bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of MicroRNAs in Resistance to Dexamethasone in Multiple Myeloma Cell Lines
Authors: Bashir Qaiser , Bemis Lynne , Robinson William , Liu Bolin , Wang Shuiliang , Amato Carol , Riley Elizabeth , Lee Choon Kee
Published: October 2019

Multiple Myeloma is a B-cells malignant disease of unknown etiology. It accounts for 1% of all cancers and 14% of hematologic malignancies. Despite the advances in treatment it remains an incurable disease. One of the commonly used drugs in the treatment of multiple myeloma is Dexamethasone. Most patients respond to this drug, but eventually most then become refractory to the treatment with steroids. MicroRNAs (miRNA) are a newly identified class of small RNAs that play important regulatory roles at the post transcriptional level. They usually act by binding to the 3′untranslated region (3′UTR) of messenger RNA (mRNA) and either block their translation or lead to degradation. MM1 cell line is a well known myeloma cell line. By continued exposure to Dexamethasone two variants of MM1 cell line i.e.; MM1.S and MM1.R were developed. MM1.S remains sensitive while MM1.R is resistant to the action of Dexamethasone. It has been reported that MM1.R cell line expresses very minimal levels of glucocorticoid receptor (GR) mRNA and protein, hence rendering it resistant to the action of steroids. We hypothesized that altered regulation of microRNAs between the two cell lines leads to the down regulation of glucocorticoid receptor hence contributing to resistance to the action of steroids. QRT-PCR and Western blot were conducted to demonstrate decreased expression of GR mRNA and protein in MM1.R cell line. We identified certain microRNAs based on bioinformatics with putative docking sites in 3′UTR of GR mRNA. DNA sequencing was used to rule out any mutation in binding sites of these microRNAs. No such mutation was found in MM1.S or MM1.R cell line. qRT-PCR was than used to analyze the differential expression of predicted microRNAs. Several microRNAs were found to be differentially expressed between the two cell lines, including microRNA-183 (p < 0.05). We then transfected inhibitors and mimics of pertinent microRNAs and were able to up or down regulate the GR mRNA. We were also able to show some evidence of apoptosis in MM1.R cell line with Dexamethasone exposure. These studies suggest that microRNAs regulate expression of GR and play an important role in conferring resistance to the action of steroids. These findings shed further light on the underlying mechanisms of steroid resistance and may have important therapeutic implications in the treatment of multiple myeloma.
Marginal Zone Lymphoma: Factors That Affect the Final Outcome, a Study of 275 Patients
Authors: Mazloom Ali , Reed Valerie K , Cabanillas Fernando , Fayad Luis , Pro Barbara , Iyengar Puneeth , McLaughlin Peter , Urbauer Diana L , Dabaja Bouthaina S.
Published: October 2019

Purpose: Retrospective review and analysis of 275 patients with marginal zone lymphoma (MZL) to determine the prognostic factors that affect outcome in an effort to find a specific prognostic score.

Materials and Methods: Two hundred and seventy five patients with pathologically confirmed MZL were studied. Their median age was 60 years (range 18–89). Male sex was found in 123 patients (45%). Ann Arbor stage I was present in 104 patients (38%), stage II in 27 (10%), stage III in 16 (6%), stage IV in 124 (45%), and unknown in 4 (1%). Most patients were asymptomatic at presentation (90%), with 96% presenting with Zubrod score of 0 or 1. Patients were divided into three groups according to sites of involvement: extranodal (211), nodal (27), and splenic (37). Treatment strategy consisted of: observation in 75 (27%), chemotherapy in 103 (37%), combined chemotherapy and radiation in 24 (9%), and definitive radiation in 71 (26%).

Results: Both univariate and multivariate analysis showed that splenic presentation had a better outcome with a hazard ratio (HR) of 0.14 and p value=0.006, while extranodal presentation with bone marrow involvement had an inferior outcome with HR of 2.27 and p value =0.045. Two factors were found to be associated with decreased overall survival (OS) in the multivariate analysis: Older age (p=0.007) and beta 2 microglobulin >1.8 mg/L (p <0.0001). On the other hand splenic presentation and being asymptomatic at presentation were associated with an improved OS with a p value of 0.002 and 0.017, respectively. Stage at presentation had no impact on the overall survival or disease free survival, and although all patients with splenic MZL were stage IV, these patients had the best outcome. Nodal presentation had a similar survival to extranodal presentation.

Conclusion: Splenic presentation even at advanced stage is associated with a better outcome. Bone marrow involvement in patients with extranodal presentation had a lower OS. Older age, high beta 2 microglobulin, and symptoms at presentation adversely affected the overall survival of patients with MZL.
Collaboration of the Meningioma 1 (MN1) Oncogene with MLL-Fusions in Pediatric Leukemia
Authors: Liu Ting , Jankovic Dragana , Brault Laurent , Ehret Sabine , Rossi Vincenzo , Biondi Andrea , Schwaller Juerg
Published: October 2019

Expression of meningioma 1 (MN1) has been proposed to be a negative prognostic marker in adult acute myeloid leukemia (AML). In pediatric leukemia, we found overexpression of MN1 in 53 of 88 cases: whereas no MN1 expression was detected in T-cell acute lymphoblastic leukemia (T-ALL), significant amounts of MN1 were found in immature B-cell ALL and most cases of infant leukemia. Interestingly, 17 of 19 cases harboring fusion genes involving the mixed-lineage leukemia (MLL-X) gene showed elevated MN1 expression. Lentiviral siRNA mediated MN1 knock-down resulted in cell cycle arrest and impaired clonogenic growth of 3 MLL-X-positive human leukemia cell lines overexpressing MN1 (THP-1, RS4;11, MOLM-13). In a mouse model of MLL-ENL-induced leukemia we found MN1 to be overexpressed as a consequence of provirus integration. Strikingly co-expression of MN1 with MLL-ENL resulted in significantly reduced latency for induction of an AML phenotype in mice suggesting functional cooperation. Immunophenotyping and secondary transplant experiments suggested that MN1 overexpression seems to expand the L-GMP cell population targeted by the MLL-ENL fusion. Gene expression profiling allowed defining a number of potential MN1 hematopoietic targets. Upregulation of CD34, FLT3, HLF, or DLK1 was validated in bone marrow transiently overexpressing MN1, in MN1-induced mouse acute myeloid leukemia, as well as in pediatric leukemias with elevated MN1 levels. Our work shows that MN1 is overexpressed in a significant fraction of pediatric acute leukemia, is essential for growth of leukemic cells, and that MN1 can act as a cooperating oncogene with MLL-ENL most probably through modification of a distinct gene expression program that leads to expansion of a leukemic progenitor population targeted by MLL-fusion genes.
Platelet-Derived Growth Factor (PDGF)-BB Mediates Survival of Leukemic Large Granular Lymphocyte Via An Autocrine Regulatory Pathway
Authors: Yang Jun , Liu Xin , Nyland Susan , Zhang Ranran , Thomas Kendall , Jarbadan Ruth Nancy , Loughran Thomas P.
Published: October 2019

Tumor cells may stimulate their own proliferation through an autocrine mechanism by simultaneously producing growth factors and growth factor receptors. Proto-oncogene Platelet derived growth factor (PDGF) is a multifunctional molecule with roles in cell growth, proliferation, chemotaxis, actin reorganization and apoptosis resistance. PDGF exerts its cellular effects by inducing homo- or hetero-dimeric complex of α- or β-tyrosine kinase receptors, mediating its receptor tyrosine kinase (PDGF-β RTK) auto-phosphorylation, and consequently initiating downstream survival signaling pathway activation cascades. The importance of PDGF ligand and receptor activities in antigenactivated lymphoid malignancies is illustrated by their expression that has only been observed in HTLV-1/2 –immortalized human T lymphocytes, but not in normal T or NK cells. Large granular lymphocyte leukemia (LGL) is similarly defined as an antigen-activated malignancy. Same data suggest that LGL leukemia may be associated with an HTLV–like retrovirus infection. Since the LGL leukemia antigen is postulated to originate from a retroviral immunogen with HTLV-like properties, we investigated the role of PDGF in T- and NK-LGL leukemia. We report that LGL leukemia cells from both T and NK subtypes simultaneously express PDGF-BB and its receptor-β at both mRNA and protein levels. PDGF-BB mediates PDGF-β RTK auto-phosphrylation and consequently initiates downstream survival pathway activation cascade. We observed that PI3 kinase, Src family kinase and their downstream PKB/Akt pathways are constitutively activated in PBMC from LGL leukemia patients. Concurrent inhibition of both PI3 kinase and Src kinase pathways results in an additive effectiveness in induction of apoptosis via inhibition of PKB/Akt and MEK/ERK phosphorylation. In conclusion, PDGF-BB and PDGF receptor-β are highly expressed in LGL leukemia cells. Constitutive expression of PDGF-BB isoform from LGL leukemia cells may possibly be the result of chronic retroviral infection or retroviral antigenic stimulation. Co-expression of PDGF-BB and its receptor-β plays a central role in mediating PDGF/RTK auto-phosphorylation, and its downstream survival pathway activation cascades via an autocrine regulatory mechanism in LGL leukemia.
Resistance to Annexin A5 Anticoagulant Activity and Specificity for Anti-B2GPI Domain I Antibodies in Obstetric Antiphospholipid Syndrome
Authors: Hunt Beverley J , Wu Xiao-Xuan , de Laat Bas , Arslan Alan A , Parmar Kiran , Stuart-Smith Sarah , Rand Jacob H
Published: October 2019

Background Obstetric antiphospholipid (aPL) syndrome (APS) is defined by the presence of persisting aPL with previous unexplained recurrent first trimester pregnancy loss, &/or intrauterine death, &/or placental dysfunction (severe pre-eclampsia, intrauterine growth retardation or placental abruption). The placental anticoagulant protein Annexin A5 (AnxA5) is expressed on the surfaces of placental syncytiotrophoblasts where it has been postulated to play a thrombomodulatory role. aPL antibodies have been demonstrated to reduce the quantity of AnxA5 on these cells and accelerate plasma coagulation. Resistance to AnxA5 anticoagulant activity has previously been demonstrated in APS patients with thrombotic histories. We have investigated whether plasma samples from patients with the obstetric APS also interfere with AnxA5 anticoagulant activity and also whether this correlated with antibody recognition of domain I on β2GPI.

Methods Plasmas were obtained from 30 healthy non-pregnant donors (median age 34, range 18–55) and 70 healthy non-pregnant women with previous obstetric APS (median age 39, range 24–58), and assayed for AnxA5 resistance and anti-β2GPI domain I antibodies, using previously published assays.

Results Women with obstetric APS had significant reduction of AnxA5 anticoagulant ratios compared to controls (median 216%, range 130–282% vs 247%, range 217–283%, p<0.0001) and also elevated levels of antidomain I IgG (median optical density: 0.056, range 0.021–0.498 vs 0.042, range 0.020–0.323, p=0.002). Patients in the lowest tertile of AnxA5 anticoagulant ratios had an odds ratio for obstetric APS of 121.1 (95% confidence interval of 6.5–2240.2) and patients in the highest tertile of antidomain I IgG had an odds ratio for obstetric APS of 3.4 (95% confidence interval of 1.2–9.7). There was a significant correlation between reduction of AnxA5 anticoagulant activity and elevation of antidomain I IgG (r=−0.49, p<0.0001).

Conclusions. Women with obstetric APS are more likely to have AnxA5 resistance and also anti-domain I IgG antibodies than the control groups. These results are consistent with the hypothesis that aPL antibody-mediated disruption of AnxA5, via recognition of domain I of β2GPI is a mechanism for pregnancy morbidity & mortality in obstetric APS.
Role and Regulation of CSF-1-Induced CSF-1 Receptor Interchain Disulfide Bonding in Receptor Activation in Macrophages
Authors: Stanley E. Richard , Xiong Ying , Song Da , Yu Wenfeng , Yeung Yee-Guide
Published: October 2019

Colony stimulating factor-1 (CSF-1) is the major regulator of tissue macrophage development and function. The effects of CSF-1 are mediated by the CSF-1 receptor (CSF-1R), a class III receptor tyrosine kinase belonging to the PDGF receptor family. To study CSF-1R structure/function in macrophages using both genetic and proteomic approaches, we developed a novel, CSF-1R-deficient, mouse bone marrow macrophage (BMM) line (MacCsf1r−/−) (Yu et al., J. Leuk. Biol.84: in press, 2008). MacCsf1r−/− macrophages are maintained in GM-CSF. Retroviral expression of the wild type CSF-1R fully rescued the CSF-1-induced survival, proliferation, differentiation and morphological characteristics, which resemble those of primary BMM. We have studied CSF-1-induced covalent modifications of the receptor in MacCsf1r−/− cells. Activation of the CSF-1R involves ligand-induced receptor dimerization and trans-phosphorylation of the cytoplasmic domains. Besides tyrosine phosphorylation, CSF-1 stimulation results in disulfide bonding of the CSF-1R dimers and cysteine mutagenesis experiments revealed that specific residues in the kinase domain are involved. Inhibition of the disulfide bond formation significantly compromised CSF-1-induced CSF-1R tyrosine phosphorylation, indicating its requirement for full receptor activation. Analysis of the regulation of CSF-1-induced CSF-1R interchain disulfide bonding revealed the participation of third party molecules with additional CSF-1R post-translational modifications. The importance of ligand-induced disulfide bonding for class III receptor tyrosine kinase activation may not be restricted to the CSF-1R as interchain disulfide bonding of PDGF receptor dimers is also ligand-induced.
Severe Malaria and Leptospirosis Are Associated with a Deficiency of the Von Willebrand Factor Cleaving Protease, ADAMTS13
Authors: Lowenberg Ester C , Cohen Sophie , van den Born Bert-Jan H , Meijers J.C.M. , Levi Marcel , Dondorp Arjen M
Published: October 2019

The thrombotic microangiopathy observed in severe malaria and leptospirosis shows resemblance to that of thrombotic thrombocytopenic purpura (TTP) and haemolytic uremic syndrome (HUS), respectively. The pathophysiology of these complications remains incompletely understood. TTP and (in some cases) HUS are associated with a deficiency of ADAMTS13, a metalloprotease that cleaves large prothrombotic VWF multimers. We hypothesized that ADAMTS13 may be deficient in cerebral malaria and leptospirosis, and that the severity of ADAMTS13 deficiency may be associated with the degree of endothelial activation. We investigated a series of patients with cerebral malaria (n=9) and leptospirosis (n=18). Patients with scrub typhus (n=12) representing a disease that is accompanied by endothelial activation, but without the clinical features of TTP-HUS, and healthy blood donors served as controls. Baseline characteristics did not significantly differ between study groups, except for BUN values which were higher in cerebral malaria than in leptospirosis (p-value 0.046). The Glasgow Coma Scale (GCS) score was evidently lower in cerebral malaria, with a median score of 4 (interquartile range 3–6) while subjects with leptospirosis, scrub typhus and normal controls had (near) maximum scores of 15. ADAMTS13 levels were significantly reduced in both cerebral malaria and leptospirosis, compared to scrub typhus and normal controls, with a significantly more severe deficiency in cerebral malaria than in leptospirosis (data in figure). VWF antigen and VWF propeptide levels were elevated in all three disease groups in comparison to healthy controls (p-values < 0.001), and most markedly increased in cerebral malaria. ADAMTS13 activity inversely correlated with both VWF antigen and VWF propeptide levels (p-values <0.001 and 0.005). No significant correlation was apparent between ADAMTS13 activity and hemoglobin (Hb) concentration or platelet count. Free Hb levels were not elevated in any of the patient groups, hence it seems highly unlikely that free Hb is involved in the observed reduction of ADAMTS13 activity in cerebral malaria and leptospirosis. The results of this study indicate that ADAMTS13 levels are decreased in patients with cerebral malaria and leptospirosis providing direct support to the hypothesis of a role of ADAMTS13 in the pathogenesis of the thrombotic microangiopathy associated with these diseases. Further research seems warranted to establish the role of ADAMTS13 deficiency in the development and the severity of these diseases, and to evaluate its prognostic significance. Potentially, ADAMTS13 function might serve as a risk marker of disease severity and it may be speculated that the administration of exogenous (recombinant) ADAMTS13 can be used as an adjunctive therapeutic strategy in the clinical management of patients with cerebral malaria and severe leptospirosis.

Figure Figure
Staphylococcal Superantigen-Like 5 Activates Platelets, and Supports Platelet Adhesion Under Flow Conditions, Which Is Mediated by GPIb- Alpha and Alpha-IIb-Beta-3
Authors: Weeterings Cees , de Haas Carla JC , Vughs Mignon M , de Groot Philip G , van Strijp Jos , Lisman Ton
Published: October 2019

Introduction - Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus Aureus and has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). Since PSGL-1 shows close homology to platelet glycoprotein Ib-alpha (GPIb), and as platelets have been shown to play an important role in neutrophil chemotaxis, we investigated whether SSL5 also interacts with platelets.

Results - When purified recombinant SSL5 was added to washed platelets in an aggregometry set-up, full and irreversible aggregation was observed even in the absence of exogenous fibrinogen. Proteolysis of the extracellular part of GPIb-alpha or the addition of dRGDW abrogated platelet aggregation. SSL5 was shown to interact with glycocalicin, a soluble GPIb-alpha fragment, and binding of SSL5 to platelets resulted in GPIb-mediated signal transduction as evidenced by translocation of 14-3-3zeta to the actin cytoskeleton. When a mixture of isolated platelets and red cells was perfused over immobilized SSL5 at a shear rate of 300 s−1, large stable platelet aggregates were observed. Platelet adhesion was substantially reduced by proteolysis of GPIb-alpha or addition of dRGDW. In addition, real-time video analysis showed that initial tethering of the platelets was abrogated by proteolysis of GPIb-alpha. Furthermore, when whole citrated blood was perfused over immobilized SSL5, real-time video analysis revealed that platelets activated with the PAR-1 activating peptide SFLLRN made transient contacts with the immobilized SSL5, although no permanent contacts were observed. Finally, SSL5 was shown to interact with endothelial cell matrix (ECM) from cultured human umbilical vein endothelial cells. The interaction of SSL5 with ECM enhanced aggregation of platelets from whole blood to this ECM.

Discussion - Here, we show that SSL5 is capable of activation, adhesion and aggregation of platelets, in which both GPIb-alpha and alpha-IIb-beta-3 play a role. We hypothesize that platelet activation by SSL5 could be important in colonization of the vascular bed and evasion of the immune system.
GP-VI Disruption Confers Additional Protection against Arterial Thrombosis in PAR-4 Deficient Mice
Authors: Cornelissen Ivo , De Candia Erica , Coughlin Shaun R
Published: October 2019

Following vascular injury, platelets are recruited and activated by adhesive proteins exposed on the subendothelium (including collagen), released agonists and locally generated thrombin. In mice, protease activated receptor 4 (PAR-4) mediates the platelet signaling response to thrombin. Previous studies showed that platelets from PAR-4 null mice do not respond to thrombin and while platelet thrombi do form at the site of injury in PAR-4 deficient animals, they do not propagate and extend into the lumen like thrombi in wild-type mice. The activation pathway responsible for this juxtamural platelet accumulation remains unknown. The collagen exposed on the subendothelium activates and recruites platelets at the site of injury by interacting with membrane glycoprotein (GP) VI. Therefore, the collagen-GP-VI pathway is a putative candidate for the platelet thrombus formation near the vessel wall. Using hirudin as a thrombin inhibitor in mice with disrupted GP-VI expression, it has been previously shown that GP-VI signaling and thrombin activation pathways may cooperate during thrombus growth. To more precisely evaluate the interplay between the thrombin/PAR-4 and collagen/GP-VI signaling pathways, we crossed mice lacking PAR-4 and GP-VI, and subsequently compared bleeding time and arterial thrombus formation in PAR-4:GP-VI double or single deficient animals. All genetic combinations were born at the expected mendelian distribution. Double deficient females carried offspring to term and no bleeding was observed during parturition. Pups lacking both receptors demonstrated transient perinatal abdominal bleeding which resolved rapidly. However, we observed prolonged tail bleeding times in double deficient animals compared to their single deficient littermates. Furthermore in a FeCl3-induced carotid thrombosis model, mice lacking both receptors were completely protected compared to their littermates. This study suggests that there is an interaction between the thrombin and collagen activating pathways in the setting of hemostasis and thrombosis.

Figure Figure Figure Figure
M1 Macrophages Eliminate Lymphoma Cells Through the Vitamin D Dependent Antimicrobial Peptide Cathelicidin
Authors: Bruns Heiko , Fabri Mario , Maurberger Anna , Pasemann Shirin , Fahrenwaldt Cornelia , Wilke Andrea , Moi Stefanie , Daudt Heidi , Amann Kerstin , Büttner Maike , Gaipl Udo , Mackensen Andreas , Gerbitz Armin
Published: October 2019

Abstract 1098

Macrophages make up the bulk of the leukocyte infiltrate in most solid tumors. These so-called tumor associated macrophages (TAMs) are thought to be recruited by tumor-derived chemokines and converted to a tumor-permissive state. TAMs are called M2, or alternatively-activated macrophages, to distinguish them from M1, or classically-activated macrophages. Of interest, these TAMs are not able to fight the tumor; instead, they play a tumor-supporting role via their ability to promote angiogenesis and metastases. In Burkitt's lymphoma infiltration of the tumor masses by macrophages is a characteristic morphological hallmark, while the phenotype and the relevance of TAMs as part of the stroma are poorly understood. Using a murine model of spontaneous Burkitt's lymphoma (λ-hu-c-myc transgenic mice), we therefore characterized macrophages infiltrating lymphoma masses and show here that infiltrating macrophages display an anti-inflammatory M2-phenotype characterized by the expression of surface markers, such as CD206. In addition, they secrete TGFβ and IL-10, which are known not only to support B-cell growth, but suppress T-cell immunity at the tumor site. In vitro analysis of human macrophages generated from PBMC either by GM-CSF to generate the M1 phenotype, or by M-CSF to obtain the M2 phenotype, revealed that both types display a distinct cytotoxic potential. When incubated together with different Burkitt's lymphoma cells, M2 macrophages did not efficiently lyse lymphoma cells. In contrast, M1 macrophages revealed a strong cytotoxic activity towards lymphoma cells, in a TNF, TRAIL, FasL and NO independent manner, as shown by antibody blocking studies. Investigating this mechanism further, we found that M1 macrophages, in contrast to M2 macrophages, express high levels of the Vitamin D inducible Cathelicidin antimicrobial peptide. Strikingly, we demonstrate that cathelicidin has a cytotoxic effect on lymphoma cells, but not on non-malignant B-cells.

Of note, excess supplementation of Vitamin-D increased the cytotoxicity of M2 macrophages against lymphoma cells. We conclude that Vitamin D may therefore point into a therapeutic option to influence a tumor stromal component that greatly impacts on survival and immunogenicity of malignant B-cells.

Disclosures:

Mackensen: Micromet Inc.: Research Funding.
Paralog 4 HOX Genes HOXA4 and HOXB4 Expand Pro-B Cells in Vitro
Authors: Fournier Marilaine , Lebert-Ghali Charles-Etienne , Hassawi Mona , Bijl Janetta Jacoba
Published: October 2019

Abstract 1299

HOX genes are known for their involvement in self-renewal of normal and malignant hematopoietic stem cells (HSC). Enforced expression of HOXB4 leads to HSC expansion in vitro and in vivo without leukemia development. We have recently shown that overexpression of its paralog HOXA4 also resulted in an increase of HSCs and myeloid progenitors in vitro. HOXA4 HSC are fully functional and reconstitute mouse chimeras with normal ratios of mature cells in the periphery. Interestingly, although the mature B-cell compartment is not enhanced, the overexpression of HOXA4 resulted in a 10-fold expansion of B-cell progenitors in the bone marrow (BM). Moreover, the number of more primitive WW-IC was not affected by the overexpression of HOXA4, indicating that this gene specifically expands IL-7 responsive B-cell progenitors. Based on these observations in vivo, we sought to determine if paralog 4 HOX genes can expand B-cell progenitors in vitro. To test this B220+ cells were sorted from BM of healthy wild type mice and transduced with MSCV retroviral vectors for HOXA4-GFP, HOXB4-GFP or control-GFP. Cells were cultured in B-cell specific medium and their growth in response to IL-7 was followed for three weeks. We observed a 15- and 10-fold increase of growth for HOXA4 and HOXB4 pro-B cells compared to control, respectively, within 16 days. FACS analysis confirmed the pro-B cell phenotype of all three cultures. These cells are currently being tested for their repopulation capacity of the B-cell compartment in irradiated hosts. To further investigate potential implication of these genes in oncogenic transformation HOXA4 and HOXB4 were overexpressed in E2APBX1 B-cells derived from E2APBX1 transgenic mice. We showed that the overexpression of HOXA4 or HOXB4 induced a strong expansion of E2APBX1 pro-B cell in vitro (2381- and 1090-fold difference over the control after 23 days, respectively), leading to an immortalization of the culture. Despite this huge expansion these cells were incapable to initiate leukemia upon transfer into irradiated recipients. B-CFC assays showed that the growth of HOXA4 or HOXB4 E2APBX1 pro-B cell cultures was supported by a strong expansion of B-cell progenitors (6138- and 15307-fold difference over the control after 20 days, respectively). Taken together, these results indicate that IL-7 responsive pro-B-cell progenitors are sensitive to paralog 4 HOX genes, an effect which is dramatically enhanced in the context of E2APBX1. Lack of clear oncogenic potential puts HOXA4 and –B4 forward as promising candidates to expand B-cell progenitors in vitro. These cells could potentially be used for B-cell complementation therapy in BM transplantation recipients.

Disclosures:

No relevant conflicts of interest to declare.
Mechanism of the Elevated UPR in CN Patients but Not in CyN Patients Carrying Same ELANE Mutations
Authors: Nustede Rainer , Kuznetsova Inna , Welte Karl , Skokowa Julia
Published: October 2019

Abstract 14

Several studies found that in patients with severe congenital neutropenia (CN) harboring mutations in the ELANE gene mutated NE protein induced unfolded protein response (UPR) leading to elevated apoptosis and diminished differentiation of myeloid cells. However, it is unclear, why UPR was not detected in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations, which have been found in CN patients. Several UPR components have been identified in mammalian cells, which include three transducers (IRE1, PERK, and activating transcription factor 6 (ATF-6) as well as one master regulator (BiP/GRP78). BiP is known to be regulated by ATF6. The activation of ATF6 and its target genes (GADD34, CHOP and BiP) in CN patients has not been studied yet. We were able to detect significantly elevated levels of ATF6 and BiP in myeloid cells of CN patients with ELANE mutations, in comparison to CyN patients and to healthy individuals. Therefore, we investigated the mechanism of UPR and activation of ATF6 and ATF6 target genes in CN patients in comparison to CyN patients. We transduced the myeloid cell lines HL60 and NB4 with lentiviral constructs contained either wild type (WT) ELANE cDNA, or mutated (MUT) ELANE cDNA and measured mRNA and protein expression of ATF6 as well as mRNA expression of ATF6 target genes. We compared the effects of three ELANE mutations: C42R, V145-C152del (both mutations presented in CN patients, but not in CyN patients) and S97L (typical for CN and CyN patients) with WT ELANE. We found that in both cell lines only C42R ELANE MUT, but not V145-C152del ELANE MUT or S97L ELANE MUT induced expression of ATF6, GADD34, CHOP and BiP, as compared to control transduced cells. Furthermore, we hypothesize that degradation of mutated NE protein by Secretory Leukocyte Protease Inhibitor (SLPI) might be involved in UPR induction. However, we detected only very low levels of SLPI mRNA in CD33+ myeloid cells and in PMNs of patients with severe congenital neutropenia (CN), as compared to patients with cyclic neutropenia (CyN) and to healthy individuals. The lack of the NE inhibitor, SLPI in CN patients may further contribute to elevated UPR triggered by ELANE MUT and normal levels of SLPI in CyN patients might protect from ELANE MUT-induced UPR. Indeed, inhibition of SLPI using SLPI-specific shRNA led to a significantly elevated expression levels of ATF6, GADD34 and BIP, as compared to ctrl shRNA transduced cells. More importantly, co-transduction of NB4 cells with SLPI shRNA in combination with ELANE S97L MUT (which is common for both CN and CyN patients), but not with WT ELANE led to elevated levels of ATF6, GADD34 and BIP. In summary, different ELANE mutations have different effects on UPR as judged by ATF6 activation and the level of ELANE-triggered UPR is regulated by SLPI.

Disclosures:

No relevant conflicts of interest to declare.
High Throughput Screening Reveals Unique Sensitivity of CD34+CD38- AML Cells to Isotretinoin and Tretinoin
Authors: Xie Wen , Hassane Duane C , Jordan Craig T. , Roboz Gail J. , Guzman Monica L.
Published: October 2019

Abstract 1430

Acute myeloid leukemia (AML) is a heterogeneous disease, originating from leukemic stem cells (LSCs). This relatively rare sub-population shows mostly a CD34+CD38- immunophenotype and has been proposed to be responsible for disease relapse and chemotherapy resistance. In order to identify effective drug(s) for clinical use that can eliminate LSCs, we have screened a library of 2000 compounds from the SPECTRUM collection (MicroSource Discovery Systems, Inc.) using a multi-parameter flow cytometry high-throughput screening. The drugs compiled in the library are 40% US FDA approved drugs, 30% natural products, 10% approved drugs for use in Europe and Japan, and the remainder consistent of bioactive compounds.

The initial screen aimed to identify potential compounds that can decrease viability or increase differentiation of AML cells at 24 and 72 hours of treatment. Compounds demonstrating autofluorescence were eliminated as false positive hits.

We found 137 compounds that decreased viability in all populations. Furthermore, 37 drugs demonstrated ability to increase cell death (more than 50%) in phenotypically described LSCs, progenitor and blasts populations, but with viability over 50% for normal residual lymphocytes within the same AML specimen. Interestingly, only 4 drugs displayed higher specificity to LSCs when compared to total blast populations. Surprisingly, two of these drugs are different forms of retinoic acid (isotretinoin and tretinoin).

The selectivity of isotretinoin and tretinoin was confirmed in different primary AML samples. We observed that phenotypically described stem cell subpopulations were affected when exposed to retinoic acid in 4 out of 5 AML samples. LSC populations display increased apoptosis and differentiation assessed by flow cytometry. Characterization of what confers sensitivity to these types of drugs is under investigation. Together, a multiparameter flow cytometry screen revealed specific anti-LSC activity for retinoid acid, which was not detectable when analyzing total populations and would have been missed using traditional screening. These studies underscore the utility of LSC-based assays for defining novel therapies and suggest that retinoid-based therapies merit further investigation in AML.

Disclosures:

No relevant conflicts of interest to declare.
High Incidence of Fanconi Anemia in Patients with a Morphological Picture Consistent with Refractory Cytopenia of Childhood
Authors: Yoshimi Ayami , Niemeyer Charlotte M. , Baumann Irith , Schwarz-Furlan Stephan , Schindler Detlev , Ebell Wolfram , Strahm Brigitte
Published: October 2019

Abstract 2780

Introduction:

Refractory cytopenia in childhood (RCC) is the most common subtype of myelodysplastic syndrome (MDS) in children. Differential diagnosis from inherited bone marrow failure (IBMF) such as Fanconi anemia (FA) remains an intriguing challenge, because most patients with RCC have a hypocellular bone marrow (BM) and dysplastic features in haematopoiesis are observed in both RCC and IBMF. Moreover the spectrum of phenotypic findings in FA is extremely wide. Some FA patients have a mild phenotype without malformation. The purpose of this study is to estimate the incidence of FA in an RCC cohort without a full clinical feature of FA, but subsequently diagnosed by chromosome breaking test.

Patients and Methods: Between 01/2007 and 12/2010 reference pathologists of the European Working Group of MDS in Childhood (EWOG-MDS) provided a morphological report consistent with RCC in 137 children studied in Germany. Seventeen patients with hypercellular BM or abnormal karyotype, 2 patients, in whom dyskeratosis congenital was diagnosed after initial inclusion and one patient, in whom chromosome breaking test was not performed, were excluded.

Results:

Seven of remaining 117 patients had facial and/or skeletal anomalies typically noted in FA and one patient had a brother with FA. In these 8 patients, FA had been suspected by their local physicians (group FA-1). Nine patients (8.3%) without these typical anomalies were subsequently diagnosed of FA by chromosome breakage test (group FA-2). The diagnosis of RCC was finally made in the remaining 100 patients with negative chromosomal breakage test (group RCC). The clinical features of patients in each group are summarized in the Table. The mean corpuscular volume of red cells (MCV) was elevated (> +2SD) for ages in all patients with FA, but only 42 % in patient with RCC. In some children of group FA-2 additional non-haematological abnormalities were also observed. However, they were not evident and or typical to prompt the treating physicians to suspect FA. A few patients in the group RCC also had some physical anomalies, not specific for any of the known IBMF disorders. Possibly that other known or not yet described IBMF disorders remain uncovered in children with “de novo” RCC.

Conclusion:

Our results illustrate that the same haematological features and congenital anomalies can be noted in FA and RCC. More importantly, they indicate that the exclusion of FA by a chromosomal breakage test or other methods is mandatory in all patients prior to diagnosis RCC. Chromosomal breakage analysis may identify patients with FA in 8% of patients with a morphological description of RCC without a full clinical picture of FA.

Disclosures:

No relevant conflicts of interest to declare.
Identification and Characterization of Human Leukemia Stem Cell Functional Regulators
Authors: Eppert Kolja , Hermans Karin G , Dick John E.
Published: October 2019

Abstract 2955

Emerging evidence shows that many cancers are organized as cellular hierarchies sustained by a subpopulation of cancer stem cells (CSC). It is thought that CSC properties influence therapy response, disease relapse, and overall survival. However, little is currently known about the molecular pathways that control stem cell behaviour. Our goal is to identify and characterize the molecular regulatory networks that govern the self-renewal and developmental program of human leukemia stem cells (LSC).

To expand our understanding of LSC function, we selected genes from our previously established LSC transcriptional program as novel candidate regulators of stem cell function. The LSC transcriptional program was identified by fractionation of 16 primary human acute myeloid leukemia (AML) samples into four populations that were subjected to sensitive in vivo LSC assays. mRNA expression analysis was performed on each fraction and a global LSC-specific signature was determined from functionally defined LSC fractions. Using similar methodology, a hematopoietic stem cell (HSC) enriched gene signature was established from human cord blood. Bioinformatic analysis identified a core transcriptional program that LSCs and HSCs share, revealing the molecular machinery that underlies stemness properties. The LSC and HSC signatures have prognostic significance independent of other factors when validated on a large cohort of cytogenetically normal AML patients. We have begun experiments to determine the role of 17 candidate stem cell regulators using functional in vivo and in vitro assays.

We first examined the effect of candidate gene overexpression in normal HSC using xenograft assays. Lineage negative cord blood cells were transduced with lentiviral overexpression vectors, injected into immune-deficient mice and engraftment of human cells was measured after 12 weeks. In the preliminary round of in vivo analysis, over-expression of GPR56 increased the engraftment capability of HSCs in immune-deficient recipients compared to control (p=0.0019). GPR56 is involved in cell adhesion and differentiation and evidence is accumulating for a role in various cancers such as melanoma, esophageal cancer, and glioma. By qPCR analysis we observed that GPR56 mRNA is more highly expressed in both LSC-enriched and primitive normal hematopoietic cell populations, including highly purified HSC, compared to more mature cell populations such as AML blasts and normal lineage committed progenitor cells.

In conclusion, our LSC and HSC data established that determinants of stemness influence clinical outcome of AML patients. We have identified multiple novel candidate stem cell-related genes and provided evidence for a role for GPR56 in hematopoietic stem cell regulation.

Disclosures:

No relevant conflicts of interest to declare.
Deficiency of CD73/Ecto-5'-Nucleotidase Enhances Acute Graft-Versus-Host Disease
Authors: Chernogorova Petya , Tsukamoto Hiroki , Ayata Korcan , Gerlach Ulrike V. , Rughani Ankur , Ganesan Jayanthi , Follo Marie , Thompson Linda F. , Idzko Marco , Zeiser Robert
Published: October 2019

Abstract 312

Acute graft-versus-host disease (GvHD) limits the success of allogeneic hematopoietic cell transplantation (allo-HCT). We have recently shown that extracellular adenosine triphosphate (ATP) enhances GvHD by activation of the purinergic receptor P2X7R. The abundance of extracellular ATP is regulated by ecto-nucleotidases such as CD39, which dephosphorylate ATP to ADP and AMP. Ecto-5'-nucleotidase (CD73) degrades AMP to adenosine, which can itself exert tolerogenic functions via activation of A2A and/or A2B adenosine receptors (AR). Extracellular ATP and adenosine have immunoregulatory roles under different inflammatory conditions. Deficiency of CD73, the enzyme that metabolizes AMP to adenosine, could therefore have pro- or antiinflammatory activity by reducing adenosine levels in the microenvironment. Currently, the roles of CD73 and adenosine in GvHD are unclear.

By using cd73−/− donor or recipient mice and a specific CD73/ecto-5'-nucleotidase inhibitor we observed enhanced GvHD severity when CD73 was genetically deleted or pharmacologically blocked. CD73 was upregulated on CD4+ and CD11c+ cells after irradiation, suggesting an inducible rescue mechanism along with CD39 to counteract ATP accumulation resulting from tissue damage due to the preconditioning treatment. CD73 deficiency led to enhanced T cell expansion and IFN-γ and IL-6 production in GvHD mice. Migratory capacity of cd73−/− T cells was increased, compatible with the ability of adenosine to inhibit transendothelial migration. A2A receptor deficiency led to increased numbers of proliferating allogeneic T cells in allo-HCT recipient mice, suggesting that signaling through the A2A receptor via CD73-generated adenosine is a significant part of the mechanism by which CD73 limits the severity of GvHD. Furthermore, pharmacological blockade of CD73 improved graft-versus-tumor activity in an in vivo B cell leukemia model.

In conclusion, we demonstrate that CD73 plays a protective role in GvHD with enhanced disease severity in its absence, while blocking CD73 led to increased graft-versus-tumor effect. These data have potential clinical implications as the alloimmune response could be enhanced by CD73 blocking in patients with residual tumor burden after transplantation. Conversely, immune modulation by addition of CD73 enzyme or an adenosine receptor agonist could improve the outcome of GvHD patients.

Disclosures:

No relevant conflicts of interest to declare.
Persistent Endothelial Activation Is Associated with An Increased Mortality During Therapy of Children with ALL – a Pilot Study
Authors: Niedzielska Ewa , Doroszko Adrian , Porwolik Julita , Krupska Ewa Szahidewicz- , Chybicka Alicja , Szuba Andrzej
Published: October 2019

Abstract 4232

Background:

Activation of endothelial cells is associated with a worse prognosis in numerous disorders treated at intensive care units. The goal of this study was to analyze if an activation of vascular endothelium despite of the treatment with the ALLIC protocol in children with ALL might have a predictive value for the survival rate.

Methods:

N=18 children at age of 4–18 years with ALL treated with the ALLIC protocol were investigated. Plasma levels of markers of endothelial activation (VCAM-1, ICAM-1, VEGF, E-selectin, P-selectin and PAI-1) were analyzed at baseline, during the 33rd and 78th day of therapy (beginning of the protocol M).

Results:

Higher baseline levels of ICAM-1 (467.1±72.8 vs. 206.3±21.7 ng/ml, p<0.05) and E-selectin (56.1±10.9 vs. 28.1±10.0 ng/ml, p<0.05) were observed in children treated unsuccessfully. Moreover, an increase in E-selectin levels and a non-decreasing trend in ICAM-1 levels within the first three days of the ALLIC protocol as well as significant increase in the PAI-1 levels were positive predictors for death in children with ALL.

Conclusions:

In this pilot study, we have demonstrated for the first time that persistent pro-inflammatory and pro-aggreagatory activation of endothelium might be a positive predictor for death in children with ALL.

Disclosures:

No relevant conflicts of interest to declare.
Combination of Mitoxantrone,Ara-C and Homoharringtonine In Treatment of Refractory and Relapsed Acute Myeloid Leukemia
Authors: Hu Jianda , Liu Tingbo , Cai Chunxia , Chen Xinji , Chen Buyuan
Published: October 2019

Abstract 4294

Advances in effective chemotherapy have improved clinical outcomes in acute leukemia in recent years. 5-year survival rate approaches 50–60% for acute myeloid leukemia (AML). However, the prognosis remains poor for patients who are relapsed or refractory to first-line therapy. Drug resistance and early disease recurrence are major contributing factors in the limited survival of patients with AML. The strategy for treating these patients is through reinduction chemotherapy followed by allogeneic stem cell transplantation. New combinations of different agents were employed in refractory patients to overcome drug resistance. The current study is to evaluate the efficacy of a MAH regimen comprising Mitoxantrone,Ara-C and Homoharringtonine in refractory or relapsed AML. 37 patients aged 14–65 years with refractory or relapsed AML (15 refractory AML patients, 22 relapsed AML patients) were treated with the MAH regimen(Mitoxantrone 10mg qd, iv.gtt, for 2□‘3 days;Ara-C 100mg bid, iv.gtt, for 5□‘7 days; Homoharringtonine 4mg qd iv.gtt, for 5□‘7 days). Chemotherapy duration lasted for 5 or 7days depended on bone marrow cellurarity. 15 (40.5%)and 1 (2.7%) patients achieved complete remission (CR) and partial remission (PR) respectively. The overall response rate was 43.2%. There was no relation between remission duration and previous chemotherapy. All patients who achieved CR received a consolidation and intensification therapy. The median overall survival (OS) for all patients was 97 days (range 18–487 d). For the patients who were in CR or PR,the median relapse-free survival(RFS) was 147 days(range 4 to 341 d). All patients experienced profound myelosuppression. The most common observed side effect of the regimen was infection because of grade ‡W neutropenia, which could be observed in 33 patients(89.1%). 4 patients died in aplasia due to severe infection and brain hemorrhage. In patients achieving remission, the median time to reach absolute neutrophil count (ANC) more than 0.5×109/l was (16.0±6.4)d. Platelet levels of more than 20×109/l were achieved in a median time of (12.7±6.2)d. Nonhematological side effects, consisting mainly of gastrointestinal toxicity(21/37,56.8%) and transient liver ALT and AST increase (4/37), were generally mild to moderate and tolerated. To a conclusion, MAH regimen can be employed in treatment of the refractory or relapsed AML patients who were not responded to other regimen. It is effective and is good tolerant.It could provide some refractory patients the chance to receive hematopoietic stem cell transplantation.

Disclosures:

No relevant conflicts of interest to declare.