bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential MLL Interaction and H3K4me3 Mark Maintenance at PU.1 Regulatory Region by AML Translocations-Associated Oncoproteins AML1-ETO and CBFβ-SMMHC.
Authors: Huang Gang , Yan Xiaomei , Zhao Xinghui , Rao Yalan , Sashida Goro , Bowie Rajeana 
Published: October 2019

Abstract
Abstract 674
The interplay between genetic and epigenetic regulators is a fundamental mechanism to ensure gene regulation and cell fate decision. Hematopoiesis and leukemia are excellent systems in which to study this process. The Mixed-Lineage Leukemia (MLL) protein, a Set1-like H3K4 methyltransferase, and the heterodimeric transcription factor RUNX1 (AML1)/CBFβ are critical for definitive and adult hematopoiesis. They are required for the generation of all hematopoietic lineages and act as tumor suppressors in human leukemia. PU.1 is a critical downstream target gene for AML1 in adult hematopoiesis. AML1 regulated PU.1 through 3 AML1 binding sites in the PU.1 URE region (1). AML1 is responsible for the H3K4me3 mark at PU.1 URE and promoter region. AML1-ETO represses PU.1 expression through PU.1 URE region (2). Dysregulation of PU.1 levels cause leukemia in both mouse and human. PU.1 is absolutely required for the normal development of monocytic and B cell lineages, in which the core binding factor (CBF) fusions (CBFβ-SMMHC or TEL-AML1) or MLL fusions (MLL-AF9 or MLL-AF4) have been high frequently identified in human acute leukemias. These suggested that AML1/CBFβ and MLL might regulate PU.1 expression and AML1/CBFβ or MLL fusions might dysregulate PU.1 expression at genetic and epigenetic levels and eventually develop similar leukemias. We found that the AML1-ETO/CBFβ complex interacts with MLL in a similar manner as AML1/CBFβ (3), while the AML1/CBFβ-SMMHC complex interacts with MLL more strongly. Surprisingly, the CBFβ-SMMHC by itself interacts with MLL through the junction region of the fusion protein. The interactions with MLL by these fusion proteins complexes, AML1-ETO/CBFβ and AML1/CBFβ-SMMHC, correlate with their ability to maintain H3K4me3 levels at PU.1 URE and promoter regions in 416B cell lines stable expressing AML1-ETO or CBFβ-SMMHC. Taken together, our data indicate that AML1-ETO/CBFβ complex preserves the interaction with MLL, while the AML1/CBFβ-SMMHC complex enhances it. This suggests that these two leukemogenic fusions downregulate PU.1 expression through different epigenetic mechanisms in the presence of H3K4me3 mark at PU.1 regulatory region. The effects of these differential interactions on the H3K4me3 mark maintenance and on target gene expression, particularly PU.1, may be critical for the aberrant regulation that underlies the etiology of M2 and M4Eo acute myelogenous leukemia (AML).
1. Huang G, et al. Nat. Genet. 2008; 40: 51-60
2. Zhang P, et al. Blood 2008; 112: 594.
3. Huang G, et al. Blood 2008; 112: 282.
Combined Modality Treatment with Intensified Chemotherapy and Dose-Reduced Involved Field Radiotherapy in Patients with Early Unfavourable Hodgkin Lymphoma (HL): Final Analysis of the German Hodgkin Study Group (GHSG) HD11 Trial.
Authors: Borchmann Peter , Diehl Volker , Goergen Helen , Mueller Horst , Mueller Rolf Peter , Eich Hans T. , Mueller-Hermelink Hans Konrad , Herrmann Richard , Markova Jana , Ho Anthony D. , Hiddemann Wolfgang , Doerken Bernd , Greil Richard , Engert Andreas 
Published: October 2019

Abstract
Abstract 717

Purpose:
Combined modality treatment consisting of 4 cycles of chemotherapy (CT) followed by involved field radiotherapy (IF-RT) is the standard treatment for early unfavourable HL. In our prior trial for this group of patients (HD8), overall survival (OS) and freedom from treatment failure (FFTF) at 5 years were 91% and 83%, respectively. The HD11 trial thus addressed two major questions: (1) improving outcome by intensifying CT (4xABVD vs. 4xBEACOPPbaseline; Bbas) and (2) defining the best radiation dose (30Gy vs. 20Gy IF-RT).

Patients and methods:
Between May 1998 and January 2003, 1395 eligible patients aged 16–75 years with untreated early unfavourable stage HL (CS I, IIA with at least one of the risk factors large mediastinal mass (a), extranodal disease (b), elevated ESR (c) or ≥ 3 nodal areas (d); IIB with risk factors c and/or d) were randomized into one of the following 4 treatment arms: 4xABVD + 30Gy (A), 4xABVD + 20Gy (B), 4x Bbas + 30Gy (C) or 4x Bbas + 20Gy (D). Since there are strong indications for an interaction between CT- and RT-doses, a comparison of pooled treatment arms (A+B vs. C+D for comparison of 4×ABVD vs. 4× Bbas and A+C vs. B+D for comparison of 30Gy IF-RT vs. 20Gy IF-RT) would be misleading. Therefore all treatment arms were analysed separately.

Results:
Patient characteristics were well balanced between the 4 arms (median age 33 years, 49% male, 6% stage I, 29% B-symptoms). CT- and RT-related acute toxicity occurred significantly more often in the arms with the more intensive therapy (CT: 74.1% vs. 51.8%; RT: 12.3% vs. 5.5%). The complete remission rate 3 months after end of therapy was 94.1% for the whole group and did not differ significantly between the 4 arms. The 5-year estimate of FFTF (primary endpoint) is 85.0% (OS 94.5%, PFS 86.0%).
Bbas is more effective than ABVD if followed by 20Gy IF-RT (5y-FFTF difference 5.7%, 95%-CI [0.1%; 11.3%]). This effect does not exist in combination with 30Gy IF-RT (5y-FFTF difference 1.6% [-3.6%; 6.9%]). Similar results are observed for the RT-question: After 4 cycles of Bbas, 20Gy is not inferior to 30Gy (5y-FFTF difference -0.1%, 95%-CI [-5.1%; 4.9%]), whereas after 4xABVD, a relevant inferiority of 20Gy cannot be excluded (-4.0% [-9.5%; 1.4%]).

Conclusion:
A reduction of RT dose from 30Gy to 20Gy IF-RT seems to be justified only in combination with Bbas, but not with a less effective chemotherapy such as 4xABVD. Patients will benefit from an intensified CT such as Bbas only in combination with 20Gy IF-RT but not with 30Gy IF-RT.

Disclosures:
No relevant conflicts of interest to declare.
Mapping the Protease Activated Receptor 4 (PAR4) Homodimer Interface.
Authors: Nieman Marvin T 
Published: October 2019

Abstract
Abstract 773
Thrombin activates platelets by binding and cleaving protease activated receptors 1 and 4 (PAR1 and PAR4). PAR1 and PAR4 communicate with each other to lower the concentration of thrombin required for PAR4 activation (Nieman Biochemistry, 2008). In addition, PAR1 and PAR4 form homo and heterodimers. However, where these receptors interact has not been defined and it is not known if dimerization influences receptor activation, downstream signaling, or both. Since PAR4 activation is important on human and mouse platelets, we sought to characterize the interaction site between PAR4 homodimers. Using bioluminescence resonance energy transfer (BRET), we mapped the PAR4 homodimer interface. The PAR4 homodimers show a specific interaction as indicated by a hyperbolic BRET signal in response to increasing PAR4-GFP expression with a fixed concentration of PAR4-Rluc. The threshold maximum BRET signal was disrupted in a concentration-dependent manner by unlabeled PAR4. In contrast, the unrelated G-protein coupled receptor, rhodopsin, was unable to disrupt the BRET signal indicating that the disruption of the PAR4 homodimer is a specific interaction. We have mapped the region required for PAR4 homodimer formation using chimeras between rhodopsin and PAR4. PAR4 does not interact with rhodopsin in BRET assays. Using a library of rho-PAR4 chimeras that have the junction at the beginning of transmembrane (TM) 2, 3, 4, 5, 6 or 7, we determined where dimer formation is restored. When the junction is placed at the beginning of TM4 or TM5, the chimera does not interact with PAR4-WT. In contrast, when the junction is moved to the end of TM2, the BRET signal is restored. These results indicate that the region on PAR4 required for homodimer formation encompasses a 63 amino acid region that includes the first extracellular loop, TM3 and the second intracellular loop. These studies establish techniques that may be used to define the interactions between other GPCRs found on the platelet surface. These receptor-receptor interactions may be another level of regulation of agonist activity and platelet function in vivo and may provide novel targets for anti-platelet therapies.

Disclosures:
No relevant conflicts of interest to declare.
Patients with Early T-Cell Precursor (ETP) Acute Lymphoblastic Leukemia (ALL) Have High Levels of Minimal Residual Disease (MRD) at the End of induction—A Children's Oncology Group (COG) Study.
Authors: Wood Brent , Winter Stuart , Dunsmore Kimberly , Raetz Elizabeth , Borowitz Michael J. , Devidas Meenakshi , Winick Naomi J. , Carroll William L. , Hunger Stephen P , Loh Mignon L. 
Published: October 2019

Abstract
Abstract 9
Current risk stratification algorithms in children with B-lineage ALL have facilitated the identification of subgroups with adverse outcomes that benefit from intensified therapy. However, many of the features that are highly prognostic of outcome in B-precursor ALL, such as the combination of age and white blood cell count (WBC) at diagnosis, have limited ability to predict outcome in T-ALL. In contrast, early response to induction therapy, as measured by bone marrow (BM) morphology at days 8, 15 or 29, and day 29 (end induction) BM MRD are highly predictive of outcome in both B-precursor and T-ALL. Coustan-Smith and colleagues (Lancet Oncology 10:147-156 2009) have recently identified a subset of children with ETP ALL that have an extremely high risk of induction failure or relapse. The ETP ALL blasts exhibit stem cell-like features as defined by the presence of antigens seen on early progenitors including CD117, CD34, HLA-DR and/or CD13/33, the absence of antigens seen on later stage immature T cells including CD1a and CD8, and decreased to absent expression of the early T cell antigen CD5.
We analyzed patients enrolled in current generation COG ALL trials, including patients with B-precursor ALL treated on AALL0232 (high risk, n=2129) or AALL0331 (standard risk, n=3747), and 416 patients with T-ALL enrolled on AALL03B1. Clinical features including age, WBC, gender, and CNS status were compared between ETP-positive (n=25) and –negative (n=391) T-ALL patients. We also compared response to four weeks of induction therapy (prednisone, vincristine, PEG-asparaginase, and daunorubicin) as measured by day 15 and 29 BM morphology and day 29 BM MRD determined by flow cytometry. Twenty-five of 416 (6%) T-ALL patients had an ETP phenotype. The ETP-positive patients were older and presented with a lower white blood cell count than the ETP-negative patients (see Table). There was no difference in the gender distribution or the presence of CNS disease at diagnosis between the two groups. Strikingly, the ETP-positive patients had a dramatically inferior early response to therapy with 100% having ≥ 0.01% day 29 MRD (vs. 46% of ETP-negative T-ALL and 23.4% of precursor B-ALL) and 74% having ≥ 1% day 29 MRD (vs. 21% of ETP-negative T-ALL and 5% of precursor B-ALL). These results confirm the poor response of ETP T-ALL and suggest that novel treatment strategies are warranted in this patient subgroup.

Disclosures:
No relevant conflicts of interest to declare.
Induction with Velcade®/Dexamethasone Partially Overcomes the Poor Prognosis of t(4;14), but Not That of Del(17p), in Young Patients with Multiple Myeloma.
Authors: Avet Loiseau Hervé , Moreau Philippe , Mathiot Claire , Charbonnel Catherine , Caillot Denis , Facon Thierry , Attal Michel , Benboubker Lotfi , Hulin Cyrille , Marit Gerald , Minvielle Stéphane , Harousseau Jean-Luc 
Published: October 2019

Abstract
Abstract 957
Translocation t(4;14)(p16;q32) has been associated with a poor outcome in multiple myeloma. This poor prognosis has been identified both in patients treated with melphalan-prednisone (MP) and in those treated with high-dose melphalan after a VAD induction. For instance, in 100 patients with t(4;14) treated with VAD and MEL200, the median PFS and OS were 21 months and 41 months, respectively, as compared to 37 months and 65 months for patients lacking the t(4;14) (Moreau et al., Leukemia 2007). Some preliminary studies have suggested that bortezomib (Velcade®) was able to overcome the poor prognosis of the translocation in elderly patients treated with MP-Velcade® (San Miguel et al., NEJM 2008). In order to address this important question, we analyzed 436 patients treated in the IFM, according to the IFM-2005-01 trial, arm B: induction with 4 cycles of Velcade®/Dexamethasone (VD), followed by one or two courses of high-dose melphalan (MEL200). A translocation t(4;14) was observed in 67 of these 436 patients treated with VD (15%), whereas del(17p) was found in 51 patients (11%). Of note, 10 patients presented both the t(4;14) and the del(17p). The median PFS was 25 and 36 months, in patients with or without the t(4;14), respectively (p=0.006). At 3 years, 76% of the patients with t(4;14) were still alive, as compared to 88% of the patients lacking the translocation (p=.003). For comparison, the OS results were respectively 62% (patients with t(4;14)) and 73% (patients lacking the translocation) in patients treated with a VAD induction. Thus, it seems that VD is able to partially overcome the poor prognosis of t(4;14). We also looked at the prognostic value of del(17p) in this series of patients treated with VD. In contrast to the t(4;14) situation, VD was enable to rescue patients with del(17p) (same PFS and OS for patients treated with VD than for those treated with a VAD induction). Thus, this study (by far the largest so far reported) shows that VD as induction before intensification is able to improve the prognosis of patients with t(4;14), but not of those with del(17p).

Disclosures:
Avet Loiseau: Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees. Moreau:Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees. Facon:Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees. Attal:Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees. Harousseau:Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees.
Snake Venom and Receptor Crosslinking.
Authors: Clemetson Kenneth J. 
Published: October 2019

Abstract
Abstract SCI-37
Nearly all poisonous snakes venoms contain proteins including snaclecs (snake C-type lectins) and metalloproteases, which influence haemostasis by interacting with coagulation factors or platelets. Like other venom constituents they weaken the prey and make it easier to swallow and digest. Although some of those that affect platelets inhibit receptors, particularly integrins, many activate platelets by clustering receptors. The main receptors involved are GPIb and GPVI and the snaclecs and metalloproteases activate platelets in the same way as their natural ligands, von Willebrand factor (VWF) and collagen. Both VWF and collagen are thought to activate platelets by clustering receptors acting in somewhat parallel ways. In both cases a mainly signalling receptor, GPIb or GPVI, respectively, is clustered to activate an integrin, aIIbb3 or a1b2, respectively. The first step can be simulated by agents that bind to and cross-link GPIb or GPVI. These include antibodies, synthetic peptides or snake venom proteins. Many of the activating snaclecs belong to the tetrameric heterodimer group such as flavocetin, convulxin and its homologues, and some use both receptors. There remain however many unanswered questions about the non-covalent structure of these molecules and how they interact with receptors on, possibly, more than one platelet to induce activation even at very low concentrations (pM). A second class of snaclecs, including aggretin (rhodocytin), which has CLEC-2 as receptor, and purpureotin that acts via GPIb, are non-covalent dimers of heterodimers but may reorganise to form higher non-covalent multimers. These non-covalent interactions are still poorly understood and may be responsible for the low activity of recombinant snaclecs. Several activating snaclecs are glycosylated. Although this may simply enhance solubility, interactions with prey molecules can not yet be excluded. Many simple heterodimeric snaclecs are superficially inhibitory of washed platelets but can be converted to activators by cross-linking, either naturally or artificially. Finally, a major problem in snake venom genomics is ascribing function, such as target molecules, to novel snaclecs identified by DNA sequence alone. Although modelling has made great progress there are still too many unknown factors.

Disclosures
No relevant conflicts of interest to declare.
Drug Inhibition of GPIb.
Authors: Deckmyn Hans , De Meyer Simon F. , Vanhoorelbeke Karen 
Published: October 2019

Abstract
Abstract SCI-38
The platelet-specific glycoprotein (GP)Ib, member of the GPIb/IX/V complex, is the main receptor for von Willebrand Factor (VWF), but also for a growing list of other ligands such as thrombin, coagulation factor XI and XII, Mac-1 and P-selectin. For VWF and GPIb to interact, VWF needs to undergo a conformational change. In the circulation, this is induced by shear forces that stretch VWF, especially when bound onto collagen at vascular lesions. As the contribution of VWF to platelet adhesion becomes more important with increasing shear forces, the idea emerged that inhibition of the GPIb-VWF interaction could actually result in an antithrombotic effect targeted to high shear arterioles or stenosed arteries, leaving hemostasis virtually unaffected in vessel beds with slower circulation. And in the mean time, a growing amount of evidence suggests that this could lead to a lower bleeding risk than what is seen with currently available antithrombotic agents. Several compounds have been tested in the past such as fragments of VWF, snake venoms and antibodies. We developed an anti-human GPIb monoclonal antibody, 6B4, that competes directly for the binding site of VWF. The humanized 6B4 Fab-fragment effectively prevented repetitive occlusions of the damaged and stenosed femoral artery in a baboon thrombosis model. Interestingly, only little prolongation of the template bleeding time and minimal increase in blood loss from a standardized incision was observed. This apparent larger therapeutic window of GPIb inhibitors could be particularly useful in ischemic stroke, where the utility of current platelet inhibitors and anticoagulants is significantly counterbalanced by the risk of intracerebral bleeding complications. Recent promising results in a mouse model of cerebral ischemia/reperfusion indeed show a significantly less severe stroke outcome using a GPIb-inhibitor, either administered before or, importantly, after cerebral artery occlusion. No increase in intracerebral hemorrhages was detected, in contrast to what is seen with e.g. GPIIb/IIIa inhibition. Finally, as antibodies may be useful for acute treatments but not for chronic applications, the identification of a GPIb-inhibiting nonamer peptide raises hopes towards development of orally available small molecule inhibitors.

Disclosures
No relevant conflicts of interest to declare.
TRALI Pathophysiology.
Authors: Stroncek David 
Published: October 2019

Abstract
Abstract SCI-48
Transfusion related acute lung injury (TRALI) is clinically defined as the new onset of acute lung injury within 6 hours of a transfusion. In TRALI a transfusion activates neutrophils leading to pulmonary leukostasis, endothelial damage, capillary leak and pulmonary edema. A number of elements (bioactive lipids, sCD40L, and leukocyte antibodies) found in blood products can active neutrophils and are risk factors for TRALI. Bioactive lipids and sCD40L accumulate in both stored red cell and platelet components. Leukocyte antibodies are most often found in blood components collected from women alloimmunized during pregnancy. A number of animal and in vitro models have shown that neutrophils activated by leukocyte antibodies cause pulmonary leukostatsis, endothelial damage and capillary leak. Far more blood components contain these TRALI factors than cause TRALI suggesting that additional patient, clinical, or blood component factors are required for the development of TRALI. For example, neutrophil-specific antibodies cause reactions in, at most, 25% of transfusion recipients. Animal models and in vitro studies have found that blood component factors are more likely to induce lung injury if the neutrophils and/or pulmonary endothelial cells are primed or activated. Endothelial cells can be primed by endotoxin and neutrophils can be primed by bioactive lipids and sCD40L. The priming of neutrophils and /or endothelium results in the tight adhesion of neutrophils to endothelial cells and enhances endothelial cell injury Between neutrophil and HLA Class I and II antibodies, neutrophil antibodies are most potent at initiating TRALI and HLA Class I antibodies are least potent. HLA Class I antigens are expressed by platelets, lymphocytes, monocytes and soluble HLA Class I antigens are present in plasma. These sources of Class I antigens likely compete with neutrophils for transfused Class I antibodies and may render them less effective at initiating TRALI than neutrophil-specific or HLA Class II antibodies. Some evidence suggests that HLA Class I antibodies induce TRALI by binding to pulmonary endothelium and activating neutrophils through their Fc portion. Class II antibodies may initiate TRALI by binding to monocytes and stimulating cytokine release. In summary, TRALI is the result of patient and blood component factors which lead to neutrophil activation and endothelial cell damage and capillary leak. Most cases likely require the confluence of multiple factors to form a “perfect inflammatory storm” which leads to significant lung injury.

Disclosures
No relevant conflicts of interest to declare.
Foxn1 and the Rise and Fall of the Thymic Microenvironment.
Authors: Manley Nancy R. 
Published: October 2019

Abstract
Abstract SCI-50
The postnatal thymus is the primary source of T cells in vertebrates, and many if not all stages of thymocyte development require interactions with thymic epithelial cells (TECs). There is abundant and growing evidence that the cellular and compartmental organization of the thymus is crucial for optimal organ function. Changes in the organization and stromal composition of the thymus as a result of genetic changes or during aging-associated involution can negatively impact both T cell production and function. The TEC-specific transcription factor Foxn1 is a critical regulator of these processes. I will present data from our Foxn1 allelic series showing that Foxn1 regulates TEC proliferation and differentiation at both fetal and adult stages, and that Foxn1-dependent TEC differentiation is required to assemble the multiple cell types that make up the complex structure of the functional thymus. Thus, Foxn1 is a central regulator throughout the life cycle of the thymus, and orchestrates both fetal organ formation and maintenance of the postnatal thymus. In vivo analysis of thymic function and stromal phenotypes is made difficult by the cellular complexity of the thymic microenvironment. I will also present data from a new quantitative, theoretical approach we are using to generate a “geographical map” of the cellular organization of the thymus. This approach will allow us to determine the spatial relationships of identifiable cell types, allowing us to generate new hypotheses about functional parameters of tissue organization at the cellular level. Thus, these results will provide a novel approach to generating quantitative and predictive models of the spatial and functional architecture of the thymic microenvironment.

Disclosures
No relevant conflicts of interest to declare.