bluuebunny/crossref_metadata_2025 huggingface dataset:
ADAR1 Is Essential For Erythroid Development
Authors: Liddicoat Brian , Hartner Jochen , Chalk Alistair , Lu Jun , Orkin Stuart H. , Walkley Carl R
Published: October 2019

Understanding the mechanisms controlling erythroid differentiation may allow therapeutic regulation of erythropoiesis. Focus so far has concentrated on the roles of transcription factors in determining erythroid fate, however one mechanism of gene regulation that has been poorly explored is that of direct modification of RNA. It is now becoming apparent that RNA sequences can be widely modified by RNA editing. Adenosine-to-Inosine (A-to-I) RNA editing is a posttranscriptional process resulting in sequence alterations to RNA transcripts, which is catalyzed by members of the adenosine deaminase acting on RNA (ADAR) family of enzymes. We have previously reported that targeted disruption of ADAR1 in hematopoietic stem cells (HSC) resulted in a global upregulation of type I and II interferon-inducible transcripts and rapid apoptosis (Hartner et al., Nature Immunology 2009). This study identified that ADAR1 is essential for maintenance of both the fetal and adult HSC compartment in a cell-autonomous fashion. Although ADAR1 is dispensable for myeloid development and B-lymphopoiesis, investigation of its role in erythropoiesis is granted owing to the severe loss erythrocytes in ADAR1 null mice. To determine the role of ADAR1 in erythropoiesis, we generated mice with an erythroid-restricted deletion of ADAR1 (EpoR-Cre Adar1fl/-). These animals were found to die in utero at ∼E14.5. The fetal liver (FL) was small and had significantly lower cellularity than in control littermates. Analysis of FL erythropoiesis demonstrated increased apoptosis and a loss of cells after the phenotypic polychromatic erythroblast stage of erythroid differentiation. When transplantation studies were done with EpoR-Cre Adar1fl/- FL and control EpoR-Cre Adar1fl/+ FL, recipients of EpoR-Cre Adar1fl/- FL could not restore erythropoiesis and were anemic in contrast to the controls. They also developed splenomegaly suggestive of extramedullary hemopoiesis occurring as a compensatory mechanism. To understand the mechanism through which ADAR1 regulates erythroid development, gene expression arrays and miRNA profiling were performed. As with HSCs, loss of ADAR1 caused a significant upregulation of IFN signatures in EpoR-Cre Adar1fl/- FL. Interestingly, despite evidence related to regulation of miRNA function by ADARs, there were only subtle changes to the expression pattern and levels of miRNAs in Adar1-deficient erythroid cells. These results demonstrate that ADAR1 is an essential in vivo mediator of erythroid cell fate and a critical negative regulator of the IFN response in cells specifically committed to erythropoiesis.

Disclosures:

Hartner: TaconicArtemis: Employment.
Iron Chelation Therapy Could Rapidly Reduce Oxidative DNA Damage In CD34+ Hematopoietic Cells Before Decrease Of Serum Ferritin Level
Authors: Kobune Masayoshi , Kikuchi Shohei , Iyama Satoshi , Takada Kohichi , Murase Kazuyuki , Ono Kaoru , Horiguchi Hiroto , Kamihara Yusuke , Hashimoto Akari , Tatekoshi Ayumi , Hayashi Tsuyoshi , Sato Tsutomu , Miyanishi Koji , Sato Yasushi , Takimoto Rishu , Kato Junji
Published: October 2019

Introduction

Myelodysplastic syndrome (MDS) is characterized by dysplastic and ineffective hematopoiesis, peripheral blood cytopenias and a risk of leukemic transformation. Most MDS patients eventually require red blood cell (RBC) transfusions for anemia and consequently develop iron overload. Excess free iron in cells catalyzes generation of reactive oxygen species (ROS) especially hydroxyl radical that cause oxidative DNA damage including formation of mutagenic 8-hydroxy-2'-deoxyguanosine (8-OHdG) and DNA double strand breaks. However, it is unclear whether iron-mediated oxidative stress affects the pathophysiology of MDS.

Methods

This study included MDS patients who visited our university hospital and affiliated hospitals (n=43). Among them, 13 patients received iron chelation therapy when their serum ferritin (SF) level was greater than 1000 ng/mL or they required more than 20 RBC transfusions (or 100 mL/kg of RBC). We analyzed hemoglobin, SF level and chromosomal abnormality and 8-OHdG levels in peripheral blood mononuclear cells (PBMC) obtained from MDS patients before and after iron chelator, deferasirox administration.

Results

We showed that reactive oxygen species (ROS) in hematopoietic cells was higher in MDS patients with high SF level. Furthermore, the 8-OHdG levels in MDS patients were significantly higher than those in healthy volunteers and the 8-OHdG levels were positively correlated with SF (p=0.003, r=0.495) and chromosomal abnormalities (p=0.019). Importantly, the 8-OHdG levels in PBMC and CD34+ cells of MDS patients dramatically decreased 3 month after deferasirox administration (the 8-OHdG levels in PBMC, 3.374±78, p<0.05) although SF level were not reduced within 3 month (Figure). However, SF level decreased to 500-600 ng/mL after 36 months of iron chelation therapy, 8-OHdG is a sensitive marker of cellular iron chelation therapy by deferasirox which readily passes through the plasma membrane. When patients were divided into the no Iron chelation group (n=10) and high Iron chelation group (n=13), the survival time was significantly longer in the latter group than the former group (P=0.046) although International prognostic scoring system (IPSS) was not significantly difference between both groups. Further, 50% of patients without iron chelation group developed into leukemia, while no patients did progress into leukemia during 36 month.

Conclusion

These results indicated that cellular excess iron could contribute to the pathophysiology of MDS and iron chelation therapy could improve the oxidative DNA damage in MDS patients.

Disclosures:

No relevant conflicts of interest to declare.
The Mutational Profile of Plasma Cell Disorders
Authors: Futreal Andy
Published: October 2019

Multiple myeloma is a plasma cell malignancy that is the second most common hematologic cancer. The genetics of the disease have been previously characterized by two major subtypes involving recurrent translocations of the immunoglobulin heavy chain, c-MAF, cyclin D (CCND1) and FGFR3/MMSET oncogenes, and the hyperdiploid group with multiple recurrent trisomies. Further, there are characterized cytogenetic/ploidy subsets that appear to carry prognostic significance. Specifically, gains of chromosomes 5, 9, 11, 15, and 19 confer a good prognosis whilst gain of 1q plus deletions of 1p, 12p, 14q, 16q, and 22q confer a poor prognosis. More recently the application of massively parallel sequencing has revealed further insights into the genetic architecture of multiple myeloma. Work from several groups has identified the contribution of mutations in genes involved in non-canonical NF-kB signaling, MAPK signaling and histone methylation, as well as infrequent mutation of cancer genes implicated in other tumor types and novel genes. These data will be presented to give an overall update on the current state of multiple myeloma genetics and our current level of insight in the genomic complexity that characterizes this disease.

Disclosures:

No relevant conflicts of interest to declare.
Hematology from the Ground Up: Lessons from C. elegans
Authors: Hamza Iqbal
Published: October 2019

Heme is a vital cofactor responsible for diverse biological functions such as gas synthesis and sensing, circadian clock control, microRNA processing, and xenobiotic detoxification. Heme synthesis occurs in the mitochondria in eukaryotes. Free heme is a hydrophobic and cytotoxic macrocycle. How then is heme transported through cellular membranes and organelles? What are the mechanisms for incorporating heme into specific hemoproteins that reside in the cytoplasm, peroxisomes, mitochondria, secretory pathway, and nucleus? Our work with the invertebrate animal model Caenorhabditis elegans has demonstrated that this roundworm is exceptional because it does not synthesize heme but rather utilizes environmental heme to manufacture heme-containing proteins that have human homologs. Genomic screens in C. elegans identified several novel genes which we termed Heme Responsive Genes (HRGs). HRG-1, the first metazoan heme importer/transporter, is a permease with four transmembrane domains, and is conserved in vertebrates. Depletion of hrg-1 paralogs in worms causes disruption of organismal heme sensing. Zebrafish with lower levels of hrg1 reveals hydrocephalus, yolk tube malformations, and profound defects in erythropoiesis. Hrg1 is strongly expressed in macrophages of the reticuloendothelial system in human and mouse tissues and is upregulated in a time- and dose-dependent manner by either erythrophagocytosis (EP) or induction of hemolytic anemia in mice. Hrg1 traffics to the phagolysosomal membranes during EP and depletion of Hrg1 in mouse macrophages causes attenuation of heme transport from the phagolysosomal compartment, concomitant with suppression of heme degradation and iron export. Importantly, studies using yeast, zebrafish, and mouse macrophages reveal that specific missense polymorphisms in Hrg1 are defective in heme transport. To uncover additional molecules in organismal heme homeostasis, we conducted a genome-wide RNAi screen in live, transgenic heme-sensor worms. The screen identified several hundred new candidates that integrate signals from different tissues to regulate systemic heme homeostasis. Our findings represent major discoveries in heme trafficking and establish a paradigm for heme transport in animals.

Disclosures:

Hamza: Rakta Therapeutics: CEO Other, Consultancy.
All Roads Lead to Rome: Phenotypic Channeling to Uniform Differentiation Structures from Diverse Leukemia Genotypes by Mass Cytometry
Authors: Nolan Garry
Published: October 2019

Emerging single-cell technologies have been pivotal in uncovering an extensive degree of heterogeneity between and within tissues (1). Analysis of single-cell data has shed light on many different cellular processes (2-7) and recent technological advances have enabled the study of a large number of parameters in single cells at unparalleled resolution. One such technology, mass cytometry (8), can measure up to 45 parameters simultaneously in tens of thousands of individual cells. Using mass cytometry and genomic sequencing of conventionally sorted subpopulations show that acute myelogenous leukemia (AML) in a given patient can simultaneously occupy multiple stages of differentiation. Occupation of these stages was correlated with the presence, or absence, of unique exonic mutation fingerprints. In another cancer, B-cell acute lymphoblastic leukemia (ALL), outgrowth of tumor at pro and pre-B cell stages was nearly always uniquely at a single stage - contrary to the results in AML. This suggests that evolutionary “niche” searching is not only for physical space in cancers, but also involves utilization of differentiation machinery as an additional elaboration mechanism. Each differentiation stage in both AML and B-cell ALL was characterized by utilization of cognate signaling networks which showed differential susceptibility to drug action. Using such deep profiling and signaling delineation approaches at the single-cell level will allow for fine structured indexing of patient disease and further tailoring of disease management. In addition, it will allow “heterogeneous” tumors to be organized by a maturation index associated with a granular catalog of mutations that drive cells to occupy these pseudo-differentiation niches.

1. Bendall, S.C., et al., A deep profiler's guide to cytometry.Trends Immunol, 2012. 33(7): p. 323-32.

2. Petilla Interneuron Nomenclature Group, et al., Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex.Nat Rev Neurosci, 2008. 9(7): p. 557-68.

3. Irish, J.M., et al., Single cell profiling of potentiated phospho-protein networks in cancer cells.Cell, 2004. 118(2): p. 217-28.

4. Sachs, K., et al., Causal protein-signaling networks derived from multiparameter single-cell data.Science, 2005. 308(5721): p. 523-9.

5. Majeti, R., C.Y. Park, and I.L. Weissman, Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood. Cell Stem Cell, 2007. 1(6): p. 635-45.

6. Tarnok, A., H. Ulrich, and J. Bocsi, Phenotypes of stem cells from diverse origin.Cytometry A, 2010. 77(1): p. 6-10.

7. O'Brien, C.A., A. Kreso, and J.E. Dick, Cancer stem cells in solid tumors: an overview.Semin Radiat Oncol, 2009. 19(2): p. 71-7.

8. Bandura, D.R., et al., Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Anal Chem, 2009. 81(16): p. 6813-22.

Disclosures:

No relevant conflicts of interest to declare.
The Role of Chromatin Factors in Hematopoietic Development
Authors: Zon Leonard I.
Published: October 2019

The initiation of blood-specific programs is orchestrated by key transcription factors. To generate a complete compendium of chromatin factors that establish the epigenetic code during developmental hematopoiesis, a large-scale reverse genetic screen was conducted targeting orthologs of 425 human chromatin factors in zebrafish. A set of chromatin regulators was identified that function at distinct steps of primitive and definitive blood formation, including factors not previously implicated in blood development. We identified 15 factors that regulate development of primitive erythroid progenitors and 29 factors that regulate development of definitive stem and progenitor cells. These chromatin factors are associated with SWI/SNF and ISWI chromatin remodeling, SET1/MLL methyltransferase, CBP/P300/HBO1/NuA4 acetyltransferase, Sin3A/NuRD deacetylase, and Polycomb repressive complexes. Knockdown of a class of chromatin factors led to an expansion of hematopoietic stem cells (HSCs). In collaboration with Nancy Speck’s laboratory, we have investigated the activity of one of these chromatin factors, CHD7, that led to an expansion of hematopoietic stem and progenitor cells in the aorta. Using a T-cell line, a CBFβ protein pulldown, and a mass spectrometric sequencing approach led to the finding that CBFβ immunoprecipitated RUNX1 and CHD7. By studying a conditional allele of murine Chd7, inactivation does not have an effect on peripheral blood counts, but Chd7-deleted HSCs purified based on phenotypic markers contain an increased number of functional HSCs. Our studies suggest that CHD7 suppresses hematopoiesis, and provides a novel control mechanism for the regulation of HSCs. Our work provides a comprehensive view of how specific chromatin factors and their associated complexes play a major role in the establishment of hematopoietic cells in vivo.

Disclosures:

Zon: Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.
TGF-Beta Signaling Favors Central Memory Phenotype Expression By Ex-Vivo Stimulated Human T Cells
Authors: Dahmani Amina , Carli Cédric , Taillefer Julie , Goupil Mathieu , Khalili Myriam , Delisle Jean-Sebastien
Published: October 2019

Adoptive immunotherapy using ex vivo differentiated and expanded T cell lines can be remarkably efficient to treat cancer and infections. Unfortunately, the process of ex vivo T cell stimulation can lead to terminal effector differentiation and functional exhaustion thereby limiting the persistence and therapeutic effects of these T cells after transfer. Accumulating evidence suggests that, owing to their proliferative capacity, self-renewal ability and long term persistence in vivo, T cells bearing a central memory (CD45RO+/CD62L+ - Tcm) instead of an effector memory (CD45RO+/CD62L- - Tem) phenotype before adoptive transfer can mediate more significant therapeutic activity. Transforming-growth factor-beta (TGF-β) is a pleiotropic cytokine that influences several aspects of T-cell biology and that is best known for its growth suppression and immunosuppressive activity. We show that TGF-β signaling can have a profound impact on the number of CD4 and CD8 T cells expressing the Tcm and Tem phenotype after anti-CD3e and anti-CD28 stimulation without altering the number of cells recovered at the end of the culture and without inducing regulatory T cells. By enhancing the percentage of the lymph-node homing receptors, L-selectin (CD62L) and CC-chemokine receptor 7 (CCR7) expressing cells, exogenous TGF-β, added to the culture medium to a concentration of 5 ng/ml, favors Tcm over Tem cell accumulation at 7 days (CD4+: 55.80 vs 35.51% (P= 0.0074); CD8+: 56.9 vs 40.3% (P= 0.0063)). Reciprocally, the inhibition of TGF-β signaling with a TGF-β receptor kinase inhibitor (GW788388) accentuated Tem phenotype acquisition. Importantly, these effects of TGF-β on Tcm marker expression were maintained in the presence of exogenous cytokines commonly used in ex-vivo cultures for adoptive immunotherapy (IL-2, IL-7 and IL-15). The manipulation of TGF-β signaling did not increase the expression of exhaustion markers (KRLG-1, CD57) but exogenous TGF-β decreased interferon-gamma (IFN-γ) expression. No effect was noted on TNF-α and IL-2 expression as well as on the percentage of polyfunctional cells generated. We also found that modulating TGF-β signaling during the course of clinically relevant cultures capable of expanding T-cells specific for Epstein-Barr virus LMP2 protein antigens, in the presence of IL-7 and IL-15, could increase the number of CD4 Tcm cells, even 2 weeks after the withdrawal of TGF-β from the culture (38.24 vs 27.82, N=3) without compromising antigen-specific IFN-γ release. In conclusion, the modulation of TGF-β signaling can significantly alter Tcm and Tem phenotype acquisition and may therefore be used to optimize Tcm phenotype expression by ex-vivo pathogen/antigen-specific T cells expanded for adoptive immunotherapy.

Disclosures

No relevant conflicts of interest to declare.
Preliminary Report of a Multicenter Prospective Phase II Study of DA-EPOCH-R in MYC-Rearranged Aggressive B-Cell Lymphoma
Authors: Dunleavy Kieron , Fanale Michelle , LaCasce Ann , Noy Ariela , Caimi Paolo , Parekh Samir , Hayslip John W , Jagadeesh Deepa , Lord Raymond S , Lechowicz Mary J , Gaur Rakesh , Lucas Andrea , Staudt Louis M , Steinberg Seth M. , Kahl Brad , Friedberg Jonathan W. , Little Richard F , Bartlett Nancy L , Wilson Wyndham H
Published: October 2019

Background: In diffuse large B-cell lymphoma (DLBCL), a MYC-rearrangement (MYC-R) is present in approximately 10% of cases and has been associated with an inferior outcome following R-CHOP chemotherapy. In a previous report that retrospectively analyzed the prognostic impact of a MYC-R on survival following DA-EPOCH-R, patients with a MYC-R had a similar outcome to MYC negative cases (4 year EFS of 83% versus 76% respectively: p=0.46 Ann Oncol 2011 (22) Suppl 4 # 71). We set out to prospectively validate these results in a multicenter study.

Methods: Patients were newly diagnosed with DLBCL or B-cell lymphoma unclassifiable (BCL-U) with features intermediate between DLBCL and Burkitt lymphoma (BL). Only cases that harbored a MYC translocation by fluorescent in-situ hybridization (FISH) or conventional cytogenetic testing were included in this report. Treatment consisted of 6 cycles of DA-EPOCH-R.

R esults: 52 patients were included in this preliminary analysis. Characteristics were median (range) age 61 (29-80) years; male sex 37 (71%); stage III or IV disease 38 (73%); IPI score 0-2 in 35% versus 3-5 in 65%; HIV positive 4 (7%). Histologic diagnosis was DLBCL in 45 (86%) and BCL-U in 7 (14%). All cases had a MYC-rearrangement. BCL2 was rearranged in 14/31 (45%) and overexpressed by IHC in 24/43 (56%) cases tested. There were 3 deaths secondary to infectious complications and otherwise toxicities were similar to previous reports of the regimen. At a median follow-up time of 14 months, progression-free survival (PFS), time to progression (TTP) and overall survival (OS) were 79%, 86% and 77% respectively for all patients. PFS was 87% and 64% in cases that were FISH positive (double-hit) and IHC positive for BCL2 respectively.

Conclusions: Albeit short follow-up, DA-EPOCH-R in MYC-R DLBCL demonstrates promising activity in a multicenter prospective setting. Further analysis of this data is planned with central pathology review of cases and longer follow-up. The principal arm of this study testing the regimen in BL remains open to accrual (NCT01092182).

Figure 1 Figure 1.

Disclosures

LaCasce: Seattle Genetics, Inc.: Research Funding.
The Combination of Belinostat with Zidovudine for Treatment of HTLV-I Related Adult T-Cell Leukemia-Lymphoma
Authors: Toomey Ngoc , Ramos Juan Carlos
Published: October 2019

Adult T-cell leukemia-lymphoma (ATLL) is an aggressive malignancy with poor prognosis that is incurable by conventional drugs. Histone deacetylase (HDAC) inhibitors (HDIs) are broadly active anti-neoplastic agents that can be cautiously exploited in the treatment of ATLL. The HTLV-I provirus is clonally integrated in virtually all ATLL cells. The HTLV-I 5'LTR promoter is trans -activated by the viral protein Tax through binding of CREB and p300/CBP. Tax binding enhances p300/CBP histone acetyl transferase (HAT) activity resulting in histone acetylation, chromatin unwinding, and transcription of the viral genome. HAT activity is countered by HDACs, and also by the viral-encoded protein HTLV-I bZip factor (HBZ), which is transcribed from the negative proviral strand at the 3' end. The 5' LTR is often mutated or deleted in ATLL cells thus HBZ is the only viral gene consistently expressed in ATLL. We hypothesized that HDAC inhibitors, which can be potent anti-neoplastic drugs, would reactivate HTLV-I in ATLL tumors containing intact provirus, thus killing virus-infected cells and provoking an immune response in vivo. Supporting this notion, adding the old generation HDI valproic acid (VPA) to AZT/IFNa in one subject suffering from aggressive acute-type ATLL resulted in HTLV-I proviral load reduction, followed by a complete molecular and prolonged clinical remission that lasted several years. We recently tested other more potent HDIs on primary ATLL cells and low-passage cell lines, which are rare and difficult to establish. In general, HDIs induced dose-dependent Tax expression and apoptosis in ATLL cells. Belinostat, a pan HDI, caused H3 acetylation, blocked HBZ protein expression, and induced Tax protein expression in ATLL cells resulting in concomitant apoptosis. ATLL cells exhibit high constitutive expression of nuclear factor kappa B (NF-kB), which is known to be activated by Tax and to play a major role in HTLV-I mediated T-cell transformation and ATLL development. Paradoxically, while belinostat induced Tax, it also inhibited NF-kB nuclear activity resulting in apoptosis. Further, adding AZT to belinostat augmented ATLL cell death. By contrast, IFNα diminished apoptosis induced by belinostat in freshly isolated ATLL cells from some of our patients. Based on these preclinical data, we have developed a clinical trial using belinostat in combination with AZT as consolidation therapy for ATLL. Our primary goal using this approach is to eradicate residual ATLL cells and HTLV-I infected reservoirs that normally persist regardless of treatment.

Disclosures

No relevant conflicts of interest to declare.
Efficacy and Bleeding Risk of Newer Anticoagulants Compared to Conventional Treatment in Patients with Chronic Kidney Disease- a Meta-Analysis of Randomized Controlled Trials
Authors: Sundararajan Srinath , Poongkunran Mugilan , Poongkunran Chithra , Kannan Arun , Balamuthusamy Saravananan
Published: October 2019

Background

Multiple newer oral anticoagulants (NOAC) are currently approved for the prevention, treatment of venous thromboembolism (VTE) and prevention of stoke in atrial fibrillation (AF). Most of the clinical trials with NOAC have excluded patients with severe renal insufficiency. There is a paucity of safety and efficacy data of NOAC in comparison with conventional treatment (CT) in patients with mild or moderate renal impairment. We performed a meta-analysis of the randomized control trials (RCTs) comparing NOAC to CT in patients with renal insufficiency to understand the safety and efficacy of NACs.

Methods

Medline, PubMed, EMBASE, EBSCO, CINAHL and Cochrane databases were queried with the keywords - "newer anticoagulants", "renal insufficiency", "dabigatran", "rivaroxaban", "apixaban", and "edoxaban". RCTs comparing NOAC to CT (warfarin, low molecular weight heparin, heparin, aspirin or placebo) were included. Non-randomized single arm trials were excluded. Authors of RCTs with missing pertinent data were contacted through electronic mail. The safety end point analyzed was clinically significant or major bleeding risk. The efficacy end points analyzed were incidence of arterial embolism in AF studies and venous thromboembolism in VTE studies. European Medical Agency classification of renal insufficiency was used, with mild renal insufficiency defined as estimated glomerular filtration rate (eGFR) from 50 to 79 and moderate renal insufficiency defined as eGFR of 30 to 49 and severe renal insufficiency defined as eGFR< 30.

Results

Sixteen RCTs with 115,147 patients fulfilled our inclusion criteria. Rivoroxaban, apixaban, edoxaban and dabigatran were the newer anticoagulants used in these 16 RCTs. Among these, 48,403 patients had either mild or moderate renal insufficiency. Patients with mild renal insufficiency treated with NAOC had lower bleeding risk [Odds ratio 0.81(CI 0.72-0.90) p<0.0001], lower incidence of arterial thromboembolism [Odds ratio 0.74(CI 0.64-0.85) p<0.0001] compared to conventional treatment. Patients with moderate renal insufficiency treated with NAOC had lower incidence of arterial thromboembolism [Odds ratio 0.74(CI 0.62-0.89) p=0.001] and a trend towards lower bleeding risk [Odds ratio 0.89(CI 0.66-1.18) p=0.41], compared to conventional treatment. Also, a trend of lowered VTE incidence was observed with NOAC compared to conventional treatment in patients with mild [Odds ratio 0.70(CI 0.46 to 1.070) p=0.1] as well moderate [Odds ratio 0.69(CI 0.34 to 1.41) p=0.31] renal insufficiency.

Conclusion:

Newer anticoagulants have decreased odds for VTE, arterial embolism and bleeding in patients with mild to moderate renal insufficiency compared to conventional treatment.

Disclosures

No relevant conflicts of interest to declare.
Involvement of the EGF Domain in the Formation of the Human Platelet Alloantigen, HPA-1a
Authors: Zhi Huiying , Lertkiatmongkol Panida , Curtis Brian R , Newman Peter J.
Published: October 2019

The human platelet alloantigen, HPA-1a (also known as PlA1), is most frequently responsible for several clinically important alloimmune platelet disorders, including neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura, and less frequently, platelet transfusion refractoriness. A single C29523T nucleotide substitution, resulting in a Leu33Pro amino acid polymorphism within PSI domain of the integrin β3 subunit, controls the expression of the HPA-1a/HPA-1b alloantigenic epitopes. Small linear or cyclic synthetic peptides encompassing amino acid 33, however, fail to bind human anti-HPA-1a alloantibodies, leading to the notion that the HPA-1a antigenic determinant is conformationally dependent, and may require noncontiguous regions of the polypeptide chain that are linearly distal to polymorphic residue 33. This concept is supported by the finding that antibodies from some alloimmunized women fail to recognize a Cys435Ala mutant form of β3, and by the observation that the PSI domain of β3 is immediately adjacent in the crystal structure of β3 to the first epidermal growth factor (EGF1 - residues 436-472) domain of the molecule. The purpose of the present investigation, therefore, was to localize the amino acid(s) within EGF1 that contribute to the HPA-1a alloantibody binding interface. Two complementary approaches were employed. In the first, site-directed mutagenesis was used to introduce four amino acid substitutions (T30A, S32P, Q33L, and N39D) into mouse β3 cDNA that have previously been shown to be necessary for reconstituting the human anti-HPA-1a epitope into the murine protein. The resulting mouse β3 integrin subunit containing this "humanized" PSI domain (termed APLDmβ3) was able to bind both the murine HPA-1a-selective monoclonal antibody, SZ21, as well as human maternal anti-HPA-1a antisera. Structural analysis indicates that S469 and Q470 at the C-terminus of EGF1 are immediately adjacent to Leu33 in human β3. Ser469 is conserved in the mouse, however residue 470 is a methionine in the murine protein. Based on these observations, we introduced an additional M470Q amino acid substitution within APLDmβ3 to produce APLDQmβ3. As predicted, binding of mAb SZ21 and human anti-HPA-1a alloantisera toHEK293 cells expressing an aIIb/APLDQmβ3 complex was increased ~30% over that which bound to cells expressing similar levels of aIIb/APLDmβ3. Conversely, substitution of M for Q at position 470 within human β3 resulted in a marked reduction in the ability of SZ21 and human anti-HPA-1a alloantisera to bind. Taken together, ourresults demonstrate that this region of the conformationally close EGF1 domain contributes importantly to the expression of the HPA-1a alloantigenic determinant. These findings may have important implications for diagnostic and therapeutic use of synthetic or recombinant HPA-1a mimetics.

Disclosures

No relevant conflicts of interest to declare.
Selective Deletion of ATG5 in Bone Marrow Leptin-Receptor-Expressing Perivascular Stromal Cells Causes Leukopenia
Authors: Arsiwala Shabnam , Ding Wei , Wang Hong-Gang
Published: October 2019

Bone marrow niche cells, specifically the Leptin-receptor-expressing perivascular stromal cells (LepR+), CXCL12-abundant reticular cells (CAR), and endothelial cells play an important role in the maintenance and self-renewal functions of hematopoietic stem cells (HSCs). This maintenance function provided by the niche cells is mediated by a number of secretory-related molecules such as stem cell factor (SCF), CXCL12 and Angiopoietin. In addition, functional autophagy within HSCs is known to play a critical role for maintaining HSC homeostasis, as the loss of ATG7 in HSCs leads to loss of normal HSC functions and severe myeloproliferation. However, it remains unclear as to how hematopoiesis will be affected if autophagy is selectively blocked in bone marrow niche cells. To investigate the effect of autophagy deficiency within bone marrow niche cells on hematopoiesis, we generated a knockout mouse model allowing for a selective ablation of an autophagy-essential protein, ATG5, in LepR+ perivascular stromal cells. We have confirmed that the deletion of Atg5 is indeed restricted to LepR+ cells, and that the loss of ATG5 is sufficient to block autophagic function, as evident through the accumulation of p62 protein. We found that the white blood cell count (WBC) is significantly reduced in 16-week old male ATG5 KO mice compare to wild type (wt) littermate control mice (p<0.05). Further, the myeloid population (CD11b+ Ly6G+) in both bone marrow and peripheral blood is significantly decreased in ATG5 KO mice (p value < 0.05, ATG KO vs. wt control). WBC count continues to decline in 24-week old ATG5 KO male mice (p=0.02) but not in female mice (p=0.58). Both LSK (Lin- Sca-1+ c-Kit+) and long term HSC (LT-HSCs, CD150+ CD48- LSK) populations are decreased in both 16-week and 24-week old male mice; however, this trend did not reach statistical significance (p>0.05). Although the absolute cell count of lymphocytes, including B and T cells (CD19+ and CD3+, respectively) in peripheral blood, is significantly dropped in ATG5 KO mice compared to wt controls, there is no significant change of mature B and T cells in bone marrow. These preliminary results suggest that deletion of a key autophagy regulator in LepR+ bone marrow niche cells will cause leukopenia. The underlying mechanism is currently under investigation and it will help us to better understand the role of autophagy in hematopoiesis within the bone marrow microenvironment.

Disclosures

No relevant conflicts of interest to declare.
Clonal Features of Hematopoietic Stem and Progenitor Cells in Aging
Authors: Sun Jianlong , Camargo Fernando D.
Published: October 2019

It is traditionally thought that Hematopoietic Stem Cells (HSCs) maintain blood homeostasis through long-term self-renewal and multilineage differentiation. This concept, however, is challenged by two recent studies in which the fundamental features of unperturbed hematopoiesis are evaluated by different approaches of lineage tracing. Both the kinetic analysis of HSC output by the Rodewald group and our clonal analysis with transposon barcoding suggest a dominant role of non-transplantable short-term HSCs and progenitors, but not the long-term HSCs, in driving native blood cell production. In addition, our longitudinal analysis of peripheral blood demonstrates extensive clonal succession in granulocyte production. These findings collectively suggest a distinct mechanism of native hematopoiesis that differs significantly from what has been learned in transplantation experiments. At the same time, they bring to light new questions regarding the ultimate fate of the progenitor population and the exact contribution of HSCs under normal physiological conditions. To address these questions, we examined clonal features of HSCs and progenitors in aged mice. Our results show a progressive reduction in clonal complexity and a concurrent increase in clonal stability when blood granulocytes are analyzed up to a hundred ten weeks after transposon barcoding. As time elapses, clonal overlapping between granulocytes and B cells become much more extensive, suggesting an increased tendency toward multilineage differentiation during aging. Analysis of stem and progenitor cells in bone marrow of aged mice reveals prevalent lineage output by multipotent progenitors (MPPs), whereas a lower fraction of HSC clones are found to produce mature progeny. While this overall pattern of differentiation is reminiscent of what has been observed in young and middle-aged animals, a two-fold increase in HSC clonal output was observed in these old mice, indicating their increased contribution to blood cell production. A comparison of clonal compositions in blood and marrow cell populations demonstrates an MPP origin of stable peripheral blood clones, and a smaller fraction of these clones can even be traced back to HSCs. These observations hence suggest extensive self-renewal and asymmetric cell division of these two cell populations in aging. Taken together, our results indicate that the aged hematopoietic system is characterized by reduced clonal complexity, increased clonal persistence, and HSC activation. The higher propensity to self-renewal during aging may also explain the elevated risk of malignant transformation in the elderly population.

Disclosures

Camargo: Cell Signaling Technologies: Consultancy; Vital Therapies: Consultancy.
The Combination of ATRA and Dasatinib for Differentiation Therapy in Acute Myeloid Leukemias with IDH Mutations
Authors: Boutzen Helena , Saland Estelle , Cathebras Mathilde , Larrue Clément , Farge Thomas , Serhan Nizar , De Toni Fabienne , David Marion , Gales Lara , Castelli Florence , Junot Christophe , Delabesse Eric , Veronique Demas , Portais Jean-Charles , Ouerfelli Ouathek , Guangli Yang , Vergez Francois , Recher Christian , Sarry Jean-Emmanuel
Published: October 2019

Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired differentiation programs due to recurrent mutations, amongst which IDH mutations occur in 15% of AML patients. Here, we show both in vitro as well as in a xenografted mouse model,

that clinically achievable doses of ATRA are sufficient to achieve a terminal granulocytic differentiation in primary AML samples and in AML cell lines harboring IDH1-R132H mutation. There is no effect at this concentration on the WT controls. This is associated with reduction of both proliferation and colony formation, and further leads to apoptosis, thereby improving overall survival of mutant xenografted mice.

We further showed, through transcriptomic and western blot analysis, that specific ATRA sensitivity is due to overexpression and activation of C/EBPα in the presence of IDH1-R132H mutation. This primes blasts into myeloid differentiation.

Moreover, IDH1 R132H mutation also reduces LYN activation, and thus, also sensitizes to clinically achievable doses of dasatinib, a LYN inhibitor. As ATRA induces a brief LYN activation, which transiently reduces ATRA activity, its combination with dasatinib synergistically increases differentiation. In vivo, the combination of ATRA and dasatinib reduces tumor growth of mutant xenografted mice.

The combination ATRA and dasatinib might also be considered for other IDH mutations that produce 2-hydroxyglutarate, since treatment with the mutant-specific oncometabolite (eg. 2-hydroxyglutarate) sensitizes AML cells to ATRA and dasatinib-induced differentiation.

Finally, ATRA also reduces BCL2 expression specifically in the presence of IDH1 R132H mutation. Since it has been shown that IDH mutations increase BCL2 dependence in leukemic cells, our results identified a subgroup of patients that is likely to respond to pharmacologic concentrations of ATRA.

To conclude, our data provide the preclinical rationale for investigating the use of the combination ATRA and dasatinib in a subgroup of patients who carry IDH1 R132H mutation, in clinical trials. The addition of a BCL2 inhibitor such as ABT-199 would also be considered.

Disclosures

Off Label Use: ATRA and dasatinib for treatment of non APL AML. Recher:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Chugai: Research Funding.
Evidence for the Role of Biologically Active Exosomes in Acute Myeloid Leukemia Relapse
Authors: Boyiadzis Michael , Hong Chang Sook , Whiteside Theresa L
Published: October 2019

Introduction: Despite of the notable achievements of multi-modality treatments in patients with acute myeloid leukemia (AML), leukemic relapse remains a formidable clinical challenge. Studies in AML patients have shown that tumor-induced impairments of immune cells contribute to leukemia progression. One recently identified immunosuppressive mechanism operating in AML is the release by leukemia blasts of virus-size (30-100nm) membrane-bound vesicles called exosomes. We have reported that plasma of patients with newly-diagnosed AML prior to any therapy contains higher levels of exosomal proteins than that of normal donors. AML exosomes carry an immunosuppressive cargo, including membrane-associated TGF-β1, MICA/MICB and markers of myeloid blasts. We hypothesize that persistence of these exosomes may contribute to the high relapse rates seen in AML.

Methods: Venous blood (20-50 mL) was obtained from patients newly diagnosed with AML prior to any treatment, from AML patients who achieved CR following initial therapy and from age-matched healthy volunteers (NC). Exosome fractions were isolated from plasma by using mini-size exclusion chromatography with Sepharose 2A. Exosome protein levels, numbers and size of exosomes (qNano) and their morphology by transmission electron microscopy were determined. Exosomes were characterized by western blots for expression of exosome markers, Tsg101 and CD81, and myeloid cell-surface markers associated with AML, interleukin-3 receptor alpha chain (CD123) and C-type lectin-like molecule-1 (CLL-1), CD44 and CD96. Isolated normal human NK cells were co-incubated with AML exosomes and multiparameter flow cytometry was used to monitor changes in expression levels (mean fluorescence intensity) of NKG2D on NK cells.

Results: Exosome fractions isolated from AML patients' plasma at diagnosis (n=16) had higher mean protein content than fractions obtained from NC plasma (96 vs 37 µg protein/mL plasma, p=0.03). In exosome fractions isolated from plasma of AML patients in CR (n=13) protein levels remained elevated (mean value=120 µg protein/mL plasma) at the time when leukemic blasts were undetectable in the bone marrow by conventional hematopathological methods. AML exosomes in patients in CR carried blast markers, CD123, CLL-1, CD44, CD96 and were enriched in TGF-β1. Co-incubation of these exosomes with activated NK cells resulted in down-regulation of NKG2D expression with a concomitant reduction of NK-cell cytotoxicity. Ten/13 AML patients in CR whose plasma exosome levels remained elevated eventually relapsed.

Conclusion: The persistently elevated levels of biologically-active TGF-β1+ exosomes carrying leukemia blast markers in plasma of AML patients in CR impair anti-leukemia immune responses and might contribute to leukemia relapse.

Disclosures

No relevant conflicts of interest to declare.
Aberrant Expression of Homeobox Gene SIX1 in Hodgkin Lymphoma
Authors: Nagel Stefan , Meyer Corinna , Kaufmann Maren , MacLeod Roderick , Drexler Hans G.
Published: October 2019

In Hodgkin lymphoma (HL) we recently identified deregulated expression of homeobox gene MSX1 which physiologically controls development of the embryonal neural plate border region. This region forms the neural crest and placodes which in turn give rise to fundamental structures and functions of the head including jaw and sensory organs. Here, we examined in HL another homeobox gene, SIX1, an additional regulator of this embryonal region mediating differentiation of placodal precursors. In silico data (GSE12453) show aberrantly activated SIX1 in 12 % of HL patient samples, indicating a pathological role in a subset of this malignancy. In addition, SIX1 expression was detected in HL cell lines which then were used as models to reveal regulators and target genes of this basic developmental factor. Cytogenetics and quantitative genomic PCR revealed copy number gains of the SIX1 locus at chromosomal band 14q23 correlating with enhanced expression while chromosomal translocations were absent. Moreover, comparative expression profiling data and pertinent gene modulation experiments indicated that the WNT-signalling pathway activates and transcription factor MEF2C suppresses SIX1 expression. MAPK7/ERK5 and HDAC9 phosphorylate and deacetylate MEF2C, respectively, boosting or constraining its inhibitory capacity. MEF2C, MAPK7 and HDAC9 show aberrant expression levels in both HL cell lines and patient samples, collectively restricting the inhibitory activity of MEF2C. Genes encoding the transcription factors GATA2, GATA3, MSX1 and SPIB - all basic lymphoid regulators - were identified as targets of SIX1 in HL, contributing to deregulated B-cell differentiation in this malignancy. In addition, cofactors EYA1 and TLE4 contrastingly mediated activation and suppression of SIX1 target gene expression, respectively. Thus, the protein domain interfaces may represent therapeutic targets in SIX1-positive HL subsets to prevent cofactor interactions and subsequent target gene regulation. Collectively, our data reveal a gene regulatory network where SIX1 centrally deregulates lymphoid differentiation. Additionally, our data strengthen the emerging concordance of lymphopoiesis/lymphomagenesis and the neural plate border region ontogeny.

Disclosures

No relevant conflicts of interest to declare.
CD30/CD16A Tandab AFM13-Induced Target Cell Lysis By NK-Cells Is Enhanced By CD137 Co-Stimulation and Blocking PD-1
Authors: Zhao Xing , Rajasekaran Narendiran , Reusch Uwe , Marschner Jens-Peter , Treder Martin , Kohrt Holbrook
Published: October 2019

Introduction: AFM13 is a CD30/CD16A bispecific tetravalent TandAb antibody that recruits and activates NK-cells by specific binding to CD16A for targeted lysis of CD30+ tumor cells. Given promising clinical activity and safety profile of AFM13 and proof-of-mechanism demonstrating dependence on the immune response, potential synergy of AFM13 and checkpoint modulators was evaluated.

Methods: Efficacy of AFM13 alone or in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies was assessed by in vitro cytotoxicity assays with human PBMCs or enriched NK-cells and CD30+ target cells as well as patient-derived xenograft in vivo models with autologous PBMC.

To evaluate NK-cell-mediated lysis of CD30+ lymphoma cell lines, 4 hour cytotoxicity assays were performed with PBMCs or enriched NK-cells as effector cells in the presence of suboptimal concentrations of AFM13 alone, and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies. For the in vivo model tumor fragments derived from surgical specimens of newly diagnosed patients with CD30+ Hodgkin Lymphoma were xenografted (PDX) in immuno-deficient mice. After 28 days mice were reconstituted with autologous patient-derived PBMC and treated with AFM13 alone and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies weekly for a total of three weeks. Tumor size, tumor-infiltrating human lymphocytes and intra-tumoral cytokines were evaluated on day 58.

Results: AFM13 as a single agent at suboptimal concentrations induced effector-to-target cell-dependent lysis of CD30+ lymphoma cells up to 40% using enriched NK-cells as effector cells in a 4 hour in vitro assay. Immune-modulating antibodies alone mediated substantially lower lysis (<25%). However, the addition of anti-PD-1 or anti-CD137 to AFM13 strongly enhanced specific lysis up to 70%, whereas the addition of anti-CTLA-4 to AFM13 showed no beneficial effect. The most impressive increase of efficacy was observed when AFM13 was applied together with a combination of anti-PD-1 and anti-CD137. In vivo, reduction of tumor growth was observed when AFM13 and anti-PD-1 were used as single agents or when AFM13 was combined with anti-CD137. Synergy was most impressive in these PDX models for the combination of AFM13 and anti-PD-1 which led to a very strong reduction of tumor size. Of note, reduction of tumor growth was strongly correlated with infiltrating NK- and T-cells and intra-tumoral cytokines.

Conclusions:

The combination trials performed with companion intra-tumoral assessment of lymphocytes and cytokines may enhance the efficacy of AFM13 in patients. This may be explained by a potential cross-talk between NK-cells and T-cell which was enhanced when AFM13 was used in combination with checkpoint modulators.

Disclosures

No relevant conflicts of interest to declare.