bluuebunny/crossref_metadata_2025_split huggingface dataset:
Vacuolin-1 Promotes Enucleation of Cultured Human and Murine Erythroblasts
Authors: Keerthivasan Ganesan , Ghinassi Barbara , Wilmes John S. , Wickrema Amittha , Migliaccio Anna Rita F , Duban Mark-Eugene , Crispino John 
Published: October 2019

Abstract
Abstract 2321
Enucleation of erythroblasts is the final step of differentiation before reticulocytes enter circulation in mammals. In culture, enucleation efficiencies vary widely, between 0 and 90%: improving this process is a necessary step for large-scale production of reticulocytes for transfusion. Recently, we demonstrated that the formation, movement and coalescence of vesicles and vacuoles play a key role in enucleation. Moreover, we showed that vacuolin-1, a small molecule that induces the formation and fusion of vacuoles, increases the enucleation of primary erythroblasts. Separately, we recently discovered that the anti-apoptotic and chromosomal passenger protein survivin plays a novel role in enucleating erythroblasts. Survivin interacts with clathrin and EPS-15, proteins that play important roles in vesicle trafficking, and together these proteins contribute to enucleation. In order to further understand the specific role played by survivin in vesicle trafficking and enucleation, we treated fetal liver Ter119- cells derived from survivin fl/fl mice with TAT-Cre to excise the survivin gene and assess the consequences of survivin loss on enucleation. We observed a significant reduction in the extent of enucleation upon deletion of survivin. Of note, survivin depleted cells harbored fewer numbers of vacuoles compared to control cells, and the vacuoles that were present were smaller than normal. Survivin deleted cells also lacked autophagolysosomes, which are formed by fusion of late endosomes/lysosomes to autophagosomes. Since vacuolin-1 is known to induce fusion of endosomes and lysosomes, we hypothesized that vacuolin-1 would rescue this enucleation defect observed in survivin KO erythroblasts. Indeed, vacuolin-1 reversed the inhibitory effect of survivin KO on enucleation. Surprisingly, we also observed that vacuolin-1 promoted the proliferation of human erythroblasts at the CFU-E stage, leading to a 4-fold increase in the percentage of glycophorin-positive cells on day 12. Thus, we conclude that vacuolin-1 acts at multiple stages of differentiation to improve the production of erythrocytes in vitro. Finally, to gain insights into the mechanism of action of the “hit” vacuolin-1, and to identify more potent small molecules that enhance enucleation in vitro, we designed and studied a further small set of vacuolin-1 structural analogs (N=12). Four vacuolin-1 analogs increase enucleation of erythroblasts with greater potency than that of vacuolin-1, providing us with initial insights into the small molecule structure-activity relationships that might govern this process. We are currently assaying the activity of these next generation compounds in erythroblast cultures as a means to improve production of red blood cells for human use.

Disclosures:
No relevant conflicts of interest to declare.
Runx1 Induced Chromatin Folding of the PU.1 Gene Locus Is Necessary for Adult Long-Term Hematopoietic Stem Cell Maintenance
Authors: Staber Philipp B. , Zhang Pu , Ye Min , Huang Gang , Ruscio Annalisa Di , Ebralidze Alexander K , Tenen Daniel G. 
Published: October 2019

Abstract
Abstract 2364

Background:
PU.1 is a hematopoietic transcription factor that is essential for normal hematopoiesis. PU.1 gene expression is mainly regulated through the proximal promoter (PrPr), and an upstream regulatory element (URE) located –14kb or –17kb upstream of the transcription start site in mice and humans, respectively. We previously demonstrated that PU.1 is a downstream target of core binding factor Runx1 (also called AML1) for megakaryocytic, lymphoid, and myeloid differentiation. Recent reports identified Runx1 to restrict proliferation of hematopoietic stem cells (HSCs) and to prevent HSC exhaustion.

Approach:
By employing conditional (Mx1-Cre) Runx1 knockout mice (Runx1Δ/Δ) and knock-in mice with targeted disruption of all three Runx1 binding sites at the −14kb URE of PU.1 (mRunxki/ki) we aimed to genetically dissect a potential functional role and mechanisms by which Runx1 might regulate PU.1 expression in HSCs.

Results:
Both, Runx1Δ/Δ and mRunxki/ki mice exhibited decreased PU.1 levels of approximately 65% in purified phenotypic HSCs (CD150+CD48-LSKs). In addition, activating histone marks H3K4me3 and H3Ac were significantly decreased at the −14kb enhancer and the PrPr. Mechanistically, we found by quantitative chromosome conformation capturing (3C) that Runx1 binding established a physical interaction of the −14kb enhancer with the PrPr thereby forming an active chromosomal conformation for proper PU.1 transcription. In concordance to Runx1Δ/Δ, phenotypic HSCs of mRunxki/ki mice were significantly increased in number. However, limiting dilution transplantations using the congenic Ly5.1/Ly5.2 system revealed a 25-fold decrease of competitive repopulating units (CRU) in phenotypic HSCs of mRunx-ki/ki mice compared to controls. Long-term reconstitution in serial transplantation assays was also severely impaired indicating HSCs exhaustion in mRunxki/ki mice. HSC homing and engraftment was not affected. Importantly, restoration of PU.1 levels by crossing to a human PU.1 transgenic fully rescued long-term HSC functions, thus demonstrating that HSC maintenance was strictly related to PU.1 levels.

Conclusion:
Runx1 binding to the −14kb enhancer of PU.1 is necessary to maintain an active chromosomal conformation for proper PU.1 transcription in order to prevent HSC exhaustion.

Disclosures:
No relevant conflicts of interest to declare.
Targeting Survivin with YM155 As a Potential Therapy in Pediatric Acute Lymphoblastic Leukemia
Authors: Jemal Abdusebur , Tyner Jeffrey W , Thayer Mathew , Muschen Markus , Druker Brian J. , Chang Bill H 
Published: October 2019

Abstract
Abstract 2490

Background:
Pediatric Acute Lymphoblastic Leukemia (ALL) remains the most common pediatric malignancy. Despite advances in treatment and outcomes, there continue to be subsets of patients that are refractory to standard intensive chemotherapy and hematopoietic stem cell transplant. Therefore, novel gene targets for therapy are needed to further advance treatment for this disease. Survivin, a member of the chromosome passenger complex and inhibitor of apoptosis has been shown to be over-expressed in malignant cells and in relapsed ALL. Therefore, survivin may be a potential target for therapy in pediatric ALL. The selective survivin suppressant, YM155 (Astellas) has been shown to inhibit survivin expression and activate cell death in multiple cell lines. Early phase I studies show promise in both tolerability and possible efficacy in B-cell malignancies. Therefore, this drug may have the potential of improving treatment for pediatric B-cell precursor ALL.

Design/methods:
Pediatric lymphoblastic cell lines, fresh primary lymphoblast cells from newly diagnosed patients with ALL and xenografted patient samples were used in this study. Cells were incubated in the presence of YM155 at doses ranging from 1nM to 10μM. Viability was measured using a standard methane-thiosulfonate viability assay. Activation of apoptosis was identified using the Guava nexin Annexin V binding assay for cell lines.

Results:
Treatment of ALL cell lines, primary patient samples and xenograft samples show a dose-dependent sensitivity to YM155 by both activation of apoptosis and by cell viability. IC50 doses for the majority of the samples are in the low nanomolar range (Table). Interestingly, there is some variability amongst patient samples suggesting possible variable responses in vivo. Ectopic expression of survivin in cell lines treated with YM155 rescues the effect of the drug. Further, t(9;22) positive ALL samples, including primary patient, xenograft, and dasatinib resistant samples remain significantly sensitive to YM155. For dasatinib sensitive Ph+ALL samples, combination therapy suggest an additive effect by isobologram analysis.

Conclusion:
Pediatric ALL samples remain sensitive to treatment with YM155 in cell lines, primary patient and xenografted samples. The results of these experiments will be used as a foundation to develop a comprehensive understanding of the mechanisms of survivin dependence in pediatric ALL. Future studies will also be designed to develop YM155 as an additional therapy for pediatric acute lymphoblastic leukemia.

Disclosures:
Druker: Cylene:; MolecularMD:; Novartis:; Bristol-Myers-Squibb:.
Fluoroquinolone Prophylaxis in Acute Myeloid Leukemia (AML) Patients Undergoing Post-Remission Chemotherapy Reduces Hospital Admission Rates
Authors: Kusick Karissa , Earl Marc , Gallagher Erika M. , Yeh Jun-Yen , Advani Anjali S. , Kalaycio Matt , Copelan Edward A. , Sekeres Mikkael A. 
Published: October 2019

Abstract
Abstract 2570

Background:
High-dose cytarabine (HIDAC) is a standard treatment for younger AML patients (pts) receiving post-remission chemotherapy. The risk for febrile neutropenia (FN) is high among these pts and considered a medical emergency, requiring prompt hospital admission and treatment. In 2004 our institution adopted the use of fluoroquinolones (FQ) for bacterial prophylaxis in AML pts receiving HIDAC. We compared rates of hospital admission due to FN before and after the initiation of bacterial prophylaxis with FQ. We also evaluated the incidence of blood stream infections (BSI) and the incidence of resistant infections among the two groups.

Methods:
We identified AML (excluding acute promyelocytic leukemia) pts receiving cytarabine-based remission induction chemotherapy at Cleveland Clinic who achieved a complete remission and then were treated with HIDAC post-remission chemotherapy (cytarabine dose of 3000mg/m2 × 6) from 1997–2008. Pts receiving FQ prophylaxis with ciprofloxacin 500mg twice daily or levofloxacin 500mg once daily comprised the treatment group, while AML patients with no prophylaxis served as controls. Pts were excluded if they developed an episode of FN during hospital admission for HIDAC or received non-FQ bacterial prophylaxis. Categorical data and continuous variables were analyzed per chemotherapy cycle using Fishers exact test and independent-sample t-tests, respectively.

Results:
80 pts received HIDAC post-remission chemotherapy during the study period; 52 received FQ prophylaxis; 28 did not. A total of 198 chemotherapy cycles were administered, 148 in the FQ group and 50 in the control group. The mean age (+/−SD) of the entire cohort was 44.8 (11.4) years; 53% were female. Despite the decreased use of granulocyte-colony stimulating factor in the FQ group (12% vs. 50%, p<0.001), the duration of neutropenia was the same (24.6 days vs. 23.8 days, p=0.5) between groups. All other baseline characteristics were similar. FQ treated pts had a 33% absolute reduction in hospital admissions due to FN (50.7% vs. 84%, p<0.001) and when admitted, hospital LOS for FN was shorter for the FQ group (7.9 days vs. 10.5 days, p=0.007). The incidence of BSI was also reduced in the FQ group (23% vs. 42%, p=0.009); however there was an increase in resistant gram-negative (5/17 vs. 0/14, p=0.04) and resistant gram-positive (15/19 vs. 3/9, p=0.03) infections among pts treated with FQ.

Conclusion:
AML pts treated with HIDAC chemotherapy followed by FQ prophylaxis had fewer and shorter hospital admissions due to FN compared to pts with no prophylaxis. FQ treated patients were at increased risk of developing antibiotic-resistant bacterial BSI.

Disclosures:
No relevant conflicts of interest to declare.
A Somatic Variant in MYD88 (L265P) Revealed by Whole Genome Sequencing Differentiates Lymphoplasmacytic Lymphoma From Marginal Zone Lymphomas
Authors: Xu Lian , Sohani Aliyah R. , Arcaini Luca , Hunter Zachary , Yang Guang , Zhou Yangsheng , Liu Xia , Cao Yang , Manning Robert , Patterson Christopher J , Ioakimidis Leukothea , Tripsas Christina , Pinkus Geraldine S. , Harris Nancy Lee , Rodig Scott J. , Treon Steven 
Published: October 2019

Abstract
Abstract 261
Lymphoplasmacytic (LPL) and marginal zone lymphoma (MZL) are distinct clinicopathological entities under the WHO classification system for B-cell lymphomas. Differentiation of LPL from MZL has been difficult due to overlapping clinical, morphological, histopathological, immunophenotypic, and cytogenetic features. We therefore sought to identify a molecular marker by which LPL could be differentiated from MZL. Using paired normal/tumor tissues from 10 LPL patients, whole genome sequencing was utilized to identify somatic variants. These studies identified a somatic variant at position 38182641 in chromosome 3p22.2 with a single nucleotide change from T→C in the myeloid differentiation primary response (MYD88) gene, and a predicted non-synonymous change at amino acid position 265 from leucine to proline (L265P) in 10 of 10 LPL patients. MYD88 L265P is an oncogenically active mutation in DLBCL ABC cell lines via activation of IRAK1/4/TRAF-6/NF-κβ signaling, and is present in tumors from 29% of patients with ABC subtype of DLBCL, and 6% of patients with MALT lymphomas (Ngo et al, Nature 2011, 470:115–119). Further to these efforts, we performed Sanger sequencing of MYD88 in malignant cells obtained from 51 patients with LPL, 49 of whom had an IgM monoclonal protein and were therefore classified as Waldenstrom's Macroglobulinemia (WM), and 2 with an IgG monoclonal protein, along with 46 patients with MZL, which included 21 Splenic (SMZL), 20 Extranodal (EMZL), and 5 Nodal (NMZL) Subtypes, as well as B-cells from 15 healthy donors. Among LPL patients, the MYD88 L265P variant was found in malignant cells from 46/51 (90.1%) cases, which included 44 patients with WM, and 2 patients with IgG LPL. Expression of the MYD88 L265P variant was heterozygous in 42, and homozygous in 4 LPL patients. By comparison, only 3/46 (6.5%) patients with MZL (1 SMZL; 1 EMZL; 1 NMZL) exhibited the MYD88 L265P variant which was heterozygous (p<0.0001), and included 2 patients (1 SMZL, 1 NMZL) with extensive bone marrow involvement, a monoclonal IgM protein, and whose clinicopathological characteristics overlapped with LPL. By comparison, the MYD88 L265P variant was absent in CD19+ cells from all 15 healthy donors. The results of this study demonstrate that the MYD88 L265P mutation is widely expressed in patients with LPL, and can be used to differentiate LPL from MZL.

Disclosures:
Treon: Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.
KPT-SINE, a Potent, Small Molecule Inhibitor of CRM1-Dependent Nuclear-Cytoplasmic Shuttling, with Potent Activity Against T-ALL and AML
Authors: Etchin Julia , Kentsis Alex , Sanda Takaomi , Kung Andrew L. , Stone Richard M. , McCauley Dilara , Kauffman Michael , Shacham Sharon , Look Thomas 
Published: October 2019

Abstract
Abstract 2622

Background:
CRM1 (XPO1) is the major nuclear exporter that mediates transport of a variety of cargos, including proteins involved in tumor suppressor and cellular proliferation pathways (e.g. p53, PI3K/AKT, and NF-kB). CRM1 is upregulated in a range of solid and hematologic malignancies and its overexpression is correlated with poor prognosis, and with resistance to chemotherapy. Recently, the crystal structure of CRM1, in complex with its export cargo, Snurportin 1, has been resolved. The structure elucidates the key contact residues required for the formation of the CRM1-cargo export complex. The molecular understanding of the CRM1-cargo binding interface has led to the development of novel small molecule inhibitors of CRM1-cargo interaction, termed KPT-Selective Inhibitors of Nuclear Export (SINE). KPT-SINE are potent, drug-like CRM1 inhibitors that irreversibly inactivate the CRM1-directed protein export by covalent modification of the essential CRM1-cargo binding residue Cys528. The inhibition of the CRM1 nuclear export has been shown to lead to selective apoptosis in cancer cells when compared to normal cells. Here, we assess the efficacy of the KPT-SINE in human T-ALL, AML, and normal hematopoietic cells. Methods: The viability of a panel of human T-ALL and AML cell lines and normal CD34+ progenitor cells upon treatment by the KPT-SINE was assessed. Dose-response measurements were done using serial dilutions of KPT-SINE from 1 μM to 0.3 nM and luminescent cell viability assay. Apoptosis was measured using Annexin V staining and TUNEL assays. Results: KPT-SINE induces rapid apoptosis in 12 of 15 T-ALL and 10 of 14 AML cell lines with 50% inhibitory concentrations (IC50s) of 15–100 nM. In the KPT-SINE-sensitive cell lines, BCL2 overexpression suppresses KPT-SINE-induced apoptosis, indicating its intrinsic pathway mediation. Importantly, KPT-SINE exhibits selective cytotoxicity against tumor cells when compared to peripheral blood mononuclear cells (PBMCs) and CD34+ progenitor cells in vitro. KPT-SINE at 57 and 150 mg/kg induced 100% inhibition of tumor growth in vivo in a MOLT-4 T-ALL xenograft study. Other xenograft studies to address the potency of the KPT-SINE on selected AML lines in vivo are ongoing. These studies emphasize the clinical promise of the KPT-SINE as a novel and selective drug candidate for the treatment of T-ALL and AML.

Disclosures:
McCauley: Karyopharm Therapeutics: Employment. Kauffman:Karyopharm: Equity Ownership. Shacham:Karyopharm: Equity Ownership.
Improved Survival in Chronic Myeloid Leukemia (CML) Since the Introductin of Imatinib Therapy - A Single Institution Historical Experience
Authors: Lee Hun , Cortes Jorge E. , O'Brien Susan , Jabbour Elias , Garcia-Manero Guillermo , Quintas-Cardama Alfonso , Shan Jenny , Rios Mary Beth , Ravandi Farhad , Faderl Stefan , Kadia Tapan , Borthakur Gautam , Huang Xuelin , Kantarjian Hagop M. 
Published: October 2019

Abstract
Abstract 2750

Background:
Outcome of CML since introduction of imatinib therapy has improved. Aims: analyze improvement of CML outcome in different phases. Study Group: A total of 1,569 patients with CML referred since 1965, within 1 month from diagnosis, were reviewed and used to identify phase-specific prognostic factors: 1,148 chronic, 175 accelerated, 246 blastic. Results: The median survival was 8.9 years in chronic, 4.8 years in accelerated, and 6 months in blastic phase. In chronic phase, the 8-year survival was ≤ 15% before 1983, 42–65% from 1983 to 2000, and 87% since 2001 (Figure 1). Survival was worse in older patients (p=0.004), but less significant since 2001 (p=0.07). Survival by Sokal risk was significantly different before 2001 (p<0.001), but not since 2001 (p=0.4). In accelerated phase, survival improved over time (p<0.001); the 8-year survival in patients treated since 2001 was 75% (Figure 2). Survival by age was not different in years < 2001 (p=0.09), but was better since 2001 in patients ≤ 70 years (p=0.004). Multivariate analysis derived adverse factors since 2001: older age (p=0.049), increased marrow blasts (p=0.03). In blastic phase, the median survival improved over time (p<0.001), although it is only 7 months since 2001. Conclusions: Survival in CML significantly improved significantly since 2001, particularly so in chronic and accelerated phases. Imatinib therapy minimized the impact of known prognostic factors and Sokal risk in chronic phase, and accentuated the impact of age in accelerated and blastic phases.

Disclosures:
Cortes: Novartis: Consultancy; Novartis: Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Kantarjian:Novartis: Consultancy; Novartis: Research Funding; Pfizer: Research Funding; BMS: Research Funding.
Resveratrol Enhances NKG2D-Mediated Cytotoxicity Against Leukemia Cells by Upregulating Both NKG2D Receptor on NK Cells As Well As NKG2D Ligands on Target Cells,
Authors: Espinoza Luis J. , Takami Akiyoshi , Nakata Katsuya , Trung Ly Quoc , Yamada Kayoko , Touda Saki , Nakao Shinji 
Published: October 2019

Abstract
Abstract 3236
NKG2D is a powerful activating receptor expressed by natural killer (NK) cells that promotes cytotoxic lysis of cancer cells expressing NKG2D ligands (NKG2D-Ls). Pharmacological induction of NKG2D-Ls in malignant cells has been an attractive therapeutic approach but has gained poor clinical utility because currently available NKG2D-Ls inducers have been hampered either by their limited efficacy or by their associated toxicity. Resveratrol (RVT), a compound derived from several natural sources, has proved in vivo and in vitro potent anti-tumor effects against various cancers. Extensive research in the last decade has shown that such effects are mediated by targeting various molecules involved in the regulation of proliferation and cell survival and those include, NFκB, STAT3, ATM/ATR and ERK1/2. To date, it is unknown whether RVT has any effect on NKG2D-Ls expression. We report here NKG2D-Ls induction by RVT in a broad range of leukemia cells. RVT upregulated the NKG2D-Ls MICA/B, ULBP1, ULBP2 and ULBP3 in the myeloid leukemia cells OUN-1, NB4, THP-1 and KG1 and upregulated MICA/B, ULBP-1 and ULBP3 ligands in the lymphoid leukemia cells Jurkat and Molt-4. The upregulation of NKG2D-Ls by RVT was associated with increased transcription of each NKG2D-L gene. Ligand upregulation induced by RVT was prevented by cell pretreatment with caffeine, and inhibitor of ATM/ATR, which is the main signal regulator of NKG2D-Ls expression. Leukemia cells treated with RVT were more susceptible to killing by NK cells than untreated cells and the enhanced cytotoxicity of NK cells was blocked by the treatment of NK cells with anti-NKG2D monoclonal antibodies. Interestingly, the same concentration of RVT that effectively induced NKG2D-Ls in tumor cells, consistently upregulated NKG2D receptor expression in primary NK cells from healthy individuals and in the NK cell lines NKL and NK-92 and this effect was also associated with enhanced NKG2D-mediated NK cells cytotoxicity. RVT-induced NKG2D receptor enhancement in NK cells associated with the activation of the MAP kinase ERK1/2 and was prevented by the ERK1/2 specific inhibitor PD98059. Thus, RVT represents the first identified agent capable of activating both arms of the NKG2D axis. Since several clinical trials on RVT are ongoing, these previously unrecognized properties of this non toxic compound have an attractive immunotherapeutic potential.

Disclosures:
No relevant conflicts of interest to declare.