bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gvhd-Associated Early Impairment of Marrow Function Is Frequent after Allogeneic Transplantation and Is a Sensitive Biomarker for Prediction of Treatment Related Mortality and of Overall Survival
Authors: Milone Giuseppe , Avola Giuseppe , Camuglia Maria Grazia , Leotta Salvatore , Cupri Alessandra , Spadaro Andrea , Tripepi Giovanni 
Published: October 2019

Abstract
INTRODUCTION: Aim of our study was to determine the role of acute-GVHD in influencing early marrow function and whether assessment of “GVHD-associated marrow impairment” may predict transplant outcome.
METHODS: We have prospectively studied 62 patients who received T-replete allogeneic stem cell transplantation because of various malignancies. At day +18/+19, we determined in bone marrow: CD34+ cells, frequency of clonogenic cell (CFU-GM and BFU-E) and, in 20 patients, also CD34+ cells showing apoptosis (AnnexinV/7-AAD). Results were related to acute-GVHD and to clinical outcome in terms of Treatment Related Mortality (TRM), Relapse Rate (RR) and Overall Survival (OS). To distinguish the effect of a-GVHD from that determined by corticosteroid, patients were divided in three groups according to time of presentation of a-GVHD: “Early a-GVHD, “No a-GVHD” and “Impending a-GVHD”. The latter group consisted of patients presenting a-GVHD after engraftment.
RESULTS: In univariate analysis “Febrile neutropenia” and” Acute GVHD” were important factors for a reduction of frequency of marrow clonogenic cells assessed on day +18/+19. However, in multivariate analysis only “Acute GVHD” was able to influence frequency of marrow CFU-GM and BFU-E at day +18/+19. Patients not developing “a-GVHD” until day +90, had on day+18 a median growth of CFU-GM of 202/10e5 plated cells while patients suffering “early GVHD” had a marrow CFU-GM growth significantly reduced: 82/10e5 plated cells, (p= 0.0009). Median CFU-GM was found significantly reduced also in patients defined as “impending GVHD” (onset of a-GVHD at day +20-/+90) (p=0.01). Apoptotic CD34+ cells in marrow cells at day +18/+19 were inversely correlated to frequency of marrow BFU-E (r= -0.5, p=0.04). Taking into account competing risks, Cumulative Incidence of TRM at 2 y in the group of patients having a frequency of marrow CFU-GM over median was 5% while it was 30% in the group having CFU-GM below the median (log-rank: p=0.004). Cumulative incidence of Relapse was not significantly different in these two groups (31% versus 34%). OS was significantly better in group having CFU-GM over median: 62% versus 35% (logrank: p=0.02). Frequency of CFU-GM over median at day +18/+19 was a factor important for a reduced risk of death (HR=0.358; p=0.004) also after adjusting, using multivariate Cox analysis, for Disease Status (Early versus Advanced).
CONCLUSIONS: acute-GVHD impairs early and significantly marrow function, marrow-GVHD is a sensitive biomarker for prediction of TRM and OS.

Disclosures
No relevant conflicts of interest to declare.
The SRC Kinase Family Member Lymphocyte Specific Kinase (p56Lck) Regulates Endothelial Cell Proliferation and Survival
Authors: Betapudi Venkaiah , Merkoulov Sergei M. , Kundu Suman , Ji Ou , Wu Meifang , McCrae Keith R. 
Published: October 2019

Abstract
Background and Objective: The Src family consists of Src, Lck (p56Lck), Fyn, Hck, Blk, Lyn, Fgr, Yes, and Yrk protein tyrosine kinases, which display cell and tissue specific expression. These non-receptor protein tyrosine kinases regulate many cellular processes including cell growth, differentiation, shape, migration, and survival. p56Lck is primarily expressed in T lymphocytes where it stimulates T cell receptor-mediated signaling to regulate thymocyte development. Similar to other SRC family members, p56Lck activity is negatively regulated by phosphorylation of Tyr505 and undergoes autophosphorylation on Tyr394 in the active state. Our previous studies showed that p56Lck activation occurred in proliferating endothelial cells exposed to cleaved high molecular weight kininogen (HKa), which induced endothelial cell apoptosis.The purpose of this study is to define the role of Lck in regulation of endothelial cell survival and angiogenesis.
Methods: Primary cultures of endothelial cells were treated with Lck siRNA or with mammalian expression vectors or lentiviral constructs containing Lck cDNA. The proliferative capacity, viability, and tube-forming ability of Lck and control endothelial cells were determined.
Results: Transfection of endothelial cells with Lck siRNA markedly changed their morphology and significantly enhanced their ability to proliferate in response to bFGF. In contrast, endothelial cells transfected with a Lck expression vector or transduced with a Lck-lentivirus not only failed to proliferate in response to growth factors, but also underwent apoptosis on several different extracellular matrix proteins. Finally, overexpression of Lck in endothelial cells led to impaired tube formation in a matrigel-based, in vitro angiogenesis assay; apoptosis of endothelial cells in this system was suggested by increased staining with Annexin V.
Conclusion: Taken together, these results suggest that regulation of Lck activation provides a key node in determination of endothelial cell proliferation and survival that is likely to be relevant to the regulation of angiogenesis. Moreover, they suggest that specific endothelial cell Src family kinase members may play contrasting roles in regulation of these processes. Activation of Lck may represent a potential mechanism for controlling aberrant angiogenesis in pathological conditions.
Figure: Effect of control or p56Lck-expressing lentiviral transduction on endothelial cell tube formation and induction of apoptosis. Figure:. Effect of control or p56Lck-expressing lentiviral transduction on endothelial cell tube formation and induction of apoptosis.

Disclosures
No relevant conflicts of interest to declare.
Calcium Calmodulin Dependent Kinase Kinase 2 Regulates Hematopoietic Stem Cell Regeneration and Quiescence
Authors: Lento William , Huang Wei , Doan Phuong , Chao Nelson J. , Racioppi Luigi 
Published: October 2019

Abstract
Extracellular free calcium (Ca2+) is the most abundant signaling molecule in the hematopoietic stem cell (HSC) niche, and intracellular calcium is an important second messenger controlling critical functions in HSCs. Accordingly, Ca2+ is a master regulator of HSC biology, and nodes of the Ca2+ signaling network are emerging as critical components of the molecular machinery governing HSC regeneration and quiescence. Ca2+ controls many cell functions by forming a complex with Calmodulin (CaM), which binds and activates a family of Calcium/Calmodulin dependent kinase (CaMK) that includes CaMKI, CaMKII, CaMKIV, CaMKK1 and CaMKK2. We have previously investigated the function of CaMKK2 in blood cells and established this protein as a critical component of the calcium signaling pathway that regulates granulopoiesis and macrophage activation. Here, we investigated the function of CaMKK2 in the regenerative response of HSCs. To determine the expression of CaMKK2 under homeostatic conditions, we examined transgenic reporter mice and identified robust reporter activity in primitive long-term hematopoietic stem cells, but reduced signal in the majority of lineage positive blood and bone marrow cells. To establish whether CaMKK2 has a functional role in controlling hematopoiesis and HSC self-renewal, we examined hematopoietic regeneration in mice deficient for CaMKK2. We demonstrate that Camkk2 -/- mice have accelerated bone marrow and peripheral blood regeneration following hematopoietic injury. Camkk2 -/- HSCs showed increased proliferative capacity in vivo and mobilized into the peripheral blood to establish extra-medullary hematopoiesis during regeneration. To identify the proximal target of CaMKK2, we assessed the phosphorylation status of CaMKI, CaMKIV and adenosine monophosphate activated protein kinase (AMPK), which are known targets of CaMKK2. Our results indicate Camkk2 -/- stem and progenitor cells (HSPC) fail to accumulate phospho-AMPK in response to ionizing radiation. Since AMPK is a negative regulator of the mammalian target of rapamycin (mTOR) pathway, we also analyzed the levels of phospho-ribosomal 6 protein (rpS6), which is a distal target of the mTOR cascade that positively regulates cell size and proliferation. We found that irradiated Camkk2 -/- HSPCs accumulate higher levels of phospho-rpS6 compared to wild type cells. This finding implicates CaMKK2 as a critical component of a negative regulatory circuit that links calcium signaling to AMPK activation and cell proliferation in HSPC. Cumulatively, our data demonstrate that CaMKK2 regulates HSC function and suggests CaMKK2 inhibition is a novel approach to stem cell expansion for clinical therapy.

Disclosures
No relevant conflicts of interest to declare.
Downregulation of Dyskerin Impairs Expansion of Normal Hematopoietic Progenitors and Erythroid Colony Formation in the Absence of Apparent Telomere Shortening
Authors: Kumari Ashu , Richards Laura A , MacKenzie Karen L 
Published: October 2019

Abstract
Dyskerin is an RNA binding protein that functions as a core component of the telomerase holoenzyme as well as in ribonuclear protein complexes involved in ribosome biogenesis and RNA processing. Within the telomerase holoenzyme, dyskerin stabilizes the telomerase RNA component (hTR) that functions as a template for synthesis of telomeric DNA at chromosome ends. Telomerase is active in malignant cells as well as normal hematopoietic stem and progenitor cells where it enables long-term cell replication.
Mutation of the gene encoding dyskerin (DKC1) is the underlying cause of the inherited disease dyskeratosis congenita (DC), which is characterized by low levels of telomerase activity and shortened telomeres. Bone marrow failure is the most common cause of mortality in DC, however since dyskerin has telomerase-independent roles in fundamental cellular processes, it is not clear whether telomerase insufficiency is the sole cause of hematopoietic dysfunction in this disorder.
To gain further insight to the consequences of deregulated expression of dyskerin in haematopoietic cells, we transduced normal human cord blood CD34+ progenitor cells with retroviral vectors encoding two different shRNAs targeting dyskerin. Transduced cells from seven independent experiments were FACS sorted then assayed for expansion and differentiation in vitro, clonogenic potential, telomerase activity and telomere length maintenance. Results from the functional assays showed that depletion of dyskerin impaired cell expansion and compromised the formation of BFU-E colonies. In contrast to BFU-E, CFU-GM colonies did not appear to be altered by dyskerin depletion. Analysis of non-transuduced cells subject to ex vivo expansion along erythroid and myelomonocytic lineages revealed that erythroid precursor cells expressed disproportionately high levels of dyskerin, and correspondingly high telomerase enzyme activity. shRNA-mediated downregulation of dyskerin lowered hTR levels and quenched telomerase activity in the erythroid precursors without altering expression of the telomerase catalytic component, hTERT. These results suggest that upregulation of dyskerin bolsters telomerase activity and renders erythroid precursors vulnerable to dyskerin depletion. However in spite of the reduction in telomerase activity induced by dyskerin shRNA, Southern blot analysis showed that the rate of telomere length shortening was not significantly altered by dyskerin depletion over the time course of these experiments. Together the data show that dyskerin is upregulated during erythroid differentiation, and has an essential function in the erythroid lineage that appears to be independent of its role in telomere length maintenance.

Disclosures
No relevant conflicts of interest to declare.
Silencing Mutations in NOTCH1 Activate Calcium Signaling in B Cells
Authors: Love Cassandra L , Healy Jane , Greenough Adrienne , Dave Sandeep S. , Li Guojie 
Published: October 2019

Abstract
The NOTCH1 pathway is known to be up-regulated in many cancer types including non Hodgkin lymphomas (NHLs), colorectal, breast and pancreatic cancers. The precise downstream effects of NOTCH1 mutations in lymphoma are not known.
We applied high throughput sequencing in three different non Hodgkin lymphomas including diffuse large B cell lymphoma (DLBCL, N=94), mantle cell lymphoma (MCL, N=58) and Burkitt lymphoma (BL, N=59). We also sequenced mutations from 208 patients with immune deficiency. In all, we identified 31 mutations in NOTCH1. Five of these mutations were either nonsense or frameshift mutations. Silencing mutations in the PEST domain have been reported in MCLs, and many other silencing mutations were observed in the other lymphoma cohorts as well as immune deficiency cases. The finding of silencing mutations was unexpected, given that in most cancers, NOTCH1 and its pathway are often constitutively active.
Calcium signaling is an important mechanism for activating B cells. Given the shared mutations between NHLs and immune deficiencies, we investigated the role of silencing NOTCH1 mutations in altering calcium signaling by engineering lymphoma cells with a deletion of the transmembrane region resulting in silencing of NOTCH1 using the CRISPR system.
We designed guide RNAs that spanned a 57 base pair region from exon 28 that encodes the transmembrane domain of the NOTCH1 gene. Each guide RNA was ligated into a pX458 Cas9 GFP expressing plasmid. Namalwa NHL cells were transfected, FACS-sorted for GFP positivity and subcloned. Successful deletion of the target region in Cas9 NOTCH1 cells was confirmed by Sanger sequencing and Western blotting.
Cells that were wild type, Cas9 control, or Cas9 NOTCH1 were incubated in a calcium free buffer containing Fluo-4 for detection of intracellular free calcium. Cells were washed and subjected to flow sorting where baseline calcium was recorded. The cells were stimulated with anti-IgM (Fab’)2 and recorded again for response. To test extracellular calcium influx, 4mM CaCl2 was added to the calcium free buffer containing cells and recorded again for response.Upon stimulation with anti-IgM (Fab’)2, we observed a significant increase (p<10-6) of free cytosolic Calcium in the Cas9 NOTCH1 cells compared to both wild type and Cas9 control. Similar results were obtained when measuring extracellular Calcium influx upon addition of CaCl2to the buffered solution of cells.
These findings indicate that silencing of NOTCH1 has an activating role in calcium signaling that could contribute to B cell survival and function in NHLs and immune deficiencies. Similar roles have been reported for activating mutations in PIK3CD and STAT3 that contribute to both immune deficiency and cancer. Our study is the first to identify the role of silencing mutations of NOTCH1 in NHLs and immune deficiencies.

Disclosures
No relevant conflicts of interest to declare.
Specific NK Cell Activation to Treat Relapsed/Refractory (r/r) Hodgkin Lymphoma (HL) Patients – Final, Updated Data on Clinical Outcome, Pharmacokinetics and Pharmacodynamics of a Phase 1 Study Investigating AFM13, a Bispecific Anti-CD30/CD16A Tandab
Authors: Rothe Achim , Sasse Stephanie , Topp Max S. , Eichenauer Dennis A. , von Tresckow Bastian , Hummel Horst-Dieter , Ravic Miroslav , Marschner Jens-Peter , Engert Andreas 
Published: October 2019

Abstract
Introduction: AFM13 is a bispecific, tetravalent TandAb antibody that targets CD16A and CD30. Preclinical data demonstrated that AFM13 specifically harnessed NK cells to exert potent cytotoxic effects on tumor cells expressing CD30. Furthermore, AFM13 showed a favorable toxicity profile in cynomolgus monkeys. HL is characterized by the presence of CD30+ Reed-Sternberg cells and by a high medical need in the r/r setting, especially since the response to brentuximab vedotin in this patient group is mostly short. Here, we present the final results of a phase 1 clinical study in r/r HL including a focus on the pre-treatment with brentuximab vedotin.
Methods: Heavily pretreated patients (pts.) with r/r HL received stepwise escalated doses of intravenous AFM13 (0.01 to 7.0 mg/kg) weekly or 4.5 mg/kg twice weekly over 4 weeks. Primary objectives were safety and tolerability, secondary objectives included pharmacokinetics (PK), pharmacodynamics (PD), and clinical efficacy measured using Cheson criteria.
Results: 28 pts. were recruited, 24 pts. received AFM13 weekly and 4 pts. received AFM13 twice weekly. AFM13 was well tolerated with mainly mild to moderate adverse events. The maximum tolerated dose was not reached. AFM13 demonstrated activity, which was consistently more pronounced for all assessed parameters at doses ≥1.5 mg/kg (n=13). 3 of 13 pts. (23%) exhibited partial responses and disease control was reached in 10 of 13 pats. (77%). Importantly, AFM13 was also active in pts. refractory to brentuximab vedotin as most recent treatment. PK data revealed an AFM13 half-life of up to 19 hours. In peripheral blood, the portion of activated NK cells (CD69+), relative to total number of NK cells, increased up to 3-fold 24 hours after infusion compared to baseline. Kinetics of NK cell activation corresponded to kinetics of AFM13 serum levels. Soluble CD30 declined to zero at the end of treatment in almost all pts.
Conclusion: Phase 1 data indicate that AFM13 is active and well tolerated in heavily pretreated r/r HL pts. including brentuximab vedotin failures. To further enhance efficacy of AFM13 both, the dose regimen (based on PK/PD profile) and the duration of treatment, will be optimized in a phase 2 study. This study is currently in preparation by the German Hodgkin Study Group.

Disclosures
Topp: Affimed Therapeutics AG: Consultancy. von Tresckow:Takeda: Honoraria, Other; Novartis: Honoraria, Research Funding. Marschner:Affimed Therapeutics AG: Employment. Engert:Millenium Takeda: Consultancy; Affimed Therapeutics AG: Consultancy.
Presence of Calreticulin Mutations in JAK2-Negative Polycythemia Vera
Authors: Girodon Francois , Broseus Julien , Park-Alexandre Ji-Hye , Hermouet Sylvie , Carillo Serge 
Published: October 2019

Abstract
Calreticulin (CALR) mutations have recently been reported in JAK2- and MPL-negative Myeloproliferative Neoplasms (MPN), particularly essential thrombocythemia (ET) and primary myelofibrosis (PMF).The clinical course of sporadic CALR-mutated patients seems to be more indolent than that of JAK2-mutated patients. In contrast, no CALR mutation has been found in the 647 published cases of Polycythemia Vera (PV) patients tested. Consequently, CALR mutations were considered exclusive to JAK2 and MPL mutations. Since 98% of PV patients harbor a JAK2 mutation (mostly the V617F mutation in exon 14 and more rarely, in exon 12), the absence of CALR mutations in PV seemed logical. Here, we describe two JAK2V617F-negative PV patients who presented with a CALR mutation at the time of diagnosis.
Patient # 1 had hemoglobin at 168 g/L, hematocrit at 51.3%, and increased red cell mass (RCM) at 128% associated with a normal erythropoietin level. The bone marrow biopsy showed hypercellularity for age, panmyelosis associated with normal megakaryocytes and rare isolated abnormal enlarged forms. Using reticulin stain, no myelofibrosis was noted. Patient # 2 had hemoglobin at 194 g/L, hematocrit at 53% and low erythropoietin level without any dehydration. Both had moderately elevated platelet counts (658 and 575 x109/L respectively) with normal leukocyte counts. They were negative for BCR-ABL. No mutation was found in JAK2 exons 12, 13 and 14 by HRM and allele-specific real-time PCR or in MPL exon 10. Using HRM analysis, CALR mutations were suspected in both patients and confirmed using Sanger sequencing and product sizing analyses: CALR mutations were in both patients type 1 deletions (52-bp deletion; c.1092_1143del). To complete genomic tests made on peripheral blood granulocytes, we performed colony assays in methylcellulose and in collagen, picked single BFU-E colonies grown after 14 days in the presence of erythropoietin, and genotyped each colony individually for CALR. Of the 27 colonies genotyped, 6 had no PCR amplification and 21 harboured the same CALR mutation observed in peripheral blood granulocytes, i.e 52-bp deletion; c.1092_1143del. BFU-E were found heterozygous for CALR, with a mean allele burden of 49%. To our knowledge, these patients are the first cases of CALR-mutated PV to be reported. However, since a biclonalJAK2V617F and CALR MPN case recently reported, we cannot rule out the possibility of a biclonal disease involving a yet unknown mutation associated with a CALR mutation. On the other hand, the presence of a CALR mutation both in peripheral granulocytes and in BFU-E suggests that the CALR mutation plays a role in the polycythemia phenotype. Our observations highlight the fact that in the absence of JAK2 mutation, CALR mutations can also be associated with PV.
In conclusion, our data indicate that testing JAK2-negative PV patients for CALR mutations may be useful.

Disclosures
No relevant conflicts of interest to declare.