bluuebunny/crossref_metadata_2025_split huggingface dataset:
Control of Neutrophil Endothelial Transmigration By EC-18 in Chemotherapy Induced Neutropenia
Authors: Kim Jae Wha , Kim Yong-Jae , Kim Young-Jun , Yoon Sun Young , Jeong Jinseon , Ko Young-Eun , Shin Su-Hyun , Yoo Nina , Kim Myung-Hwan , Suh Cheolwon , Sohn Ki-Young , Kim Heung-Jae , Han Yong-Hae , Chong Saeho 
Published: October 2019

Abstract

Chemotherapy-induced neutropenia (CIN) is a common dose-limiting side-effect that often compromises chemotherapy resulting in dose reduction and/or delayed treatment. Chemotherapy-induced myeloid suppression is a prevalent mechanism to describe CIN. However, diapedesis of neutrophils from the blood is also a possible mechanism of CIN. Cytokines, chemokines, and damage- (or danger-) associated molecular pattern molecules (DAMPs) are produced from the damaged tissues by chemotherapy, which accelerates neutrophil transmigration. EC-18 is a synthetic monoacetyldiglyceride (MW=635) currently under development as an oral agent to treat and potentially prevent CIN. Here, we showed that neutrophil transmigration is induced in CIN model, and the efficacy and potential mechanism of EC-18 have been evaluated in mice and various cell models. Gemcitabine (50mg/Kg), cyclophosphamide (100mg/Kg) or tamoxifen (50mg/Kg) was administered daily for 3 days in mice (C57BL/6), followed by oral administrations of EC-18 (50mg/Kg) for 21 days. Blood neutrophils were analyzed by complete blood count (CBC) and fluorescence-activated cell sorting (FACS). All three chemotherapeutic agents significantly reduced absolute neutrophil count (ANC) in the blood; 40.4% decrease in gemcitabine, 19.8% in cyclophosphamide, and 21.8% in tamoxifen, which were recovered by the co-administration of EC-18. Microarray analysis of the lymph nodes from gemcitabine-treated mice revealed that the expression of DAMP molecules, S100A8 and S100A9, was induced by gemcitabine and decreased by the co-treatment of EC-18. Using immune cell lines, the expression of neutrophil transmigratory molecules, such as C3 anaphylatoxin, IL-6 and IL-8, was increased by gemcitabine treatment, and EC-18 blocked the expression of those cytokines in dose-dependent manners. Western blot analysis showed that EC-18 regulated STAT3 and STAT6 signaling pathways to modulate the expression of DAMPs, C3, IL-6, and IL-8. The decrease of C3 and IL-6 levels by EC-18 treatment was also verified in PBMCs from normal adults. In a pilot clinical study (WJON 2015 accepted, ASCO e20697), orally administrated EC-18 (1,000mg/day) alleviated neutropenic phenotypes in patients with pancreatic cancer who had been treated with gemcitabine (1,000mg/m2) and erlotinib (100mg daily). As a supportive oncology agent, EC-18 can maximize the antitumor efficacy of myelo-suppressive chemotherapeutic agents by enabling the treatment to continue or even higher doses to be administered.

Disclosures
No relevant conflicts of interest to declare.
Engineered Donor Marrow Macrophages Phagocytose Cancer Cells and Aggressively Shrink Solid Tumor Xenografts Compared to Tumor Associated Macrophages
Authors: Alvey Cory M , Spinler Kyle , Tewari Manu , Discher Dennis 
Published: October 2019

Abstract
Cell-based immunotherapies such as those based on engineered T-cells appear safe and often effective against liquid tumors. In solid tumors, macrophages are typically abundant, but the density of tumor associated macrophages (TAMs) correlates with poor clinical outcomes as they promote tumor growth and immunosuppression. In our studies, less differentiated donor marrow phagocytes are engineered in order to target tumors and selectively phagocytose cancer cells. Xenograft tumors were first made on the flanks of NSG mice using a tdTomato human lung carcinoma cell line (A549). Systemic injections of anti-human IgG (anti-hum) with large tumors (~70 mm2) showed no effect on tumor growth. However, systemic injection of bone marrow from donor NSG mice together with biweekly anti-hum treatments effectively stopped growth of the solid tumors. Replacing anti-hum with a non-specific antibody had no effect on tumor growth. Based on tdTomato signal intensity within macrophages isolated from tumors, 5-10-fold more donor macrophages are phagocytic compared to resident TAMs (2-3% are phagocytic). Since cancer cells express on their surface 'self' markers that limit the phagocytosis of these cells, we inhibited the 'self' receptors on the injected donor phagocytes prior to systemic injection of the donor marrow. This combination of 'self'-receptor inhibition with anti-hum causes a rapid decrease in tumor burden, shrinking tumors by ~40% in just 10 days compared to a similar growth of untreated tumors in the same time period. The anti-hum injection was again necessary as injection of a non-specific antibody failed to affect tumor growth. Tumor analysis showed that >65% of macrophages that were 'self'-receptor inhibited had phagocytosed the tdTomato A549 cells, which is ~20-fold greater than resident macrophages. Importantly, these cell therapy treatments appear safe with no significant decreases in hematocrit or platelets, which is unlike the anemia that has been reported upon systemic injection of 'self' inhibitors. Our results thus suggest that therapies based on engineered macrophages can be safe and effective against solid tumors if three requirements are met: a phagocytic phenotype, target opsonization, and inhibition of 'self' signaling. Future experiments will involve characterizing macrophage phenotypes of donor versus resident macrophages as well as efforts to maintain the necessary phagocytic phenotype.

Disclosures
No relevant conflicts of interest to declare.
TET2 Regulates CD8+ T Cell Responses to Acute and Chronic Viral Infection
Authors: Carty Shannon A. , Gohil Mercy , Stelekati Erietta , Banks Lauren B. , Wherry E. John , Koretzky Gary A. , Jordan Martha S 
Published: October 2019

Abstract
DNA methylation is one of the major epigenetic mechanisms that controls cellular differentiation. The ten-eleven translocation (TET) family of methylcytosine dioxygenases mediates active DNA demethylation through the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and subsequent intermediates. Here we demonstrate that TET2 regulates CD8+ T cell differentiation in vivo following acute and chronic viral infection. At steady-state, mice with a T-cell specific deletion of TET2 have intact thymic and peripheral T cell populations. Following acute viral infection with LCMV-Armstrong, TET2 loss enhances LCMV-specific CD8+ T cell memory differentiation in a cell-intrinsic manner without disrupting antigen-specific cell expansion or cytokine production. However, TET2-deficient memory CD8+ T cells exhibit altered recall responses with blunted re-expansion, retained expression of phenotypic memory markers and restricted re-expression of activation markers. During chronic viral infection with LCMV-clone 13, TET2 controls CD8+ T cell expansion and alters differentiation. Importantly, though mice with T-cell specific loss of TET2 developed similar levels of CD8+ T cell exhaustion as wild-type mice, TET2 loss specifically reduced PD-1 expression suggesting that TET2 may direct DNA demethylation of the PD-1 locus. Together, our data indicate that TET2 is an important regulator of CD8+ T cells following both acute and chronic viral infections and suggest targeting epigenetic regulators have potential for enhancing antiviral immunity.

Disclosures
No relevant conflicts of interest to declare.
Non-Proteolytic Regulation of the Multimerization of Von Willebrand Factor By ADAMTS13 Based on a Novel Cysteine Thiol Redundancy
Authors: Rottensteiner Hanspeter , Skalicky Susanna , Seyfried Birgit , Kaufmann Stefan , Fiedler Christian , Hasslacher Meinhard , Dong Jing-fei , Zheng X. Long , Plaimauer Barbara , Scheiflinger Friedrich 
Published: October 2019

Abstract
The platelet dependent-clotting process is a highly complex pathway that needs to be tightly controlled by a variety of soluble and cellular components. Failure to regulate the activity of multimeric von Willebrand factor (VWF) by ADAMTS13 for instance causes thrombotic thrombocytopenia purpura (TTP). Regulation is achieved through the established function of ADAMTS13 as a VWF-cleaving protease; however, it has been reported that ADAMTS13 may additionally curtail VWF multimer fibrillation by forming disulfide bridges with VWF. Since the latter mechanism remained ill-defined, we aimed to directly visualize disulfide bond formation between ADAMTS13 and VWF using multimer gel electrophoresis. Covalent interaction between ADAMTS13 and VWF was time-, concentration-, temperature-, and shear-stress dependent. The covalent linkage was independent of the proteolytic activity of ADAMTS13 and could be assigned to a C-terminal fragment comprising TSP1 domains 5 to 8 plus the CUB domains. This interaction was blocked by thiol-reactive agents, indicating that association was accomplished by a thiol/disulfide exchange reaction. Furthermore, a polyclonal anti-ADAMTS13 antibody that inhibited the protease in a FRETS-VWF73 activity assay also inhibited the formation of VWF-ADAMTS13 adducts.
Using a coupled reversed phase HPLC-ESI-QTOF-MS system, several partially reduced free thiols were identified in ADAMTS13, with cysteines 1254 and 1275 being the most prominent. Together with the observation that a C1275S point mutation retained the ability to form covalent linkages with VWF, this result indicated that ADAMTS13 has disulfide plasticity. The same conditions that led to VWF complex formation also caused a hitherto undetected homo-oligomerization of ADAMTS13. An even more pronounced oligomerization was noted in the presence of thiol-sensitive agents such as glutathione.
We propose a dynamic network of redundant, partially free thiols in ADAMTS13 that undergo intra- and inter-molecular redox reactions capable not only of depolymerizing VWF, but also to prevent further VWF multimerization by facilitating the aggregation of ADAMTS13 around the monomers.

Disclosures
Rottensteiner: Baxalta Innovations GmbH: Employment. Skalicky:Baxalta Innovations GmbH: Employment. Seyfried:Baxalta Innovations GmbH: Employment. Kaufmann:Baxalta Innovations GmbH: Employment. Fiedler:Baxalta Innovations GmbH: Employment. Hasslacher:Baxalta Innovations GmbH: Employment. Plaimauer:Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment.
Widespread and Dynamic Translational Control of Red Blood Cell Development
Authors: Hu Wenqian , Zhang Xu , Alvarez-Dominguez Juan 
Published: October 2019

Abstract
Cell development requires tight yet dynamic control of protein production. Here, we use murine erythropoiesis as a model to study translational regulatory dynamics during mammalian cell differentiation. We uncover pervasive translational control of protein synthesis, including widespread alternative translation initiation and termination, stoichiometric synthesis, and dynamic use of upstream reading frames. In addition, we identify hundreds of mRNAs with dynamic translation efficiencies during erythropoiesis. Interestingly, these mRNAs have significantly enriched binding motifs of many mRNA-binding proteins that are expressed with high specificity in hematopoietic cells, suggesting that those mRNA-binding proteins are important players in the translational regulatory programs in erythropoiesis. A major such program involves enhanced decoding of specific mRNAs depleted in terminally differentiating/enucleating cells with decreasing transcriptional capacity. We functionally characterized one such protein, Rbm38, which was previously identified as a splicing factor in terminal erythropoiesis, can enhance the translation of a subset of those mRNAs by interacting with the general translation initiation factor eIF4G in erythroid cells. Altogether, our findings reveal widespread and dynamic translational control in supporting specialized mammalian cell formation.

Disclosures
No relevant conflicts of interest to declare.
DYRK2 Inhibits the Self-Renewal of Leukemic Stem Cells in Chronic Myeloid Leukemia By Inducing Degradation of c-Myc Downstream of the Reprogramming Factor KLF4
Authors: Park Chun Shik , Shen Ye , Suppipat Koramit , Lewis Andrew , Tomolonis Julie , Puppi Monica , Mistretta toni-Ann , Ma Leyuan , Green Michael R , Lacorazza Daniel 
Published: October 2019

Abstract
Chronic myeloid leukemia (CML) is a blood cancer originated by expression of BCR-ABL, a constitutively activated kinase product of the chromosomal translocation t(9;22), in hematopoietic stem cells (HSC). Although tyrosine kinase inhibitors (TKI) can efficiently induce molecular remission in CML patients, drug discontinuation often leads to relapse caused by reactivation of leukemic stem cells (LSC) spared from TKI therapy via BCR-ABL-independent mechanisms of self-renewal and survival. Thus, there is a need for alternative drugs for relapse patients to prevent expansion of BCR-ABL-positive LSC during discontinuation of chemotherapy or emergence of chemoresistance. We found that somatic deletion of the reprogramming factor Krüppel-like factor 4 (KLF4) in BCR-ABL(p210)-induced CML severely impaired disease maintenance. This inability to sustain CML in the absence of KLF4 was caused by a progressive attrition of LSCs in bone marrow and the spleen and impaired ability of LSCs to recapitulate leukemia in secondary recipients. Analyses of global gene expression and genome-wide binding of KLF4 revealed that the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) is repressed by KLF4 in CML LSCs. Immunoblots revealed elevated levels of DYRK2 protein that were associated with a reduction of c-Myc protein and increased levels of p53 (S46) phosphorylation and PARP cleavage in KLF4-deficient LSCs purified from the bone marrow of CML mice. Genomic silencing of KLF4 in the murine CML cell line 32D-BCR-ABL resulted in increased levels of DYRK2 and phosphorylated c-Myc (S62) leading to diminished levels of c-Myc protein, which was reverted by treatment with a proteasome inhibitor, suggesting that KLF4 prevents c-Myc degradation triggered by DYRK2-mediated priming phosphorylation. Consistent with an inhibitory role in leukemia, DYRK2 levels are significantly reduced both in CD34+CD38+ and CD34+CD38− cells from CML patients compared to normal stem/progenitor cells. Aiming at pharmacological activation of DYRK2 to abrogate self-renewal and survival of CML cells, we treated CML cells with vitamin K3 that inhibits Siah2, an ubiquitin E3 ligase involved in Dyrk2 proteolysis. Vitamin K3, and not Vitamin K1 and K2, induces dose-dependent cytotoxicity in a panel of human-derived CML cell lines by stabilizing Dyrk2 protein and consequently promoting c-Myc degradation. Interestingly, combination of vitamin K3 with Imatinib exhibit additive effect inducing cytotoxicity in CML cells. Collectively, the identification of Dyrk2 as a critical mediator of LSC downfall is a novel paradigm poised to support the development of LSC-specific therapy to induce treatment-free remission in conjunction with Imitinib in CML patients.

Disclosures
No relevant conflicts of interest to declare.
Individual Variation in Hemostatic Alterations Caused By Tyrosine Kinase Inhibitors - a Way to Improve Personalized Cancer Therapy?
Authors: Lotfi Kourosh , Deb Suryyani , Sjöström Clara , Tharmakulanathan Anjana , Boknäs Niklas , Ramström Sofia 
Published: October 2019

Abstract
Introduction
During the last two decades, Bcr-Abl tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myelogenous leukemia (CML), and are now considered standard treatment for this disease. However, TKIs can induce serious hemostatic side effects including cardiovascular disease and bleeding disorders. Blood platelet aggregation and formation of pro-coagulant platelets are important to allow a well-balanced hemostatic response. Therefore, a detailed understanding of what effect different TKIs exert on platelets and hemostasis could help to understand if there are differences of importance to minimize the risk of bleeding complications in treated patients.
Aim
To investigate how TKIs used in CML (imatinib, dasatinib, nilotinib, bosutinib, and ponatinib) affect platelet activation and hemostasis.
Materials and Methods
We have developed a multi-parameter six color flow cytometry protocol to study different aspects of platelet function upon activation, e.g. formation of aggregatory (PAC-1-positive) and pro-coagulant (phosphatidylserine-exposing) platelets, exocytosis of alpha- and lysosomal granules and mitochondrial membrane potential.This protocol was performed in presence or absence of TKIs in blood from normal donors and in treated patients. Whole blood aggregometry (Multiplate®), thrombin generation in platelet-rich plasma and in vitro thrombus formation by free oscillation rheometry (ReoRox G2) was further evaluated in some situations.
Results
At clinically relevant concentrations, dasatinib significantly decreased the formation of procoagulant platelets. Ponatinib induced a slight decrease in formation of procoagulant platelets, whereas bosutinib and nilotinib showed opposite tendencies (n=7). Dasatinib also decreased platelet aggregation (n=4-6) and in vitro thrombus formation (n=3). Thrombin generation was not significantly affected by therapeutic levels of TKIs, whereas higher doses of dasatinib, bosutinib, ponatinib and imatinib significantly changed one or several of the thrombin generation parameters (n=7-8). Interestingly, large differences in response to the drugs were observed among the healthy donors, especially for dasatinib and bosutinib. Major inter-individual variations were also observed in dasatinib-treated patients.
Conclusions
Different TKIs show varying potency to affect platelet-based hemostasis. In addition, we found large inter-individual variations in how some drugs affected platelet function. Therefore, we suggest that development of a clinically useful protocol for platelet function testing could help to identify patients more susceptible to adverse drug reactions. Such a protocol could potentially help clinicians to gain insight into the risk of side effects, which could help to choose the most suitable drug for each individual patient.

Disclosures
No relevant conflicts of interest to declare.
CML Treatment in Dutch Hospitals with Low Patient Volumes Is Associated with a Substandard Quality of Molecular Response Monitoring
Authors: Geelen Inge G.P. , Thielen Noortje , Janssen Jeroen J.W.M. , Roosma Tanja , Visser Otto , Cornelissen Jan J. , Westerweel Peter E. 
Published: October 2019

Abstract
Introduction
Chronic myeloid leukemia (CML) is a relatively rare condition with approximately 170 new patients per year in the Netherlands. Patients at risk of progression to advanced stage disease need to be identified based on their molecular BCR-ABL1response, for which detailed guidelines are available. We evaluated the quality of molecular monitoring in Dutch clinical practice.
Methods
Chronic phase CML patients with a minimum of one year available follow-up were abstracted from a real-world population based registry of patients newly diagnosed between January 2008 and April 2013 in the Netherlands (PHAROS-CML). Hospitals were categorized into three groups based on the average number of CML-treatment initiations per year: low (<1/year), intermediate (1-2/year) and high volume (>2/year). The minimum standard of care was defined as molecular monitoring of response at least three times in the first year of treatment.
Results
In 253 eligible patients, molecular response assessment within the first year after diagnosis was performed a minimum of three times in 78%, twice in 15%, once in 4% and not performed in 3% of patients. Treatment for these patients was initiated in 54 different hospitals, corresponding to an average just below 1 new CML patient per year per hospital. In univariate logistic regression analysis treatment in an academic hospital, clinical trial inclusion, hospital volume and first line second generation TKI (2GTKI) treatment proved to be significant predictors of the minimum standard of care. Only the latter two remained significant predictors in multivariate analysis.
Treatment was initiated for 153 patients (61%) in high volume hospitals (n=16), 66 patients (26%) in intermediate volume hospitals (n=18) and 34 patients (13%) in low volume hospitals (n=20). In the high volume hospitals, 90% of patients received the minimum standard of care, compared to 67% in the intermediate (p<.001) and 50% in the low volume hospitals (p<.001). Moreover, monitoring in imatinib treated patients was poorer than in patients initially treated with 2GTKI (74% vs 94%, p=.002).
Conclusion
Approximately 40% of newly diagnosed CML patients in the Netherlands are treated within centres that see less than two new patients per year. Such low treatment volumes are associated with a substandard quality of molecular response monitoring. Surprisingly, patients treated with imatinib as first line treatment are monitored less stringent, even though the rate of treatment failure is known to be higher than 2GTKI treated patients. Our observations support a potential patient benefit of centralization of CML treatment.

Disclosures
Janssen: Novartis: Research Funding; ARIAD: Consultancy; BMS: Honoraria; Pfizer: Honoraria.