bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Antibody Targeted To The Anionic Region Of Human Protease Activated Receptor 4 Inhibits Thrombosis Without Influencing Bleeding
Authors: Mumaw Michele M. , Fuente Maria de la , Arachiche Amal , Nobel Daniel N. , Nieman Marvin T. 
Published: October 2019

Abstract
Protease activated receptors (PARs) are G-protein coupled receptors which are activated by cleavage of their N-terminus by thrombin. This generates a tethered ligand which is then able to activate the corresponding receptor. Human platelets express PAR1 and PAR4, which both have crucial roles in mediating the response of platelets to injury. Our hypothesis is that PAR4 is an ideal target for new anti-platelet therapies because it is required for stable clot formation and has limited tissue distribution. We have previously determined a region on PAR4 that is required for efficient activation by thrombin. A polyclonal antibody (CAN12) targeted to this region of the PAR4 exodomain does not cross react to PAR1. Initial studies determined that CAN12 is able to block thrombin-induced human platelet aggregation with an IC50 of 10 ng/ml. Control IgG does not inhibit aggregation at 2 mg/ml. In mouse platelets, CAN12 inhibits P-selectin expression and integrin activation. In the Rose-Bengal mouse model of carotid artery thrombosis, CAN12 (1 mg/kg) administered 10 minutes prior to injury was able to completely inhibit the formation of a thrombus in a dose dependent manner. The antibody delayed thrombosis to greater than 90 min; the experiment was terminated at 90 minutes. In contrast, control treatment (2 mg/kg IgG or saline) resulted in complete occlusion at ∼40 minutes. Further, the minimal dose of CAN12 required for complete inhibition of thrombosis (0.5 mg/kg) administered fifteen minutes after injury also delayed thrombosis from ∼50 minutes to ∼80 minutes. This indicates that CAN12 is able to disrupt a thrombus after it has been initiated. Preliminary evidence indicates that CAN12 is able to delay the cleavage of PAR4. Importantly, CAN12 (2 mg/kg) treatment does not increase bleeding time or blood loss in the tail clip assay compared to control IgG (2 mg/kg) treatment. There was also no significant increase in bleeding in the saphenous vein assay. The mice treated with CAN12 (2 mg/kg) had an average bleeding time of 102 seconds for 12 clot formations in 20 minutes compared to the control mice (IgG 2 mg/kg) which had an average bleeding time of 143 seconds for 11 clot formations. These data demonstrate that we are able to inhibit platelet aggregation in vitro and thrombosis in vivo without influencing bleeding time. Overall, these studies provide insight towards the development of new anti-platelet therapies and, specifically, PAR4 as an antiplatelet therapy target.

Disclosures:
No relevant conflicts of interest to declare.
MSC-Derived HSCs Can Reconstitute Hematopoietic Function In Patients With Severe Aplastic Anemia
Authors: Yin James Q , Gao Chunji , Han Bing , Sheng Jianliang 
Published: October 2019

Abstract

Introduction
Naturally-occurring regeneration of cells and tissues is generally involved in four working mechanisms such as directed differentiation, dedifferentiation, trans-differentiation and transdetermination. The better exploring of these mechanisms could be beneficial to develop clinical strategies for regenerative medicine and to reduce the likelihood of immune rejection and relevant complications Recently, “trans-determination” has attracted great controversy, mostly in regards to whether adult stem cells can colonize other tissues after transplantation. More importantly, how to generate large amounts of a particular stem cell type through a transdetermination process remained to be unsolved. Similarly, it is unclear whether mesenchymal stem cells (MSCs) can transdeterminate into hematopoietic stem cells (HSCs).

Methods
Many technologies were used to validate the transdetermination of adipose-derived mesenchymal stem cells (AD-MSCs) into hematopoietic stem cells (HSCs) from different aspects. They include FACS analysis, PCR tests, immunostaining, expansion and repopulating assays, transplantation analysis and others, showing their in vivo and in vitro potentials for long-term self-renewal and differentiation into multi-lineages of blood cells. Moreover, these AD-HSCs can reconstitute hematopoietic function in six patients.

Results
We report firstly here that a huge number of human AD-MSCs that are CD44+,CD29+, CD105+, CD166+,CD133-,CD34- could rapidly transdifferentiate into hematopoietic stem cells (CD49f+/CD133+/CD34+) and their descending blood cells in vitro, after transfected with two small RNAs. The sRNAs were high-effectively delivered into MSCs by a novel peptide means. These adipose-derived HSCs (AD-HSCs) could form different types of hematopoietic colonies as nature-occurring HSCs did. Upon the primary and secondary transplantation into sublethally or lethally irradiated mice, these MSC-HSCs engrafted and differentiated into all hematopoietic lineages such as erythrocytes, lymphocytes, myelocytes and thrombocytes. Furthermore, we demonstrated the first evidence that the transdetermination of MSCs was induced by acetylation of histone proteins and activation of many transcriptional factors. More excitingly, these MSC-derived HSCs can reconstitute hematopoietic function in six patients with severe aplastic anemia.

Conclusion
our findings identify the molecular mechanisms that regulate the directed transdifferentiation of MSCs toward HSCs, create a new source for individual HSC transplantation used for the treatment of blood diseases and cancers, and break the stalemate caused by bone marrow match and graft-versus-host disease.

Disclosures:
No relevant conflicts of interest to declare.
Outcome Of Aplastic Anemia In Adolescence. A Survey Of The Severe Aplastic Anemia Working Party (SAAWP) Of The European Blood and Bone Marrow Transplant Group (EBMT)
Authors: Dufour Carlo , Pillon Marta , Passweg Jakob , Socié Gérard , Bacigalupo Andrea , Franceschetto Genny , Oneto Rosi , Risitano Antonio M. , Peffault de la Tour Regis , Tichelli Andre , Rovo Alicia , Peters Christina , Hochsmann Britta , Schrezenmeier Hubert , Aljurf Mahmoud D. , Marsh Judith 
Published: October 2019

Abstract
We analyzed the outcome according to different treatments of 537 adolescents (age 12-18 years) with acquired aplastic anemia from the data-base of the SAAWP of the EBMT.
OS after front-line matched family donor (MFD) hematopoietic stem cell transplantation (HSCT) was 86% whereas after front-line immunosuppressive therapy (IST) was 82% (p= 0.53). EFS (events being death, relapse/no response, PNH/MDS/AML evolution, transplant) was 83% after MFD HSCT and 37% after IST (p=0.0001).
To evaluate the outcome of adolescents who were transplanted after failure of IST upfront (HSCT after failed IST) we extrapolated these patients from the original population receiving front-line immunosuppression and compared with those undergoing MFD HSCT upfront and IST upfront but who were not subsequently transplanted (IST up-front alone in whom transplant was not an event). OS in MFD HSCT was 86%, in IST up-front alone 90% and in HSCT post failed IST 78% (p=0.14). EFS was respectively 83%, 64% and 71% (p= 0.04) with no difference in post hoc analysis between IST alone and HSCT post IST (p=0.17). In the transplanted population, OS and EFS were significantly higher with bone marrow vs peripheral blood as source of cells. No differences in OS and EFS were seen in patients transplanted in adult, pediatric or mixed centres.
Cumulative incidence (CI) of rejection was 8 % in MFD HSCT and 9% in HSCT post failed IST (p=0.62). CI of Ac GVHD was 12% in MDF HSCT and 18% in HSCT post failed IST (p=0.18). CI of Chronic GVHD was 8% in MFD HSCT and 20% in HSCT post failed IST (p=0.0009). CI of secondary malignancies was 0.7% in MDF HSCT, 7% in HSCT post-failed IST and 21% after IST up-front alone (p= 0.0017).
In the whole cohort in multivariate analysis, the diagnosis to treatment interval of ≤2 months positively affected OS whereas IST alone negatively affected EFS. In transplanted patients interval diagnosis-treatment ≤2 months, first line MDF HSCT and BM as source of cells provided a significant advantage in OS and EFS. Aplastic anemia in adolescents has a very good outcome. If a MFD is available, HSCT using BM cells is the first choice treatment. If a MFD is not available, IST looks a reasonable second choice. If IST fails HSCT represents a very good rescue option.

Disclosures:
Dufour: Pfizer: Consultancy, Research Funding. Marsh:Sanofi: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.
Disturbed CXCR4/CXCL12 Axis In Pediatric Precursor B-Cell Acute Lymphoblastic Leukemia
Authors: van den Berk Lieke C.J. , Veer Arian van der , Willemse Marieke E. , Theeuwes Myrte J.G.A. , Luijendijk Mirjam W. , Tong Wing H. , van der Sluis Inge M. , Pieters Rob , den Boer Monique L. 
Published: October 2019

Abstract
Malignant cells that infiltrate the bone marrow (BM) interfere with the normal cellular behavior of supporting cells, thereby creating an alternative malignant niche. This intercellular communication is mostly mediated by cytokines and their receptors. In this study, we find that expression of the CXCR4 receptor is significantly increased in pediatric precursor B-cell acute lymphoblastic leukemia (BCP-ALL) cells compared with normal mononuclear hematopoietic cells derived of the bone marrow (p=0.016). Furthermore, we show that high CXCR4 expression is correlated with an unfavorable clinical outcome in BCP-ALL (5-yr CIR ±SE: 38.4% ±6.9% in CXCR4-high versus 12.0% ±4.6% in CXCR4-low expressing patients, p<0.001). Interestingly, BM serum levels of the CXCR4 ligand (CXCL12) are 2.7-fold lower (p=0.005) in samples taken at initial diagnosis of BCP-ALL compared with the levels in samples taken of non-leukemic controls. We show that induction chemotherapy restores CXCL12 levels in the BM to normal levels. Blocking the CXCR4 receptor with Plerixafor (FDA-approved drug) showed that the lower CXCL12 serum levels at initial diagnosis could not be explained by consumption by the leukemic cells, nor did we observe an altered CXCL12-production capacity of BM-MSC at this time-point. We rather observed that a very high density of leukemic cells negatively affected CXCL12 production by the BM-MSC while stimulating the secretion levels of G-CSF. These results suggest that highly proliferative leukemic cells are able to down-regulate the production of cytokines involved in homing (CXCL12), while simultaneously up-regulating the production of cytokines involved in hematopoietic mobilization (G-CSF). This disbalance may stimulate the spreading of BCP-ALL outside the BM.
The data presented here suggest that interference with the CXCR4/CXCL12 axis (for instance by using Plerixafor) may be an effective way to mobilize BCP-ALL cells; the more ALL cells become mobilized, the less ALL cells may escape from combination chemotherapy. In proof-of concept studies, this hypothesis needs to be validated to pave the way for implementation in future treatment protocols for children with ALL.

Disclosures:
No relevant conflicts of interest to declare.
BCR-ABL1+ Leukemic Stem Cells Are Dependent On Selectin-Ligand Interactions For Engraftment In The Bone Marrow Niche
Authors: Krause Daniela S. , Lazarides Katherine , Lewis Juliana B. , von Andrian Ulrich H. , Scadden David T. , Van Etten Richard A. 
Published: October 2019

Abstract
Leukemic progenitors from patients with chronic myelogenous leukemia (CML) have defects in the adhesive function of β1 integrins and in their response to the chemokine CXCL12 (Stem Cells 2002;3:259), pathways that are critical for engraftment and maintenance of normal hematopoietic stem cells (HSC) in the bone marrow (BM) niche. Previous studies in a mouse BCR-ABL1 retroviral transduction/transplantation model of CML demonstrated that BCR-ABL1+ leukemic stem cells, but not normal murine HSC, are dependent on expression of the CD44 adhesion molecule for BM homing and engraftment (Nat Med 2006;12:1175). To investigate further other adhesion molecules required for stable engraftment of CML stem cells, we employed donor and recipient mice with targeted mutations in genes encoding selectins and those required for expression of selectin ligands in the mouse retroviral CML model. Neither the β1 integrin ligand VCAM-1 nor P-selectin was required in the BM endothelium of recipient mice for efficient engraftment and induction of CML-like myeloproliferative neoplasia (MPN) by BCR-ABL1-expressing stem cells. By contrast, loss of recipient E-selectin significantly impaired engraftment of BCR-ABL1+ stem/progenitor cells, as demonstrated by decreased frequency of BM proviral leukemia-initiating clones, a defect overcome by direct intrafemoral injection of the leukemic cells. We also demonstrated a requirement for expression of selectin ligands on the leukemic stem cells, as BCR-ABL1-expressing stem cells lacking enzymes contributing to biosynthesis of selectin ligands (Core2 or Fucosyltransferases IV and VII) exhibited decreased efficiency of engraftment, resulting in attenuated disease. Further, donor cells deficient in P-selectin glycoprotein ligand-1 (PSGL-1) or in both PSGL-1 and CD44, a glycoform of which can function as a selectin ligand on hematopoietic stem cells (J. Biol Chem 2001;278:47623), displayed intermediate or profound impairment in engraftment of CML-like leukemia, respectively. Removal of selectin ligands by neuraminidase treatment of BCR-ABL1-transduced HSC blocked engraftment of leukemic stem cells and resulted in long-term survival of transplant recipients. Together, these results demonstrate that BCR-ABL1-expressing stem cells are dependent on selectin ligands, expressed on PSGL-1 and possibly on CD44, and their interaction with BM E-selectin for engraftment and retention in the BM microenvironment. Disrupting these niche interactions could represent new avenues for targeting CML stem cells that are resistant to ABL kinase inhibitors like imatinib.

Disclosures:
Van Etten: Bristol Myers Squibb: Consultancy; Deciphera Pharmaceuticals: Consultancy; TEVA Pharmaceuticals: Consultancy, Research Funding.
A Phase I Study Of Nilotinib As a c-Kit Inhibitor Combined With Re-Induction Chemotherapy For Relapsed and Refractory c-Kit Positive Acute Myeloid Leukemia
Authors: Brandwein Joseph M. , Hedley David , Chow Sue , Yee Karen , Schuh Andre C , Gupta Vikas , Schimmer Aaron , Minden Mark D. 
Published: October 2019

Abstract

Background
A previous study in relapsed/refractory AML, combining re-induction chemotherapy with imatinib as a c-kit inhibitor, demonstrated that effective inhibition of downstream pAkt was associated with a higher CR rate (Brandwein et al, 2011). Nilotinib is also a c-kit inhibitor which diffuses into cells and is therefore not affected by potential defects in active cellular influx mechanisms.

Methods
Patients age 18-65 with acute myeloid leukemia (AML) who had persistent leukemia after 3+7 induction with cytarabine and daunorubicin, or relapsed within 24 months of achieving CR, and whose blasts were c-kit (CD117) positive, were enrolled in a phase I clinical trial with nilotinib on Days 1-12 combined with mitoxantrone 10 mg/m2/day IV Days 6-10, etoposide 100 mg/m2/day IV Days 6-10 and cytarabine 1.5 g/m2 IV q12h x 4 doses on Days 11-12. None of the cases harbored c-kit mutations. Nilotinib was escalated through 2 dose levels (400 mg daily and 400 mg BID) in successive 6 patient cohorts; up to 2/6 dose-limiting toxicities (DLTs) were permitted.

Results
At the 400 mg once daily nilotinib dose there were 2 DLTs, both hematologic and consisting of prolonged platelet and neutrophil recovery. At the 400 mg BID dose there was one DLT, consisting of grade 3 transient liver enzyme elevation; 5/6 patients experienced transient grade I-II bilirubin elevations at this dose. The median time to ANC > 0.5 x109/L was 38 days (range 28-54) and to platelets > 20 x109/L was 30 days (range 28-54). Complete responses were seen in 10 patients, 5/6 at each dose level.
AML blasts from peripheral blood were assayed for pAKT, pERK and pS6 by flow cytometry, after stimulation with stem cell factor (SCF), on Days 1 and 6, pre- and 1.5 and 3 hours post-nilotinib dosing on each day. A decrease in the level of SCF-stimulated pAkt of at least 50% on Day 6 post-nilotinib, as compared to baseline, was seen in 1/6 patients at the 400 mg BID dose; the remaining 11 cases did not demonstrate an appreciable decrease in pAkt and other intermediates as compared to pre-nilotinib levels. Ex vivo dose titration curves showed that nilotinib inhibited c-kit mediated ERK and Akt activation in the blast cells of all cases, but concentrations >10 micromolar were needed to produce 50% inhibition in most cases.

Conclusions
The results indicate that nilotinib can be safely combined with this re-induction chemotherapy regimen, at doses up to 400 mg BID. Although the CR rate was high, in most cases nilotinib did not demonstrate significant inhibition of the c-kit pathway in leukemia cells in vivo. Dose titration curves showed that concentrations needed to produce 50% inhibition were generally above those achievable in vivo.

Disclosures:
Brandwein: Novartis: Honoraria, Research Funding. Off Label Use: nilotinib for acute myeloid leukemia.
HLA Ligandome Analysis Of Chronic Myeloid Leukemia (CML), Revealed Novel Tumor Associated Antigens For Peptide Based Immunotherapy
Authors: Stickel Juliane S. , Kowalewski Daniel J. , Schemionek Mirle , Kanz Lothar , Brümmendorf Tim H. , Salih Helmut R. , Rammensee Hans-Georg , Stevanovic Stefan 
Published: October 2019

Abstract
Despite the success of targeted therapy using tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable, most likely due to the treatment resistance of leukemic stem cells. Specific T-cell based immunotherapies for patients with CML might be able to eliminate these residual CML cells. For this goal the identification of tumor associated HLA-presented peptides, which are able to induce a tumor-specific T cell response, is indispensable. However, only few tumor associated antigens for CML are described till date. Thus the aim of this study was to identify for the first time naturally processed and presented HLA ligands from the cell surface of primary CML cells. HLA class I ligands from primary CML cells as well as from bone marrow mononuclear cell (BMNCs) and peripheral blood PBMCs of healthy donors were analyzed using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry (LC-MS/MS). LC-MS/MS peptide analysis provided qualitative and semi-quantitative information regarding the composition of the respective ligandomes. Comparative analysis of malignant and benign samples served to identify ligandome-derived tumor associated antigens (LiTAAs) and to select peptide vaccine candidates. So far 10 CML patients (8 chronic phase, 2 accelerated phase) and 30 healthy donors were analyzed within this study. We were able to identify a total of more than 8200 CML derived HLA ligands representing >4500 different source proteins, of which 734 were exclusively represented in CML, but not in healthy PBMCs/BMNCs. 55 of these CML exclusive antigens are presented on 3 or more of all examined CML patients, representing, as broadly represented LiTAAs, promising targets for peptide vaccination. For the first time, previously predicted CML tumor associated antigens for example Myeloperoxidase (10 peptide sequences on 7 CML patients) and Proteinase 3 (5 peptide sequences on 4 CML patients) were here confirmed by direct elution from primary CML cells, which also served to validate our methodological approach. Notably, beyond that we also identified a vast array of novel proteins (e.g. Carcinoembryonic antigen-related cell adhesion molecule 8, CEACAM8; Matrix metallopeptidase 8, MMP8; intracellular adhesion molecule 3, ICAM3) that are broadly and exclusively represented in the ligandome of CML cells.
By providing for the first time HLA class I tumor associated antigens, directly obtained from the HLA ligandomes of CML patients, this study may pave the way for the future development of a multipeptide-based immunotherapy approach to eradicate residual CML cells after conventional TKI therapy.

Disclosures:
No relevant conflicts of interest to declare.
Activating Mutations In Fyn Kinase In Peripheral T-Cell Lymphomas
Authors: Couronne Lucile , Palomero Teresa , Khiabanian Hossein , Kim Mi-Yeon , Ambesi Alberto , Carpenter Zachary , Abate Francesco , Allegretta Maddalena , Lossos Izidore S. , Nicolas Concha , Balbin Milagros , Bastard Christian , Bhagat Govind , Piris Miguel A. , Campo Elias , Bernard Olivier A. , Rabadan Raul , Ferrando Adolfo A. 
Published: October 2019

Abstract
Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of aggressive non Hodgkin lymphomas with poor prognosis. Molecular and cytogenetic studies have shown a prominent role for kinase fusion oncogenes, mostly NPM-ALK, in ALK+ anaplastic large cell lymphomas (ALCLs) and ITK-SYK kinase in unspecified PTCLs. To gain further insight on the genetics and pathogenic mechanisms of aggressive PTCLs we performed an integrated mutation analysis using whole exome sequencing (n=12) and RNAseq (n=35) data. This analysis identified 288 candidate coding somatic mutations in 268 genes including known recurrent mutations in the TET2, DNMT3A and IDH2 epigenetic factor genes and pointed to the FYN kinase gene as a new recurrently mutated oncogene in T-cell transformation. The FYN tyrosine kinase is, with LCK, the predominant SRC family kinase found in T lymphocytes and plays an important role in T-cell activation upon T-cell receptor (TCR) stimulation. FYN mutations in PTCL included a FYN L174R mutation detected in one AITL patient sample, a FYN R176C allele recurrently found in two PTCL NOS cases and a FYN Y531H mutation present in a PTCL NOS sample. Notably, each of these alleles are predicted to specifically disrupt the inhibition of FYN kinase activity by the C terminal SRC kinase (CSK). Thus, structure analysis of FYN and FYN mutant proteins predicted that FYN L174R and, most prominently, FYN R176C and FYN Y531H can disrupt the inhibitory interaction of the FYN SH2 domain with the CSK-phosphorylated Y531. Consistently, pull down assays using GST-FYN-SH2 recombinant proteins and biotinylated C-terminal FYN peptides encompassing Y531 showed abrogation of the interaction between FYN-SH2 and P-Y531 in each of these mutants. In agreement with these results, expression of FYN L174R, FYN R176C and FYN Y531H resulted in increased levels of FYN activation. Moreover, CSK expression effectively inhibited wild type FYN, but failed to abrogate FYN L174R, FYN R176C or FYN Y531H activation. In contrast, pharmacologic kinase inhibition with dasatinib, a multikinase inhibitor which blocks ABL1 and SRC kinases, effectively abrogated the activity of FYN L174R, FYN R176C and FYN Y531H mutant proteins and suppressed the growth of cells transformed via expression of activated FYN mutant alleles. Overall these results support an oncogenic role for FYN activating mutations in the pathogenesis of PTCL and support a role for SRC kinase inhibitors for the treatment of this disease.

Disclosures:
No relevant conflicts of interest to declare.