bluuebunny/crossref_metadata_2025 huggingface dataset:
Lymphoid Precursors Arise Independently at Multiple Sites During Development.
Authors: Porayette Prashanth , Yoshimoto Momoko , Glosson Nicole , Ferkowicz Michael , Shelley William C. , Kaplan Mark , Yoder Mervin C.
Published: October 2019

Abstract 699

Primitive hematopoietic cells arise on embryonic day 7.0 (E7.0) within the murine extra-embryonic yolk sac (YS) followed by formation of definitive hematopoietic cells on E8.25. The first cells capable of long term multi-lineage repopulation in an adult irradiated mouse arise in the embryo proper on E10.5. The temporal and spatial emergence of lymphoid precursors capable of forming mature T and B lymphocytes remains controversial. Cardiac contractions begin on E8.25, leading to the circulation of cells between various sites of hematopoiesis thus making it difficult to identify definitively the site where lymphoid precursors emerge. We have previously utilized a transgenic knock out murine mutant in which no heartbeat develops to examine hematopoietic cell emergence. NCX1 is a protein required for the initiation of cardiac contractions. The ncx1 null embryos develop normally until about E10.5, and then succumb to the lack of a systemic circulation. Single cell suspensions from within the para-aortic splanchnopleure (P-Sp) of the embryo proper and from YS at E8.5 and E9.5 were isolated and plated on the OP9-Dl1 stromal cell line that is known to support T lymphopoiesis. We observed that both YS and P-Sp independently displayed the potential to produce mature T cells including CD4 and CD8 DN, DP, and SP cells. We observed that both sites can give rise to both αaβ and γδ T cells independently at a frequency approaching 1:165 hemogenic endothelial cells plated. Cells derived from both YS and P-Sp demonstrated lymphoid specific VDJ rearrangement. Adoptive transfer of the lymphoid cells derived from these sites into NOD/SCID/IL2Rg null recipient mice revealed engraftment within multiple lymphohematopoietic organs including thymus, spleen and liver in the recipient mouse and the presence of circulating donor T cells. Stimulation of splenocytes from transplanted animals with anti-CD3 antibodies induced proliferation of these cells suggesting intact antibody responses in YS and P-Sp derived T cells. Development of mature functional T lymphocytes from precursors isolated from YS and P-Sp before the presence of a functional thymus brings forward the hypothesis for an extra-thymic multi-site origin of T cells in the early embryo. The presence of prospective lymphoid precursors more than a day earlier than the adult reconstituting hematopoietic stem cell (HSC) first observed at E10.5 during development suggests that either the HSC may be present at earlier points in development than is believed or that there might be a HSC independent emergence of lymphoid precursors in the developing embryo.

Disclosures:

No relevant conflicts of interest to declare.
Two Cycles of ABVD Followed by Involved Field Radiotherapy with 20 Gray (Gy) Is the New Standard of Care in the Treatment of Patients with Early-Stage Hodgkin Lymphoma: Final Analysis of the Randomized German Hodgkin Study Group (GHSG) HD10. Study Supported by the Deutsche Krebshilfe and in Part by the Competence Network Malignant Lymphoma.
Authors: Engert Andreas , Diehl Volker , Pluetschow Annette , Eich Hans T. , Herrmann Richard , Doerken Bernd , Kanz Lothar , Greil Richard , Markova Jana , Fuchs Michael , Borchmann Peter , Mueller-Hermelink Hans Konrad , Mueller Rolf Peter
Published: October 2019

Abstract 716

Background:

There has been an ongoing debate on the best treatment for patients with early favourable Hodgkin lymphoma (HL). Open questions include the choice between combined modality treatment or chemotherapy only, the number of chemotherapy cycles needed and the optimal radiation dose. The GHSG thus conducted a randomized study for patients with early-stage favourable Hodgkin lymphoma (HD10) in which these questions were addressed.

Methods:

HD10 was an international prospectively randomized multicenter trial comparing 2 and 4 cycles of ABVD as well as 20Gy or 30Gy involved field radiotherapy (IFRT) in a 2 × 2 statistical design. Between 5/1998 and 1/2003, a total of 1370 patients from 329 centers were randomized into four arms: 4 × ABVD + 30Gy; 4 × ABVD + 20Gy; 2 × ABVD + 30Gy; 2 × ABVD + 20Gy. All patients had their initial histology reviewed by a lymphoma expert panel. Documentation was complete in more than 99,1% of cases for this final analysis.

Results:

Patients were equally balanced for age, gender, stage, histology, performance status and risk factors between arms. There were significant differences in major toxicity (WHO grade III/IV) between 4 × ABVD and 2 × ABVD in the overall number of events (52% vs 33%) including leukopenia (24% vs 15%) and hair loss (28% vs 15%). In terms of radiation dose, there also was a difference in toxicity between 30Gy and 20Gy IFRT (all events: 8.7% vs 2.9%), dysphagia (3% vs 2%), mucositis (3.4% vs 0.7%). Complete remission was achieved in 97% of patients treated with 4 × ABVD, 97% with 2 × ABVD, 99% after 30Gy and 97% after 20Gy. With a median follow-up of 79–91 months, there was no significant difference between 4 × ABVD and 2 × ABVD in terms of overall survival at 5 years (OS: 4 × ABVD 97.1%; 2 × ABVD: 96.6%), freedom from treatment failure (FFTF: 93.0% vs 91.1%) and progression free survival (PFS: 93.5% vs 91.2%). For the radiotherapy question, there were also no significant differences between patients receiving 30Gy IFRT and those with 20Gy IFRT in terms of OS (97.6% vs 97.5%), FFTF (93.4% vs 92.9%) and PFS (93.7% vs 93.2%), respectively. Importantly, there was also no significant difference in terms of OS, FFTF and PFS when all four arms were compared.

Conclusion:

Two cycles of ABVD followed by 20Gy IFRT is the new GHSG standard of care for Hodgkin patients in early favourable stages.

Disclosures:

No relevant conflicts of interest to declare.
The MAPK ERK1 Is a Sensor of the Steady-State Splenic Erythropoiesis.
Authors: Guihard Soizic , Clay Denis , Cocault Laurence , Opolon Paule , Souyri Michele , Porteu Francoise , Gaudry Murielle
Published: October 2019

Abstract 73

In different culture models, conflicting results have been obtained with respect to the role of the ERK/MAPK pathway and the ERK kinases on erythropoiesis. There is no in vivo experimental data on the role of these kinases in adult erythropoeisis. The existence of two ERK isoforms (ERK1 and ERK2) suggests that they could play specific role, based on their expression, their activation level and/or the ratio between both of them. The ERK1−/− mice were used to study this hypothesis. Increased number of circulating erythrocytes, increased hemoglobin level and hematocrit were found in these mice. The deletion of ERK1 leads to an uncontrolled splenic erythropoiesis while the bone marrow erythropoiesis remains normal. The ERK1−/− mice display splenomegaly characterized by a marked expansion of the red pulp and an increased number in basophilic (Ery.A) and late basophilic (Ery.B) erythroblasts. This impaired erythropoiesis in ERK1−/− mice is cell autonomous as shown by bone marrow transplantation experiments. This splenic erythropoiesis is not due to an overexpression or overactivation of the ERK2 isoform in erythroblasts. It has been shown that Fas-mediated apoptosis of erythroblasts would limit the basal erythropoietic rate. In ERK1−/− mice, Ery.A expansion is associated with a decrease in cell surface expression of both Fas and FasL as compared with wild-type mice. This fall in Fas/FasL expression is correlated with a decrease in Annexin V binding on splenic Ery.A and Ery.B. In addition, cell cycle analysis revealed an increased S-phase in ERK1−/− Ery.A cells compared with wild-type Ery.A. In conclusion, these data demonstrate for the first time the in vivo involvement of the ERK/MAPK pathway in adult splenic erythropoiesis and underlies the specific role of ERK1 in this function. By regulating the cell surface expression of Fas and FasL on splenic erythroblasts, ERK1 acts as a sensor of the basal erythropoietic rate.

Disclosures:

No relevant conflicts of interest to declare.
Dysregulation of TGF-Beta Stimulated Smad Signaling Is Seen in Myelodysplasia and Points to the Potential Therapeutic Efficacy of TGF-Beta Receptor I Kinase Inhibition in Low Grade Disease.
Authors: Zhou Li , McMahon Christine , Bhagat Tushar , Alencar Christina , Yu Yiting , Fazzari M. , Sohal Davendra P. , Gundabolu Krishna , Ng Chun , Mo Yongkai , Wickrema Amittha , Kong G. , Friedman Ellen , Sokol Lubomir , Burnette Pearlie K , Steidl Ulrich , Yan L. , Yingling Jm , Lahn MM , List Alan F , Bitzer Markus , Verma Amit
Published: October 2019

Abstract 737

Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis that leads to peripheral cytopenias. TGF-beta is a hematopoietic inhibitory cytokine that has been indirectly linked to the pathogenesis of some subsets of MDS and acute leukemias. We have shown (Blood, 112(8):3434; 2008) that smad2, a component of the TGF-beta signaling pathway, is constitutively activated and upregulated in MDS progenitors. Since there is conflicting data about upregulation of TGF-beta levels in MDS, we next sought to determine the molecular basis of TGF-beta receptor-I (TBRI) overactivation and subsequent smad2 phosphorylation / activation in this disease. We observed that smad-7, a negative regulator of TBRI kinase, is markedly down regulated in a meta-analysis of gene expression studies from 89 MDS bone marrow derived CD34+ cells when compared to 61 normal controls (Adjusted P<0.001, Benjamini Hochberg). Smad-7 protein was also found to be significantly decreased in MDS BM progenitors (n=14) when compared to anemic controls (n=18) when examined immunohistochemically in a bone marrow tissue microarray (P=0.04, T test). siRNA mediated knockdown of smad-7 levels led to increased TGF mediated gene transcription over baseline, that could be effectively inhibited by a TBRI (ALK5 kinase) inhibitor. TBRI kinase inhibition effectively abrogated TGF-beta mediated activation of smad-2 in hematopoietic cells. Most importantly, treatment with the TBRI inhibitor increased erythroid and myeloid colony formation from primary MDS bone marrow specimens in a concentration-dependent fashion. Furthermore, the hematopoietic potentiating effect of the TBRI kinase inhibition was validated in a TGF-overexpressing transgenic mouse model. This mouse constitutively expresses TGF-beta through an albumin promoter, develops progressive anemia and dysplasia and thus serves as a novel model of human bone marrow failure. Treatment with the TBRI inhibitor by oral lavage at 100 mg/kg/d for two weeks led to significant increases in hematocrit when compared to placebo (Mean increase in hematocrit=48%, P value=0.01, T test). Taken together, these studies demonstrate an important role for reduction in smad-7 in contributing to ineffective hematopoiesis in MDS by activating TGF-beta signaling in hematopoietic cells. These studies also illustrate the promising therapeutic potential of TBRI inhibitors in this disease.

Disclosures:

No relevant conflicts of interest to declare.
Multiple Myeloma Cell Induction of GFI-1 in Stromal Cells Suppresses Osteoblast Differentiation in Patients with Myeloma.
Authors: D'Souza Sonia , Del Prete Davide , Esteve Flavia , Sammut Benedicte , Yu Shibing , Xiao Guozhi , Galson Deborah , Roodman G. David
Published: October 2019

Abstract 742

Multiple Myeloma (MM) is a plasma cell malignancy characterized by formation of lytic bone lesions in approximately 90% of the patients which do not heal even after prolonged complete remission. The basis for this selective and protracted suppression of osteoblast (OBL) differentiation from immature bone marrow stromal cells (MSC) is unknown. Although factors that inhibit OBL differentiation in MM have been identified, such as DKK-1, sFRP2, IL-3, IL-7, and TNF-a, none of these factors have been shown to be responsible for the protracted suppression of OBL differentiation in MM. Further, inhibition of Runx2 activity, a critical transcription factor required for OBL differentiation has been reported in MM, but the mechanisms responsible are still unclear. To address the basis for the protracted inhibition of OBL differentiation in MM, we have developed a murine model of MM-induced OBL suppression using a genetically modified murine myeloma cell line that expresses GFP and thymidine kinase (5TGM1-GFP-TK MM cells). Injection of these 5TGM1-GFP-TK MM cells into SCID mice resulted in persistent inhibition of OBL differentiation even when the MM cells were totally depleted by ganciclovir treatment. The MSC from these mice had selective inhibition of OBL differentiation, but not adipogenesis, and minimally differentiated to OBL even when treated with BMP2. These MSC expressed elevated levels of the SNAG family Zn-finger containing transcriptional repressor, Gfi-1, which we found can cause both acute and protracted suppression of RUNX2. In support of these results, decreased RUNX2 expression and elevated GFI-1 levels were also protracted in MSC from 7 MM patients with impaired OBL differentiation compared to normals. Further, 5TGM1 inhibition of OBL differentiation in vitro was dependent on TNF-a and IL-7, and neutralizing antibodies to TNF-a and IL-7 blocked MM-induced Runx2 suppression. In addition, TNF-a and IL-7 increased Gfi-1 in a murine OBL precursor cell line (MC4). Deletion analysis of the Runx2 P1 promoter revealed that a 943-bp region containing 27 putative Gfi-1 binding sites (AA(T/G)C core) was responsible for MM repression of Runx2 expression. Importantly, siRNA knockdown of GFI-1 expression restored RUNX2, OCN, BSP and OSX expression in both MM exposed MC4 cells and in MSC from MM patients. These results support an important role for GFI-1 in repressing RUNX2 expression in MSC exposed to MM cells, thereby inhibiting osteoblastogenesis in MM.

Disclosures:

Roodman: Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy; Acceleron: Consultancy.
Follicular Lymphoma Cell Niche: Identification of a Preeminent IL-4-Dependent TFH- B Cell Axis.
Authors: Tarte Karin , Celine Pangault , Ame-Thomas Patricia , Ruminy Philippe , Rossille Delphine , Caron Gersende , Bahia Maryse , De Vos John , Roussel Mikael , Monvoisin Celine , Lamy Thierry , Gaulard Philippe , Tilly Herve , Fest Thierry
Published: October 2019

Abstract 757

Follicular lymphoma (FL) B cells contract tight connections with their microenvironment, which governs the pathogenesis and progression of the disease. Indeed, specific immune response gene signatures, obtained on whole biopsy samples, have been associated with patient survival, independently of classical clinical features. In this study we performed, using Affymetrix U133 Plus 2.0 oligonucleotide microarrays, the gene expression profiling of purified CD19pos B-cell and CD19negCD22neg non-B cell compartments, prospectively obtained from FL and reactive lymph nodes. Unsupervised analyses of B-cell compartment in one hand (n=21) and non-B cell compartment in the other hand (n=14) allowed to differentiate FL from reactive samples. We then identified 677 nonredundant genes defining the FL synapse, i.e. the list of genes involved in the crosstalk between B cells and their microenvironment in FL. Using Ingenuity pathway analysis we pointed out 26 FL-specific functional networks among this FL synapse, including an IL-4-centered pathway. Interestingly, whereas in tonsils and reactive lymph nodes only exceptional scattered phospho-STAT6pos cells were observed, a high number of CD20posphospho-STAT6pos cells were evidenced within FL biopsies. In addition, several IL-4-target genes, including IL4I1 and HOXC4, were overexpressed in malignant B cells. Altogether, these results demonstrated that the upregulation of IL-4 within FL microenvironment was associated to a strong activation of FL B cells. In addition, FL microenvironment was characterized by a strong enrichment in follicular helper T cells (TFH), as demonstrated through both Gene Set Enrichment Analysis (GSEA)-based transcriptomic approach and flow cytometry analysis of the CD4posCXCR5hiICOShi cell compartment. The majority of phospho-STAT6pos B cells were located at the vicinity of cells expressing the PD-1 TFH marker. Moreover, purified FL-derived TFH, unlike non-TFH T cells, expressed IL4. Finally, whereas chronically inflamed tonsils also exhibited an increase percentage of TFH cells, tonsil-derived TFH did not express IL4. Altogether, our study demonstrated that tumor-infiltrating TFH specifically express functional IL-4 in FL, creating therefore an IL-4-dependent TFH-B cell axis. This crosstalk could sustain FL pathogenesis and represent a new potential therapeutic target.

Disclosures:

No relevant conflicts of interest to declare.
Filamin A Deficiency in Platelets Reveals Functional Impairment in ITAM-Based Signaling.
Authors: Falet Hervé , Pollitt Alice Y , Weber Sarah E , Duerschmied Daniel , Wagner Denisa D , Watson Steve P , Hartwig John H
Published: October 2019

Abstract 769

Filamin A (FlnA), encoded by the FLNA gene on the X-chromosome, crosslinks F-actin and controls the topology of the Von Willebrand factor (VWF) receptor and the integrin αIIbβ3 on platelets by connecting them to the underlying cytoskeleton. In mice, FlnA deficiency is embryonic lethal to hemizygous males. Heterozygous (HT) females are born, but experience premature mortality. FlnA confers survival value to platelets since HT female mice have a mild thrombocytopenia and 95% of their circulating platelets are FlnA positive. Mice specifically lacking FlnA expression in platelets were generated by breeding FlnAloxP/loxP females with GATA1-Cre males. FlnAloxP/y GATA1-Cre males have a severe macrothrombocytopenia and increased tail bleeding time. Their platelets lack a normal cytoskeleton and have decreased GPIbα expression, which lacks cytoskeletal linkage and has an altered surface distribution by electron microscopy. Binding of VWF to FlnA-null platelets was decreased in response to botrocetin. Under arterial shear conditions (shear rate 1,500/s), fluorescent FlnA-null platelets covered ∼70% less of the collagen-coated area via collagen-bound VWF than WT platelets. Unexpectedly, immunoreceptor tyrosine-based activation motif (ITAM)-mediated signals were severely compromised in the absence of FlnA. FlnA-null platelets had decreased α-granule secretion and integrin αIIbβ3 activation in response to stimulation through the collagen receptor GPVI and the C-type lectin-like receptor 2 (CLEC-2). Protein tyrosine phosphorylation, including that of the protein tyrosine kinase Syk and phospholipase C-γ2, was severely impaired in FlnA-null platelets stimulated through GPVI and CLEC-2. Interaction between FlnA and Syk was detected using immunoprecipitation and purified proteins. The data reveal a novel interaction between FlnA and Syk regulating ITAM-containing receptor signaling and platelet function.

Disclosures:

No relevant conflicts of interest to declare.
Coagulation Factor XIIa Activates Platelets and Is the Physiological Agonist of Protease-Activated Receptor 3.
Authors: Mao Yingying , Getz Todd M , Jin Jianguo , Kunapuli Satya P.
Published: October 2019

Abstract 770

Protease-activated receptors (PARs) are G-protein coupled receptors that are activated by proteases. Thrombin is the major agonist for PAR1 and PAR4, whereas tryptase and coagulation factor Xa are the agonists for PAR2. In addition to these major agonists, PARs can be activated by other coagulation proteases. The physiological agonist of PAR3 has not been identified to date; as a result, the molecular pharmacology and physiology of PAR3 remain poorly understood. The purpose of this study is to identify a physiological agonist to PAR3. We used PAR4 null murine platelets, which are known to express only PAR3. In this study, we tested the effect of several coagulation proteases and found that only coagulation factor XIIa (FXIIa) activated PAR4-/- murine platelets, in a concentration-dependent manner. FXIIa caused murine platelet shape change, aggregation, secretion and thromboxane A2 generation and this activation was abolished by C1 esterase inhibitor, a FXIIa inhibitor. FXIIa-induced murine platelet activation was completely abolished by BMS200261, a PAR1 antagonist, without affecting the catalytic activity of FXIIa. As murine platelets do not express PAR1, these data indicate that BMS200261 acts as an antagonist of PAR3 and hence inhibits FXIIa-induced platelet activation. FXIIa also caused mobilization of intracellular calcium from murine platelets and this calcium increase is abolished by BMS200261 in the presence or absence of the PAR4. PAR1 and PAR4 couple to Gq to cause intracellular calcium increases. YM-254890, a Gq inhibitor, abrogates PAR1- or PAR4-mediated calcium mobilization. However, YM-254890 did not affect FXIIa –induced platelet calcium mobilization in murine platelets. FXIIa caused activation of Gq-/- mice platelets similar to wild -type platelets, suggesting that FXIIa -induced calcium mobilization in platelets is independent of Gq pathways. Furthermore, FXIIa-induced platelet activation was completely abolished by BAPTA-AM, which indicates that calcium is required for FXIIa-induced platelet activation. Furthermore, FXIIa caused phosphorylation of Erk and Akt in PAR4 null murine platelets and this phosphorylation was abolished by BMS200261, but not by YM-254890. These observations may explain previous reports that demonstrated lack of stable thrombus formation in FXII null mice. We conclude that FXIIa activates platelets through PAR3 independently of Gq pathways leading to calcium mobilization and activation of Erk and Akt.

Disclosures:

No relevant conflicts of interest to declare.
Rituximab Administration within 6 Months of Allogeneic SCT Carries Risk of Prolonged and Life Threatening Cytopenias.
Authors: McIver Zachariah A. , Stephens Nicole , Naguib Nicolas , Grim Andrew , Barrett A. John
Published: October 2019

Abstract 793

The use of the monoclonal anti-CD20 antibody Rituximab to treat chronic graft-versus-host disease (c-GVHD) occurring after stem cell transplantation (SCT) is generally effective and well tolerated. However this agent can cause neutropenia. Here we describe cytopenias following Rituximab treatment in patients developing c-GVHD after allogeneic SCT. Between 2006-2009, 50 patients received a myeloablative matched-sibling T-cell depleted or selectively-depleted SCT for a variety of hematological malignancies, mainly AML, ALL, CML, and MDS. Median age was 43 years (range, 13 – 68), and median CD34+ cell dose was 6.1 × 106/kg (range, 3.1-10.1). Thirty-one patients developed acute GvHD; 13 grade 1, 16 grade 2, and 1 patients with grade 3. Twenty-seven patients had chronic GvHD (17 limited, 10 extensive). Nineteen patients were treated with Rituxmab; 15 within 180 days, and 4 between 1- 7 years following SCT. Patients receiving Rituximab one year after SCT experienced only moderate neutropenia 3-5 months after treatment lasting 10-20 days while maintaining ANC > 1.0× 109/L. In contrast, neutropenia occurring within 4 weeks of treatment occurred in all 15 patients given Rituximab within the first 180 days after SCT. Twelve of these patients developed severe neutropenia (ANC < 0.5K/μL) lasting 2-8 months in duration, with 10 out of the 12 patients requiring hospitalization to treat neutropenic infections. Six of the 12 patients died of infection complicationg GVHD treatment. Six patients treated with Rituximab within 120 days of SCT experienced the worse complications. All 6 developed neutropenia within 4 weeks of treatment lasting 3- 7 months. Five required recurrent hospitalization for neutropenia associated infections; one died of invasive fungal pneumonia, one died of gram negative bacteremia and relapsed disease, and one required prolonged intubation for parainfluenza type 3 infection associated ARDS. Recovery of both neutrophil and lymphocytes was extremely delayed in this group, with a significantly lower ANC and ALC at 9 months and 12 months post SCT compared to patients with GvHD not treated with Rituximab (p < 0.02). Investigation of the etiology of neutropenia revealed evidence of T cell clonality in 4 patients; 2 patients were diagnosed with LGL with evidence of restricted rearrangement patterns detected by PCR, one patient had 95% NK cell dominated bone marrow infiltrate seen on flow cytometry, and one patient had idiopathic thrombocytopenia purpura and responded to appropriate immunosuppression. Although Rituximab resulted in rapid control of c-GVHD, we conclude that administration of Rituximab early after SCT is associated with prolonged, possibly autoimmune mediated profound cytopenias, and should be avoided.

Disclosures:

No relevant conflicts of interest to declare.
In Vivo Lentiviral Marking Demonstrates Long-Term Myeloid and Lymphoid Lineage Contribution of Individual Hematopoietic Stem Cell Clones to Murine Steady-State Hematopoiesis.
Authors: Zavidij Oksana , Ball Claudia R , Fessler Sylvia , Belle Daniela , Schmidt Manfred , von Kalle Christof , Glimm Hanno
Published: October 2019

Abstract 813

Most of the knowledge to date on the in vivo blood forming activity of individual hematopoietic stem and progenitor cells was gained in transplantation experiments of defined cell populations into syngeneic or xenogeneic murine hosts. Consequently, stem and progenitor cells are solely defined by their role in post-transplant reconstitution and very little is known on their clonal activity in steady-state hematopoiesis. To gain new insights into the clonal activity of stem and progenitor cells under steady-state conditions we used a genetic in vivo lentiviral marking strategy and subsequently monitored the clonal activity of marked hematopoietic cells for up to one year by highly sensitive integration site amplification using LAM-PCR. Highly concentrated GFP-expressing lentiviral vectors (LV) were injected intravenously (IV, n=10) or intrafemorally (IF, n=15) into GFP-tolerant B6.Cg-Tg (Krt1-15-EGFP) 2Cot/J (Krt15) mice to directly mark hematopoietic stem and progenitor cells. 5 mice from each of the two cohorts were treated with 5-Fluorouracil (5-FU, 150 mg/kg) to mobilize hematopoietic stem cells prior to LV-marking. The clonality of the transduced myelopoiesis and lymphopoiesis was analyzed by LAM-PCR. A small proportion of all peripheral blood cells in LV-injected mice consistently expressed GFP for up to one year (5-100 GFP+ cells per 20000 PB cells analyzed). Pre-treatment with 5-FU did not affect the percentage or lineage distribution of marked blood cells even when the vector was injected intravenously. Even though the initial percentage of marked cells was similar after IV and IF vector injection (p>0.05) the marking kinetics were different. Whereas the percentage of GFP expressing cells in PB of IF-marked mice remained stable over the whole observation period for up to 1 year, a 2-fold decline of the percentage of marked cells was detected two weeks after IV-marking indicating that predominantly short-lived more mature cells were transduced after IV vector injection. LAM-PCR analyses of sorted cell lineages showed that multiple clones contributed to the marked myeloid and lymphoid long-term hematopoiesis after IF-injection. In summary, our data demonstrate stable marking of steady-state hematopoiesis for up to one year. Our results demonstrate that remarkably stable stem cell clones with myeloid and lymphoid differentiation potential contribute to murine steady-state long-term hematopoiesis. In vivo marking will further allow to directly address the response of individual stem cell clones to hematopoietic stress including chemotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Increased Immune Suppressive CD14+ hla-DRneg Circulating Monocytes Are Found in Aggressive Non-Hodgkin's Lymphoma and Correlated with Increased Arginase I Level.
Authors: Lin Yi , Gustafson Michael P. , Bulur Peggy A , Maas Mary , Mohr Amy , Witzig Thomas E , Dietz Allan
Published: October 2019

Abstract 970

Despite advances in treatments for patients with non-Hodgkin's lymphoma (NHL), the relapse rates remain high and 40% of diffuse large B-cell NHL (DLBCL) patients die of disease. These patients have variable levels of immunodeficiency that may impact their prognosis. Previously we reported that monocytes in relapsed NHL patients suppress both autologous and allogeneic T cell proliferation and that these monocytes were unable to mature into dendritic cells in vitro. These suppressive functions were associated with increased frequency of CD14+HLA-DRneg monocytes in NHL patients (NHL: 29.4 ± 3.2%, n = 36; healthy donors 7.54 ± 0.97%, n = 27; p < 0.0001). This phenotype is distinct from other myeloid suppressors (Lin−CD33+HLA-DR−) or non-classical monocytes (CD16+), neither of which was different in numbers between NHL and healthy donors. We have now expanded our study cohort and found that the percentage of CD14+HLA-DRneg cells among circulating monocytes was increased in patients with more aggressive disease: DLBCL 37.5 ± 5.11%, n = 16 vs SLL and MALT lymphoma 15.8 ± 4.02%, n = 7; p = 0.01. Two newly diagnosed patients and three patients with localized disease amenable to resection all had frequencies of CD14+HLA-DRneg monocytes closer to that of healthy donors whereas 5 of the 6 patients with low grade NHL that transformed to DLBCL had significantly elevated percentage of these suppressive monocytes (49.1 ± 4.70%). To better understand the mechanism of immune suppression by CD14+HLA-DRneg monocytes, we investigated production of IFN-alpha induced by CpG oligodeoxynucleotide. Monocytes from NHL patients were less effective at IFN-alpha production (0.54 ± 0.22%, n = 4) compared to healthy donors (1.37 ± 0.25%, n = 4; p = 0.04). The corresponding culture supernatants also had a 4-fold reduction in IFN-alpha level by ELISA (p = 0.01). Reduced IFN-alpha production and subsequent decreased stimulatory response may contribute to diminished treatment response to CpG in NHL. Finally, we also found that the frequency of CD14+HLA-DRneg monocytes correlated with increasing plasma levels of arginase I, a known mediator of T cell suppression (n = 16, p = 0.06). Such correlation was not observed for circulating VEGF, IL-6 or IL-10. Taken together we have described a population of immunosuppressive CD14+HLA-DRneg monocytes in NHL patients that is more prevalent in aggressive disease and likely suppresses T cell function through multiple mechanisms including arginine metabolism. This work was supported through the University of Iowa and Mayo Clinic Lymphoma SPORE CA097274 and the Gerstner Family Foundation.

Disclosures:

Witzig: Novartis: Research Funding.
Mitochondrial Physiology in Health and Disease: Changes with Aging.
Authors: Wallace Douglas C.
Published: October 2019

Abstract SCI-1

The incidence and cost of the age-related metabolic and degenerative diseases is rising in the face of ever increasing investment. Therefore, something must be missing in our current hypotheses about these diseases. Until now, the prevailing paradigms in Western medicine have been the anatomical paradigm of disease which posits that tissue-specific symptoms are due to tissue-specific structural defects and the Mendelian paradigm of genetics which posits that if a disease is inherited according to the Laws of Mendel it is genetic and if not it is environmental. The epigenome is invoked to explain the environmental modulation of the nuclear gene expression. However, life requires both structure and energy and the over 1500 mitochondrial energy genes are dispersed across the chromosomes plus the maternally inherited mitochondrial DNA. Moreover, the cells and tissues most affected by aging and the age-related disease are those most reliant on mitochondrial energy and the mitochondrial lies at the interface between environmental calories and human physiology, diabetes and obesity. The epigenome and the signal transduction pathways are regulated by protein phosphorylation by ATP, acetylation via acetyl-CoA, and methylation by S-adenosylmethionine, all driven by mitochondrial high energy substrates modulated by available calories. Furthermore, mitochondrial redox chemistry regulates reactive oxygen production and thiol/disulfide chemistry and these also regulate cellular signaling and function. Therefore, bioenergetics and mitochondrial genetics are the missing factors which have inhibited our capacity of address the biology and genetics of the age-related metabolic and degenerative diseases, cancer and aging.

Disclosures

No relevant conflicts of interest to declare.
Telomere Dysfunction in Bone Marrow Failure.
Authors: Lansdorp Peter M.
Published: October 2019

Abstract SCI-12

In order to distinguish a normal telomere from a double strand break, a minimum number of telomere repeats must “cap” each chromosome end. The length of each repeat array will reflect a unique history of addition and losses. Telomere losses are known to occur sporadic as well as with every replication cycle. Losses of telomeric DNA are countered by the telomerase enzyme containing telomerase RNA (encoded by the TERC gene) and a reverse transcriptase protein (encoded by TERT gene) as minimal components. Telomerase levels are high in cells of the germline and immortal cellines and the telomere length is typically maintained in such cells. In contrast, telomerase activity is limiting in most human somatic (stem) cells and as a result the average length of telomere repeats in most somatic cells shows a highly significant decline with age. The hypothesis that loss of telomere repeats acts as a “mitotic clock” and a tumor suppressor mechanism in stem cells is strongly supported by recent studies of patients with mild telomerase deficiency resulting from haplo-insufficiency for either the TERC or TERT gene. Such genetic defects can give rise to various disorders including autosomal dominant Dyskeratosis Congenita (DKC), aplastic anemia, liver fibrosis and pulmonary fibrosis. Other recent studies have revealed that amplification of the hTERT gene is one of the most common genetic abnormalities in various cancers. Paradoxically, it is becoming clear that SNPs within the TERT locus are among the most reproducible risk factors for the development of different types of cancer including lung cancer, acute myeloid leukemia and chronic lymphocytic leukemia. The links between hypo- and hyperproliferative consequences of inborn telomerase deficiencies and SNP's in the TERT gene are poorly understood. It seems plausible that the increased risk of leukemia development in aplastic anemia, myelodysplastic syndrome and Dyskeratosis Congenita, results from stem cell failure. Could reduced stem cell numbers by itself provide a risk factor for tumor development? More direct measures of stem cell numbers in vivo are needed to examine this possibility and the relationship between stem cell numbers and tumor development in patients with defective telomere maintenance as well as in normal individuals as a function of age. Measurements of the average telomere length as well as the length of telomere repeats at individual chromosome ends in specific cells and tissues will further calrify the involvement of telomeres in bone marrow failure, normal aging and tumor biology.

Disclosures

Lansdorp: Repeat Diagnostics Inc.: Equity Ownership.
Nuclear Transcription Factories.
Authors: Fraser Peter
Published: October 2019

Abstract SCI-16

The discovery of long-range intrachromosomal and interchromosomal interactions in higher eukaryotes (1, 2) points to a functional interplay between genome architecture and gene expression, challenging the view of transcription as a one-dimensional process. However, the extent of such interactions and the underlying mechanisms are unknown. Here we present the first genome-wide analysis of interactions between transcriptionally active genes at transcription factories using the mouse globin genes in erythroid tissues. Our results show that the transcripitonally active globin genes associate with hundreds of other transcribed genes, revealing extensive and preferential intra- and interchromosomal transcription interactomes. We show that the erythroid-specific transcription factor Klf1 mediates preferential co-associations of Klf1-regulated genes at a limited number of specialized transcription factories. Our results establish a new gene expression paradigm, suggesting that active co-regulated genes and their regulatory factors cooperate to create specialized nuclear hotspots optimized for efficient and coordinated transcriptional control. 1. Carter D, Chakalova L, Osborne CS, Dai YF, Fraser P. Long-range chromatin regulatory interactions in vivo. Nat. Genet. 2002;32:623-626. 2. Osborne CS, Chakalova L, Brown KE, et al. Active genes dynamically co-localize to shared sites of ongoing transcription. Nat. Genet. 2004;36:1065-1071.

Disclosures

No relevant conflicts of interest to declare.
Mast Cells and Tumor Progression.
Authors: Clapp D. Wade , Yang Fengchun , Ingram David A. , Robertson Kent A. , Hutchins Gary D. , Parada Luis F.
Published: October 2019

Abstract SCI-33

Interactions between tumorigenic cells and their surrounding microenvironment are critical for tumor progression yet remain incompletely understood. Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a common genetic disorder characterized by complex tumors called neurofibromas. Neurofibromas form in association with peripheral nerves and are composed of Schwann cells, blood vessels, fibroblasts and degranulating mast cells. Genetic studies in engineered mice indicate that biallelic loss of Nf1 is required in the tumorigenic cell of origin in the embryonic Schwann cell lineage. However, in the physiologic state, Schwann cell loss of heterozygosity is not sufficient for neurofibroma formation in a genetically engineered murine model and Nf1 haploinsufficiency in at least one additional nonneoplastic lineage is required for tumor progression. Recent studies in our group have focused on evaluating the role of bone marrow-derived cells, and particularly mast cells, in the tumor microenvironment. Previous work by our group established that human and murine Nf1 deficient Schwann cells secrete high concentrations of kit-ligand. Kit-ligand has a central role in multiple mast cell functions including chemotaxis, proliferation and degranulation. In a series of bone marrow transplantation studies, we established that Nf1 haploinsufficiency in bone marrow is sufficient to allow neurofibroma progression in the context of Schwann cell Nf1 deficiency. Further, genetic or pharmacologic attenuation of c-kit signaling diminishes neurofibroma initiation and progression. Collectively, the studies implicate mast cells as active participants in tumor formation and identify therapeutic targets for human phase 1-2 clinical trials.

Disclosures

Off Label Use: The drug imatinib mesylate was used to treat plexiform neurofibromas in genetically engineered mice, in a child with a plexiform neurofibroma, and it is currently being tested in a phase 2 clinical trial.
Ontology of Megakaryopoesis.
Authors: Geddis Amy E.
Published: October 2019

Abstract SCI-8

Compared to red cells, whose passage from embryonic to adult stages is marked by the expression of distinct forms of hemoglobin, the development of megakaryocytes during embryogenesis is less well understood. However, certain shared characteristics between megakaryocytes, endothelial cells, hematopoietic stem cells and erythrocytes infer developmental relationships between these lineages. Recent data support the model that hematopoietic stem cells derive from the hemangioblast, and that megakaryocytes and erythrocytes develop from a common precursor both in primitive and adult hematopoiesis. Evidence of these common origins can be found in the genetic programs that are activated during hematopoiesis, in that many of the cell surface markers and transcriptions factors that are characteristic of megakaryocytes can also be found in endothelial cells, stem cells and erythrocytes. In this session I will review current views on developmental thrombopoiesis, key megakaryocytic transcription factors and the experimental and clinical phenotypes associated with their disruption, and current controversies in lineage choice during megakaryocyte differentiation.

Disclosures

Geddis: Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding.
Whole Kinome Profiling In Pediatric Acute Leukemia.
Authors: Gombert Michael , Krell Pina Fanny Ida , Linka René Martin , Knapp Stefan , Borkhardt Arndt
Published: October 2019

Abstract 1029

With almost 130.000 entries in PubMed by today, the kinase family represents one of the gene families with the best documented history in malignant diseases.

Acute myeloid leukemias account for 4.7% of all childhood cancers and 13.7% of all childhood leukemias. Recent german statistics (Kinderkrebsregister Mainz) indicate a 5-year survival rate of only 66%, emphasizing the need to discover novel therapeutic targets and prognostic markers. Here we present an adaptable workflow to detect mutations in kinase family members on genomic and expression level as well as in silico assessment of their functional consequences.

To undertake the venture of resequencing the human kinome of leukemia patients, we constructed a custom SequenceCapture microarray (NimbleGen) based on all exons of all 518 members of the kinase family previously reported by Greenman and colleagues (Greenman et al, Nature, 2007) as well as a set of TaqMan® low density arrays (Life Technologies) to confirm the expression of potential candidates in tumor material. Genomic DNA from the initial tumor sample and germline material from a 16 year old female patient diagnosed with a AML FAB1 with Auer bodies, 93% blasts, CD33+ CD117+ CD38+ and partial CD15+ and from a 16 year old female patient with a bilinear CD22+ CD33+ leukemia was enriched on SequenceCapture arrays and sequenced on the Roche GS FLX Titanium platform. Acquired sequences were mapped against the current human reference (hg19/GRCh37). RNA derived from the initial leukemic blasts was used for kinome-wide expression profiling on TaqMan low density arrays.

From sequencing the enriched DNA, we recieved between 85 and 129 megabases of tumor and germline sequence, of which 97.7–99.4% mapped to the human reference. About 90% of the targeted regions were covered at least once. Mapping and comparison to the germline material led to 28 and 46 tumor specific non-synonymous single nucleotide variants sequenced at a depth of ≥10x that were not previously reported as SNPs (dbSNP130). Analyzing these candidates with UniProt revealed that 6 and 12 were located in or very close to important functional domains of their respective protein. Structural protein models of one patients remaining mutations underscore the potential influence of detected mutations on the protein function. TaqMan® expression profiling of all kinases present on the SequenceCapture Array identified 3 and 4 of the remaining genes to be expressed in the tumor on mRNA level. In conclusion, starting from 518 candidates, we present a powerful workflow for high throughput analysis of the whole kinome in leukemic samples as well as CDK4, DYRK1B, GSG2 (pt1) and MASTL, MARK3, RP6KA5, CLK4 (pt2) as potential targets for further functional analysis.

Disclosures:

No relevant conflicts of interest to declare.
Pharmacokinetic, Pharmacodynamic and Pharmacogenetic Determinants of Osteonecrosis In Children with Acute Lymphoblastic Leukemia.
Authors: Kawedia Jitesh D. , Kaste Sue , Pei Deqing , Panetta John C , Cai Xiangjun , Cheng Cheng , Neale Geoffrey A. , Howard Scott C , Evans William E. , Pui Ching-Hon , Relling Mary V.
Published: October 2019

Abstract 1033

Osteonecrosis is a debilitating corticosteroid-induced toxicity in patients treated for acute lymphoblastic leukemia (ALL). Our goal was to ascertain genetic and non-genetic risk factors for this complication. In St. Jude Total XV protocol for children with newly diagnosed ALL, we prospectively screened 365 patients with magnetic resonance imaging of hips and knees, irrespective of symptoms. We determined whether age, race, sex, ALL treatment arm, body mass index, serum lipids, albumin and cortisol levels, dexamethasone pharmacokinetics and genome-wide germline genetic polymorphisms were associated with symptomatic (grade 2–4) osteonecrosis. Sixty-eight patients developed symptomatic osteonecrosis with a cumulative incidence of 17%. Age greater than 10 years (odds ratio 4.8; 95 % confidence interval [CI], 2.5–9.2; p = 0.00001) and treatment arm (standard/high risk vs. low risk) (odds ratio 2.5; 95% CI 1.2–4.9; p = 0.011) were associated with increased risk of osteonecrosis, and were included as covariates in all other analyses. Lower serum albumin (p = 0.04) and higher serum cholesterol levels (p = 0.02) were associated with symptomatic (grade 2–4) osteonecrosis. In addition, higher dexamethasone plasma exposure (area-under-the-curve) was associated with severe (grade 3–4) osteonecrosis (p = 0.0007). After adjusting for clinical features (age and treatment arm), we identified several single nucleotide polymorphisms (SNPs) associated with the risk of developing osteonecrosis. These include: ACP1 (rs12714403, p = 1.03 × 10-5; odds ratio 4.8; 95% CI 2.4–9.6), a gene involved in regulation of lipids and osteoblast differentiation; and SH3YL1 (rs4241316, p = 5.7 × 10-6; odds ratio 5.0; 95% CI 2.5–10.1). These SNPs were also associated with lower albumin and elevated serum cholesterol levels. In conclusion, older age, lower albumin levels, increased dexamethasone systemic exposure, and higher cholesterol levels were associated with osteonecrosis. Inherited genomic variation that predisposes to osteonecrosis may do so via pleiotropic effects on dexamethasone pharmacokinetics, serum cholesterol and serum albumin.

Disclosures:

Pui: EUSA Pharma: Honoraria; Enzon: Honoraria; Sanofi-Aventis: Honoraria. Relling:St. Jude Children's Research Hospital: Employment, Patents & Royalties; Enzon Pharmaceuticals: Research Funding.