bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interaction of MLL Amino Terminal Sequences with Menin Is Required for Transformation.
Authors: Hess Jay L. , Yang Zhaohai , Wang Haoren , Chen Ya-Xiong , Milne Thomas A. , Martin Mary Ellen , Slany Robert K. , Hua Xianxin 
Published: April 2020

Abstract
Rearrangements of the mixed lineage leukemia gene MLL are associated with aggressive lymphoid and myeloid leukemias. The resulting MLL fusion proteins enforce high-level expression of HOX genes including HOX A7 and HOX A9 and the HOX cofactor MEIS1, which is pivotal for leukemogenesis. The mechanism by which this occurs and the relationship to normal MLL function is unknown. MLL and MLL fusion proteins bind with a similar distribution in hematopoietic cells at both promoters and coding sequences of target genes. Our studies suggest that a major mechanism of regulating MLL, which is expressed throughout hematopoiesis, is through modulating it’s binding to target promoters. MLL binds directly to the promoters and coding regions of HOX A7, HOX A9, and MEIS1 only in myeloblasts and not in neutrophils, indicating MLL is physically associated with genes only when they are actively transcribed. Expression of A cluster HOX loci and MEIS1 remains persistently elevated when MLL-ENL or dimerized MLL fusion proteins are expressed. Expression of either fusion protein is associated with increased binding of wild type MLL accompanied by increases in histone acetylation and histone H3 lysine 4, marks that are normally almost completely erased during myeloid differentiation. In addition MLL-ENL induces increased lysine 79 methylation. Both MLL and MLL fusion proteins interact with the tumor suppressor menin via sequences in the extreme amino terminus of MLL. In addition both proteins physically interact with RNA polymerase II, which shows abnormal pausing in the coding regions of HOX genes in Mll null cells. Genetic ablation of menin or expression of a dominant negative inhibitor of the MLL-menin interaction inhibits the growth of MLL fusion protein transformed cells. These findings suggest MLL fusion proteins act in concert with menin, MLL and other coactivators to deregulate HOX gene expression pivotal for transformation.
c-Myb Is an Essential Downstream Target for Homeobox Mediated Transformation of Hematopoietic Cells.
Authors: Slany Robert K. , Bittner Claudia B. , Zeisig Deniz T. , Bach Christian , Hess Jay L. 
Published: April 2020

Abstract
Overexpression of Hox genes is frequently associated with leukemogenic transformation of hematopoietic cells. In particular the abdominal-type HoxA genes together with their homeodomain containing dimerization partners of the TALE (three amino acid loop extension) family are well documented oncogenes in various hematologic malignancies. Paradigmatic examples are Hoxa9 and Meis1 that are coactivated by retroviral insertions in the BXH2 mouse leukemia model. In addition Hoxa9 is a fusion partner of NUP98 in acute myeloid leukemias with a t(7;11) and Meis1 as well as Hoxa9 are critical targets of MLL (mixed lineage leukemia) fusion proteins. Despite their important role in leukemogenesis little is known how they exert their transforming function. Here we used a novel system of transient transformation by an inducible oncoprotein to generate populations of primary hematopoietic cells that overexpress Hoxa9 in combination with Meis1. The RNA inventory of these cells was compared to control cells by Affymetrix array analysis. Interestingly the RNA of c-Myb (myeloblastosis leukemia virus oncogene) and of a known c-myb downstream target (Gstm1) was amongst the top ten genes upregulated by Hoxa9/Meis1. c-Myb overexpression was verified by Northern blot and quantitative RT-PCR. c-Myb levels also responded to the introduction of MLL-ENL, an oncoprotein that upregulates Hoxa9 and Meis1. Small interfering RNAs directed against c-Myb suppressed transformation by MLL-ENL but had no effect on transformation of hematopoietic precursors by E2A-HLF an oncogene that does not work by increasing Hoxa9/Meis1 levels. The anti c-Myb siRNA effect was abrogated by coexpression of a c-Myb derivative with a mutated siRNA target site. The coexpression of a dominant negative c-Myb mutant had a similar but weaker effect on MLL-ENL mediated transformation. Ectopic expression of c-Myb in hematopoietic precursor cells induced a mild differentiation block. However, c-Myb alone was not able to give a positive readout in a serial replating assay under conditions where the combined overexpression of Hoxa9 together with Meis1 was clearly transforming. In summary the results suggest that c-Myb is essential but not sufficient for the oncogenic activity of Hoxa9/Meis1.
Minimal Residual Disease Monitoring by Four Color Flow Cytometry before Second Induction Is an Independent Prognostic Factor in Childhood Acute Myeloid Leukemia - AML-BFM MRD Study Group.
Authors: Langebrake Claudia , Dworzak Michael , Hrusak Ondrej , Mejstrikova Ester , Griesinger Frank , Zimmermann Martin , Creutzig Ursula , Reinhardt Dirk 
Published: April 2020

Abstract
Monitoring of minimal residual disease (MRD) by flow cytometry in childhood acute myeloid leukemia (AML) is still a topic of discussion in terms of the optimal time of analysis and the best antibody combinations. Here we report the largest prospective international series of MRD monitoring (395 samples) in children with AML (n=124).
All children have been treated according to the AML-BFM 98 study. Based upon a CD33/CD34 backbone, a wide panel of antibodies, independent from the initial immunophenotype, was applied at up to four defined time points during treatment (MRD1: day 15, MRD2: before 2nd induction, MRD3: before 3rd block, MRD4 before 4th block). For each of the 12 leukemia associated immunophenotypes (LAIP) and time points, a threshold level based upon a previous retrospective analysis of 149 other children with AML as well as analysis of normal bone marrow was determined.
Regarding all four time points there is a statistically significant difference in the 3-year event free survival (EFS) in those children presenting with MRD positivity at ≥ 3 time points (pEFS 68% vs. 33%, p=0.01). The MRD level at time point 2 has turned out to have the most significant predictive value for 3-year EFS: 66% (n=104) vs. 35% (n=17), plogrank=0.006. Regarding the standard risk group (defined by FAB subtype [M1, M2± Auer rods, M3, M4eo], favorable cytogenetics and morphologically determined treatment response at day 15) only, it is possible to identify a subgroup with poor outcome (20% [n=5] vs. 88% [n=40], plogrank &lt;0.0001). In accordance with this, a multivariate analysis proved the MRD2 as a significant prognostic factor (pChi2=0.023) independent from the known risk factors like FAB subtype, cytogenetics or morphological blast percentage at day 15. As a conclusion, (A) MRD monitoring before second induction has the same predictve value as examining MRD level at four different time points during intensive chemotherapy and (B) the immunologic analysis of MRD before second induction is particulary suitable for risk group stratification and therapeutic consequences in addition to the known risk factors.
Molecular Switch between Tissue Factor-Mediated Signaling and Coagulation.
Authors: Ahamed Jasimuddin , Vesrteeg Henri H. , Kerver Marjolein , Hogg Philip J. , Mueller Barbara M. , Ruf Wolfram 
Published: April 2020

Abstract
Tissue factor (TF) forms a catalytic complex with coagulation factor VIIa and thus triggers coagulation as well as cell signaling through protease activated receptor (PAR) 2. The molecular switch between these two functions remains elusive. We demonstrate in growth arrested keratinocytes that TF’s coagulant function remains unchanged when TF expression levels and TF-VIIa signaling are concordantly downregulated. Thus, coagulation and signaling are mediated by two distinct and differentially regulated pools of TF. TF-VIIa signaling, but not coagulation, is blocked by bacitracin, an inhibitor of protein disulfide isomerase, and TF co-immunoprecipitates protein disulfide isomerases. To test the role of isomerization, we expressed single thiol containing mutants of the cross-stranded, surface exposed TF Cys186-Cys209 disulfide bond. Retaining Cys186, but not Cys209, preserves signaling properties with high affinity for VIIa, but eliminates the TF-dependent coagulation. Cys186-containing TF is surface expressed predominantly in a dimeric form, indicating a trapped isomerization intermediate. Signaling by wild-type, but not the Cys186 dimer, is inhibited by blocking free thiols indicating that thiol-isomerization plays an essential role in regulating TF signaling. Furthermore, we identified a monoclonal antibody that blocks TF-VIIa signaling of wild-type, but not Cys186 dimer TF, suggesting interference with the dynamics of TF isomerization. This human TF specific antibody does not interfere with coagulation, but inhibits the growth of human xenograft tumors in mice. Thus, TF-VIIa signaling, not coagulation, is the primary pathway for TF-driven tumorigenesis. These experiments resolve the molecular mechanism that switches TF-VIIa from coagulation to signaling and show feasibility to block signaling TF for the therapeutic intervention in cancer.
Altered Oncogenic Fitness of Imatinib- and Dasatinib-Resistant BCR-ABL Mutants Is Due to Differential Intrinsic Kinase Activity and Signaling Pathway Selection Defined by Phosphoproteome Profiling.
Authors: Skaggs Brian J. , Burgess Michael R. , Gorre Mercedes E. , Graeber Thomas G. , Sawyers Charles L. 
Published: April 2020

Abstract
Point mutations in the kinase domain of BCR-ABL are the most common cause of resistance to imatinib therapy in CML. E255K and T315I are detectable before the initiation of imatinib therapy in a small population of CML patients, suggesting that certain imatinib-resistant BCR-ABL mutants display altered biological fitness relative to wild-type p210 BCR-ABL. To test this hypothesis, we performed primary murine bone marrow transformation assays using BCR-ABL-expressing retroviruses. In a survey of six clinically imatinib-resistant mutations (Y253F, Y253H, E255K, E255V, T315I, M351T) as well as five additional mutations that confer in vitro resistance to the dual SRC/ABL inhibitor dasatinib (L248R, V299L, T315A, F317L, F317V), we identified several that altered oncogenic fitness either positively or negatively relative to p210. To elucidate the molecular basis for altered transformation potential, we examined the kinase activity of p210 versus mutant full-length BCR-ABL. Enzyme kinetics studies with an optimized Abl peptide substrate as well as gel-based in vitro kinase assays illustrated that intrinsic kinase activity does explain some, but not all, of the mutant oncogenic potency phenotypes. The imatinib- and dasatinib-resistant mutant T315I, for instance, was a gain-of-fitness mutant in the bone marrow assay but exhibited a three-fold lower Vmax than p210. In contrast, T315A, a dasatinib-resistant mutation at the T315 gatekeeper residue, exhibits reduced transforming potential relative to wt p210 and is completely unable to phosphorylate Abl substrates in the kinase assays, although it does retain the ability to autophosphorylate. Subsequent kinase assays on immunoprecipitations from stably expressing IL-3-independent Ba/F3 cells illustrated that certain mutants bind and phosphorylate distinct substrates from p210. We further delineated signaling differences between p210 and the mutant panel by utilizing a mass spectrometry-based global phosphorylation profiling technique. Trypsinized proteins from stably expressing p210 and mutant Ba/F3 cells were immunoprecipitated using anti-phosphotyrosine antibodies, then identified and quantified by two dimensional mass spectrometry. Phosphorylation of specific substrates correlates with gain- or loss-of fitness observed in the bone marrow assay. We conclude that certain BCR-ABL kinase domain mutations, in addition to conferring drug resistance, also affect the oncogenic potency of BCR-ABL by altering intrinsic kinase activity as well as downstream targets. Elucidation of alternative signaling pathways activated by these mutants by the combination of biochemical and mass spectrometric methods may provide novel therapeutic targets for imatinib- and dasatinib-resistant CML.
Donor Lymphocyte Transfusion (DLT) in the Treatment of Relapsed CML after Allogeneic PBSCT - A Retrospective Analysis of 346 Patients by the EBMT Chronic Leukemia Working Party.
Authors: Schmid Christoph , Biezen Anja v. , Porkka Kimmo , Chalandon Yves , Urbano Alvaro , Olavarria Eduardo , de Witte Theo , Hertenstein Bernd , Archese Wiliam , Clark Richard , Kolb Hans-Jochem , Niederwieser Dietger , Guglielmi Cesare 
Published: April 2020

Abstract
DLT has become the gold standard for the treatment of reccurrent CML after allogeneic stem cell transplantation. However, so far, published data are based on DLT following bone marrow transplantation (BMT). During recent years, mobilized peripheral blood stem cell transplantation (PBSCT) have been increasingly performed. To evaluate the efficacy of DLT following PBSCT, we retrospectively analysed the results of 86 patients treated with DLT for CML relapse after PBSCT from HLA-identical family donors. Results were compared to 260 patients receiving DLT for relapse after related BMT. Reduced intensity transplants were excluded.
BMT and PBSCT groups were balanced in terms of patient age (median: 40 vs 42 y), conditioning, stage at transplant (84% vs 76% in first chronic phase (CP) p=.2), and stage at relapse (molecular/cytogenetic: 59% both, CP: 28% vs 20%, advanced: 13% vs 21%, p=.2). However, BMT patients had been transplanted in earlier years (median: 1994 vs.1998, p&lt;.001), had a longer remission after transplantation (median: 677 vs. 275 d, p&lt;.001), and had experienced more aGvHD after transplantation (58% vs. 34%, p&lt;.001). Complete molecular or cytogentic remission was achieved in 71% of the patients, with a trend towards better response in BMT recipients (p=.06). Overall (OS) and event free survival (EFS) at five years from DLT are shown in table 1. Outcome was significantly superior in BMT recipients, in particular if DLT was given for hematological, rather than cytogenetic or molecular relapse. In a cox regression model, four factors were associated with better OS/EFS: Transplantation in CP1 (p&lt;.001/.002), early stage at relapse (molecular/cytogenetic vs. hematological, p&lt;.00/&lt;.001), a longer remission after transplantation (p=.005/.028), and BM vs. PBSC for transplantation (p= .003/.004). In contrast, age of patient and donor, acute or chronic GvHD after transplantation, the use of chemotherapy or imatinib before DLT, and the initial number of transfused CD3+ cells were not significant.
The results of this retrospective study suggest a reduced efficacy of DLT in CML relapse following related allogeneic PBSCT as compared to BMT. This effect seems to be independent from established risk factors as disease stage and remission duration. However, since several adverse factors are not equally distributed among the two groups, a bias might be induced, although no statistically significant imbalance could be detected. Therefore, the observation needs to be confirmed in a larger cohort or a prospective study.
Table 1. Outcome after DLT No BMT PBSCT p Total 346 260 86 OS at five years from DLT 71% 75% 66% &lt; .001 EFS at five years from DLT 61% 64% 41% &lt; . 001
Relationship between Conditioning Intensity and Comorbidity in Patients (Pts) with Acute Myeloid Leukemia (AML) or Myelodysplasia (MDS) Receiving Allogeneic Hematopoietic Cell Transplantation (HCT).
Authors: Sorror Mohamed , Storer Barry , Sandmaier Brenda M. , Maris Michael , Baron Frederic , Maloney David , Scott Bart , Deeg H. Joachim , Storb Rainert 
Published: April 2020

Abstract
We developed a new HCT-specific comorbidity index (HCT-CI) as a sensitive tool to capture pretransplant comorbidties among pts given allogeneic HCT (Sorror et al, online, 2005; DOI 10.1182/blood-2005-05-2004). In the current report, we used the HCT-CI to explore differences in the tolerability of nonablative compared to ablative HCT in MDS or AML pts with pretransplant comorbidities. Consecutive pts (n=88) receiving nonablative conditioning with 2 Gy total body irradiation (TBI) alone (6%) or with 90 mg/kg of fludarabine (94%) were compared to concurrent pts (n=361) conditioned with cyclophosphamide plus busulfan (70%) or 12–14 Gy TBI (30%). All pts were classified into low (AML-first complete remission or MDS-refractory anemia) and high-risk disease (all others). Overall, 76% of nonablative pts had AML compared to 66% of ablative pts. At HCT, nonablative pts differed substantially from ablative pts with respect to age (median 60 vs 46 years), use of unrelated donor grafts (57% vs 42%), use of marrow grafts (5% vs 20%), and HCT-CI scores of ≥2 (75% vs 45%). Smaller differences existed for high-risk disease (64% vs 58%) and pt cytomegalovirus positive sero-status (59% vs 55%). Proportional hazards models were used to estimate the hazard ratio (HR) for non-relapse mortality (NRM) and survival for pts receiving nonablative compared to ablative conditioning regimens; these models were adjusted for stem cell source, pt age, donor type, pt cytomegalovirus sero-status, diagnoses (MDS vs AML), and HCT-CI scores (Table). Adjusted HRs were not statistically significantly different, except among pts with high-risk disease and HCT-CI scores of ≥2 where nonablative pts had lower HR for NRM (0.34, P=0.005) and worse survival (0.51, P=0.006, Fig.1). These results suggest that pts with high-risk MDS or AML and HCT-CI scores of ≥2 might have less NRM and better survival with nonablative than with ablative HCT. Prospective randomized trials are warranted to confirm this retrospective observation. Additional data are needed for AML and MDS pts with low comorbidities or low disease risk in order to clarify the usefulness of nonmyeloablative conditioning.
Table: Adjusted hazard ratios for pts receiving nonablative relative to ablative conditioning Nonablative/ablative NRM Survival Number HR* (95% CI) P HR* (95% CI) P *HR &lt;1 indicates more favorable outcome for pts receiving nonablative conditioning Low-risk disease HCT-CI scores 0–1 7/86 0.0 (undefined) 0.33 0.8 (0.1–6.3) 0.83 HCT-CI scores ≥2 25/64 1.97 (0.7–5.9) 0.23 1.86 (0.8–4.1) 0.13 High-risk disease HCT-CI scores 0-1 15/112 0.60 (0.1–2.7) 0.51 0.96 (0.5–2.0) 0.92 HCT-CI scores ≥2 41/99 0.34 (0.2–0.7) 0.005 0.51 (0.3–0.8) 0.006
Figure Figure
Severe Thrombocytopenia with Impaired Megakaryocytopoiesis in a Family with von Willebrand Disease Type 2B.
Authors: Nurden Paquita , Debili Najet , Vainchenker William , Pasquet Jean-Max , Bobe Regis , Bredoux Raymonde , Nurden Alan T. , Enouf Jocelyne 
Published: April 2020

Abstract
Thrombocytopenia associated with type 2B von Willebrand disease (VWD) is said to result from an increased elimination of platelets due to an abnormal binding of the higher molecular weight VWF multimers. Here we report a family (brother and sister) with severe thrombocytopenia, presence of agglutinated platelets and a very severe spontaneous bleeding syndrome since childhood. Their mutation concerns the VWF A1 domain, with a R1308P substitution which corresponds to an interactive site for GPIb (French VWD network). Electron microscopy showed agglutinates composed of adjacent discoid platelets with plasma membrane domains either in very close contact or showing signs of being fusioned. By flow cytometry, increased amounts of VWF and sometimes also fibrinogen were found at the platelet surface, but not P-selectin. Binding of antibodies to the GP Ibalpha N-terminal domain was decreased whereas GPIX and GPV were normally detected. In Western Blotting (WB), GPIbalpha was normally present but there were signs of filamin degradation. Basal Ca2+ levels were elevated. Platelet proteins involved in Ca2+ regulation were analysed by quantitating mRNA or by WB. Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)-type 3b was found to be increased but not the isoforms 2b or 3a. Furthermore, plasma membrane Ca2+ ATPase (PMCA)-4b as well as Inositol triphosphate receptor (InsP3-R3) were also increased in concentration. Interestingly, this platelet profile, mimics that observed in normal immature MK before proplatelet formation. As PMCA4b is a target of caspase-3 in MK, another substrate the polyadenosine diphosphate (ADP)-ribose polymerase protein (PARP) was evaluated. For both patients, platelets contained increased PARP and a degradation product indicating an abnormal persistance of caspase-3 activity. In vitro, megakaryocyte (MK) culture from peripheral blood CD34+ cells in the presence of SCF and TPO showed impaired MK maturation and the production of self-associated proplatelets. All these results are in favour of a role for abnormal platelet production with abnormal apoptosis that can explain at least in part the marked thrombocytopenia in this family with VWD type 2B.
SP1 and NF-Y-Dependent Gene Transactivation Defines a Gain-of-Function for the PML-RARα Oncoprotein.
Authors: van Wageningen Sake , Breems-de Ridder Marleen C. , Nikoloski Gorica , Erpelinck-Verschueren Claudia A.J. , Nigten Jeannet , Löwenberg Bob , de Witte Theo , van der Reijden Bert A. , Jansen Joop H. 
Published: April 2020

Abstract
PML-RARa is the product of the chromosomal translocation t(15;17) and is the causative oncogene in more than 98% of the cases of acute promyelocytic leukemia. PML-RARa interferes with the transcription of retinoic acid receptor target genes in a dominant manner, contributing to the transformation of the cells. RARa regulates transcription via heterodimerisation with RXR. PML-RARa can bind as a homodimer to the same sequences as RARa/RXR. At supra-physiological dosis of all-trans retionoic acid (ATRA), the block in differentiation in APL can be overcome. ATRA releases co-repressors from PML-RARa and activates RARa target genes.
The ID1 gene is rapidly induced by ATRA in acute promyelocytic leukemia cells. Promoter analysis showed that the ID1 promoter was activated by PML-RARa but, unexpectedly, not by wild type RARa/RXR. Surprisingly, PML-RARa lacking the DNA binding domain could still transactivate the ID1 promoter indicating that transactivation was mediated without direct DNA binding. Promoter deletion studies showed that adjacent NF-Y and SP1 binding sites were essential for this transactivation. A direct physical interaction was shown between PML-RARa and SP1 in vitro and chromatin immunoprecepitation assays confirmed that a complex of PML-RARa/NF-Y/Sp1 is present on the ID1 promoter in vivo. In addition, we found that ectopic expression of PML-RARa induced expression of the endogenous ID1 gene in response to retinoic acid. We propose a novel, gain-of-function model for PML-RARa in which the fusion protein interferes with the transcription of SP1-NF-Y regulated genes. Interference is mediated without DNA-binding, through direct interaction with SP1. This implicates that apart from the previously described deregulation of retinoic acid receptor target genes, PML-RARa may deregulate an additional class of genes that are nomally not regulated by retinoid receptors.