bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Novel CD94+ Immature NK Cell Population Based on NKp80 Expression with ILC3-like Features in Human Secondary Lymphoid Tissue
Authors: Scoville Steven D , Keller Karen , Mundy-Bosse Bethany , Caligiuri Michael A. , Freud Aharon G. 
Published: October 2019

Abstract
Natural killer (NK) cells are important for their innate ability to identify and kill cancer cells without previous stimulation. However, many of the mechanisms concerning their differentiation from immature precursor cells are still unknown. Elucidation of these pathways are critical to improving targeted therapies that can both activate as well as overcome inhibitory pathways that prevent an effective response. NK cells are thought to develop in secondary lymphoid tissue (SLT) through five stages distinguished by the expression of CD34, CD117, CD94, and CD16. While virtually all NK cells in blood express the pan-NK cell activating receptor, NKp80, we have discovered a distinct Lin CD94+NKp80-CD16- population that is selectively enriched in SLT. The NKp80- population appears to comprise those cells “in transit” between stage 3 (CD117+CD94-) and stage 4 (CD117loCD94+) when evaluating CD94 and CD117 expression among Lin-CD34- cells by flow cytometry. These ex vivo data suggest that this NKp80- population may represent a novel NK cell developmental intermediate (NKDI). SLT-derived NKp80- cells lack cytolytic granules, show minimal cytotoxicity, and produce negligible amounts of interferon-gamma when compared to NKp80+ NK cells in SLT and blood. In addition, NKp80- cells have lower mRNA and protein expression of TBET and EOMES compared to NKp80+ NK, while expressing these factors at higher levels compared to stage 3 cells. Unexpectedly, the NKp80- population also demonstrates Group 3 innate lymphoid cell (ILC3)-like features ex vivo with expression of surface interleukin-1 (IL-1) receptor 1 and CD127 and mRNA and protein expression of the ILC3-associated transcription factors, AHR and RORC. Moreover, following overnight stimulation with IL-1-beta and IL-23, NKp80- cells produce IL-22, albeit at lower concentrations compared to stage 3 cells. Collectively, these data identify NKp80 as a surrogate marker of functional competence during in vivo human NK cell development and provide evidence for the existence of at least two distinct maturation steps within the previously described stage 4 NK cell population in SLT. Since CD94 expression is currently considered to be NK-restricted, these findings raise new questions regarding the developmental relationship between NK cells and ILC3 in humans. In addition, as ILC3s are primarily used to stimulate an anti-microbial defense while NK cells are functional anti-cancer effector cells, defining the differentiation patterns of these cells can have impacts in both of these areas of research. Studies are ongoing to investigate the in vitro differentiation potential(s) of this novel SLT-derived Lin-CD94+NKp80-CD16- population.

Disclosures
No relevant conflicts of interest to declare.
Improved Immune Recovery after Transplantation of TCRαβ/CD19 Depleted Allografts from Haploidentical Donors in Pediatric Patients
Authors: Lang Peter , Feuchtinger Tobias , Teltschik Heiko-Manuel , Schwinger Wolfgang , Schlegel Patrick , Schumm Michael , Lang Anne-Marie , Lang Barbara , Schwarze Carl Philipp , Ebinger Martin , Urban Christian E. , Handgretinger Rupert 
Published: October 2019

Abstract

Transplantation of haploidentical stem cells has become an accepted option for pediatric patients and adults with high risk malignancies who lack a matched related or unrelated donor. In recent years, the majority of pediatric transplant centers chose the CD34 positive selection of peripheral stem cells, which allowed minimizing GvHD by effective reduction of T cells in the graft. However, infectious complications caused by delayed immune recovery were a major reason for transplant related mortality (TRM). In order to improve the immune recovery, we have established a new T-cell depletion method which removes αβ+ T-lymphocytes via a biotinylated anti-TcRαβ antibody followed by an anti-biotin antibody conjugated to magnetic microbeads while retaining γδ+ T-lymphocytes, natural killer (NK) cells and other cells in the graft. In addition, CD19+ B-lymphocytes were concomitantly depleted for the prevention of post-transplant EBV-associated lymphoproliferative disease.
Immune recovery was retrospectively analyzed in a cohort of 41 patients with acute leukemia, MDS and non-malignant diseases, who received αβ T and B cell depleted allografts from haploidentical family donors. Conditioning regimens consisted of fludarabine or clofarabine, thiotepa, melphalan and serotherapy with OKT3 or ATG-Fresenius®. Graft manipulation was carried out with anti TCRαβ and anti CD19 antibodies and immunomagnetic microbeads. γδ T cells and NK cells remained in the grafts. Primary engraftment occurred in 88%, acute graft versus host disease (aGvHD) grade II and III-IV occurred in 10% and 15%. Immune recovery data were available in 26 patients and comparable after OKT3 (n=7) or ATG-F® (n=19). Median time to reach > 100 CD3+/µl, > 200 CD19+ cells/µl and > 200 CD56+ cells/µl for the whole group was 13, 127 and 12.5 days. Compared to a historical control group of patients with CD34 positive selected grafts, significantly higher cell numbers were found for CD3+ at days +30 and +90 (267 vs. 27 and 397 vs. 163 cells/µl), for CD3+4+ at day +30 (58 vs. 11 cells/µl) and for CD56+ at day +14 (622 vs. 27 cells/µl). The clinical impact of this accelerated immune recovery will be evaluated in an ongoing prospective multi-center trial.

Disclosures
No relevant conflicts of interest to declare.
Dysregulation of SHFM1, a Novel Target for Prevention of Genomic Instability in Myeloma, Is Associated with Epigenetic Changes at Specific CpG Sites
Authors: Shammas Masood , Kumar Subodh , Pal Jagannath , Nanjappa Puru , Samur Mehmat , Gkotzamanidou Maria , Shi Jialan , Munshi Nikhil C. 
Published: October 2019

Abstract
We have previously demonstrated that elevated homologous recombination (HR) activity mediates genomic instability and progression in myeloma. We, therefore, hypothesized that mechanisms underlying dysregulation of HR, drive genomic instability and have potential to serve as novel markers of progression and target to make cancer cells static. In an attempt to identify molecular intermediates underlying increased HR, we conducted a high-throughput shRNA screen using HR activity in MM cells as an endpoint, and identified split hand/split foot malformation locus 1 - SHFM1 - as a novel regulator of HR in myeloma. We demonstrate that suppression of SHFM1 using specific lentiviral shRNA knockdown inhibits HR activity in myeloma as well as other (esophageal) cancer cells. Suppression of HR activity is also associated with reduction in spontaneous DNA breaks thus having a positive impact on genomic integrity. We have also observed that suppression of SHFM1 gene lead to reduction in acquisition of new genomic changes by both copy number alteration as well as SNPs. Evaluation of expression profile in 330 MM patients and 18 normal plasma cells indicated that the gene is overexpressed in myeloma, relative to normal plasma cells (P = 0.0059). Expression of this gene in myeloma also significantly correlates with its copy number (R=0.43; P<0.00000004). To understand the molecular mechanism of its overexpression in myeloma, we evaluated DNA methylation changes associated with altered expression of this gene. We investigated CpG sites in the promoter region of SHFM1 gene using primers with sodium bisulfite converted DNA-specific design. The primers were designed such that they avoid annealing to CpG sites at the region of interest to the maximum extent possible. Samples were bisulfite converted and amplification of all samples using specific primer pairs was performed, and then massively parallel sequenced and analyzed. We observed methylated CpG at two SHFM1 gene locations in PBMC but unmethylated in all 12 out of 12 myeloma cell lines at first location and in 9 out of 12 myeloma cell lines at second cite (Fisher’s exact test for comparisons of PBMC vs. average methylation ratios in caner samples for two locations was P=1.38E-13 and 0.05, respectively).
In summary our results highlight SHFM1 as an important gene modulating genomic instability and its control by methylation provides an insight into epigenomic control of DNA stability.

Disclosures
No relevant conflicts of interest to declare.
Leukemia-Associated Oncometabolite 2-Hydroxyglutarate Regulates Epigenetic Modifications in Response to Oxygen Availability in Cells without IDH1/2 Mutations
Authors: Intlekofer Andrew M. , Dematteo Raymond G. , Finley Lydia W. , Cross Justin R. , Thompson Craig B. 
Published: October 2019

Abstract
Mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) result in production of the 'oncometabolite' D-2-hydroxyglutarate (D-2HG). D-2HG blocks differentiation of leukemia cells by functioning as a competitive inhibitor of alpha-ketoglutarate (alpha-KG) dependent enzymes, including Jumonji family histone lysine demethylases and TET family DNA methylcytosine dioxygenases, which function as important regulators of chromatin structure and gene expression. 2HG is a chiral molecule that can exist in either the D- or L- enantiomer. Although leukemia-associated IDH1/2 mutations exclusively produce D-2HG, biochemical studies have demonstrated that L-2HG functions as a more potent inhibitor of alpha-KG-dependent enzymes. However, biologic sources and/or activities of L-2HG remain unknown. Possible physiologic roles for both 2-hydroxyglutarate enantiomers are suggested by the existence of evolutionarily conserved 'waste disposal' enzymes, known as D2HGDH and L2HGDH, whose sole function is to eliminate D-2HG or L-2HG, respectively. Indeed, children born with genetic deficiency of either enzyme develop severe developmental disorders known as D- and L-2HG acidurias, characterized by systemic elevation of 2HG. We sought to investigate physiologic sources and functions of D-2HG and L-2HG by manipulating D2HGDH and L2HGDH in cells without IDH1/2 mutations. Using this approach, we found that under conditions of oxygen limitation, cells potently and selectively produce L-2HG via enzymatic reduction of alpha-KG. Hypoxia-induced L-2HG production is not mediated by IDH1 or IDH2, but instead appears to occur via promiscuous substrate usage by lactate dehydrogenase A (LDHA), and to a lesser extent, malate dehydrogenase 2 (MDH2). Hypoxic induction of L-2HG increases repressive histone modifications, including H3K9me3 and H3K27me3, chromatin marks associated with an undifferentiated cellular state. Thus, L-2HG appears to function as a metabolic signaling intermediate, translating information about oxygen availability into epigenetic modifications that might prevent a cell from undergoing differentiation when it lacks the capacity for oxidative metabolism. As hematopoietic stem cells (HSCs) reside in hypoxic niches, oxygen-regulated L-2HG might influence self-renewal of HSCs and represent a physiologic system exploited by IDH1/2 mutant-derived D-2HG in leukemia.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.
Iron Overload in the Face of Anemia
Authors: Shah Yatrik 
Published: October 2019

Abstract
Several distinct congenital disorders can lead to tissue-iron overload with anemia including β-thalassemia and sickle cell disease. We show that intestinal absorption of iron is highly increased and significantly contributes to tissue iron accumulation in these disorders. The present work describes a novel pathway by which oxygen sensing transcription factors are highly upregulated in iron overload anemias and are subsequently essential for the increase intestinal iron absorption. Oxygen signaling is mediated through well-conserved hypoxia driven transcription factors, hypoxia-inducible factor (HIF)1a and HIF2a. In the intestine, HIF2a directly activates divalent metal transporter 1 (DMT1), duodenal ferric reductase (DcytB), and Fpn1, which are iron transporters critical for adaptive changes in iron absorption. We demonstrate that HIF2a and its downstream target gene, DMT1 are essential for iron accumulation in mouse models of β-thalassemia and sickle cell disease. Furthermore, studies of thalassemic mouse model with established iron overload demonstrated that loss of intestinal HIF2a and DMT1 signaling led to decreased tissue iron accumulation in the livers. Interestingly, disrupting intestinal HIF2a not only improves tissue iron accumulation, but a marked improvement of anemia was also observed. These novel findings suggests that inhibition of HIF2a signaling pathway could be a novel and robust treatment strategy for several conditions that cause iron overload with anemia.

Disclosures
No relevant conflicts of interest to declare.
The Somatic Cancer Genome
Authors: Golub Todd R. 
Published: October 2019

New genome sequencing technologies have led to an explosion of cancer genome sequence information, resulting in new insights into the genetic basis of cancer. This talk will review recent advances in somatic cancer genome analysis, with particular emphasis on hematological malignancies including diffuse large B-cell lymphoma and multiple myeloma. New analytical approaches have led to both a refined ability to distinguish true drivers of cancer from mere passenger mutations and to a better understanding of what scale of cancer genome sequencing will be required to “complete” a cancer genome atlas of mutations. While many of the recurrently mutated genes in cancer have known function, many remain uncharacterized. As such, a new kind of follow-up research activity is required – one that can bring systematic, high throughput biology methods to bear on elucidating mutant allele function and their potential as therapeutic targets. Efforts to pilot such a “Target Accelerator” will be presented, with particular emphasis on use of the Connectivity Map/Library of integrated cellular signatures (LINCS) resource to systematically connect mutant alleles to pathways and to potential therapeutic strategies.

Disclosures
Golub: Foundation Medicine, Inc.: Consultancy, Equity Ownership.
Inhibition of the NAMPT-Triggered Deacetylation of HCLS1 Protein: A New Therapeutic Option in Chronic Myeloid Leukemia
Authors: Samareh Bardia , Zimmer Inna , Zikic Andrew , Klimenkova Olga , Loghmani-Khouzani Houra , Suttorp Meinolf , Kanz Lothar , Welte Karl , Skokowa Julia 
Published: October 2019

Abstract
G-CSFR-triggered phosphorylation and activation of the hematopoietic-specific lyn-substrate 1 (HCLS1) protein is important for the granulocytic differentiation of hematopoietic stem cells (HSCs) (Skokowa J. et al., Nat Med 2012). G-CSF also activates protein deacetylation via Nicotinamide phosphoribosyltransferase (NAMPT)/NAD+/sirtuin 1 (SIRT1) pathway (Skokowa J. et al., Nat Med 2009). SIRT1 is NAD+-dependent protein deacetylase playing an important role in the regulation of gene transcription. Interestingly, having strong pro-differentiation effects on myeloid progenitors, both HCLS1 and NAMPT are markedly hyper-activated in myeloid leukemias, inducing proliferation and survival of undifferentiated blasts.
Knowing this, we investigated whether HCLS1 could be activated by NAMPT/NAD+/SIRT1 pathway via deacetylation in HSCs. Indeed, we found that HCLS1 is deacetylated in HSCs by NAMPT/SIRT1 and that G-CSF treatment of these cells induced deacetylation and phosphorylation of HCLS1 via NAMPT/SIRT1. Moreover, we found that deacetylation of HCLS1 is important for myeloid differentiation and proliferation of HSCs.
We further demonstrated that HCLS1 protein was activated by deacetylation in blasts of chronic myeloid leukemia (CML) patients, as compared to HSCs of healthy individuals. This was in line with elevated mRNA and protein levels of NAMPT and HCLS1. We further investigated, whether inhibition of NAMPT-triggered deacetylation of HCLS1 has any effect on the survivial and differentiation of CML blasts in vitro. We found that treatment of CML blasts with the specific NAMPT inhibitor FK866 resulted in increased acetylation of HCLS1. In addition, treatment of CML blasts with the FK866 in a dose of 10nM resulted in marked inhibition of proliferation and elevated cell death, which was consistent between different patients and was comparable to the effects of Imatinib treatment. In CFU assay in the presence of FK866 (10nM) no CFU colonies were grown. Even at a dose of 3nM FK866 drastically suppressed total colony counts. This inhibitory effect was further amplified by combination of FK866 with Imatinib. Addition of 10nM of FK866 to CFU culture medium was not toxic for CD34+ cells of healthy donors.
Taken together, we demonstrated that NAMPT and HCLS1 have dose-dependent effects on hematopoietic cells and that NAMPT activates HCLS1 functions by SIRT1-triggered deacetylation. Inhibition of NAMPT in CML blasts markedly reduced their survival, proliferation and differentiation. In conclusion, we suggest a possible therapeutic application of NAMPT inhibition in the treatment of CML patients via deactivation of HCLS1 function by acetylation. This therapy could be applied in combination with Imatinib, or in Imatinib-resistant CML patients.

Disclosures
No relevant conflicts of interest to declare.
Qbleed Score in an African American Cohort
Authors: Benyounes Amin , Candelario Nellowe , Braitman Leonard , Leighton John C. , Tester William , Varadi Gabor 
Published: October 2019

Abstract
Introduction/Background:
The Qbleed score is an algorithm that calculates the absolute risk of gastrointestinal and intracranial bleed in patients on anticoagulation that was published in the British Medical Journal in July 2014.
It was developed using a UK general practice database. African Americans were not represented in the development of the algorithm. We intended to investigate if the score is applicable to African Americans.
Materials and Methods:
Our retrospective cohort study was conducted at Albert Einstein Medical Center in Philadelphia, USA. The study's duration was 7 years between 1 January 2007 and 31 December 2014. Inclusion criteria to our study were African Americans admitted to the hospital for gastrointestinal bleeding (GIB) event requiring an esophagogastroduodenoscopy (EGD) or for an intracranial hemorrhage (ICH) event. Study participants on anticoagulants were identified by chart review. The Qbleed score of African Americans on anticoagulation admitted for GIB and ICH was calculated at the time of study entrance and compared with a control group; the control group was African Americans on anticoagulation that did not experience any of the above bleeding events.
Results:
1288 study participants were admitted for a GIB event requiring EGD. Of these, 107 (8.3%) were on anticoagulants.
The average Qbleed value in this group was 1.36 compared to 0.78 in the control group. On average, this group had 0.57 higher Qbleed score than controls (95% confidence interval 0.13 to 1.01, p=0.01). The receiver operating curve statistic value was 0.6698 (95% confidence interval 0.60 to 0.74).
1169 study participants were admitted for an ICH event. Of these 59 (5%) were on anticoagulants.
The average Qbleed value in this group was 0.85 compared to 0.48 in the control group. On average, this group had 0.36 higher Qbleed score than controls (95% confidence interval 0.16 to 0.57, p=0.0004). The receiver operating curve statistic value was 0.65 (95% confidence interval 0.56 to 0.74).
Conclusion:
In our African American cohort the absolute risk of gastrointestinal and intracranial bleeding calculated by the Qbleed score was significantly higher in patients who experienced a bleeding event. Our study suggests that the application of the Qbleed score to African Americans allows the identification of patients with higher risk of bleeding while on anticoagulation.

Disclosures
No relevant conflicts of interest to declare.
Upregulation of Indoleamine 2,3-Dioxygenase Enzymes in Leukemic Mesenchymal Stromal Cells (MSCs) Can Influence MSC/Acute Myeloid Leukemia Cell Cross Talk
Authors: Ciciarello Marilena , Corradi Giulia , Baldazzi Carmen , Testoni Nicoletta , Lemoli Roberto Massimo , Cavo Michele , Curti Antonio 
Published: October 2019

Abstract
Mesenchymal stromal cells (MSCs), an essential element of both normal and leukemic hematopoietic microenvironment, are multipotent cells with a unique immune-modulating ability. Thus, MSCs play a crucial role for both the proliferation and differentiation of hematopoietic stem cells (HSCs) and induce an immune-tolerant milieu. Indoleamine 2, 3-dioxygenase (IDO1 and IDO2) enzymes catabolize tryptophan to kynurenines and play a key role in the induction of immune tolerance in different settings, including acute myeloid leukemia (AML). Furthermore, IDO1/IDO2 pathway is a well described mechanism by which MSCs exert their mmunomodulatory properties. We hypothesized that: 1) MSC-dependent mechanisms are involved in leukemia initiation, maintenance and progression; 2) the expression of IDO1 and IDO2 by MSCs is part of a MSC-dependent mechanism able to create a tumor-supportive milieu. To this aim, we isolated MSCs from the bone marrow of AML patients (AML-MSCs) at diagnosis. We first analyzed their phenotypic and functional properties compared to that of healthy donor-derived MSCs (HD-MSCs). We found that AML-MSCs showed a reduced proliferative capacity but normal immunophenotype, differentiative and immunomodulatory capacity as compared to HD-MSCs. Furthermore, AML-MSCs did not show the chromosomal abnormalities identified in the primary blast counterpart (FISH analysis). We next investigated IDO1/2 expression and functions in MSCs. We demonstrated that IDO enzymes are expressed in AML-MSCs as well as in HD-MSCs. IDO1 is efficiently upregulated by different inflammatory stimuli, and IDO1 protein expression parallels mRNA in both HD-MSCs and AML-MSCs. Interestingly, IDO2 mRNA is expressed at low basal level in all analyzed conditions in HD-MSCs, while it is upregulated, in particular after IFN-gamma stimulation, in AML-MSCs, although the level of induction varies between different patients. When T-cell proliferation was tested in MSC co-cultures, w/or w/out IDO1/2 inhibitor, 1-methyltryptophan, we found that MSC immunomodulatory potential is IDO-dependent both in HD-MSCs and AML-MSCs. Finally, we found that in co-culture assay with primary AML blasts, MSCs stimulated blast proliferation and this effect is, at least in part, IDO-mediated. These data suggested that IDO enzymes, in particular IDO2, may be differentially expressed in AML-MSCs as compared to HD-MSCs and IDO inhibition has an impact on MSC/AML cell cross talk. These findings may help to discover novel niche-target prognostic/therapeutic factors and to provide novel applications for drugs already under active clinical investigation (i.e. IDO-inhibitors).

Disclosures
Cavo: Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.