bluuebunny/crossref_metadata_2025_split huggingface dataset:
Residual Plasmatic Activity of ADAMTS13 in Congenital Thrombotic Thrombocytopenic Purpura Correlates with Disease Phenotype
Authors: Lotta Luca A , Wu Haifeng M , Scully Marie A , Noris Marina , Veyradier Agnes , Mackie Ian J , Remuzzi Giuseppe , Coppo Paul , Liesner Ri , Donadelli Roberta , Loirat Chantal , Gibbs Richard A , Horne April , Yang Shangbin , Palla Roberta , Peyvandi Flora 
Published: October 2019

Abstract
Abstract 2219

ABSTRACT
Congenital thrombotic thrombocytopenic purpura (TTP) (OMIM #274150) is a rare, recessively inherited thrombotic microangiopathy characterized by the congenital deficiency of ADAMTS13 due to mutations in the corresponding gene. The clinical phenotype of congenital TTP is variable, encompassing neonatal-onset disease and adult-onset disease, forms with a single disease episode and chronic-relapsing forms. A review of the ∼100 published cases of congenital TTP showed similar age of first disease episode in patients carrying the same ADAMTS13 gene suggesting that different ADAMTS13 mutations might influence the severity of clinical phenotype, conceivably by determining different patterns of residual plasmatic activity of ADAMTS13. However, the quantification of residual ADAMTS13 activity in severely deficient patients was blunted by the relatively high limit of detection (LOD) of commonly used ADAMTS13 activity assays (varying between 3% and 6%). We report 29 cases of congenital TTP from 4 European cohorts, with 32 mutations of ADAMTS13 including 8 not previously published. To assess if residual ADAMTS13 activity was detectable in these patients and correlated with their clinical history, ADAMTS13 activity was measured by SELDI-TOF mass spectrometry (LOD=0.5%).
ADAMTS13 activity above 0.5% was measurable in 26 (90%) patients, and lower levels of activity correlated with an earlier age of disease onset (r=0.51; p=0.006) and with the need for regular fresh frozen plasma prophylaxis (2.24% vs 3.88%; p=0.03). Mutations at the highly conserved N-terminal domains of the protein were associated with lower residual ADAMTS13 activity (1.48% vs 4.89%, p=0.02) and earlier disease onset (3.2 years vs 24.2 years, p=0.003). Our results show that residual ADAMTS13 activity correlates with the severity of clinical phenotype in congenital TTP and provide insights into genotype-phenotype correlations.

Disclosures:
No relevant conflicts of interest to declare.
The Earliest Thymic T Cell Progenitors Sustain B Cell and Myeloid Lineage Potentials
Authors: Luc Sidinh , Macaulay Iain C , Buza-Vidas Natalija , Bouriez-Jones Tiphaine , Lutteropp Michael , Woll Petter , Mead Adam J. , Boukarabila Hanane , Luis Tiago C , Mizukami Takuo , Matsuoka Sahoko , Brown John , Ferry Helen , Anderson Kristina , Duarte Sara , Atkinson Deborah , Soneji Shamit , Domanski Aniela , Farley Alison , Sanjuan-Pla Alejandra , Carella Cintia , Patient Roger , de Bruijn Marella , Enver Tariq , Nerlov Claus , Blackburn Clare , Godin Isabelle , Jacobsen Sten Eirik W 
Published: October 2019

Abstract
Abstract 2335
The stepwise lineage-commitment from hematopoietic stem cells (HSCs) in the bone marrow (BM) to T-lymphocyte-restricted progenitors in the thymus represents a paradigm for how distinct stages of lineage restriction from a multipotent to a lineage-restricted progenitor require different extrinsic cues. However, the commitment stage at which progenitors migrate from the BM to the thymus remains unclear. Previous studies demonstrated the existence of adult early thymic progenitors (ETPs) restricted to T lymphocyte and granulocyte-monocyte (GM) fates (Bell and Bhandoola, Nature 2008; Wada et al., Nature 2008). The challenge remains to couple this thymic T–GM restricted progenitor to candidate thymus seeding progenitors (TSPs) identified in the BM, such as common lymphoid progenitors, lymphoid-primed multipotent progenitors (LMPPs) and HSCs, which all also possess B cell potential.
Using high resolution FACS and sensitive clonal assays, we have identified the most primitive ETPs in the neonatal thymus and demonstrate at the single cell level that rare Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs possess combined granulocyte-monocyte (GM), T and B lymphocyte but not megakaryocyte-erythroid (MkE) lineage potentials, identical to closely molecularly related thymus-seeding progenitors in the BM.
Moreover, global molecular profiling demonstrates that neonatal Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs cluster closest to multipotent progenitors in the BM, rather than subsequent (DN1 and DN2) progenitor stages in the thymus. Finally, in support of Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs with combined T, B and GM lineage potentials, being the most multipotent progenitors in the thymus, phenotypic and functional studies demonstrate that the neonatal thymus is not seeded by HSCs or MPPs with MkE potential. These findings establish for the first time a distinct lineage commitment stage for the transition of T-lineage commitment from the BM to the remote thymus.

Disclosures:
No relevant conflicts of interest to declare.
Src Kinases Act Downstream Akt and Are Required for the Transforming Properties of Constitutively Activated Forms of STAT5A and STAT5B
Authors: Nyga Remy , Savay Lea , Marcq Ingrid , Regnier Aline , Damaj Gandhi , Rochette Jacques , Gouilleux Fabrice , Lassoued Kaiss 
Published: October 2019

Abstract
Abstract 2378

Background:
Stat5A and Stat5B transcription factors play an important role in the control of hematopoietic cell proliferation and survival and are constitutively activated in number of hematological neoplasia. Moreover, expression of constitutively active Stat5 (caStat5) mutants in primary hematopoietic cells can lead to leukaemia. The transforming properties of CaStat5 have been previously shown to require Akt activation. However, the mechanisms by which CaStat5 mutants and Akt are activated remain enigmatic. In this study we aimed to determine the roles of src kinases in these processes,

Methods:
For this purpose, we used murine Ba/F3 cells transfected or not with CaSta5A (Stat5A1*6) or CaStat5B (Stat51*B6) or with the oncogenic fusion proteins Tel-JAK2 and Tel-Abl as controls. Primary hematopoietic progenitors obtained from mouse bone marrow were also used in this sutdy. Cells were treated or not with the pan-src inhibitors PP1, PP2, and SU 6656 (and with PP3 an inactive analogue of PP1 as a control), imatinib (Abl inhibitor) and AG490 (JAK inhibitor). In some experiments cells were treated with U0126 (MEK inhibitor) or with the highly transducible TaT-dominant negative (dn) Akt fusion protein. Cell proliferation was assessed by cell counting. Cell death was evaluated using the trypan blue dye exclusion test. Cell apoptosis was assessed by analysing annexin V staining using flow cytometry and PARP cleavage using western blot. Expression of kinases and their phosphotylated forms were examined by western blot.

Results:
CaStat5A and CaStat5B cells were shown to express constitutively phosphorylated Lyn, Hck, Src kinases as well as Akt, IKK and ERK1/2. Stat1, Stat3, and JAK family members (TYK2 and JAK1-3) were not phosphorylated in these cells. Use of pan-Src inhibitors inhibited proliferation of CaStat5 cells and induced apoptosis of a fraction of these cells, in a dose-dependent manner. By contrast, imatinib and AG490 had no effect on the growth and survival of CaStat5 cells. Moreover, pan-src inhibitors prevented Erk but not Akt, IKK or STAT5 phosphorylation. By contrast, inhibition of Akt by means of TaT-dnAkt abolished Lyn, Src and ERK1/2 phosphorylation. Importantly, inhibition of MAPK did not interfere with Akt phosphorylation and did not interfere with CaStat5 cell survival.

Conclusion:
Altogether our data indicate that Akt acts upstream in the CaStat5-associated signalling cascade, then activates src kinases and thereby promotes MAPK-dependent cell proliferation and MAPK-independent cell survival. These results shed light onto a hitherto undescribed role of Src kinases acting downstream Akt in the mechanisms of action of CaStat5.

Disclosures:
No relevant conflicts of interest to declare.
Survivin Is Highly Expressed in AML Stem Cells and Predicts Poor Clinical Outcome
Authors: Carter Bing Z , Qiu Yihua , Huang Xielin , Diao Lixia , Zhang Nianxiang , Coombes Kevin R , Mak Duncan H , Konopleva Marina , Cortes Jorge E. , Kantarjian Hagop M. , Andreeff Michael , Kornblau Steven M. 
Published: October 2019

Abstract
Abstract 238
Survivin, a member of the inhibitor of apoptosis (IAP) protein family, plays important roles in cell proliferation and survival and is highly expressed in various malignant cells, including leukemic cells. We showed previously that targeting survivin expression in AML cells induces a cell proliferation defect and subsequent cell death involving the mitochondrial pathway. To assess its usefulness as a prognostic marker in AML, we profiled survivin expression in samples from 511 newly diagnosed AML patients using a validated reverse-phase protein array and correlated its protein expression levels with clinical outcomes. We found that survivin levels were higher in the bone marrow (BM) than in peripheral blood (PB) leukemic cells in 140-paired samples (p = 0.0001) consistent with its function in cell proliferation. Survivin levels did not correlate with cytogenetic groups, the mutational status of nucleophosmin or FLT-3, BM or PB blast counts, white blood count, or patient performance status. Interestingly, survivin levels were found to significantly predict shorter overall (P = 0.016) and event-free (P = 0.023) survival in multivariate Cox model analysis. Age, gender, white blast count, BM blasts, cytogenetics, and survivin levels were included in the final model. The groups expressing low and high survivin were defined by the median value. This was also true when data sets from BM and PB were analyzed separately. Further, we found that survivin levels were significantly higher in CD34+38− AML stem/progenitor cells than in bulk AML blasts and total CD34+ cells (2.06 and 1.91 fold, respectively, P < 0.001) when we analyzed survivin expression in 37 samples of isolated CD34+38− AML cells (see Figure).
These results suggest that survivin expression levels are prognostic in AML and that survivin is overexpressed in AML stem/progenitor cells, may play an important role in AML stem cells, and may thus be an important target in AML therapy.

Disclosures:
No relevant conflicts of interest to declare.
Selective Damage to Antioxidant Gene Promoters in FA Cells
Authors: Du Wei , Rani Reena , Sipple Jared , Schick Jonathan , Pang Qishen 
Published: October 2019

Abstract
Abstract 2413
Oxidative stress has been implicated in the pathogenesis of many human diseases including Fanconi anemia (FA), a genetic disorder associated with bone marrow failure and progression to leukemia and other cancers. Here we show that several major anti-oxidant defense genes, including Glutathione peroxidase 1, Peroxiredoxin 3, Thioredoxin reductase 1, Superoxide dismutases 1, NAD(P)H:quinone oxireductase and Catalase, are down-regulated in bone marrow cells of FA patients. This gene down-regulation is selectively associated with increased oxidative DNA damage in the promoters of these anti-oxidant defense genes. Further, we show that both increased initial damage and reduced repair rate contribute to augmented oxidative DNA damage in FA cells. Using cell-based assays to assess promoter activity and damage repair kinetics, we demonstrate that FA proteins function to protect the promoter DNA from oxidative damage. Mechanistically, FA proteins appeared to act in concert with Brg1, a chromatin-remodeling ATPase subunit of the BAF complex. Specifically, Brg1 binds to the promoters of the anti-oxidant defense genes in steady state. Upon challenge with oxidative stress, FANCA and FANCD2 proteins are recruited to the promoter DNA, which correlates with significant increase in the binding of Brg1 within the promoter regions. Intriguingly, the formation of the FA-Brg1-promoter complex results in a marked decrease in nuclease hypersensitivity and oxidative damage in the promoter DNA in normal cells compared to FA cells. Finally, disassociation of the FA proteins from the Brg1-promoter complex parallels Pol II loading, suggesting a regulatory role for the FA proteins in transcription. Taken together, the study identifies a role of FA proteins in protecting anti-oxidant genes from oxidative damage.

Disclosures:
No relevant conflicts of interest to declare.
MicroRNA Expression in Acute Myeloid Leukaemia and Their Effects on the Differentiation of Acute Myeloid Leukaemia Cells
Authors: Palma Catalina A , Tonna Elise , Bryant Adam J , Jayaswal Vivek , Agapiou David , Yang Yee Hwa , Ma David , Lutherborrow Mark 
Published: October 2019

Abstract
Abstract 2430
Acute myeloid leukaemia (AML) is typified by an aberrant halt in maturation of myeloid progenitor cells, leading to uncontrolled proliferation of immature blasts. In the majority of cases of AML with normal karyotype, the underlying cause of the maturation arrest remains unclear. MicroRNAs, small inhibitory RNAs, are known to be dysregulated in cancers and have been postulated to play a causative role in leukaemogenesis.
We aimed to investigate the contribution of aberrant microRNA expression to the inhibition of maturation in AML cells. MicroRNA expression profiling was performed on the bone marrow of 28 AML patients with normal karyotype and 8 normal controls. Differential expression was confirmed by qRT-PCR. We found that the expression of a panel of 12 microRNAs was able to accurately separate AML-M1 and AML-M5 subtypes. The AML-M1 subtype represents a more immature cell population when compared to the monoblast morphology observed in AML-M5. Four candidate microRNAs, miR-181a, -146a, -130a and -135b were selected for further investigation based in their putative targeting of key monocytic transcription factors as determined by in silico modelling followed by luciferase assays.
In vitro monocyte and macrophage differentiation of HL60 and NB4 cell lines with 1,25-dihydroxyvitamin D3 and/or phorbol-12-myristate-13-acetate treatment resulted in a significant decrease in all four candidate microRNAs (measured by qRT-PCR), supporting the hypothesis that candidate microRNA over-expression in AML-M1 may contribute to its maturation arrest. Over-expression of the candidate microRNAs by transfection with Pre-miR microRNA precursor molecules (Ambion) into the HL60 and NB4 monocyte differentiation model, resulted in the significant suppression of CD14 (Figure, *p <0.05 compared to Scrambled control) and CD15 expression, markers of monocytes and granulocytes respectively. Conversely, knockdown of miR-181a, -146a, -130a and -135b with miRCURY LNA microRNA knockdown probes (Exiqon) induced an increase in CD14 expression in HL60 cells compared to non-targeting Scrambled control.
An important regulatory role of these microRNAs in myeloid/monocytic differentiation is strongly suggested by their targeted suppression of the key transcription factors KLF4, MAFB, IRF8, HOXA10, MCL1 and PU.1 which was confirmed by luciferase reporter assay. This study provides evidence that the over-expressed microRNAs discovered in our profiling work between AML-M1 and AML-M5 are biologically relevant microRNAs, which have the potential to affect the maturation potential of AML cells.

Disclosures:
No relevant conflicts of interest to declare.
Reduced c-FOS Is Associated with Poor Prognosis and Clinical Course in Chronic Lymphocytic Leukemia
Authors: Oakes Christopher C , Claus Rainer , Schmidt Christopher , Park Yoon Jung , Boutros Michael , Stilgenbauer Stephan , Plass Christoph 
Published: October 2019

Abstract
Abstract 2433
We have previously shown that silencing of the tumor suppressor gene, DAPK1, by genetic and epigenetic mechanisms is associated with the pathogenesis of CLL. To elucidate genes and pathways involved in DAPK1 transcriptional regulation, genome-wide siRNA screens were performed using a novel transgene reporter system involving a stably-integrated BAC construct expressing luciferase under the control of the DAPK1 locus. c-FOS and other members of the FOS gene family were identified as positive regulators, implicating the AP-1 pathway in DAPK1 transcription. A subsequent comprehensive examination of the FOS, JUN and ATF gene families in CLL and healthy B cells was performed. As the expression of the AP-1 gene family is highly subject to fluctuations in stress and mitogenic stimuli, freshly isolated cells were cultured using autologous serum conditions, allowing for a uniform establishment of baseline gene expression. This work reveals an alteration of the relative composition of AP-1 transcription factors in CLL, demarked by a substantial reduction in c-FOS gene expression. We find that the inducibility of c-FOS in CLL following MAPK activation by TPA (ERK-MAPK) is impaired 7.0-fold relative to healthy B cells and is completely abrogated following anisomycin (p38-MAPK) stimulation. The level of c-FOS induction following TPA activation can be used to clearly segregate CLL patients into two non-overlapping groups, those with low (mean=4.0-fold reduced expression; range=3.0–7.2; n=9) and very low (mean=21.7-fold reduced expression; range=17.6–36.7; n=8) following one hour induction versus healthy B cells. The very low c-FOS induction cases are characterized by poor prognostic indicators (IGHV unmutated (0/9 in low vs. 5/8 in very low); 17p, 6q deletion (0/9 in low vs. 3/8 in very low); and a substantially shorter time to progression (102.7±40.4 months in low vs. 10.8±8.6 in very low). Patients with very low c-FOS induction also demonstrate elevated c-MYC induction following activation. TPA-dependent ERK phosphorylation and activation of other immediate early genes, such as EGR1 and c-JUN, is intact in both CLL groups, absolving MAPK pathway dysfunction as a relevant c-FOS silencing mechanism. Reduced c-FOS expression can be partially explained, in the majority of very low c-FOS inducible cases, by elevated levels of miR-155 and miR-221 targeting the c-FOS transcript. Reduced expression of c-FOS and the resulting reconfiguration of AP-1 transcription factor composition may be involved in the pathogenesis of CLL and the subsequent silencing of DAPK1.

Disclosures:
No relevant conflicts of interest to declare.
CBL Deletion Mutant Found in AML Patients Cause Transformation of Receptor Tyrosine Kinase Class III Expressing Cells by Activation of the AKT Pathway
Authors: Polzer Harald , Janke Hanna , Hiddemann Wolfgang , Eick Dirk , Spiekermann Karsten 
Published: October 2019

Abstract
Abstract 2447
We examined the oncogenic potential of CBL deletion mutant found in AML patients in cytokine receptor and receptor tyrosine kinase (RTKs) expressing cells. In addition, we analyzed the interaction sites of FLT3/CBL and the critical pathways activated by CBL deletion mutants.
RTK, CBL and AKT constructs were expressed in Ba/F3 cells via a retroviral expression vector. Stable protein expression after transduction and fluorescence-activated cell sorting (FACS) was confirmed by western blotting and cellsurface-marker expression of receptors by flow cytometry. Cell Proliferation and apoptosis assays were done in presence and absence of IL-3 or receptor-ligands.
Coexpression of RTK III-WT (PDGFRA, PDGFRB, FLT3, KIT) and CBL deletion mutants cause IL-3 independent and ligand dependent growth of Ba/F3 cells. RTK III-WT/CBLΔexon8 cells show a more than 10 fold hyperproliferation in response to ligand stimulation. In contrast Non-class III receptor tyrosine kinases (EGFR, EPOR, MPL, IGF1R) and CSF1R show just a very weak hyperproliferation if coexpressed with the CBL deletion mutant. Selective protein tyrosine kinase inhibitors abrogate this proliferation. In cells coexpressing RTK-III receptor and CBLΔexon8 the receptor internalization is delayed and cells were protected from apoptosis after cytokine withdrawal. Ba/F3 cells after ligand stimulation and AML cell lines coexpressing CBL deletion mutants and FLT3 show an enhanced AKT phosphorylation. The PI3K inhibitor LY294002 and the AKT inhibitor MK2206 abolish the CBL mutant mediated hyperproliferation. Furthermore, a combined pharmacological inhibition of PI3K/AKT pathway and RTK shows an additive effect. The transforming potential of the CBL mutant is completely abolished by a mutated PTB domain of CBL (G306E) and decreased by mutation of tyrosines 589 and 591 in the juxtamembrane domain of FLT3. A constitutive active AKT mutant (E17K) recapitulates the CBL deletion mutant induced phenotype in Ba/F3 cells.
CBL is a selective negative regulator of class III RTK receptors and the PI3K/AKT pathway is critical for the transforming potential of the CBL oncogene. An alternative mechanism for the constitutive activation of RTKs in tumors occurs through inactivation of a negative regulator. CBL mutants mirror the phenotype of oncogenic RTK and cause an enhanced AKT phosphorylation. Targeted inhibition of FLT3 and AKT might be of therapeutic value in AML patients carrying CBL deletion mutants.Figure:Hyperproliferation of Ba/F3 cells coexpressing indicated receptors and CBL deletion mutant is quoted as X-fold of CBL wildtype coexpressing cells.Figure:. Hyperproliferation of Ba/F3 cells coexpressing indicated receptors and CBL deletion mutant is quoted as X-fold of CBL wildtype coexpressing cells.

Disclosures:
No relevant conflicts of interest to declare.