bluuebunny/crossref_metadata_2025 huggingface dataset:
Reactive Oxygen Species Might Have Important Roles On Angiogenesis of Endothelial Progenitor Cells Derived From HCB(Human Cord Blood) Which Are Stimulated by VEGF.
Authors: Yoo Eun-Sun , Mun Yeung-Chul , Lee Kyoung-Eun , Nam Eunmi , Ahn Jee-Young , Oh Soo-Ah , Seong Chu-Myong
Published: October 2019

Abstract 1445

Poster Board I-468

Purpose

VEGF is a key angiogenic growth factor stimulating proliferation, migration, and tube formation on endothelial cells (ECs), that works through the VEGF receptor type 2 (VEGR2, KDR/Flk1). Reactive oxygen species (ROS) such as superoxide and H2O2 have roles signaling for molecules on angiogenesis. In present study, the aim is to investigate the roles of reactive oxygen species on neovascularization in endothelial progenitor cells.

Methods

Mononuclear cells isolated from UCB were cultured using EGM-2 medium with VEGF, IGF-1 and FGF or basal medium in presence or absence of VEGF for 7 days. Outgrowing endothelail progenitor cells (Yoo et al, STEM CELLS 21:228-235, 2003) at first week of culture were analyzed ROS production by dichlorofluorescein (DCF) fluorescence by use of 2,7-dichlorodihydro-fluoresceine-diacetate (H2DCF-DA). In order to determine that ROS production might involve in EPC proliferation and migration, we had analyzed the impact of N-acetyl-L-cysteine (NAC), broad spectrum ROS scavenger, and NAD(P)H oxidase inhibitor, diphenylene iodonium (DPI) using the proliferation, in vitro tube formation matrigel assay, migration assay with SDF-1/VEGF. We also analyzed the expression of NOX2-based NADPH oxidase (gp91phox) and activation of ERK2 and Akt (Thr308 and Ser473) using VEGF with or without DPI.

Results

Intracellular ROS level were increased during endothelial progenitor cell culture and were higher in UCB compared to that of BM and increased by VEGF treatment. Proliferation, in vitro tube formation matrigel assay and migration assay on endothelial progenitor cells using SDF-1/VEGF were decreased with additions of ROS scavenger DPI when compared with that of control group. In western blot data, NOX2-based NADPH oxidase(gp91phox) was increased by VEGF and decreased by addition of DPI. VEGF induced pERK2 expression was also decreased by DPI and that finding was correlated on down-regulation of endothelial cell proliferation by DPI. Activation of Ser473 Akt was found in control group and decreased by VEGF and rebounded by VEGF and DPI. But Thr308 Akt was not activated in our experiments.

Conclusions

These results suggested that NOX2-based NADPH oxidase(gp91phox)-induced ROS might play important roles on EPCs migration and proliferation by VEGF. Namely, manipulating the level of ROS biochemically may alter the pathogenesis in cardiovascular and in ischemic limb diseases. In results, these data may be useful to develop new therapeutic strategies.

Disclosures

No relevant conflicts of interest to declare.
The Role of HDAC1 Acetylation in Hematopoiesis and GATA-1 Function.
Authors: Luo Yi , Bungert Jorg , Huang Suming , Qiu Yi
Published: October 2019

Abstract 1459

Poster Board I-482

Histone deacetylases (HDACs) play important roles in transcriptional regulation in eukaryotic cells. Several lines of evidence also link HDACs to cancer. HDAC inhibitors have shown preclinical promise when combined with other therapeutic agents in the treatments of leukemia and solid tumors. HDAC1 often presents in repressor complexes, such as Sin3, NuRD and CoREST complexes. We previously found that HDAC1 can be acetylated in vivo by acetyltransferase p300 and that acetylated HDAC1 completely loses its deacetylase activity (Qiu et al, 2006). In this study, we investigate the role of HDAC1 during erythroid differentiation. HDAC1 deacetylase activity gradually reduced upon the induction of differentiation. However, the HDAC1 protein level remains unchanged, suggesting that HDAC1 deacetylase activity, but not its protein level, is regulated. To further test the role of HDAC1 in erythroid differentiation, stable cell lines that overexpress HDAC1 and mutants mimicking acetylated or unacetylated HDAC1 were established. We also generated stable HDAC1 and HDAC2 knock down cell lines. The results showed that HDAC1 deacetylase activity is required to promote erythroid proliferation and to inhibit differentiation. Next, we studied whether HDAC1 modulates erythroid differentiation through regulating the activity of key erythroid transcription factor GATA-1. GATA-1 interacts with many other key hematopoietic transcription factors, as well as chromatin remodeling/modifying coactivators and corepressors. It is suggested that GATA-1 mediates gene activation through its association with coactivator complexes. However, recent studies indicated that GATA-1 associates with HDAC1/2 containing corepressor complexes (NuRD) throughout differentiation of erythroid cells. We investigated the deacetylase activity of the GATA-1 associated NuRD complex during erythroid differentiation. We found that the deacetylase activity of the complex decreased and further diminished at day 5 of DMSO induced MEL cells. Accordingly, the acetylated form of HDAC1 within the GATA-1 complex increased during erythroid differentiation. We further demonstrated the role of HDAC1 in GATA-1 mediated gene transcription in transient transfection assays. These studies indicate that HDAC1 plays an important role in regulating GATA-1 activity and the deacetylase activity of the GATA-1 associated NuRD complex is also regulated. This complex may play different roles in undifferentiated and differentiated erythroid cells. Thus, our results suggest a novel but rather general regulatory mechanism of histone deacetylase containing protein complexes.

Disclosures

No relevant conflicts of interest to declare.
Klf1 Regulatory Networks in Primary Erythroid Cells.
Authors: Tallack Michael , Whitington Thomas , Gardiner Brooke , Wainwright Eleanor , Keys Janelle , Monet Marion , Nourbakhsh Ehsan , Cloonan Nicole , Grimmond Sean , Bailey Timothy , Perkins Andrew Charles
Published: October 2019

Abstract 1462

Poster Board I-485

Klf1/Eklf regulates a diverse suite of genes to direct erythroid cell differentiation from bi-potent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which Klf1 works, we performed Klf1 ChIP-seq using the SOLiD deep sequencing platform. We mapped more than 10 million unique 35mer tags and found ∼1500 sites in the genome of primary fetal liver erythroid cells are occupied by endogenous Klf1. Many reside within well characterised erythroid gene promoters (e.g. b-globin) or enhancers (e.g. E2f2 intron 1), but some are >100kb from any known gene. We tested a number of Klf1 bound promoter and intragenic sites for activity in erythroid cell lines and zebrafish. Our data suggests Klf1 directly regulates most aspects of terminal erythroid differentiation including synthesis of the hemoglobin tetramer, construction of a deformable red cell membrane and cytoskeleton, bimodal regulation of proliferation, and co-ordination of anti-apoptosis and enucleation pathways. Additionally, we suggest new mechanisms for Klf1 co-operation with other transcription factors such as those of the gata, ets and myb families based on over-representation and spatial constraints of their binding motifs in the vicinity of Klf1-bound promoters and enhancers. Finally, we have identified a group of ∼100 Klf1-occupied sites in fetal liver which overlap with Klf4-occupied sites in ES cells defined by Klf4 ChIP-seq. These sites are associated with genes controlling the cell cycle and proliferation and are Klf4-dependent in skin, gut and ES cells, suggesting a global paradigm for Klfs as regulators of differentiation in many, if not all, cell types.

Disclosures

No relevant conflicts of interest to declare.
The MiR-23a MicroRNA Cluster Inhibits B Cell Development.
Authors: Mullenix Jason , Kong Kimi Y , Owens Kristin Severns , Rogers Jason , FitzPatrick Shannon , Dahl Richard E.
Published: October 2019

Abstract 1465

Poster Board I-488

The miR-23a microRNA (miRNAs) cluster inhibits both [ITALIC]in vitro[/ITALIC] and [ITALIC]in vivo[/ITALIC] B cell development. When murine hematopoietic progenitor cells expressing the 23a cluster miRNAs were cultured in B cell promoting conditions we observed over a five-fold decrease in the generation of CD19+ B cells compared to control cultures. Conversely, we observed over a five-fold increase in CD11b+ myeloid cells. When irradiated mice were transplanted with bone marrow expressing the miR-23a cluster we observed a two-fold decrease in bone marrow and splenic B cells, 8 weeks post-transplant compared to control mice. The miR-23a cluster codes for a single pri-transcript, which when processed yields three mature miRNAs: miR-23a, miR-27a, and miR-24-2. All three mature miRNAs are more abundant in myeloid cells compared to other hematopoietic cells. In vitro miR-24 alone is necessary and sufficient to inhibit B cell development. The promoter for the cluster contains conserved binding sites for the essential myeloid transcription factors PU.1 and C/EBP alpha. Chromatin immunoprecipitations demonstrated that PU.1 and C/EBP alpha are associated with the promoter in myeloid cells. In addition, C/EBP alpha is bound to several highly conserved regions upstream of the promoter. Both PU.1 and C/EBP alpha promote myeloid development at the expense of lymphopoiesis. Our work suggests that the miR-23a cluster may be a critical downstream target of PU.1 and C/EBP alpha in the specification of myeloid cell fate. Although miRNAs have been identified downstream of PU.1 and C/EBP alpha in mediating the development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in the regulating lymphoid cell fate acquisition. We are currently identifying targets of miR-24 that may mediate the inhibitory effect on B lymphopoiesis.

Disclosures

No relevant conflicts of interest to declare.
Controlling Hematopoiesis through Sumoylation-Dependent Regulation of a GATA Factor.
Authors: Lee Hsiang-Ying , Johnson Kirby D. , Fujiwara Tohru , Boyer Meghan E. , Kim Shin-Il , Bresnick Emery H.
Published: October 2019

Abstract 1467

Poster Board I-490

GATA factors function via distinct modes to establish transcriptional networks that control fundamental developmental processes including hematopoiesis. Whereas the master regulator of hematopoiesis GATA-1 is subject to multiple posttranslational modifications, how these modifications influence GATA-1 activity at endogenous loci is poorly understood. GATA-1 is sumoylated at K137, which resides in the N-terminus, but how the N-terminus contributes to GATA-1 function remains unclear. Expression of a GATA-1 mutant lacking amino acids 1-83 of the N-terminus is linked to the development of acute megakaryoblastic leukemia [Wechsler et al. (2002) Nat. Genet. 32, 148], and deletion of the N-terminus preferentially deregulates a subset of target genes [Johnson et al. (2006) PNAS. 103, 15939]. We demonstrate that sumoylation at K137 promotes transcriptional activation only at a subset of its target genes – those requiring the cell type-specific coregulator, Friend of GATA-1 (FOG-1). Interestingly, a GATA-1 mutation that disrupts FOG-1 binding (V205G) and K137 mutations yielded similar phenotypes, although FOG-1 was not required for K137 sumoylation. Both V205 and K137 mutations dysregulated GATA-1 chromatin occupancy at select sites, FOG-1-dependent target gene expression, and were rescued by tethering SUMO-1. While FOG-1- and SUMO-1-dependent genes migrated away from the nuclear periphery upon erythroid maturation, FOG-1- and SUMO-1-independent genes localized at the periphery independent of maturation. These results illustrate how sumoylation of a critical developmental regulator selectively controls its function at specific loci, and members of a target gene ensemble with distinct coregulator and posttranslational modification requirements reside in different subnuclear compartments.

Disclosures:

No relevant conflicts of interest to declare.
High Incidence of Arterial Thrombosis in Young Patients Treated for Multiple Myeloma:Results of a Prospective Cohort Study.
Authors: Libourel Eduard J , Sonneveld P. , van der Holt Bronno , Leebeek F. W.G.
Published: October 2019

Abstract 149

It is well known that patients with multiple myeloma (MM) are at high risk for venous thromboembolism (VTE). Thrombotic complications are most frequently seen in patients treated with thalidomide based regimens. Several other risk factors for VTE have been identified including abnormalities in coagulation factors, such as high factor VIII levels, APC resistance and hypofibrinolysis. Recently a few cases of MM patients and arterial thromboembolism (ATE) have been reported. Therefore we prospectively studied the risk of ATE in 195 consecutive patients aged <66 years with previously untreated MM. All patients were treated according to the HOVON 50 study protocol, a prospective randomized phase III study on the effects of 3 courses of doxorubicin and dexamethasone with thalidomide (TAD) or vincristine (VAD) followed by high dose melphalan and thalidomide or interferon-alpha2 maintenance or the HOVON 65 study protocol, a prospective randomised phase III study on the effects of 3 courses doxorubicin and dexamethasone with vincristine (VAD) or bortezomib (PAD), followed by high dose melphalan and bortezomib or thalidomide maintenance. All patients treated with TAD courses received prophylaxis for VTE with low molecular-weight heparin. During a follow-up of 522.4 patient-years 11 patients (5.6%) developed ATE. Most events occurred within one year after start of treatment (64%). Median age at onset of ATE was 59 years (range 43–65). For myocardial infarction we demonstrated an annual incidence of 0.77% in men and 0.19% in women compared to an equal age group, 55–59 years, described by Parikh et al in the Framingham Heart Study population who reported annual incidences of 0.22% in men and 0.04% in women, respectively. For cerebral ischemic events we demonstrated an annual incidence of 0.77% in men and 0.19% in women compared to 0.29% in men and 0.22% in women in the general Dutch population, aged 55–59, as described by Gijsen and Poos. Of thrombophilic factors a statistically significant higher factor VIII:C (FVIII:C) was observed in symptomatic patients, 2.93 IU/ml (1.16–6.45) versus 2.15 (0.74–6.48) (p=0.03). FVIII:C correlated significantly with age (p=0.02) and ISS stage (p=0.001). Adjustment for age and ISS stage, revealed a RR for ATE of 11.67 (95% CI: 2.23–61.18) for hypertension, a RR of 15.17 (1.78–129.54) for smoking and a RR of 1.85 (0.99–3.47) for each IU/ml increase in FVIII level, respectively. In conclusion, MM patients are at high risk for arterial thrombotic events during and following induction chemotherapy. High factor VIII levels, which may reflect disease activity, contribute to the risk of ATE especially in patients with known risk factors for cardiovascular disease.

Disclosures:

Sonneveld: Johnson&Johnson: Consultancy.
Inhibition of HIV-1 in Sickle Cell Disease Peripheral Blood Mononuclear Cells.
Authors: Ammosova Tatiana , Charles Sharroya , Rotimi Jamie , Foster Altreisha , Diaz Sharmin , Kim Bak , Gordeuk Victor R. , Nekhai Sergei
Published: October 2019

Abstract 1509

Poster Board I-532

Background

The hypoxic response is an important component of the body 's reaction to impaired tissue oxygenation associated with the anemia and vasoocclusive episodes of sickle cell disease (SCD). It has been reported that HIV infection progresses relatively slowly in patients with SCD (Am J Hematol 1998; 59:199-207). We recently showed that HIV-1 transcription and replication is significantly reduced in cells cultured at 3% versus 21% oxygen (J Cell Physiol 2009; in press). Our previous studies indicated that protein phosphatase-1 (PP1) interacts with HIV-1 transcriptional activator, Tat, and thereby participates in the regulation of HIV-1 transcription. Sickle cell patients are in chronically hypoxic state and we hypothesized that HIV-1 replication in their peripheral blood mononuclear cells (PBMCs) would be slower then in controls.

Methods

We isolated PBMCs from patients with SCD and from normal subjects, activated the cells with phytohemagglutinin and IL-2 for 24 h, and infected with pseudotyped HIV-1 virus expressing Luciferase. The infected cells were cultured at 3% of oxygen for 72 h.

Results

We show here that PP1 association with cellular regulatory subunits is modified and that PP1 activity is significantly reduced by 20-40% in different cell lines at 3% versus 21% oxygen. One round of replication of pseudotyped HIV-1 Luciferase virus normalized to the number of the cells in culture was significantly reduced in SCD PBMCs comparing to normal controls.

Conclusions

Our results provide a direct evidence of that HIV-1 replication may be slower in SCD-derived PBMCs. In future, we will analyze PP1 activity and the association of PP1 with regulatory subunits in SCD PBMCs. Understanding of how oxygen status influences HIV-1 replication might open new possibilities for treatment of hidden HIV-1 reservoirs that harbor non-replicating HIV-1 virus.

Acknowledgments

This work was supported by NHLBI Research Grant 2 R25 HL003679-08 from the National Institutes of Health and The Office of Research on Minority Health.

Disclosures

No relevant conflicts of interest to declare.
Cytoprotective Mechanisms of the Lung in Sickle Cell Disease.
Authors: Ghosh Samit , Tan Fang , Monsunjac Mario , Ofori-Acquah Solomon F
Published: October 2019

Abstract 1538

Poster Board I-561

Circulating plasma hemoglobin contributes to major vasculopathies including pulmonary hypertension in patients who have sickle cell disease (SCD). There is an emerging concept that such vasculopathies are relatively mild because of activation of several cytoprotective pathways in SCD. The biochemical profile of plasma and the transcriptome of peripheral blood cells in patients who have SCD offer indirect support for this concept. Indeed, heme oxygenase-1 (HO-1), an acute phase enzyme that degrades heme into intermediates and byproducts with vasculoprotective properties is markedly elevated in mononuclear leukocytes of patients who have SCD. Nonetheless, the scope of the cytoprotective mechanisms of the lung and other organs impacted directly by sickle vasculopathies remain poorly appreciated. We previously identified an array of cytoprotective enzymes in lung endothelial cells chronically exposed to non-toxic concentrations of hemin in vitro. In this study, we examined the expression of NAD(P)H oxidase and candidate cytoprotective enzymes in two models of transgenic mice with SCD, and examined HO-1 expression in sickle chronic lung disease. Although NAD(P)H oxidase catalyzes reactive oxygen species generation by heme and is responsible for increased adhesion of leukocytes to the endothelium in SCD mice, there was no elevation of any of its subunits (gp91Phox and p22Phox, p47Phox, p67Phox and p40 Phox) in sickle mice lungs compared to hemizygote control mice lungs. Quantitative RT-PCR analysis revealed unexpectedly no difference in HO-1 mRNA level in sickle and non-sickle control lungs. On the contrary, analysis of the same tissues showed significantly higher NAD(P)H quinone oxidoreductase-1 (NQO1) mRNA level in both Berkeley and Townes knock-in sickle mice compared to controls (p<0.001). Enhanced expression of NQO1 but not HO-1 in sickle mice lungs was confirmed at the protein level by western blot analysis and by immunohistochemistry. Finally, we studied cases of sickle chronic lung disease (n=17) and discovered that NQO1 is expressed in the pulmonary endothelium at significantly higher levels than in normal lung tissues (p<0.002). In agreement with the data in mice, we found no difference in HO-1 staining in sickle human lung and normal lung tissues. Our findings indicate that despite its established role in acute elimination of excess heme, HO-1 is not elevated in sickle lungs. This study highlights the importance of dissecting the cytoprotective phenotype of individual organs impacted by SCD towards a rationale approach to developing efficacious antioxidant therapy for this disorder.

Disclosures

No relevant conflicts of interest to declare.
Improved Patient Survival and Cure for Hodgkin Lymphoma: A Population-Based Study of 6,136 Patients Diagnosed in Sweden 1973-2005.
Authors: Sjoberg Jan , Halthur Cat , Kristinsson Sigurdur Y , Landgren Ola , Dickman Paul W , Bjorkholm Magnus
Published: October 2019

Abstract 1553

Poster Board I-576

We evaluated survival for all patients diagnosed with Hodgkin lymphoma (HL) in Sweden 1973-2005 (n=6,136; 3,515 men and 2,621 women; median age 40 years). Patients were categorized into six age groups and four calendar periods (1973-1980, 1981-1988, 1989-1996, and 1997-2005). Relative survival ratios (RSRs) were computed as measures of patient survival. Cumulative relative survival improved over time in all age groups with the greatest improvement in patients 51-65 years. Also in patients 66-80 years, significant improvements were recorded in both five- and ten year RSRs. Importantly, a plateau in relative survival was observed after 5 years in patients below ≤ 35 years of age during the last calendar period suggesting a lack of long-term treatment-related mortality. The ten-year RSRs in this calendar period were 0.95, 0.95, 0.92, 0.80, and 0.51 for the age groups 0-18, 19-35, 36-50, 51-65, and 66-80, respectively. Thus, despite this progress, age at diagnosis remains an important predictor of outcome. We also found a significantly better survival for women when adjusted for age and calendar period. During the study period, refined treatment options for patients with limited and advanced-stage HL have contributed to an increasing cure rate. In addition, our findings support that long-term mortality of HL therapy has decreased. Elderly patients still do poorly and targeted treatment options associated with fewer side effects will advance the clinical HL field.

Cumulative relative survival stratified by age at diagnosis and calendar period of diagnosis Cumulative relative survival stratified by age at diagnosis and calendar period of diagnosis

Disclosures

No relevant conflicts of interest to declare.
The Cell Polarity PTK7 Receptor Is Expressed in Myeloid Cells and Acts as a Modulator of the Chemotherapeutic Response in AML Patients.
Authors: Prebet Thomas , Lhoumeau Anne Catherine , Arnoulet Christine , Aulas Anais , Marchetto Sylvie , Sebbagh Michael , Esterni Benjamin , Vey Norbert , Borg Jean Paul
Published: October 2019

Abstract 1571

Poster Board I-596

The pseudo tyrosine kinase receptor 7 (PTK7) is an orphean tyrosine kinase receptor assigned to the planar cell polarity pathway (PCP). It has been recently described and plays a major role during embryogenesis and epithelial tissue organisation. To date there is no report in the litterature considering a potential implication in hematopoiesis. In silico and in vitro analysis found that PTK7 was also expressed in normal myeloid progenitors and CD34+ CD38- bone marrow cells in humans. Preliminary results from our team showed that PTK7 was also expressed in various leukemic cell lines such Jurkat, TF-1 or KG-1a. We decided to perform a wide range multicolour immunophenotyping screen on patients with acute myeloid leukemia (AML) at diagnosis and to investigate the role of PTK7 in AML in vitro. More than 250 patient samples were evaluated and we demonstrated that PTK7 was largely expressed in AML as 72% of the samples were PTK7 positive. Its expression mostly correlates with granulocytic lineage differentiation. PTK7 expression was associated with a lower WBC count at diagnosis and a lower frequency of extramedullary disease whatever was FAB subtype. Interestingly, PTK7 expression was associated with some cytogenetic subgroups including CBF-AML and APL. There was no correlation with molecular subgroups (i.e. FLT3-ITD/NPM1/CEBPA status). Overall Survival and Relapse Free Survival were evaluated in non-APL patients treated with induction chemo (n=182). Patients with PTK7 positive AML are more resistant to anthracycline-based frontline therapy with a significantly reduced Relapse Free Survival in a multivariate analysis model integrating all pre treatment variables (2 year probability of RFS= 29% vs 66% for PTK7 negative patients, p= 0.003). Forrest plot analysis showed that the negative impact of PTK7 expression was the most significant in intermediate cytogenetic risk subgroup and when PTK7 was aberrantly expressed in M4-M5 FAB subtypes. There was no demonstrated impact on CR. In cultured cells, expression of PTK7 promotes leukemia cell migration, cell survival and resistance to anthracyclin-induced apoptosis. There was no effect of PTK7 expression on cell proliferation in tritiated thymidine assay. In the absence of known inhibitor of PTK7, we produced a soluble recombinant PTK7-Fc protein that efficiently competes for PTK7 functions in cell migration and survival assays in cell lines and primary AML samples. These data were confirmed using a shRNA strategy. We conclude that PTK7 is a PCP component expressed in the myeloid progenitor compartment that conveys promigratory and anti-apoptotic signal to leukemia cells. Its use as a potential biomarker or therapeutical target should be investigated.

Disclosures

No relevant conflicts of interest to declare.
Expression Profiling of a Hemopoietic Cell Survival Transcriptome Identifies a Functional Prognostic Gene Signature in Normal Karyotype AML.
Authors: Guthridge Mark , Thomas Daniel , Barry Emma F , Chung Kok H , McClure Barbara J , To L. Bik , Goodall Greg , Speed Terry P , Osato Motomi , Haylock David , Nilsson Susie , D'Andrea Richard , Lopez Angel , Powell Jason A
Published: October 2019

Abstract 1618

Poster Board I-644

Deregulated cell survival programs are a classical hallmark of cancer. We have previously identified a serine residue (Ser585) in the cytoplasmic domain of the bc subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) receptors that selectively and independently promotes hemopoietic cell survival. We now show that Ser585 phosphorylation is constitutive in the majority of acute myeloid leukemia (AML) patient samples indicating that this hemopoietic “survival-only” pathway is frequently deregulated in leukemia. This constitutive Ser585 phosphorylation is refractory to tyrosine kinase inhibitors allowing the survival of a quiescent population of leukemic progenitor cells in vitro. We performed a global expression screen to identify gene targets of this survival-only pathway and report a unique bc Ser585-regulated transcriptome. Pathway analysis defines a gene network enriched for PI 3-kinase target genes as well as a cluster of genes involved in cancer and cell survival. From this panel of Ser585-regulated genes, we have validated by Q-RT-PCR a “six-gene Ser585-signature” that is deregulated in AML blasts exhibiting constitutive Ser585 phosphorylation. We show that siRNA-mediated knockdown of key components of this Ser585-signature induces apoptosis in CD34+CD38-CD123+ leukemic stem and progenitor cells. Furthermore, multivariate analysis indicates that the Ser585-signature is an independent prognostic indicator of overall patient survival in normal karyotype AML (n=56, HR=2.0, p=0.01). These results delineate a novel cytokine-regulated Ser585/PI3-kinase signalling network that is deregulated in AML and identify new prognostic targets with therapeutic potential.

Disclosures

No relevant conflicts of interest to declare.
Final Results of a Phase 2 NCI Multicenter Study of Romidepsin in Patients with Relapsed Peripheral T-Cell Lymphoma (PTCL).
Authors: Piekarz Richard , Wright John , Frye Robin , Allen Steven L , Joske David , Kirschbaum Mark , Lewis Ian D. , Prince Miles , Smith Sonali , Jaffe Elaine S , Bates Susan
Published: October 2019

Abstract 1657

Poster Board I-683

Background

The histone deacetylase (HDAC) inhibitors are a class of epigenetic agents undergoing clinical testing. HDAC inhibitors modulate expression of genes involved in cell cycle regulation, leading to induction of differentiation or apoptosis. Romidepsin, a novel pan-HDAC inhibitor, has previously demonstrated activity as a single agent in patients (pts) with cutaneous T-cell lymphoma (CTCL) in two phase 2 studies.

Aims

To evaluate the efficacy and tolerability of romidepsin in the treatment of advanced PTCL and as an exploratory endpoint, to examine the molecular effects of romidepsin in both PTCL and CTCL.

Methods

This Phase 2, open-label, multi-arm, multicenter study enrolled 46 PTCL pts from the NCI and 9 extramural sites. CTCL pts were also enrolled in this study. This study is now closed to accrual. Pts with relapsed or refractory PTCL who had received at least one prior standard chemotherapy regimen were eligible. Pts received romidepsin 14 mg/m2 as a 4-hr infusion on days 1, 8 and 15 every 28 days. Responses were assessed using elements of the IWG criteria and RECIST, as appropriate, for pts with lymph node involvement and extranodal disease.

Results

46 pts (24 [52%] men and 22 [48%] women) with a mean age of 59 (range: 28 to 84) years were treated with romidepsin. 32 pts received ≥ 2 cycles of therapy. Mean number of prior therapies was 4.8 (range 1 to 14). Objective disease response rates (ORR) are summarized in the table. The mean number of cycles of treatment was 6.8 (range 1-47) and the overall median duration of response was 9.0 months (range 1.8 months to 5.8 yrs) for all pts. The median duration of response for the 5 pts who achieved CR was 6.0 months (range 2.8 months to 5.8 yrs).The most frequent drug-related AEs (all grades, all cycles) were generally mild and included: nausea (74%; 9% ≥grade 3), fatigue (72%; 20% ≥grade 3), decreased platelets (72%; 35% ≥grade 3), cardiovascular/general-other (72%; 0% ≥grade 3) and decreased AGC (65%; 43% ≥grade 3). One death, in a pt with significant cardiovascular disease who had achieved a CR, was considered possibly related to treatment.

Conclusions

This study demonstrates tolerability and durable clinical benefit (ORR of 33% and median duration of response of 9.0 months) of romidepsin in pts with recurrent or refractory PTCL. Based on these promising results, a Phase 2B protocol investigating romidepsin in pts with relapsed or refractory PTCL is ongoing at multiple international centers.

Disclosures

Kirschbaum: Gloucester Pharmaceuticals: Consultancy.
Efficacy of Abbreviated Stanford V Chemotherapy and Involved Field Radiotherapy in Early Stage Hodgkin's Disease: Mature Results of the G4 Trial.
Authors: Advani Ranjana H , Hoppe Richard T , Baer David M. , Mason Joseph , Rosenberg Saul A , Horning Sandra J
Published: October 2019

Abstract 1670

Poster Board I-696

The standard management for early stage Hodgkin's disease (HD) is combined modality therapy. In the G4 protocol, patients (pts) with non-bulky, supra-diaphragmatic stage I-IIA disease received 8 weeks of Stanford V chemotherapy + 30 Gy involved field radiotherapy (IFRT).

87 pts were enrolled and treated between 4/1996 and 4/2001. Median age was 30 years (16-59) and stages were IA (n=23), IIA (n=64). Unfavorable risk factors were present in 47 patients (54%) according to German Hodgkin Study Group (GHSG) criteria (> 2 AA sites, ESR > 50 or EN involvement) and 38 (44%) according to EORTC criteria (> 3 AA sites, ESR > 50). Therapy was well tolerated with grade 3-4 non-hematologic toxic events in 7 % of pts. These included constipation (n=3), peripheral neuropathy (n=1), generalized weakness (n=2), chest pain (n=1), mylagias (n=1), abdominal pain (n=1) and an allergic reaction to etoposide (n=1). 42 patients received cytokine support for grade 3-4 neutropenia however only 2 pts developed fever with neutropenia. No cases of clinical bleomycin toxicity or radiation pneumonitis were observed. At a median follow-up of 9 (2-12) years, freedom from progression (FFP) and survival (OS) are 94% and 96% respectively. FFP was 100% for favorable and 89% for unfavorable patients by GHSG criteria (p=0.04) with no differences in OS (96.9% versus 95.7%). All relapses (n=5) occurred in the RT field: limited in 2 patients and combined with distant disease in 3 pts. All relapses were in “unfavorable” risk patients: 5 per GHSG and 4 per EORTC criteria. Secondary therapy included chemotherapy followed by high dose therapy and stem cell support (n=3) and ABVD (n=2). Three pts died, 2 due to disease progression after second-line therapy and one due to metastatic colon cancer. 5 patients developed a second cancer (2 breast, 2 melanomas at unirradiated sites and 1 colon cancer). The cases of breast cancer were considered unrelated to RT as both occurred within 5-years of therapy in women who were > 30 yrs at time of treatment. No secondary AML and no late cardiac or pulmonary toxicities have been observed.

In conclusion, for pts with non-bulky stage I/II A HD, abbreviated Stanford V with 8 weeks of chemotherapy and 30 Gy IFRT is a safe and highly effective regimen. It is still too early to assess potential RT-related complications. Our outcomes in “unfavorable” stage I-IIA patients without bulky or symptomatic disease compare favorably with more intensive or prolonged regimens employed by the GHSG and EORTC.

Disclosures

No relevant conflicts of interest to declare.
A Recombinant Human Antibody to EphA3 with Pro-Apoptotic and Enhanced ADCC Activity Shows Selective Cytotoxicity against Myeloid Leukemia Cells and CD123-Positive Leukemic Stem Cells.
Authors: Palath Varghese , Vekhande Rohini , Lee Andreia , Williams Jason , Zhang Ling , List Alan F , Boyd Andrew , Lackmann Martin , Scott Andrew M , Cilloni Daniela , Yarranton Geoffrey T. , Bebbington Christopher
Published: October 2019

Abstract 1728

Poster Board I-754

Members of the Eph family of receptor tyrosine kinases play important roles in embryonic development but have restricted tissue distribution and activity in adult tissues. The EphA3 receptor is an oncofetal antigen expressed at high levels on the surface of several solid tumor types and certain leukemias. KaloBios is in pre-clinical development with KB004, a high-affinity recombinant human antibody, derived from a monoclonal anti-EphA3 antibody by Antibody HumaneeringTM Technology. KB004 binds EphA3 and stimulates apoptosis in primary cells from myeloid leukemia patients. The cell-surface expression of EphA3 was analyzed by flow cytometry on primary cells from chronic myeloid leukemia (CML) [n=10], acute myeloid leukemia (AML) of various sub-types [n=29], and myelodysplastic syndromes (MDS) [n=7]. EphA3 surface expression was detected in at least 50% of patient samples from each of the diseases analyzed. CD123-positive leukemia stem cells (CD34+ CD38- CD123+) also displayed surface expression of EphA3 whereas normal bone marrow CD34+ stem/ progenitor cells lacked detectable EphA3. KB004 was shown to stimulate apoptosis in EphA3+ primary leukemia cells, including CD123+ leukemic stem cells with no activity in EphA3-negative specimens, including normal CD34+ bone marrow cells. Cross-linking of the antibody was not required for induction of apoptosis. KB004 also inhibited myeloid leukemia colony formation (CFU-L) from primary AML samples in methylcellulose colony assays without affecting normal hematopoietic colony formation. In addition to its direct pro-apoptotic effect, KB004 induced potent antibody-dependent cellular cytotoxicity (ADCC) activity against EphA3-positive cells, mediated by CD16-expressing effector cells. CD16-dependent ADCC activity was further enhanced by expression of the antibody in CHO cells that lack a1,6 fucosyl transferase, generating afucosylated antibody with higher affinity for CD16a. The data support the development of an anti-EphA3 antibody for the treatment of hematologic malignancies in addition to solid tumors. In particular, the selective activity against leukemic stem cells supports a novel strategy for therapeutic targeting of leukemia-initiating cells that merits clinical investigation.

Disclosures

No relevant conflicts of interest to declare.
Conventional and Molecular Cytogenetic Responses in Patients with Myelodysplastic Syndrome (MDS) with Deletion 5q Treated with Lenalidomide.
Authors: Coutinho Rita , Costa Dolors , Carrió Ana , Vidal Ana , Belkaid Mohammed , Campo Elias , Montserrat Emili , Nomdedeu Benet
Published: October 2019

Abstract 1746

Poster Board I-772

Background and objectives

Interstitial deletions of the long arm of chromosome 5 - del (5q) - are the most common chromosomal abnormalities in MDS. Del (5q) is detected in 14 % of the patients. Lenalidomide induces complete cytogenetic remissions and abolishes transfusion dependence in a significant proportion of these patients, apparently correlated with the malignant clone suppression. The objectives of this study were to assess clinical and cytogenetic responses to Lenalidomide and to establish the relationship between the number of 5q deleted cells detected by conventional cytogenetics (CG) and by fluorescence in situ hybridization (FISH) previously and after treatment. Due to its larger sensitivity, the FISH technique might be able to predict an eventual loss of response to treatment.

Patients and methods

Bone marrow samples were collected at diagnosis and every 6 months from the beginning of treatment with lenalidomide. CG and FISH (with LSI EGR1 (5q31)SO/D5S23, D5S721 (5p15.2) probe, from Vysis) studies were performed in all samples, even in the cases that a complete cytogenetic remission was confirmed by karyotype during the treatment follow-up.

Results

We have treated 9 transfusion dependent MDS patients with lenalidomide, with a median age of 76 years (45-92). Four of the patients had one additional cytogenetic anomaly besides 5q deletion. Due to high clinical and cytological suspicion, one of the patients had a trisomy 9 by karyotype (2/20 metaphase) and was diagnosed of del (5q) by FISH, with a low clonal load (9,4% of deleted nuclei). Six patients were low IPSS risk, while 2 were Int-1 and one was Int-2 risk IPSS. All patients achieved complete erythroid response and transfusion independence (in a median of 8 weeks of treatment) and are still in response. Treatment was stopped in two patients after achieving complete hematologic response, maintaining normal hemoglobine levels for > 24 months. Five patients achieved cytogenetic remission, but 2 of them relapsed in a latter phase of the treatment (25 and 30 months). FISH analysis could not predict cytogenetic relapse in these two cases.

Conclusions

Our results support the established high efficacy of lenalidomide in MDS with del (5q). In our experience a single additional cytogenetic anomaly did not compromise hematologic response. Moreover, in our study FISH analysis did not bring additional information to CG analysis in prediction of response and follow-up in the majority of the cases.

Disclosures

No relevant conflicts of interest to declare.
Ribosomal Protein Genes S10 and S26 Are Commonly Mutated in Diamond-Blackfan Anemia.
Authors: Gazda Hanna T. , Sheen Mee Rie , Doherty Leana , Vlachos Adrianna , Choesmel Valerie , O'Donohue Marie-Francoise , Schneider Hal E. , Clinton Catherine , Sieff Colin A. , Newburger Peter E. , Ball Sarah E. , Niewiadomska Edyta , Matysiak Michal , Glader Bertil , Atsidaftos Eva , Lipton Jeffrey M. , Gleizes Pierre-Emmanuel , Beggs Alan H.
Published: October 2019

Abstract 175

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by anemia usually presenting during infancy or in early childhood, birth defects, and increased risk of cancer. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial, upper limb, heart and urinary system defects, that are present in ∼30–50% of patients. To date, DBA has been associated with mutations in seven ribosomal protein (RP) genes, S19, S24, S17, L35A, L5, L11, and S7 in about ∼43% of patients. To complete our large scale screen of RP genes in a DBA population, we sequenced 49 ribosomal protein genes in our DBA patient cohort of 117 probands. Together with our previous efforts of screening 29 RP genes, this completes the analysis of all known RP genes (excluding RPS4Y on the Y chromosome).

Here we report probable mutations in four more RP genes, RPS10, RPS26, RPL19, and RPL26, and variants of unknown significance in six more, RPL9, RPL14, RPL23A, RPL7, RPL35, and RPL3. RPS10 and RPS26 are now confirmed DBA genes as we identified five mutations in RPS10 in five probands and nine mutations in RPS26 in 12 probands. We found single small frameshifting deletions in RPL19 and RPL26 in single DBA families. Pre-rRNA analysis in lymphoblastoid cells from patients bearing mutations in RPS10 and RPS26 showed elevated levels of 18S-E pre-rRNA. This accumulation is consistent with the phenotype observed in HeLa cells after knock-down of RPS10 or RPS26 expression with siRNAs, which indicate that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins. This brings to 11, the total number of RP genes mutated in ∼54% of patients with DBA, with nine more genes harboring variants of unknown significance requiring further study.

Disclosures:

No relevant conflicts of interest to declare.
Secretome Analyses of Primary Bone Marrow Fibroblasts Isolated From MGUS and Multiple Myeloma Show a Stepwise Occurrence of Alterations.
Authors: Drach Johannes , Slany Astrid , Mohr Thomas , Griss Johannes , Zielinski Christoph C , Gerner Christopher
Published: October 2019

Abstract 1801

Poster Board I-827

The microenvironment of tumor cells in the bone marrow was demonstrated to contribute to tumor promotion and survival. The role of bone marrow fibroblasts (BMFs) in supporting the malignant plasma cell clone in multiple myeloma (MM) has been established, but it remains unclear to which extent the BM microenvironment in general and BMFs in particular are involved in the progression of monoclonal gammopathy of undetermined significance (MGUS) to MM. Therefore we performed proteomics studies on the secretome of BMFs isolated from healthy donors, patients suffering from MGUS and patients suffering from MM. Compared to normal background, BMFs derived from MGUS secreted elevated levels of proteins indicating mitogenic activity and moderate inflammation. These proteins included periostin, IL-6, CXCL5 and CSF-1. Insulin-like growth factor II, which is normally not expressed by normal BMFs, was secreted by BMF cells derived from MGUS as well as from MM. In addition to those and other proteins, BMF cells derived from MM were found to specifically secrete stem cell growth factor, MMP-28 and stanniocalcin-1. These data indicate a step-wise alteration of BMF secretion activity related to the stage of the underlying plasma cell dyscrasia. Therefore BMF might support the progression from MGUS to MM. In order to correlate the secretion performance of BMF with blood serum levels of candidate marker proteins, Luminex assays are employed. Based upon these results, it is our aim to identify serum biomarkers which allow to assess the functional state of BMF and thus the risk for the progression of MGUS to MM.

Disclosures

No relevant conflicts of interest to declare.
Clonal Expansions of Cytotoxic T Cells in the Blood of Patients with Waldenstrom's Macroglobulinaemia Are Anergic and Disappear After Nucleoside Analogue Therapy.
Authors: Brown Ross D , Li Jia , Sze Daniel , Cowley Mark , Kaplan Warren , Yang Shihong , Aklilu Esther , Kabani Karieshma , Ho Phoebe Joy , Loh Yen S , Yamagishi Tetsuo , Mo Suilin , Chen Yuling , Joshua Douglas
Published: October 2019

Abstract 1820

Poster Board I-846

T cells contribute to the immunomodulatory control of the tumor in patients with monoclonal gammopathies. We previously found that CD3+CD8+CD57+TCRVβ+restricted cytotoxic T cell expansions were present in 48% of patients with multiple myeloma (n=221) and conferred a significant favorable prognosis. We now report the presence of these expansions in 70% of patients with Waldenstrom's Macroglobulinaemia (WM) (n=20) with a wide spectrum of the TCRVβ repertoire represented. Previous nucleoside analogue (NA) therapy, known to be associated with an increased incidence of transformation to aggressive lymphoma, significantly influenced the presence of TCRVβ expansions (χ2=11.6; p<0.001) as 5 of the 6 patients without and only 1 of the 14 patients with TCRVβ expansions had received NA. Clonality of CD3+CD8+CD57+TCRVβ-restricted cytotoxic T cells was confirmed by determining the size of the TCRVβ chain CDR3 region and by direct sequencing. We identified differential gene expression by using microarray analysis (Affymetrix GeneChip Human Genome U133 plus 2.0 Arrays) and confirmed the expression of selected genes by real-time qPCR and flow cytometry. Data was analysed by paired moderated t-test and then Gene Set Enrichment Analysis (GSEA) for pathway analysis. Clonal T cells not only had upregulated genes from cytotoxic pathways (granzyme B, perforin and FGFBP2) but also genes which suppress apoptosis, proliferation, cell cycle G1/S transition arrest and T cell activation (RAS, CSK and TOB pathways) indicating anergy which was validated by CFSE tracking of proliferation after antiCD3+antiCD28 stimulation. The current studies provide further evidence for the persistence and relevance of clonal T cells in monoclonal gammopathies.

Disclosures

No relevant conflicts of interest to declare.
MPT Vs ThaDD in Very Elderly Patients with Multiple Myeloma: a Case-Match Study.
Authors: Offidani Massimo , Bringhen Sara , Corvatta Laura , Petrucci Maria Teresa , Gentili Silvia , Musto Pellegrino , Polloni Claudia , Gentilini Fabiana , Falcone Antonietta , Caravita Tommaso , Liberati Anna Marina , Brunori Marino , Callea Vincenzo , Piro Eugenio , Grasso Mariella , Cangialosi Clotilde , Galieni Piero , Rossini Fausto , Galli Monica , Montanaro Marco , Alesiani Francesco , Stefano Valerio De , Catarini Massimo , Elice Francesca , Zambello Renato , Visani Giuseppe , Griso Luciano , Angelucci Emanuele , Boccadoro Mario , Leoni Pietro , Palumbo Antonio
Published: October 2019

Abstract 1835

Poster Board I-861

Five studies demonstrated the superiority of MPT over MP regimen in elderly patients with MM not eligible for transplantation. In particular, one of these studies, showed this figure in patients aged more than 75 years who represent more than one third of MM patients. Nevertheless, in this latter study 42.5% of patients withdrawn from the MPT protocol because of toxicity. Therefore, there is a wide room of improving these results in this troublesome patient population. Using ThaDD regimen, including liposomal pegylated doxorubicin, we reported low haematological and non-hematological toxicity in elderly and