bluuebunny/crossref_metadata_2025_split huggingface dataset:
Adenosine Signaling-Mediated Metabolic Reprogramming Regulates Erythropoiesis
Authors: Liu Hong , Zhang Yujin , D'Alessandro Angelo , Nemkov Travis , Couturier Jacob , Zhao Shushan , Hansen Kirk C , Lovering Andrew T. , Roach Robert , Kellems Rodney E. , Eltzschig Holger K. , Blackburn Michael R. , Xia Yang 
Published: October 2019

Abstract
Erythropoiesis is an extremely dynamic process finely regulated by cytokines, hormones, and growth factors at transcriptional and translational levels. Stress-induced erythropoiesis is defined as a stimulated basal erythropoiesis with expansion of the erythroid progenitor pool, associated with reticulocytosis and splenomegaly. Stress erythropoiesis is stimulated under the condition of insufficient oxygen availability such as high altitude, blood loss, infection, and anemia. Thus, stress erythropoiesis is an important stress adaptive response for survival. Although stress erythropoiesis has been long speculated to be linked with increased metabolic requirements, until recent two years with innovative metabolomics profiling and state of art isotopically labelled metabolic flux approaches, the filed has evolved and revealed that enhanced glucose and glutamine metabolism is essential for stress erythropoiesis. However, molecular basis underlying metabolic reprogramming to enhance glucose metabolism and subsequently stress erythropoiesis remains unclear.
To address this question, we conducted both human and mouse studies. First, we found that plasma adenosine is rapidly induced and associated with stress erythropoiesis features including increased hematocrit (HCT), hemoglobin (Hb) mass and reticulocytes in healthy human volunteers at high altitude and in mice exposed to hypoxia mimicking high altitude. Follow-up mouse genetic studies showed that activation of adenosine signaling via erythroid ADORA2B promotes the survival and expansion of proerythroblasts both in spleen and bone marrow and in this way contributes to hypoxia-induced stress erythropoiesis independent of erythropoietin. Using unbiased high-throughput metabolic profiling, we identified that erythroid ADORA2B contributes to an overall hypoxia metabolic reprogramming with substantial increased glycolysis in proerythroblast progenitors in mice. Finally, using primary human CD34+ hematopoietic stem cells culture, we showed that adenosine analogue and ADORA2B agonist promote the survival and expansion of erythroid progenitors in a time and dose-dependent manner. Taken together, both human and mouse studies identify that adenosine ADORA2B is a previously unrecognized purinergic signaling underlying hypoxia-induced erythropoiesis by facilitating expansion and survival of proerythroblasts, and highlight that enhancing this pathway is a potential strategy to induce erythropoiesis.

Disclosures
No relevant conflicts of interest to declare.
Sargramostim Accelerates Leukocyte Recovery and Improves Mortality Rate at Day 60 in a Non-Human Primate Model of Hematopoietic Acute Radiation Syndrome When Administered 48 h after Total Body Irradiation
Authors: Clayton Nicholas P. , Charpentier Eric J.J. , LaCasse Emily R. , Khan-Malek Richard C. , Keutzer Joan M. 
Published: October 2019

Abstract
Background: Hematopoietic acute radiation syndrome (H-ARS) occurs in individuals that are exposed to high levels of radiation over a short period of time. The destruction of bone marrow leads to pancytopenia, which increases the incidence of infections and accounts for the majority of morbidity and mortality. Sargramostim (yeast-derived rhGM-CSF) is a leukocyte growth factor that promotes differentiation, maturation and activation of granulocytes, monocytes and macrophages.
Methods: We evaluated the efficacy of repeat administrations of sargramostim (7 μg/kg/day) in a blinded, GLP study in total body irradiated non-human primates (NHPs) when administered 48 h post-irradiation with minimal supportive care (e.g., no blood products or individualized antibiotics). The primary objective was to assess the efficacy of sargramostim versus vehicle on mortality rate at Day 60 in irradiated male and female NHPs at LD50-60/60 (n=36/group). Secondary objectives included the efficacy of sargramostim on overall survival and its effect on hematology parameters. The study included an exploratory arm of male and female NHPs that were irradiated at a LD70-80/60(n= 18/group) and received the same treatments.
Results: Sargramostim decreased the mortality rate at Day 60 by 36% in the LD50-60/60 group (p=0.0018) and by 44% in the LD70-80/60 group (p=0.0076). Additionally, neutrophil, platelet, lymphocyte, and white blood cell levels in survivors demonstrated accelerated recovery in the sargramostim-treated NHPs in the LD50-60/60 and LD70-80/60 groups.
Conclusion: Treatment with sargramostim (7 μg/kg/day) beginning at 48 h, in the absence of blood products and individualized antibiotics, suggests that the use of sargramostim is a viable therapeutic strategy for H-ARS in a mass casualty event.
Funding: This project has been funded in whole or in part with Federal funds from the Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority, under Contract No. HHSO100201300005I.

Disclosures
Clayton: Sanofi Genzyme: Employment. Charpentier:Sanofi: Employment. LaCasse:Sanofi Genzyme: Employment. Khan-Malek:Sanofi: Employment. Keutzer:Sanofi Genzyme: Employment.
L-DEP Is an Effective Treatment for Adult and Adolescent Patients with Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis
Authors: Lai Wenyuan , Wang Yini , Wang Jingshi , Wang Zhao 
Published: October 2019

Abstract
Epstein-Barr virus-associated hemophagocytic lymphohistocytosis (EBV-HLH) is the most frequent subtype of secondary HLH with infectious triggers. In addition to meet the HLH-2004 diagnostic criteria, the diagnosis of EBV-HLH requires the evidence of EBV infection and exclusion of primary HLH and lymphoma-associated HLH. The prognosis of EBV-HLH is generally dismal. To assess the efficacy of current treatment protocols in adult and adolescent patients with EBV-HLH, we retrospectively analyzed 171 HLH patients presented at our institution from March 2009 to April 2016. Of those, 133 patients were EBV-HLH; 35 patients were lymphoma-associated HLH; 3 of 68 patients who underwent genetic test were diagnosed with primary HLH including one with homozygous mutation in UNC13D, one with homozygous mutation in RAB27A, and one with hemizygous mutation in SH2D1A. The rest of the 68 patients exhibited various levels of genetic defects. Of the 133 EBV-HLH patients, 91 male and 42 female cases (2.2:1) were aged between 14 - 77 years with a median age of 26 years. EBV-DNA load on admission was at a median of 6.6 × 105 copies/ml (range: from 5.0 × 103 - 7.8 × 109 copies/ml). One-year mortality rate for them was 66.4%. One hundred and twelve cases were initially treated by HLH-94 or HLH-2004 regimen. Of those, 52 cases (46.4%) had response including 22 cases (19.6%) of complete response (CR) and 30 cases (26.8%) of partial response (PR). Six patients received L-DEP regimen as initial treatment, of which 5 cases had response consisting 3 cases of CR and 2 cases of PR. The overall response rate (ORR) was 83.3%. The rest 15 patients received initial treatment without etoposide, 5 patients (33.3%) achieving PR and 10 patients (66.7%) showing no response. Sixty-nine refractory or relapsed cases received DEP or L-DEP regimen, ORR of which was 79.7%. For those cases, 26 cases received DEP regimen, of which 17 cases (65.4%) had response including 5 cases of CR and 12 cases of PR. Forty-three patients received L-DEP as salvage therapy, of which 38 cases (88.4%) had response including 13 cases of CR (30.2%) and 25 cases of PR (58.1%). Compared to DEP treatment, who received L-DEP had a higher effective rate (65.4% versus 88.4%, p=0.031). Of 133 EBV-HLH patients, only 36 patients received allogeneic hematopoietic stem cell transplantation, the survival rate was 61.1%. In conclusion, EBV-HLH is a fatal disease. Our results indicate DEP or L-DEP is an effective salvage therapy. Additionally, L-DEP may be a more effective therapy than HLH-94/2004 regimen to be used as an initial treatment. Finally, allogeneic hematopoietic stem cell transplantation is an effective approach for the treatment of EBV-HLH.

Disclosures
No relevant conflicts of interest to declare.
Clinical Dosing Regimen of Selinexor Maintains Normal Immune Homeostasis and T Cell Effector Function in Mice: Implications for Combination with Immunotherapy
Authors: Tyler Paul M , Servos Mariah M , Klebanov Boris , Kashyap Trinayan , Shacham Sharon , Landesman Yosef , Dougan Michael , Dougan Stephanie K 
Published: October 2019

Abstract
Selinexor (KPT-330) is a first in class nuclear transport inhibitor of exportin-1(XPO1) currently in advanced clinical trials to treat patients with solid and hematological malignancies. To determine how selinexor might impact anti-tumor immunity, we analyzed immune homeostasis in mice treated with high selinexor doses (15 mg/kg, three times a week: M, W, F) and found disruptions in T cell development, a progressive loss of CD8 T cells and increases in inflammatory monocytes. Antibody production in response to immunization was mostly normal. Precursor populations in bone marrow and thymus were unaffected by high doses of selinexor, suggesting that normal immune homeostasis could recover. We found that high dose of selinexor given once per week preserved nearly normal immune functioning, whereas a lower dose given 3 times per week (7.5 mg/kg, M, W, F) was not able to restore immune homeostasis. Both naïve and effector CD8 T cells cultured in vitro showed impaired activation in the presence of selinexor. These experiments suggest that XPO1 function is required for T cell development and function. We then determined the minimum concentration of selinexor required to block T cell activation, and showed that T cell inhibitory effects of selinexor occur at levels above 100nM, corresponding to the first 24 hours post-oral dosing of 10 mg/kg. In a model of implantable melanoma, we used selinexor treatment at the clinically relevant dosing regimen of 10 mg/kg with a 5-day drug holiday (M, W selinexor treatment). After two weeks of treatment, tumors were harvested and tumor infiltrating leukocyte (TIL) populations were analyzed. This treatment led to intratumoral IFNg+, granzyme B+ cytotoxic CD8 T cells that were comparable to vehicle treated mice. Overall, selinexor treatment leads to transient inhibition of T cell activation but the clinically relevant once and twice weekly dosing schedules that incorporate sufficient drug holidays allow for normal CD8 T cell functioning and development of anti-tumor immunity. These results provide additional support to the recommended selinexor phase 2 dosing regimen, as was determined recently (Razak et al. 2016).

Disclosures
Klebanov: Karyopharm Therapeutics: Employment, Equity Ownership. Kashyap:Karyopharm Therapeutics: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics: Employment, Equity Ownership. Dougan:Karyopharm Therapeutics: Consultancy. Dougan:Karyopharm Therapeutics: Consultancy.
Impaired Metabolic Fitness in T Cells in Chronic Lymphocytic Leukemia
Authors: van Bruggen Armando , Endstra Sanne , van der Windt Gerritje J.W. , Kater Arnon P. 
Published: October 2019

Abstract
Quiescent T cells primarily use oxidative phosphorylation (OXPHOS) to generate ATP, while in response to activation T cells switch to high rates of aerobic glycolysis, also known as the Warburg effect. While less efficient in overall ATP production, this switch to aerobic glycolysis is essential to produce cellular biomass needed for proliferation, and is required for T cell effector functions. In chronic lymphocytic leukemia (CLL) acquired T cell dysregulation occurs independent of treatment with functional impairment and exhaustion of T cells. As tumor-imposed metabolic restrictions in mouse models can mediate T cell hyporesponsiveness during cancer, we hypothesized that in the context of CLL T cell metabolism might be altered.
Comparison of gene expression profiles of T cells from patients with CLL and age-matched healthy donors (HD) revealed a highly significant increase in the expression of genes in the OXPHOS pathway (P=3.4*10-15) in the CD8 T cell compartment. In corroboration with these array results, we found that in naïve CD8 T cells in CLL, mitochondrial mass and respiration were significantly increased. In addition, increased mitochondrial ROS production was observed in the naïve CD8 T cell subset.
Using Seahorse EFA technology on sorted CD8 T cells from CLL and age-matched HD, we found increased oxygen consumption rates in CLL derived CD8 T cells, indicating increased OXPHOS, while the spare respiratory capacity was lower in these cells, indicating impeded ability to cope with cellular stress. Extracellular acidification rates (ECAR; indicating glycolysis), and uptake of fluorescently labeled glucose were similar in CD8 T cells from CLL patients and HD.
Next, we studied the metabolic plasticity of CLL derived T cells by stimulation of PBMCs from CLL patients and age-matched HD using anti-CD3/CD28 antibodies. Two days after stimulation, CLL derived T cells had diminished expression of activation markers CD25 and CD38, which correlated with reduced uptake of fluorescently labeled glucose. Moreover, preliminary Seahorse analysis of the immediate response to stimulation with anti-CD3/CD28 showed a reduced increase in ECAR in CLL derived CD8 T cells, indicating an impairment of the glycolytic switch in these cells.
Taken together, these results demonstrate that the metabolic fitness of CD8 T cells is impaired in CLL at resting state and after activation. Boosting T cell metabolism in CLL might therefore improve existing immunotherapies in CLL such as CAR-T cell therapy.
This work is funded by VENI and VIDI grants from the Dutch Organisation for Scientific Research, and a Marie Curie Career Integration Grant from the European Union.

Disclosures
Kater: Celgene: Research Funding.
A Novel Monoclonal Antibody Against A2 Domain of Von Willebrand Factor Reduces Its Proteolysis By ADAMTS13
Authors: Zhang Lulu , Su Jian , Shen Fei , MA Zhenni , Zhao Yiming , Yu Ziqiang , Ruan Changgeng 
Published: October 2019

Abstract
Introduction:ADAMTS13 metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. A minimal region as a functional substrate for ADAMTS13 consisted of 73 amino acid residues from D1596 to R1668 of VWF, designated VWF73, and deletion of the E1660-R1668 region (the C-terminal α helix of VWF A2 domain) led to the loss of cleavage by ADAMTS-13. This suggested that the C-terminal α helix of VWF A2 domain contributes to ADAMTS13 binding to substrate. We will explore whether murine monoclonal antibody (mAb) against the C-terminal α helix of human VWF A2 domain affects the susceptibility of VWF to proteolysis by ADAMTS13 in vitro.
Methods:Balb/c mice were immunized with R1659-R1668 of the VWF polypeptide. Monoclonal antibodies (mAbs) were developed by standard hybridoma technology and identified with ELISA. The epitope of mAbs were mapped using recombinant VWF A2 and a series of its truncated mutants with western blot analysis. The effect of mAbs to VWF on its proteolysis by ADAMTS13 was investigated under static/denaturing condition by 1.3% agarose gel electrophoresis and immunologic analysis. The effect of mAbs to GST-VWF73-His on its proteolysis by recombinant ADAMTS13 was measured by 15% SDS-PAGE under reducing condition and western blot analysis.
Results: One mAb, designated SZ-179, was found to have high affinity (50 ng/ml) with both a synthetic peptide encompassing residues 1659-1668 and purified pVWF. SZ-179 has isotype of IgG1. It was shown that SZ-179 reacted specifically with recombinant VWF A2 and GST-VWF73-His, respectively, but not with recombinant VWF A1 nor with recombinant VWF A3. Epitope mapping experiments revealed that SZ-179 bound to the distal portion of the VWF A2 domain (E1660-L1666). SZ-179 inhibited proteolytic cleavage of GST-VWF73-His by recombinant ADAMTS13 in a concentration-dependent manner, with a maximal inhibition at concentrations of 50 ug/ml. SZ-179 also reduced degradation of high molecular-weight (HMW)-VWF-multimers under static/denaturing condition dose-dependently, with a maximal inhibition at concentrations of 1 ug/ml.
Conclusion:SZ-179, a novel mAb to the A2 Domain of VWF, inhibits the interactions between VWF and ADAMTS13, and prevents excessive degradation of HMW-VWF multimers. This mAb might be useful for the development of therapeutic options to treat bleeding episodes among hemorrhagic diseases.

Disclosures
No relevant conflicts of interest to declare.
TOP2A a New Predictive Marker of Response to Carfilzomib in Multiple Myeloma
Authors: Reale Antonia , Khong Tiffany , Mithraprabhu Sridurga , Reynolds John , Ramachandran Malarmathy , Savvidou Ioanna , Bergin Krystal , Hocking Jay , Spencer Andrew 
Published: October 2019

Abstract
Introduction/background: Multiple myeloma (MM) remains incurable despite the introduction of novel therapeutic agents. Microarray-based technologies were adopted in our study to determine if a genetic signature associated with resistance to carfilzomib (CZ), a second-generation proteasome inhibitor (PI) currently used in clinical settings, could be identified.
Materials and Methods: Nine human myeloma cell lines (HMCLs) were treated with carfilzomib and a cell viability profile was assessed categorizing the HMCLs as sensitive or resistant to carfilzomib. Subsequently, the gene expression profiles (GEP) of untreated resistant versus sensitive HMCLs were compared. GEP-identified differences were validated on a panel of 17 HMCLs with q-RT-PCR and siRNA knockdown assays. Topoisomerase 2-alpha (TOP2A) immunohistochemistry was performed on a panel of fully annotated trephine biopsy specimens acquired prior to treatment with PIs.
Results: 206 genes were differentially expressed between the sensitive and resistant HMCLs. Gene ontology analysis identified two pathways that were significantly different: pathogenic Escherichia Coli infection (p=0.002) and lysosome (p=0.006). Eight GO terms were enriched: 4 related to biological processes and 4 related to cellular components. TOP2A, an enzyme that controls and alters the topologic states of DNA during transcription and is involved in cell cycle and proliferation, was overexpressed in carfilzomib-resistant HMCLs. It functions as the target for several anticancer agents and a variety of mutations in this gene have been associated with the development of drug resistance. TOP2A has also been noted to be overexpressed in the 'proliferation cluster' of published MM GEP profiles and associated with poor outcome. Following suppression of TOP2A by siRNA, carfilzomib-resistant HMCLs were re-sensitized to carfilzomib, moreover, the combination of carfilzomib with topoisomerase inhibitors known to target directly TOP2A, demonstrated synergistic cytotoxic effects against HMCLs. Trephine-derived TOP2A protein expression levels were shown to be higher in patients treated with PIs including bortezomib, carfilzomib, ixazomib, marizomib, failing to achieve a response when compared to responding patients. Finally, logistic regression analysis confirmed that TOP2A protein expression was a highly significant predictor of response to PIs (AUC 0.764, p=0.048).
Conclusion: TOP2A status may be used as a predictive factor for patient response to PIs including carfilzomib.

Disclosures
No relevant conflicts of interest to declare.
Feasibility of a Cell-Based Oncolytic Virotherapy Targeting Bone Marrow Lesions of Multiple Myeloma
Authors: Sato Kota , Futami Muneyoshi , Chit Ung Weng , Nakamura Takafumi , Tojo Arinobu 
Published: October 2019

Abstract
Virotherapy is a potentially promising modality to treat intractable malignant tumors that are resistant to conventional chemotherapy and molecularly targeted therapy. From a viewpoint of clinical application, vaccinia virus (VV) has several advantages over other oncolytic viruses, such as a short life cycle, potent tissue penetration and robust lytic activity. We showed that intravenous injection of miRNA (let-7a)-regulated and thymidine kinase-deleted VV (MDVV) efficiently infects and kills multiple myeloma cells with much less adverse effects on normal cells and tissues in SCID mice carrying a subcutaneous xenograft (Blood 2014 124:2082). However, in another xenograft model carrying bone marrow (BM) myeloma lesions, MDVV failed to reduce tumor burden or prolong survival probably due to its poor penetrance to BM. Then, for an alternative strategy, we tested the feasibility of a cell-based virus delivery which targets BM lesions using myeloma cells infected ex vivo with MDVV.
A human myeloma cell line KPMM2 is absolutely dependent on interleukin-6 or a murine stromal cell line such as HESS-5 for its continuous growth and survival (Blood 1997 90:2437). It recapitulates BM myeloma lesions and IgG paraproteinemia when transplanted into SCID mice intravenously. Furthermore, KPMM2 cells are highly susceptible to and efficiently killed by MDVV. First, we tested whether KPMM2 cells can serve as MDVV carriers to cause secondary infection. MDVV was originally labeled by F-luc, and KPMM2 cells were transduced with R-luc (target: T), then seeded onto a HESS-5 monolayer. Twenty-four hrs later, MDVV-infected KPMM2 cells (effector: E) were added at various E/T ratios. Dual F/R-luc assay showed that target cell viability was significantly reduced at E/T ratio ≥ 1 along MDVV propagation. This lytic effect was dependent on time and a multiplicity of infection (MOI), but not perturbed by the co-culture with cord blood mononuclear cells (bystander). Next, we validated the possibility that MDVV-infected KPMM2 cells can deliver MDVV to BM after intravenous injection into SCID mice. Most of KPMM2 cells show high level expression of CXCR4, a critical regulator of myeloma cell homing to BM, and retain comparable CXCR4 expression even after MDVV infection. Actually, 48 to 72 hrs after 1x107 MDVV-infected KPMM2 cells (labeled by F-luc) were injected into SCID mice, in vivo imaging analysis detected the accumulation of prominent F-luc signal at the BM following early phase trap in the lung (Figure). Taken together, the present cell-based strategy is a unique way to enable targeted delivery of MDVV to BM and application of oncolytic virotherapy to hematological malignancies, especially multiple myeloma. The therapeutic efficacy of MDVV-infected KPMM2 cells on BM myeloma lesions in SCID mice is currently under investigation.
Figure Figure.

Disclosures
No relevant conflicts of interest to declare.