bluuebunny/crossref_metadata_2025_split huggingface dataset:
P53 is Activated without RPL11 Upregulation in Diamond-Blackfan Anemia,
Authors: Du Hong-Yan , Elghetany M. Tarek , Alter Blanche P , Shimamura Akiko 
Published: October 2019

Abstract
Abstract 3432
Diamond-Blackfan anemia (DBA) is an autosomal dominantly inherited bone marrow failure syndrome characterized by red cell aplasia, physical anomalies, and cancer predisposition. DBA is caused by mutations resulting in haploinsufficiency of genes encoding ribosomal proteins. p53 is activated in the erythroid lineage following reduction of ribosomal protein expression; however the mechanism whereby ribosomal stress results in p53 activation in DBA remains unclear. RPL11 has been proposed to play a central role in p53 activation following ribosomal stress. Reduced expression of individual small ribosomal subunit proteins in a tumor cell line resulted in increased translation of RPL11. Excess free RPL11 can bind and inactivate HDM2, an E3 ubiquitin ligase targeting p53 for degradation. The recent demonstration that cellular responses to ribosomal perturbations vary widely between different tissues raised the question of whether RPL11 upregulation contributes to p53 activation following ribosomal stress in hematopoietic progenitors. To address this question, we modeled DBA in human CD34+ cells. Since RPS19 is the most commonly mutated gene in DBA, we used lentiviral vectors expressing short hairpin RNAs to knock down RPS19 expression in primary human CD34+ cells. RPS19 protein levels were reduced to about 50% of control levels in a manner reflecting the haploinsufficient state in DBA. RPS19 depletion resulted in elevated p53 protein levels and increased mRNA levels of p21, a transcriptional target of p53. Total p53 mRNA levels and p53 mRNA translational activity remained unchanged consistent with a post-transcriptional mechanism for p53 activation. Although total RPL11 mRNA levels were not diminished following RPS19 depletion, RPL11 protein levels were significantly decreased consistent with post-transcriptional downregulation. Depletion of RPS19 in human CD34+ cells did not affect polysome loading of RPL11 mRNA. Reduction of additional ribosomal proteins also accompanied RPS19 knockdown consistent with coordinate regulation of multiple ribosomal protein levels. Corticosteroids, which improve anemia in the majority of DBA patients, did not prevent p53 activation, nor did this improve RPS19 or RPL11 protein levels. Expression of p53 was also assessed in bone marrow biopsy slides from 26 DBA patients with the following genotypes: RPS19 (18), RPS24 (2), RPS26 (2), RPS10 (1), RPS17 (1), RPS7 (1), and RPL11 (1). p53 was over-expressed in all but one patient (RPS26), and was clearly over-expressed in the DBA patient harboring the RPL11 mutation. In summary, we find that p53 activation in DBA does not require upregulation of RPL11 translation or elevated RPL11 protein levels. p53 activation persists in DBA caused by RPL11 deficiency. Corticosteroids do not improve ribosomal protein levels nor do they prevent p53 activation.

Disclosures:
No relevant conflicts of interest to declare.
Cytokine/Chemokine Expression with Protein Arrays Identifies Three Subsets of T-Cell Lymphomas with Differented Immunological Profiles,
Authors: Mercadal Santiago , Martinez Antonio , Colomo Lluis , Campo Elias , Lopez-Guillermo Armando 
Published: October 2019

Abstract
Abstract 3473

Background:
Only a few studies have addressed the expression of cytokine/chemokine in T-cell lymphomas. The aim of this study was to analyze the cytokine/chemokine protein expression profiles and to correlate them with the pathological manifestation of T-cell lymphomas. Patients and methods: Samples at diagnosis from 19 patients were T-cell lymphoma (unspecified peripheral T-cell lymphomas (PTCL), 10 cases; angioimmunoblastic (AITL), 4; ALK-negative anaplastic large-cell (ALCL), 3 and ALK-positive ALCL, 2) and were studied by protein microarrays. The characteristics of the patients were as follows: 11/8 M/F; median age, 55 years. 50% of patients were stage IV, 69% had extranodal involvement, 79% high LDH and 66% high β2 microglobulin. The high or high-intermediate risk IPI and PIT score were 62% and 61%, respectively. We have analyzed the expression of 174 cytokines by using a highly sensitive and specific antibody array containing single band specific primary antibodies duplicate. Chemolumiscence detection was performed by a HRP-conjugated streptavidin in an image analysis system. Densitometry analysis of unsaturated pixels was performed with Image Gauge software and an endogenous housekeeping gene was used to normalize results. Unsupervised clustering analysis was then performed. Results: Three clusters were obtained. Cluster 1 included 5 ALCL, 2 PTCL and 1 AITL, with a cytokine/chemokine profile that had high expression of IL21R, MIG, PDGF AA/AB, LIGHT, IL10, MIP1γ, MIP3α and IL13. Cluster 2 was composed by 5 PTCL and 1 AITL and exhibited a profile characterized by the high expression of L-SELECTIN, MMP1, IL1RII, SIGLEC 5, IL10R, IL1β, IL18BPA, FGF7, IL13RAα2, GMCSF, SDF1β, LIF, IL1Rα and IL11. Finally, the analysis defined a cluster 3 constituted by 2 AITL and 3 PTCL with a cytokine/chemokine profile that showed the high expression of TIMP1, NT4, ITAC and IGBP6. Detailed initial features and outcome of the patients according to the clusters are detailed in the table. Cluster 1 had better survival than the other groups (p=0.01). Summary: T-cell lymphomas show three different cytokine/chemokine patterns as was determined by protein microarrays. Cluster 1 was well characterized with the presence of all ALCL and better survival.

Disclosures:
No relevant conflicts of interest to declare.
Treatment with the PI3K/mTOR Inhibitor, NVP-BEZ235, Overcomes Resistance to Imatinib in Quiescent or T315I-Mutated Ph+ Leukemia Cells,
Authors: Minami Yosuke , Minami Miho , Fukushima Nobuaki , Kuwatsuka Yachiyo , Naoe Tomoki 
Published: October 2019

Abstract
Abstract 3488
Ph+ leukemia is effectively treated with BCR-ABL kinase inhibitors such as imatinib (IM). However, recent studies suggest that IM therapy does not eradicate Ph+ leukemia stem cells (LSCs). In order to examine the mechanisms of Ph+ LSC resistance to IM and seek strategies to overcome this, Ph+ ALL patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice and abundant spleen cells derived from leukemic NOG mice were co-cultured with S17-stromal cells using methods previously reported (Minami et al., Proc Natl Acad Sci USA, 2008). Slow-cycling (Hoechst-33342low/Pyronin-Ylow) CD34+ cells were insensitive to IM despite complete dephosphorylations of BCR-ABL and its substrate CrkL, which indicated that addiction of BCR-ABL activity is lower for survival in such quiescent cells. From comprehensive drug screening of other small compounds using this co-culturing system, we found that inhibitors of PI3K/AKT/mTOR-axis signaling, including rapamycin, were promising candidates. Aberrant activation of mTOR signaling has also been reported to be involved in some systems of leukemia initiating cells. In vitro, combination treatment with IM and rapamycin analogue, everolimus (RAD001), induced substantial cell death in the slow-cycling CD34+ population with p70-S6K dephosphorylation and decreased expression of the anti-apoptotic BCL-2 family protein, MCL-1. In the leukemic NOD/SCID mouse ALL model, in vivo RAD001-treatment showed a decrease of tumor burden including slow-cycling CD34+ cells. However, during the cell death by RAD001-treatment, however, negative feedback-loop effects were detected such as reversely increased phosphorylations of AKT (Ser473) and FOXO1/3a. In vitro, the dual PI3K/mTOR inhibitor, NVP-BEZ235 (BEZ), induced substantial cell death including slow-cycling CD34+ cells at lower doses than those required by RAD001. In biomarker analyses, BEZ-treatment inhibited not only p70-S6K phosphorylation but also phosphorylations of mTOR (Ser2481) itself, 4E-BP1, AKT (Ser473) and FOXO1/3a. At the same time, expression of MCL-1 decreased during the cell death by BEZ-treatment. In T315I-mutated BCR-ABL dependent cell lines such as murine Baf3/p210-T315I and human-derived TCC-Y/T315I, treatment with BEZ induced growth inhibition and cell death. The effects of BEZ on normal stem cells and in mouse Ph+ leukemia models are under investigation. These results suggest that treatment with BEZ might have potential for overcoming the resistance to IM due to quiescent property in Ph+ LSCs or T315I-mutation.

Disclosures:
Naoe: Zenyaku-Kogyo: Research Funding; Novartis Pharma.: Research Funding; Chugai Pharma.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Kyowa-Hakko Kirin.: Research Funding; Otsuka Pharma.: Research Funding.
Platelet Factor 4 (PF4) Levels Are Inversely Correlated with Steady-State Platelet Counts and with Nadir Counts in Chemotherapy-Induced Thrombocytopenia (CIT),
Authors: Lambert Michele , Reznikov Alisa , Xiao Liqing , Poncz Mortimer 
Published: October 2019

Abstract
Abstract 3527
PF4 (CXLC4) is a member of the C-X-C family of chemokines and has been shown to be a negative autocrine of megakaryopoiesis in vitro and in animal models of CIT and radiation-induced thrombocytopenia (RIT). In animal models, steady-state platelet counts, as well as duration of thrombocytopenia and nadir platelet count after chemotherapy or radiation therapy were inversely correlated with platelet PF4 levels. We designed a clinical study to examine the role of PF4 in human subjects treated with chemotherapy. We examined platelet PF4 levels in 97 pediatric patients treated for standard risk leukemia in a single institution study. All patients completed therapy since January 1, 1999. Samples were collected at the completion of therapy from all patients, and a subset of randomly selected patients had multiple samples collected to determine intra-subject variability of assessment. PF4 levels were no different in this cohort from age matched normal controls. While there was significant inter-individual variability in the cohort, there was no significant difference in an individual between samples collected up to 1 year apart. There was an inverse correlation between platelet PF4 levels and steady-state platelet count (r=-0.2107 95% CI: −0.39 to −0.01, p=0.0383), yet we found no correlation with plasma thrombopoietin (TPO) or CXCL11 (stromal derived factor-1) levels, both of which are known positive regulators of megakaryopoiesis. We then examined the effect of PF4 on CIT by reviewing medical records of these patients, extracting the nadir platelet count and transfusion records for delayed intensification. There was an inverse correlation between platelet PF4 content and nadir platelet count during delayed intensification (r=-0.2087 95% CI: -0.41 to 0.01, p=0.0316). In addition, subjects who required platelet transfusion during delayed intensification had significantly higher platelet PF4 levels when compared to those subjects who did not require transfusion (11.48 ± 8.2 IU/108 platelets versus 5.23 ± 4.7 IU/108 platelets, p=0.003). All of the patients with PF4 levels > 1SD above the mean (>18 IU/108 platelets) were transfused, while only 28% of patients with PF4 levels > 1 SD below the mean (< 4 IU/108 platelets) (overall 58% of patients were transfused). In contrast, there was no difference between plasma TPO or CXCL11 levels between those patients who did and did not require transfusion. These clinical data are not only consistent with our previously published animal studies; they show that platelet PF4 levels may be the first identified, patient-specific predictor of risk of transfusion during chemotherapy and may allow for stratified dose-intensification in patients with cancer. These studies also suggest new approaches for modifying megakaryopoiesis and developing novel strategies to moderate CIT and possibly RIT.

Disclosures:
Lambert: Cangene: Honoraria.
Sequential Acquisition of AML1-ETO and c-Kit Mutation Leads to Formation of Human t(8;21) Acute Myelogenous Leukemia Stem Cells,
Authors: Shima Takahiro , Kikushige Yoshikane , Miyamoto Toshihiro , Akashi Koichi 
Published: October 2019

Abstract
Abstract 3584
The 8;21 translocation, one of the most general chromosomal abnormalities in acute myelogenous leukemia (AML), encodes the AML1-ETO chimeric fusion gene. Because AML1-ETO can inhibit the CBF complex to transactivate myeloid-lineage genes in a dominant negative fashion, the high level expression of this gene plays a critical role in inhibiting differentiation of target cells, which leads to progression of AML. We, however, have reported that patients maintaining a long-term remission retain AML1-ETO expression at a very low level that can be detected by nested RT-PCR. The AML1-ETO transcripts in these patients were derived from a small fraction of t(8;21)+ hematopoietic stem cells (HSCs) capable of multilineage differentiation (PNAS 2000). In fact, previous data shown that AML1/ETO knock-in or AML1/ETO transgenic mice did not develop AML. These data suggest that acquisition of the AML1-ETO fusion is not sufficient to develop t(8;21) AML. Since t(8;21) AML cells frequently possess constitutive active mutation of c-Kit, we hypothesized that the c-Kit mutation may work as a second oncogenic hit in t(8;21)+ HSCs to transform into AML. To test the hypothesis, we extensively analyzed the existence of c-Kit mutation within AML1-ETO+ HSCs from patients maintaining remission for a long-term. CD34+CD38− HSCs were purified from the bone marrow of patients in long-term remission, and were cultured in vitro to form colonies. These HSC-derived colonies were picked up, and tested for the presence ofAML1-ETO and c-Kit mutation. Five t(8;21) AML patients with c-Kit mutation were enrolled in this study. All of 1020 blastic colonies at diagnosis were positive for both AML1-ETO and c-Kit mutation. In 7187 colonies formed in the culture of remission marrow, almost 1% (89 colonies) of these colonies expressed AML1-ETO. Surprisingly, none of these colonies possessed c-Kit mutation, indicating that AML1-ETO+ clones in remission are not identical to these in t(8;21) AML. Accordingly, it is highly likely that HSCs first acquire AML1-ETO, and a fraction of these cells additionally mutated c-Kit, resulting in transformation into AML stem cells. This is the first clear-cut evidence that human HSCs transform into AML via multi-step oncogenesis in vivo.

Disclosures:
No relevant conflicts of interest to declare.
A Phase 1 Study to Assess the Absolute Bioavailability and Safety of An Oral Solution of Decitabine In Subjects with Myelodysplastic Syndromes (MDS),
Authors: Mistry Bipin , Jones Mark M , Kubiak Peter , Garcia-Manero Guillermo , Litzow Mark R. , Mesa Ruben A. , Rifkin Robert , Tarassoff Peter , Cortes Jorge E. 
Published: October 2019

Abstract
Abstract 3801

Purpose:
Decitabine is a deoxycytidine nucleoside derivative inhibitor of DNA-methyltransferases indicated for treatment of myelodysplastic syndromes (MDS) including previously treated and untreated, de novo and secondary MDS of all French-American-British subtypes. The aim of this study was to determine the absolute bioavailability and safety of a single oral dose of decitabine. Methods: This is an open-label, Phase 1, dose-escalation absolute bioavailability trial in which decitabine was administered orally on Cycle 1 Day 1, and followed by a 1-hour IV infusion of the currently approved regimen (20 mg/m2) on Cycle 1 Days 2–5. Serial blood samples for decitabine pharmacokinetic (PK) assessment were collected up to 6 h post-dosing during Cycle 1 on Days 1 and 2. Three subjects were recruited at each oral dose level of 30, 60, 120 and 240 mg. Subjects were evaluated for adverse events (AEs). Results: Study results are summarized in Table 1. Sixteen subjects were screened and 12 were enrolled. Nine subjects had de novo MDS and 3 had secondary MDS (IPSS score Int-2 = 6, Int-1 = 5, Low = 1). Following oral dose, Cmax was reached within 0.50 h (tmax) and after Cmax the plasma profile showed biphasic decline with terminal half-life ranging between 0.36 to 0.93 hr. Increase in oral dose did not result in proportional increase in Cmax and AUC. The PK parameters following oral doses showed very high variability. Based on comparison of dose normalized exposure, the absolute bioavailability ranged between 3.9 to 14.1% and increased proportionally with dose. The non-dose normalized bioavailabilities were calculated to find a dose at which mean oral bioavailability reaches ≥80% of the current IV regimen of 20 mg/m2. The non-dose normalized bioavailability ranged between 11.7 and 80.5% with highest oral bioavailability observed at 240 mg dose. The most common SAEs were pneumonia (n=4) and neutropenic fever (n=4). Five patients (41.7%) experienced diarrhea and nausea (all grades). Conclusion: The study demonstrates that the absolute bioavailability ranged between 3.9 and 14.1%. Oral decitabine was generally well tolerated at doses between 30–240 mg in MDS subjects and exhibited a safety profile similar to the profile following intravenous administration. The bioavailability and safety data may allow for further testing in the phase 2 setting.

Disclosures:
Mistry: Eisai Inc.: Employment. Jones:Eisai Inc.: Employment. Kubiak:Eisai Inc.: Employment. Garcia-Manero:Eisai Inc.: Research Funding. Litzow:Eisai Inc.: Research Funding. Mesa:Eisai Inc.: Research Funding. Tarassoff:Eisai Inc.: Employment. Cortes:Eisai Inc.: Research Funding.
Marked Upregulations of Survivin and Aurora-B Kinase Are Associated with Disease Progression in the Myelodysplastic Syndromes,
Authors: Yoshida Akira , Zokumasu Kouichi , Yamauchi Takahiro , Takagi Kazutaka , Kishi Shinji , Urasaki Yoshimasa , Tohyama Kaoru , Ueda Takanori 
Published: October 2019

Abstract
Abstract 3823
Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffctive hematopoiesis. Survivin belongs to the inhibitors of apoptosis (IAP) family and inhibits apoptosis. Besides its role as IAP, Survivin appears to function as a subunit of the chromosomal passenger complex (CPC) for regulating mitosis with other CPC proteins including Aurora-B. As the CPC, Survivin and Aurora-B play an important role in the maintenance of a stable genome. Until now, there has been no report to examine whether expression of Survivin and Aurora-B kinase may be increased in CD34(+) cells prepared from the patients with MDS during disease progression. To investigate the contribution of Survivin and Aurora-B to the progression and prognosis of MDS, we evaluated the expression levels of these two genes in CD34(+) cells prepared from 57 patients with MDS and 50 patients with de novo AML using real-time quantitative polymerase chain reaction. The degree of Survivin and Aurora-B expression was highly correlated with the type of MDS, was much higher in RAEB-1, RAEB-2 and secondary AML following MDS (s-AML) compared with normal control, and increased during disease progression. Expression levels of both Survivin and Aurora-B were significantly correlated with International Prognostic Scoring System (IPSS). Furthermore, the amount of Aurora-B kinase was significantly correlated with Survivin expression in MDS and s-AML, but not in de novo AML. Interestingly, the levels of Survivin and Aurora-B were remarkably higher in patients with s-AML when compared with de novo AML. We demonstrated for the first time that high levels of Survivin and Aurora-B kinase expression are distinctive molecular feature of high-risk MDS and s-AML. Marked up-regulations of Survivin and Aurora-B Kinase may contribute to genetic instability and disease progression of MDS.

Disclosures:
No relevant conflicts of interest to declare.
Platelet Doubling After the First Azacitidine Cycle Is a Promising Predictor for Response in MDS, CMML and AML Patients in the Dutch Azacitidine Compassionate Patient Named Program,
Authors: Huls Geert A , van der Helm Lieke H. , Alhan Canan , Wijermans P.W. , van Marwijk Kooy Marinus , Schaafsma Martijn R. , Biemond Bart J , Beeker Aart , Hoogendoorn Mels , van Rees Bastiaan P. , de Weerdt Okke , Wegman Jurgen , Libourel Eduard J , Luykx-de Bakker Sylvia A. , Minnema Monique C. , Brouwer Rolf , Croon-de Boer Fransien , Eefting Matthias , Jie Kon-Siong G. , van der Loosdrecht Arjan A. , Koedam Jan , Veeger Nic J.G.M. , Vellenga Edo 
Published: October 2019

Abstract
Abstract 3841
The efficacy of azacitidine in the treatment of high risk myelodysplastic syndromes (MDS), chronic myelomonocytic leukaemia (CMML) and acute myeloid leukaemia (AML) (20–30% blasts) has been demonstrated. To investigate the efficacy of azacitidine in daily clinical practice and to identify predictors for response, we analyzed a cohort of 90 MDS, CMML and AML patients who have been treated in a Dutch compassionate patient named program.
Patients received azacitidine for a median of 5 cycles (range 1–19). The overall response rate (CR/PR/HI) was 57% in low risk MDS, 53% in high risk MDS, 50% in CMML, and 39% in AML patients. Median overall survival (OS) was 13.0 (9.8–16.2) months. In multivariate analysis we confirmed that circulating blasts (HR 0.48, 95% CI 0.24–0.99; p=.05) and poor risk cytogenetics (HR 0.45, 95% CI 0.22–0.91; p=.03) are independent predictors for OS. Interestingly, in this analysis we also identified platelet doubling after the first cycle of azacitidine as a simple and independent positive predictor for OS (HR 5.4, 95% CI 0.73–39.9; p=.10).
Of the 90 treated patients, 14 (16%) had an at least two-fold increase in platelet count after the first cycle of azacitidine, which was associated with significant better OS (p=.01, according to logrank test) (figure). Of these 14 patients 13 could be classified according the azacitidine prognostic scoring system for OS as recently proposed by Itzykson et al. (Blood:2011;117:403); 6 patients belonged to the low risk and 7 to the intermediate risk group. Median baseline platelet count of these patients was 35 x109/L (range 2–290 x109/L). Characteristics of this subgroup of patients were not significantly different from the patients without platelet doubling. Interestingly, platelet doubling was observed in all cytogenetic risk groups, in patients with and without circulating blasts, and in patients who are transfusion dependent and independent.
In conclusion, routine administration of azacitidine to patients with variable risk groups of MDS, CMML and AML is feasible and subgroups with distinct efficacy of azacitidine treatment can be identified.

Disclosures:
Wijermans: Centocor Ortho Biotech Research & Development: Research Funding.