15). We recorded mean WBC counts of 28.6 x 109/L vs 34.5 x 109/L (P=0.051), and a mean WBC-index, expressed as WBC x (% Bone Marrow blasts/100), of 37.2 vs 15.5 (P=0.0395), for KIT(+) and KIT(−), respectively. Seven out of 50 patients experienced an EMD: in 6 cases (28.57%) this event occurred in the KIT(+) group. 40 patients (age < 60 years) were evaluable for clinical response; 18 out of them were KIT mutated. At a median follow-up of 24 months (range 10–107), we recorded for KIT(+) and KIT(−), respectively: CR incidence of 88.8 % (16/18) vs 100% (22/22) (P=0.38); Relapse Incidence 81.3% (13/16) vs 31.81% (7/22), P=0.0072; OS 27.7% (5/18) vs 77.3% (17/22), P=0.0049; DFS 16.7% (3/18) vs 77.3% (17/22), P=0.0005. Using a log-it linear model for univariate and multivariate regression analysis, we found a significant contribution to death (P=0.048) and relapse (P= 0.045) exerted by mutation in the whole group of patients; this effect was more evident when the analysis was restricted to patients <60 years (P=0.003 and P=0.014, respectively). In conclusion, this study confirms the correlation between c-KIT mutational status and high WBC-index in CBFL patients. Furthermore, our data indicate a statistical correlation between the presence of c-KIT mutations and both the OS and the DFS.
Id1 and Id2 Are Retinoic Acid Responsive Genes and Induce a G0/G1 Arrest in Acute Promyelocytic Leukemia Cells.
Authors: Nigten Jeannet , Nikoloski Gorica , De Witte Theo , Van der Reijden Bert A. , Jansen Joop H. 
Published: October 2019

Abstract
Acute promyelocytic leukemia (APL) is uniquely sensitive to treatment with all-trans retinoic acid (ATRA), which overcomes the differentiation arrest and induces terminal granulocytic differentiation of the leukemic blasts. In 98% of the cases of APL, the leukemic cells express a promyelocytic leukemia (PML)- retinoic acid receptor a (RARa) fusion protein as a result of a t(15;17) chromosome translocation. Previously, we have identified Id1 and Id2 as direct retinoic acid target genes being upregulated after ATRA stimulation in the APL cell line NB4 as well as in primary leukemic cells from APL patients. Id (inhibitor of DNA-binding) proteins act as antagonists of basic helix-loop-helix (bHLH) transcription factors by trapping them in heterodimeric complexes, thereby inhibiting DNA-binding and gene transactivation. Various bHLH proteins are pivotal in the control of differentiation and proliferation in various tissues (like muscle and nerve). We have studied the expression pattern of E2A, an ubiquitously expressed bHLH protein, which is generally considered as a promiscuous heterodimerization partner of other, more tissue restricted bHLH proteins. The expression of E2A was high in untreated APL cells and strongly downregulated upon ATRA stimulation. The simultaneous upregulation of Id1 and Id2, and the downregulation of E2A suggest a role for bHLH proteins in the induction of differentiation of APL cells upon treatment with ATRA. To assess the importance of Id1 and Id2 induction for the neutrophilic differentiation of the cells, we have overexpressed both proteins in the APL cell line NB4 using amphotropic retroviral transduction. Ectopic expression of Id1 and Id2 resulted in respectively 27% (n=3, SD= 9%) and 48% (n=3, SD=25%) inhibition of clonogenic growth in semi-solid medium, compared to vector-transduced control cells. Apart from the reduction in the number of colonies, overexpression of Id1 and Id2 did not alter the ATRA sensitivity of APL cells. NB4 liquid cultures revealed that Id1 and Id2 overexpression resulted in inhibition of proliferation and an increase of the percentage of cells in G0/G1, without having an effect on differentiation or apoptosis. These results indicate that Id1 and Id2 are important retinoic acid responsive genes in APL, and suggest that the inhibition of specific bHLH transcription factor complexes may play a role in the therapeutic effect of ATRA in APL.
Proteomic Identification of the C/EBPalpha Multiprotein Complex Reveals That JNK1, an Activator of C/EBPalpha, Is Inactive in Acute Myeloid Leukemia.
Authors: Trivedi A. , Bararia D. , PeerZada A. , Hiddemann W. , Behre G. 
Published: October 2019

Abstract
CCAAT Enhancer Binding Protein Alpha (C/EBPalpha) is one of the key myeloid transcription factors whose function is impaired in Acute Myeloid Leukemia (AML). This is either due to mutation or inhibition by other proteins via protein-protein interaction. Here, we hypothesised that protein-protein interaction of C/EBPalpha with other proteins might be important for its activation. Based on our hypothesis, we analysed the multiprotein complex of C/EBPalpha. After GST-pull down from U937 nuclear extract and using a proteomic approach we were able to identify JNK1, PAK6, MADP, ZNF45 and others as interacting proteins of C/EBPalpha. Following confirmation of physical interaction of JNK1 with C/EBPalpha, we were able to show that JNK1 phosphorylates C/EBPalpha, increases its protein half-life and thereby enhances its transactivation and DNA binding capacity. However, in various AML subtypes, JNK1 mRNA and Phospho-JNK1 protein expression is downregulated as compared with normal bone marrow which is one of the possible reasons why C/EBPalpha is inactive in AML. Thus, here we report that C/EBPalpha is impaired in AML due to inactivation of JNK1.
Identification of Distinct inv(16) Subclasses in Adult Acute Myeloid Leukemia Based on Gene Expression Profiling.
Authors: Bullinger Lars , Scholl Claudia , Bair Eric , Dohner Konstanze , Frohling Stefan , Schlenk Richard F. , Tibshirani Robert , Dohner Hartmut , Pollack Jonathan R. 
Published: October 2019

Abstract
Recurrent cytogenetic aberrations have been shown to constitute markers of diagnostic and prognostic value in acute myeloid leukemia (AML). However, even within the well-defined cytogenetic AML subgroup with an inv(16) we see substantial biological and clinical heterogeneity which is not fully reflected by the current classification system. To better characterize this cytogenetic group on the molecular level we profiled gene expression in a series of adult AML patients (n=26) with inv(16) using 42k cDNA microarrays.
By unsupervised hierarchical clustering we observed that samples with inv(16) separated primarily into two different subgroups. These showed no significant differences regarding known risk factors like age, WBC, LDH, etc. However, these newly defined inv(16)-subgroups were characterized by distinct clinical behavior. There was a strong trend towards unfavorable outcome with shorter overall survival times in one group (P=0.09, log rank test). Since the primary translocation/inversion events themselves are not sufficient for leukemogenesis, distinct patterns of gene expression found within each of these cytogenetic groups may suggest alternative cooperating mutations and deregulated pathways leading to transformation. Therefore, we performed a supervised analysis to determine the characteristic gene expression patterns underlying the cluster-defined subgroups. This Significance Analysis of Microarrays (SAM) method identified 260 genes significantly differentially expressed between the two newly defined inv(16)-subgroups (false discovery rate = 0.002). High expression levels of JUN, JUNB, JUND, FOS and FOSB characterized the first inv(16) subgroup (having less favorable prognosis). FOS gene family members can dimerize with proteins of the JUN family, forming the transcription factor complex AP-1 which has been implicated in the regulation of cell proliferation, differentiation, and transformation. Among the second subgroup, the proto-oncogene ETS1,displayed elevated expression, possibly resulting from aberrant MEK/ERK pathway activation as these cases also showed an over-expression of MAP3K1 and MAP3K2.
In conclusion, both supervised and unsupervised methods provide numerous insights into the pathogenesis of AML with inv(16), identifying clinically significant patterns of gene expression, as well as candidate target genes involved in leukemogenesis.
Arginine Methylation of Runx1 Regulates Its Biological and Transcriptional Activities.
Authors: Zhao Xinyang , Parkanani Animesh , Zhang Jin , Dunne Richard , Xiao Andrew , Allis C. David , Nimer Stephen 
Published: October 2019

Abstract
The AML1/Runx1 protein is required for definitive hematopoiesis and for the maturation of adult megakaryocytic cells. Alterations of the Runx1 gene by mutations, deletions and chromosome translocations are associated with several types of acute leukemia. Runx1 functions as both an activator and repressor of gene transcription with promoter and cell type context dependency. Likely, this relates to the ability of Runx1 to interact with a variety of transcription factors such as MEF, C/EBPa, Ets-1 and GATA-1 and also with repressor proteins such as Groucho, mSin3 and HDACs. Runx1 is post-translationally modified through acetylation and phosphorylation, and the acetylable and phosphorylable forms of Runx1 can activate transcription to higher level in Runx1 dependent reporter assays when HATs or Erk2 are coexpressed. Runx1 has also been shown to be methylated on lysine residues by SUV39H1 methyltransferase in fibroblasts. Based on the presence of a SGRGK motif in the runt domain of Runx1, we have been examining whether Runx1 is methylated on arginine residues by the protein arginine methyltransferases (PRMT). We have found that PRMT1 and PRMT5 are associated with Runx1 in AML cells by co-immunoprecipitation assays and using in vitro by GST-pulldown assays with in vitro translated PRMT(s) have shown that the interactions are direct. Using a luciferase gene reporter assay, we show that PRMT1 acts synergistically with p300 to activate Runx1 mediated transcription in response to cell proliferation signals. We have mapped the arginine methylation sites in Runx1 using GST-Runx1 fusion proteins, site-specific mutagenesis and mass spectrometry analysis. We have found three potential arginine methylation sites, one in the Runt domain, and two in the Runx1 carboxy-terminal region. Interestingly, one of these sites is in the region shown to interact with both the mSIN3A transcriptional repression complex and with p300. This suggests that arginine methylation of Runx1 may affects its transcriptional activating and repressing functions. Chromatin immunoprecipitation assays are underway to show how arginine methylation of Runx1 affects its activities in hematopoietic cells. Additional studies examining the effects of cross-talk between arginine methylation, lysine acetylation and serine phosphorylation has on Runx1 functions (biological and biochemical) will be presented.