bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evolution of the Donor T Cell Repertoire In Allogeneic Stem Cell Transplant Recipients In the Second Decade After Transplantation
Authors: Le Robert Q. , Melenhorst J. Joseph , Hill Brenna , Memon Sarfraz , Battiwalla Minoo , Savani Bipin N. , Shenoy Aarthi , Hensel Nancy F. , Koklanaris Eleftheria K. , Keyvanfar Keyvan , Hakim Frances T. , Douek Daniel C. , Barrett A. John 
Published: October 2019

Abstract
Abstract 831
After allogeneic stem cell transplantation (SCT), donor T lymphocyte immune function is slowly re-established in the recipient through reconstruction of the donor's post-thymic T cell repertoire and from T cell neogenesis in the thymus. Although long-term survivors from SCT appear healthy, their immune repertoire and differences from that of their donors have not been characterized. We studied 38 healthy patients surviving more than 10 years from a myeloablative SCT for hematological malignancy (median follow-up 12 years, range 10–16 years). T cell and natural killer (NK) cell repertoires in these patients were compared with cells from their stem cell donors cryopreserved at time of transplant and from the same donors at 10 year after SCT. The median age of both recipients and their sibling donors at time of transplant was identical (36 years). Patients received cyclosporine GVHD prophylaxis and delayed add-back of donor lymphocytes 30–90 days post transplant. Only one patient was on continued immunosuppressive treatment at the time of study. Compared with the donor pre-transplant counts there was no significant difference in the absolute lymphocyte, neutrophil, monocyte, CD4+ and CD8+ T cell, NK cell, and B cell subset counts. However, compared to their donors, recipients had a) significantly fewer naïve CD4+ and CD8+ T cells; b) lower T cell receptor excision circles levels; c) fewer CD4+ central memory T cells; d) more effector CD8+ T cells; e) and more FOXP3+ regulatory T cells. These data suggest that the patient had a persistent deficiency on T cell neogenesis. Molecular examination of the T cell receptor Vbeta (TCRBV) repertoire by spectratype analysis showed that there was no significant difference in total complexity score, defined as the sum of the number of discrete peaks for each Vbeta subfamily, between the patients and their donors. TCRBV subfamily spectratyping profiles of patients and donors, however, had diverged, with both gains and losses of peaks identifiable in both patient and donor. In conclusion, patients surviving 10 or more years after allogeneic SCT still show a T cell repertoire that reflects expansion of the donor-derived post thymic T cell compartment, with a limited contribution by new T cell generation and persistently increased Tregs. It therefore appears that a diverse TCRBV repertoire predominantly derived from the memory T cell pool is compatible with good health.

Disclosures:
No relevant conflicts of interest to declare.
Growth Factor Independence 1 b (Gfi1b) as a New Regulator of Hematopoietic Stem Cell Fate
Authors: Khandanpour Cyrus , Vassen Lothar , Gaudreau Marie-Claude , Kosan Christian , Moroy Tarik 
Published: October 2019

Abstract
Abstract 837
Donor matched transplantation of bone marrow or hematopoietic stem cells (HSCs) are widely used to treat hematological malignancies, but are associated with high mortality. Methods for expansion of HSC numbers and their mobilization into the bloodstream of a donor could significantly improve therapy. We show here that the zinc finger transcriptional repressor Gfi1b is highly expressed in hematopoietic stem cells (defined as CD 150+, CD 48-, Lin-, Sca1+ and c-kit+) cells and is down-regulated more than 10 fold upon differentiation into multipotential progenitors (defined as CD 150+ or CD150-, CD 48+, Lin-, Sca1+ and c-kit+). Constitutive germline deletion of Gfi1b is lethal at midgestation due to impaired development of erythrocytes and megakaryocytes. We have therefore developed a conditional knock-out of Gfi1b to study its role specifically in the adult hematopoietic system. Deletion of Gfi1b leads to a 30-fold increase of HSC numbers in bone marrow and around a100 fold increase in spleen and peripheral blood. This was due to a higher rate of HSCs undergoing cell cycling. Concomitantly, the number of quiescent HSCs was reduced 5–6 times.
We then performed an gene expression array of wt and Gfi1b deficient HSCs and observed that loss of Gfi1b leads to an altered RNA expression of integrins and adhesion molecules, for instance CXCR4, VCAM-1 and Tenascin C, which usually retain HSCs in a dormant state in the endosteal niche. These changes were also confirmed on protein level. Finally, we could observe a higher levels of Reactive Oxygen Species (ROS) in the Gfi1b deficient HSCs compared to wt HSCs. We verified whether elevated level of ROS are causative for the expansion of HSCs and noticed that application of N-Acetyl-Cystein, which counteracts the effects of ROS, limits significantly the expansion of HSCs, underscoring the important role of ROS in the expansion of Gfi1b deficient HSCs. Despite markedly increased proliferation, Gfi1b-/- HSCs can reconstitute lymphoid and myeloid lineages to the same extent as wt HSCs when transplanted in competition with wt HSCs. Furthermore, Gfi1b deficient HSCs also feature an expansion after transplantation and expand 5–10 fold more than wt HSC when transplanted initially in equal numbers with wt HSCs. It is possible that lower expression of CXCR4, VCAM-1 and other surface proteins leads to release and egression of Gfi1b deficient HSCs from the hypoxic endosteal stem cell niche and exposes the HSCs to more oxygen which in turn increases ROS levels. Elevated ROS could promote entry of Gfi1b-/- HSCs into cell cycle. In conclusion Gfi1b regulates HSC dormancy, pool size and potentially also the egress and mobilization of HSCs and might offer a new therapeutic approach to improve human HSC transplantation.

Disclosures:
No relevant conflicts of interest to declare.
14-3-3 Regulates the Lnk/JAK2 Pathway In Hematopoietic Stem and Progenitor Cells
Authors: Balcerek Joanna , Jiang Jing , Bersenev Alexey , Song Yiwen , Wu Chao , Tong Wei 
Published: October 2019

Abstract
Abstract 86
Hematopoietic stem and progenitor cell (HSPC) homeostasis is regulated by intricate signaling networks. The tyrosine kinase JAK2 plays an essential role in cytokine signaling during hematopoiesis and its dysregulation can lead to hematologic malignancies. Recently activating mutations in JAK2 were found in a large fraction of patients with myeloproliferative neoplasms (MPNs). We previously demonstrated that lymphocyte adaptor protein (Lnk) binds JAK2 and attenuates its activity, thereby limiting HSPC expansion (Bersenev et al., JCI, 2008;118:2832-2844). We further showed that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced MPNs in mice (Bersenev et al., JCI, 2010;120:2058-2069). Lnk directly inhibits oncogenic JAK2 as well as acting through JAK2- independent pathways to constrain MPN development. Consistently, aged Lnk–/– mice spontaneously developed a CML-like MPN (Bersenev et al., JCI, 2010;120:2058-2069). More importantly, loss-of-function mutations in Lnk are found in human MPN patients (Oh, et. al., Blood, 2010, in press). This work suggests that Lnk plays a pivotal role in regulating both normal and malignant HSPC expansion. However, it remains to be determined how Lnk attenuates JAK2 activity since Lnk itself does not possess any enzymatic activity. Therefore, we began exploring Lnk regulatory mechanisms by identifying novel Lnk partners through protein purification and mass spectrometric analysis. This resulted in the identification of a number of novel Lnk binding proteins, which include the 14-3-3 proteins as the most robust interactors. 14-3-3s are a group of scaffold proteins that regulate many disease-relevant gene products and play important functions in many aspects of cellular processes. We found that Lnk is phosphorylated at two serine residues, which serve as the critical binding sites for 14-3-3. 14-3-3 abrogates the Lnk-JAK2 interaction thereby alleviating Lnk inhibitory function in both JAK2 signaling and cell growth. Furthermore, 14-3-3 binding is necessary and sufficient to maintain Lnk in an inactive state. We also investigated the signals that regulate Lnk phosphorylation, revealing Lnk as a signaling nodal point that integrates multiple signaling pathways in controlling HSPC homeostasis. The physiological significance of the Lnk-14-3-3 interaction in HSPC development will be discussed. Thus, our data implicate previously unappreciated serine phosphorylation events in Lnk-dependent hematopoietic function and regulation of JAK2. In addition, identification of novel signaling molecules that influence hematopoiesis might facilitate stem cell therapies and provide novel therapeutic targets for the treatment of MPNs.

Disclosures:
No relevant conflicts of interest to declare.
Loss of the Cell Cycle Regulator p26INCA1 Induces Exhaustion of Leukemic Stem Cells
Authors: Tschanter Petra , Baeumer Nicole , Lohmeyer Lisa , Berkenfeld Frank , Tickenbrock Lara , Diederichs Sven , Stehling Martin , Köhler Gabriele , Serve Hubert , Berdel Wolfgang E. , Koschmieder Steffen , Müller-Tidow Carsten 
Published: October 2019

Abstract
Abstract 96
Acute myeloid leukemia is a clonal disease characterized by a malignant proliferation and accumulation of myeloid progenitor cells. Current therapeutic strategies are often not able to eradicate the leukemic cells. Malignancy is associated with deregulation of cell cycle check- points and the deregulation of checkpoints is associated with altered stem cell properties. A better understanding of malignant stem cells and their cell cycle regulation might help to develop new therapies. Recently, we identified p26INCA1 as a novel cell cycle regulator. GST pulldown assays revealed binding of INCA1 predominantly to CDK2- specific Cyclins and we demonstrated an inhibitory effect of INCA1 on CDK2/ CyclinA complexes in kinase activity assays. The loss of INCA1 and its inhibitory effect on the cell cycle regulation led to an increased cell cycling and consequently to an enlarged stem cell pool in vivo. Upon cytotoxic stress, the loss of Inca1 enhanced cell cycling and bone marrow exhaustion. To analyze a potential role of INCA1 in leukemogenesis we retrovirally transduced wildtype and Inca1−/− bone marrow cells with AML1-ETO9a (A1E9a) and transplanted these cells into wildtype recipients. Most of the wildtype cell- transplanted recipients died due to AML. In contrast, only one of the Inca1−/− cell- transplanted mice developed AML. Engraftment was higher upon transplantion of Inca1−/− cells but engraftment was not sustained. To consider the repopulation capacity of the leukemic cells we performed transplantation of primary leukemic cells into secondary recipients. A1E9a induced leukemia in Inca1 wildtype cells was transplantable and lethal. However Inca1−/− leukemic cells were severely impaired in leukemia development in secondary recipients. Colony forming units and replating capacity were significantly reduced in A1E9a Inca1−/− bone marrow cells although these cells showed increased CDK2 activity. Exhaustion of leukemic cells in the absence of Inca1 was confirmed by cloning efficiency assays. Further analyses were performed with c-myc induced leukemias. Interestingly, Inca1 deletion precluded the development of leukemias in secondary recipients. Taken together, these findings identify an important role for p26INCA1 in the maintenance of leukemia and potentially the self-renewal capacity of leukemic stem cells.

Disclosures:
No relevant conflicts of interest to declare.
Genomics in 2K10: Fulfilling the Promise of a Sequenced Human Genome
Authors: Green Eric D. 
Published: October 2019

Abstract
Abstract SCI-16
The Human Genome Project's completion of the human genome sequence in 2003 was a landmark scientific achievement of historic significance. It also signified a critical transition for the field of genomics, as the new foundation of genomic knowledge started to be used in powerful ways by researchers and clinicians to tackle increasingly complex problems in biomedicine. To exploit the opportunities provided by the human genome sequence and to ensure the productive growth of genomics as one of the most vital biomedical disciplines of the 21st century, the National Human Genome Research Institute (NHGRI) is pursuing a broad vision for genomics research beyond the Human Genome Project. This vision includes facilitating and supporting the highest-priority research areas that interconnect genomics to biology, to health, and to society.Current efforts in genomics research are focused on using genomic data, technologies, and insights to acquire a deeper understanding of biology and to uncover the genetic basis of human disease. Some of the most profound advances are being catalyzed by revolutionary new DNA sequencing technologies; these methods are already producing prodigious amounts of DNA sequence data, including from large numbers of individual patients. Such a capability, coupled with better associations between genetic diseases and specific regions of the human genome, are accelerating our understanding of the genetic basis for complex genetic disorders and for drug response. Together, these developments will usher in the era of genomic medicine.

Disclosures:
No relevant conflicts of interest to declare.
DNMT3A and IDH1/2 Mutations Are Stable During the Progression of Acute Myeloid Leukemia
Authors: Kihara Rika , Hoshino Hideaki , Kiyoi Hitoshi , Naoe Tomoki 
Published: October 2019

Abstract
Abstract 1466
Several genetic alterations, which are involved in the pathophysiology of acute myeloid leukemia (AML), have been identified. Recently some mutations in genes affecting DNA and histone methylation have been reported. Their stabilities during the disease progression are not fully clarified. We analyzed mutations of FLT3, NRAS, NPM1, TP53, CEBPA, IDH1/2 and DNMT3A genes in paired initial diagnosis and the first relapse samples from 44 adult AML patients. Informed consent was obtained from all patients, and approval was obtained from the ethics committee of Nagoya university school of medicine. At diagnosis, we identified FLT3 mutation in 12 patients, NPM1 mutation in 17, N-RAS mutation in 7, TP53 mutation in 2, CEBPA mutation in 7, IDH1/2 mutations in 10 and DNMT3A mutation in 6. At relapse, the acquisition and loss of FLT3 mutation were observed in 4 and 1 patients, respectively. Those of N-RAS mutation were in 2 and 3 patients, respectively, and those of TP53 mutation were in each one. No patient acquired NPM1 mutation, but one patient lost it at relapse. And no patient acquired CEBPA mutation, but 2 patients lost it at relapse. One patient acquired IDH1/2 mutation, but no patient lost them at relapse. The acquisition and loss of DNMT3A mutation was not observed. Notably, one patient harboring IDH1 and FLT3 mutations at the diagnosis lost FLT3 mutation, but not IDH1 mutation at relapse; one patient harboring IDH1, DNMT3A and NRAS mutations at the diagnosis lost NRAS mutation, but not IDH1 and DNMT3A mutations at relapse and one patient harboring IDH2 and CEBPA mutations at the diagnosis lost CEBPA mutation, but not IDH2 mutation at relapse. These results indicated that Class 2 mutation, which is more stable than Class 1 mutations, may be lost during the progression of AML. However, IDH1/2 and DNMT3A mutations were stable during the disease course of AML suggesting aberrations of the DNA methylation is an early event in the leukemogenesis.Type of changeFLT3 (n = 44)NRAS (n = 44)NPM1 (n = 44)TP53 (n = 27)CEBPA (n = 34)IDH1/2 (n = 44)DNMT3A (n = 44)Gain at relapse4301010Loss at relapse1311200Total no. of changes(%)5(11)6(14)1(2.2)2(7.4)2(5.9)1(2.2)0(0)
Table Frequencies of gain and loss of gene mutations at relapse in AML

Disclosures:
Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.
WT1 Gene Expressions Predicting Posttransplant Outcome
Authors: Kim Heeje , Choi Hee-Baeg , Sohn Hyun-Jeong , Lee Jong-Wook , Min Woo-Sung , Kim Tai-Gyu 
Published: October 2019

Abstract
Abstract 1499
Patients with AML show over expression of Wilms' tumor gene 1 (WT1). WT1 is a valuable prognostic parameter for minimal residual disease (MRD). Quantitation of this gene transcript by real-time quantitative PCR (RQ-PCR) may be useful for predicting prognosis in AML. We prospectively investigated the prognostic potential of the leukemia-associated antigen, WT1. We simultaneously determined the expression of the gene in bone marrow (BM) from 160 adult patients with newly diagnosed AML and healthy normal controls using RQ-PCR. The molecular profiles were then evaluated with multiple parameters. We evaluated whether the expression level of the gene in the patients affected complete remission (CR) and survival rates before and after hematopoietic stem cell transplant (HSCT). The median follow-up period for all patients who were event-free survivors was 30 months (range: 2–56). The Kaplan-Meier estimated overall survival (OS) and event-free survival (EFS) rates in the whole population of evaluable patients were 52% (95% CI, 31%–41%) and 50% (95% CI, 31%–43%), respectively. The median expression levels of the gene in normal control and patients were 7.4 (range 0.74–30.3), 58.6 (range 0.9–1950.5), respectively. Of note, WT1 levels of expression at initial diagnosis revealed influential to the EFS after HSCT (P =0.041). Interestingly, there were no correlated results with CR, OS, and EFS rates for treated AML, specifically before HSCT. This finding may suggest that initial expressions of WT1 possibly in association with the leukemic stem cells remained post-transplant can be the critical target of future immunotherapeutic trial.

Disclosures:
No relevant conflicts of interest to declare.
AZD1208, a Novel, Potent and Selective Pan PIM Kinase Inhibitor, Demonstrates Efficacy in Models of Acute Myeloid Leukemia
Authors: Keeton Erika , Palakurthi Sangeetha , Alimzhanov Marat , Grondine Michael , Chen Yuching , Brown Jeffrey , McEachern Kristen , Cao Yichen , Chinnappan Dharmaraj , Shen Minhui , Dakin Les , Zheng Xiao , Lamb Michelle , Wu Allan , Chen Huawei , Lyne Paul , Huszar Dennis 
Published: October 2019

Abstract
Abstract 1540
The PIM serine/threonine kinase family is composed of three highly homologous members; PIM1, PIM2 and PIM3, identified by the ability of the prototype member PIM1 to drive lymphomagenesis in mice. Upregulation of PIM1 and PIM2 is observed in leukemias and lymphomas, including AML, NHL and CLL, highlighting the potential of these kinases as therapeutic targets in these indications. PIMs are downstream effectors of many cytokine and growth factor signaling pathways and are direct transcriptional targets of STAT transcription factors activated by these pathways. PIMs can phosphorylate multiple substrates to mediate cell proliferation and survival. Here we describe the function of AZD1208, an orally available, potent and highly selective PIM inhibitor that effectively inhibits all three isoforms. AZD1208 inhibits the growth of several AML cell lines and sensitivity correlates with the level of PIM1 expression, STAT5 activation and presence of protein tyrosine kinase mutation. AZD1208 causes cell cycle arrest and apoptosis in MOLM-16 cells in culture. This is accompanied by a dose-dependent reduction in pBAD serine 112 and other substrates, as well as an increase in cleaved caspase 3. AZD1208 suppresses the growth of MOLM-16 and KG-1a xenograft tumors in vivo in a dose proportional manner. In addition, AZD1208 leads to potent inhibition of colony growth of primary AML cells from bone marrow aspirates and downregulates phosphorylation of PIM targets. These results underscore the therapeutic potential of PIM kinase inhibition by AZD1208 for the treatment of AML.

Disclosures:
Keeton: AstraZeneca: Employment, Equity Ownership. Palakurthi:AstraZeneca: Employment, Equity Ownership. Alimzhanov:AstraZeneca: Employment, Equity Ownership. Grondine:AstraZeneca: Employment, Equity Ownership. Chen:AstraZeneca: Employment, Equity Ownership. Brown:AstraZeneca: Employment, Equity Ownership. McEachern:AstraZeneca: Employment, Equity Ownership. Cao:AstraZeneca: Employment, Equity Ownership. Chinnappan:AstraZeneca: Employment, Equity Ownership. Shen:AstraZeneca: Employment, Equity Ownership. Dakin:AstraZeneca: Employment, Equity Ownership. Zheng:AstraZeneca: Employment, Equity Ownership. Lamb:AstraZeneca: Employment, Equity Ownership. Wu:AstraZeneca: Employment, Equity Ownership. Chen:AstraZenenca: Employment, Equity Ownership. Lyne:AstraZeneca: Employment, Equity Ownership. Huszar:AstraZeneca: Employment, Equity Ownership.
Innmune-Related Risk Factors for Non-Hodgkin Lymphoma in Twins
Authors: Wang Jun , Mack Thomas M , Hwang Amie , Nathwani Bharat N. , Hamilton Ann , Cozen Wendy 
Published: October 2019

Abstract
Abstract 1588
Acquired or inherited immune deficiency is an established risk factor for non-Hodgkin lymphoma (NHL), but does not account for the majority of cases. Recent epidemiologic evidence from the InterLymph Consortium suggests that subclinical, subtle alterations in immune response may also affect risk. Specifically, atopic conditions have been associated with a protective effect and infections at an early age with risk. Unaffected twins of patients are ideal controls because they are partially (fraternal) or completely (identical) matched on genetic factors. We evaluated these factors in twin pairs in whom one member was diagnosed with NHL. At least one member of 197 twin pairs returned detailed questionnaires; in 75 pairs, both the case and unaffected co-twin returned the questionnaire and in 122 pairs only one twin (usually the unaffected co-twin) returned the questionnaire. NHL diagnosis in the case-twin was confirmed by medical record and pathology review. Multivariable conditional logistic regression adjusted for education was used to estimate the effect of the exposures on risk with odds ratios (ORs) and 95% confidence intervals (CI). Eczema in childhood was associated with a 60% decreased risk of NHL (OR= 0.4, 95% CI= 0.2, 0.9). Allergic asthma (OR= 0.2, 95% CI= 0.1, 0.6), hay fever (OR= 0.4, 95% CI= 0.2, 0.8) and allergies to plants (OR= 0.3, 95% CI= 0.1, 0.9) were protective. Compared to no atopic conditions, the twin with ≥ 3 atopic conditions had a 90% decreased risk of NHL (95% CI= 0.0, 0.5). Tonsillectomy and appendectomy were each associated with a significant, ∼ 2-fold increased risk. The twin with more early childhood exposures (more contact with pets, young children, sucking thumb or pacifier) likely to transmit infection was also at an increased risk of NHL. The study confirms the inverse association between allergy and NHL risk in genetically matched cases and controls and provides further evidence that childhood exposures are important mediators of risk.

Disclosures:
No relevant conflicts of interest to declare.