bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of Decitabine and Homoharringtonine in Combination in Leukemia Cell Lines
Authors: Geng Suxia , Yao Han , Weng Jianyu , Huang Xin , Wu Ping , Deng Chengxin , Lu Zesheng , Du Xin 
Published: November 2019

Abstract
Background: The methylation inhibitor decitabine (DAC) has great therapeutic value for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, DAC monotherapy is associated with relatively low rates for overall response and complete remission. We aimed to investigate the effects of the combination of DAC and homoharringtonine (HHT) in the SKM-1 and Kg-1a cell lines and explore their associated mechanisms.
Methods: Cell viability was estimated by MTS assay. Cell apoptosis were detected by flow cytometry analysis and PI/Annexin V staining. Western blot and quantitative reverse transcription-polymerase chain reaction assays were performed to detect the expression of apoptosis-related genes including caspase-3, caspase-9, BCL-2 and BCL-XL and DNA methyltransferases(DNMT) including DNMT1, DNMT3A and DNMT3B. Changes in LINE-1 gene methylation were assessed by quantitative polymerase chain reaction after bisulfite conversion. The effects of the combinations were estimated using CalcuSyn software.
Results: The combination of DAC and HHT showed synergistic effects for inhibiting cell viability in SKM-1 and KG-1a cell lines. This combination can enhance inhibition of colony formation and apoptosis induction of DAC alone in SKM-1 cell line. However, in Kg-1a cells, this combination only enhanced the apoptosis induction of DAC alone. For SKM-1 cells, further study found that different doses of DAC and HHT alone have different effects on the expression of the apoptosis-related genes caspase-3, caspase-9, BCL-XL and BCL-2. The combination of 0.4 μM DAC and 5 nM HHT treatment significantly increased the mRNA expression of caspase-3 (P=0.0005) and caspase-9 (P=0.0075) and decreased the expression of BCL-2 (P=0.0331), but has no significantly effect on the BCL-XL (P=0.3436) compared with 0.4 μM DAC alone. However, 4 μM DAC plus 50 nM HHT had no significant effects on the mRNA and protein expression of the apoptosis-related genes examined compared with 4 μM DAC alone (P>0.05). Low-dose DAC induced greater hypomethylation than higher doses of the drug. Whereas HHT had no demethylation effects, and also had no enhancement effects with DAC on the hypomethylation and mRNA and protein expression of DNMT1, DNMT3A and DNMT3B in SKM-1 cells.
Conclusion: The combination of DAC and HHT has synergistic effects on cell viability inhibition and enhancement effects on cell apoptosis in SKM-1 and KG-1a cell lines. But this combination did not enhance the hypomethylation effect of DAC.These data suggest that DAC used in combination with HHT has clinical potential in the treatment of MDS and AML.

Disclosures
No relevant conflicts of interest to declare.
Chidamide, a Novel Histone Deacetylase Inhibitor, Displays Potent Antitumor Activity Against MDS Cells Mainly through JAK2/STAT3 Signaling Inhibition
Authors: Chang Chunkang , Zhao Sida , Guo Juan , Zhao Youshan , Fei Chengming , Gu Shucheng , Li Xiao 
Published: November 2019

Abstract
Background:
Many studies have indicated that histone deacetylases (HDAC) activity is always increased in a lot of human tumors, and inhibition of HDAC activity are promising new strategies in the treatment of cancers. Chidamide (CS055/HBI-8000), a novel histone deacetylases inhibitor (HDACi) of the benzamide class, is currently under clinical trials. Despite emerging information on the effect of chidamide in multiple cancers, little is yet known about mechanism of action, and there were few reports about this drug's effects on myelodysplastic syndromes (MDS). In this study, we aimed to investigate the antitumor activities of chidamide on MDS cell line SKM-1 and to explore the possible mechanism.
Methods:
We treated MDS cells with different concentrations of chidamide. The effect of chidamide on HDAC activity of MDS cells was studied by using a HDAC Fluorometric Activity Assay Kit. The induction of histone acetylation was confirmed by detecting acetylated histone H3 and H4 using Western blot analysis. The effect of chidamide on the proliferation of MDS cells was analyzed by Cell Counting Kit (CCK-8) assay. Apoptosis were detected by Annexin V/propidium iodide (PI) double-labeled cytometry. Cell cycle was analyzed by a PI method. The acetylation levels of genes promoterassociated histone H3 and H4 were examined by chromatin immunoprecipitation (ChIP) and quantitative real-time PCR. Quantitativereal-time PCR and Western blot were used to detect the expression of signaling pathway factors (SOCS3, JAK2, STAT3,Bcl-XL,Bcl-2 and Mcl-1).
Results:
Our results demonstrated chidamide suppressed MDS cells growth in a time- and dose-dependent manner, and induced cell apoptosis and cell cycle arrest at G0/G1phase. Chidamide was able to significantly inhibit the HDAC activity and increase the acetylated histoneH3 and H4 of MDS cells. ChIP analysis further indicated that chidamide induced acetylated histones accumulation in the promoter of SOCS3.Moreover, chidamide upregulated the mRNA and protein expression ofSOCS3, and also significantly downregulated JAK2/STAT3 signaling. Further, we identified reduced expression of STAT3 downstream targets with treatment of chidamide, including Bcl-XL, Bcl-2 and Mcl-1, which may explain the cytotoxic effects of chidamide on MDS cells.
Conclusion:
These results demonstrate that chidamide possesses significant cytotoxic effects on MDS cells mainly through histone modifications of SOCS3 and consequently JAK2/STAT3 signaling inhibition. Therefore, Our data provide rationale for clinical investigation of chidamide in MDS.

Disclosures
No relevant conflicts of interest to declare.
Interlaboratory Variation in CD34+ Cell Quantification in Low Risk Myelodysplastic Syndromes
Authors: Font Patricia , Subira Dolores , Matarraz Sergio , Benavente Celina , Cedena Teresa , Morado Marta , Perez Corral Ana , Bellon Jose Maria , Diez-Martin Jose Luis 
Published: November 2019

Abstract
Background: Accurate bone marrow (BM) blast count is essential for establishing prognosis in lower-risk myelodysplastic syndromes (MDS). Flow cytometry immunophenotyping (FCI) helps to evaluate the most immature BM cell compartment, but it is not known whether the CD34+ myeloid cell count in MDS are comparable among different observers. We investigated the FCI approaches used for enumeration of CD34 + myeloid cells in MDS in the routine practice in 6 clinical Spanish centers, and then explored if data were reproducible among participants.
Methods: In this prospective study, participants exchanged 47 FCI files from MDS BM samples with <5% blasts, and quantified the CD34+ myeloid cells in a blind manner. No agreement regarding cytometers, number, reagents, fluorochromes and events recorded were previously established.
Results: The mAb combinations selected to identify BM myeloid CD34+ cells was highly variable: CD45 was included in 45/47 (95.7%) files, CD34/CD45/CD117 in 37 (78.7%), and HLA-DR in 19 files (40.4%). The FCI assays used in every centre were quite different, but a very good concordance among the 6 FCI observers for CD34+ cell enumeration was observed with the intraclass correlation coefficient (0.720). Considering the specific analysis of each sample, 2 groups were distinguished. Twenty-five cases (53.2%) exhibited the highest concordance, with an interlaboratory coefficient of variation (CV) <30%, or a difference among observers <0.5%. In the remaining 22 cases, the concordance among participants was low. Comparison of the characteristics of high and low agreement assays showed no statistical differences. In order to determine whether the lower concordance obtained among observers after the analysis of these 22 samples could be improved, we designed a protocol with specific guidelines with gating strategies for identification of the CD34+ myeloid cells in several consecutive steps. The strategy was designed for samples with the CD34/CD45/CD117 mAb combination (n=15), with or without HLADR. Using this strategy, the interlaboratory CV improved in 7/15 cases (46.66%), but no improvement was obtained in the remaining 8 cases. Finally, the percentage of CD34+ myeloid cells was reproducible in 32/47 cases (68%) diagnosed with low risk MDS. Conclusions: Despite different methodology, accuracy among observers in CD34 cell quantification is feasible. Selection of appropriate protocols and targeted training would help to enhance the reproducibility of the technique among different observers. However, the intrinsic heterogeneity of these cells in MDS might justify the difficulty for their identification in some cases.

Disclosures
No relevant conflicts of interest to declare.
Cytogenetics of Patients with Myelodysplasic Syndrome in Venezuela
Authors: Vizcaino Jennifer , De Salvo Cardullo Luigi , Salas-Auvert Rodofo J , Hassanhi Manzur , De Salvo Dalila del V , Herrera-Merchan Humberto C 
Published: November 2019

Abstract
The myelodysplastic syndromes are clonal hematological disorders of the stem Cell, and clinically characterized by ineffective hematopoiesis and morphological dispoyesis. The affected patients have a high risk of leukemic transformation. In a total of (n=416) patients studied, (n=247) patients (59.3%) corresponded with a female gender, with an average age of = 59 years (limits 15-99 yrs), and (n=169) male patients (40.6%), with average age of =62 years (limits 09-89 yrs). In (n = 295) studied patients, 71% had normal karyotype 46, XX or 46, XY, where in 16% (n = 66) of the samples cell growth was not attained. In (n = 54) of patient samples, 13% showed karyotype changes, including those frequently reported for MDS; and 11.1% (n = 6) with -Y, 11q 1.8% (n = 1), del20q 3.7% (n = 2), +8 3.7% (n = 2), - 21 1.8% (n = 1), del 7q 1.8% (n = 1), inv3 1.8% (n = 1), complex karyotypes 12.9 % (n = 7) cases. Other alterations include, simple anomalies: +2 1.8% (n = 1), +13 5.5% (n = 3), del17q 1.8% (n = 1), del4p 1.8 %(n=1), del9q 3.7% (n = 2), der21q 3.7 %(n = 2), del17p3 3.7% (n = 2), t (1; 7) 1.8% (n = 1), t (4; 12) 1.8% (n = 1), rob13 1.8% (n = 1); 14, r2 1.8%(n = 1); double anomalies, delXq and del10q (n = 1), del18p and del13q (n = 1). Among other findings, polymorphisms in (n=15) patients (27.7%), such as 1qh + (n = 2), 9qh + (n = 4), 16qh + (n = 3), 14PS + (n = 1), 15ps + (n = 2) , 22ps + (n = 1), inv9 (n = 2). According with IPSS-R karyotypes, samples were classified as very favorable 12.9% (n = 7), pro 3.7% (n = 4), Intermediate 40.7% (n = 22), 1.8% unfavorable ( n = 1) and very favorable 18.5% (n = 12.9).

Disclosures
No relevant conflicts of interest to declare.
Phenotypic Characteristics of Tumor Cells in Patients with Chronic Lymphocytic Leukemia into Different Prognostic Groups
Authors: Kuvshinov Aleksei , Voloshin Sergei , Martynkevich Irina , Martynenko Ludmila , Garifullin Andrei , Kleina Elizaveta , Abdulkadyrov Kudrat 
Published: November 2019

Abstract
Background. The presence or absence of certain cluster of differentiation on the tumor cells of chronic lymphocytic leukemia may affect the course of the disease. Influence of genetic abnormalities on the prognosis of the disease was also proved.
Aim. To determine the relationship of the phenotype of tumor cells with genetic prognostic groups of patients with chronic lymphocytic leukemia (CLL).
Methods. Thirty-five adult pts (median age 61 year, range 44 - 82; male 24, female 11) with diagnosed CLL were included in the study. The CLL was diagnosed according to the standard basic examination (complete blood count with differential, multicolor flow cytometry (MFC) of blood and bone marrow (BM), lymph node and BM immunohistochemistry (IHC), computered tomography). Cytogenetic studies were performed on blood samples using standard GTG-method. Interphase FISH analyses were performed according to the manufacturer's protocol using DNA probes: LSI 13(RB1)13q14, LSI ATM (11q22), CEP12, LSI TP53 (17p13.1) (Abbott). Immunophenotype (IFT) of CLL cells assessed with combinations: CD3/CD19, CD19/CD5, CD19/CD11c, CD19/CD20, CD19/CD22, CD19/CD23, CD19/CD25, CD19/CD38, CD19/CD43, CD19/CD81, CD19/HLA-DR, and CD19/CD5/CD23.
Results. Stratification of patients into prognostic groups was performed based on identified GA. Favorable prognosis - patients with del(13q) (n = 9); neutral prognosis - normal karyotype (n = 14) or trisomy of chromosome 12 (n = 4); unfavorable prognosis - del(17p) (n = 3), del(11q) (n = 3) and the complex karyotype (n = 2).
Expression of CD20 was lower, and CD38 - higher in adverse group (51.0±16.31 % and 36.02±10.35 %, respectively) versus neutral or favorable groups (CD20+ - 83.17±5.52 % and 84.41±4.7 %; CD38 - 10.46±4.8 %, and 12.44±4.1 %, respectively, p <0.05). The expression level of CD20 and CD38 did not differ between the neutral and favorable groups.
The number of patients with CD38 expression more than 10% was higher in the unfavorable group (7/8) versus favorable (4/8) (p <0.05). At the same time overexpression of CD38 was observed more frequently in patients with a lack of expression of CD23 on CD5 tumor cells (CD5+CD23-) (p<0.05).
Expression of HLA-DR was higher in patients with MRD-negative remissions (3/4) versus patients with MRD-positive remissions (1/8) (p<0.05).
Conclusions. The study of the influence of various factors on the prognosis and course of CLL requires a comprehensive approach. Further researches are needed to determine the relationship between CLL affecting factors like genetic abnormalities, phenotypic characteristics of the tumor cells and MRD.

Disclosures
No relevant conflicts of interest to declare.
The BET Protein BRD2 Cooperates with CTCF to Enforce a Transcriptional Boundary in Erythroid Cells
Authors: Hsu Sarah C. , Bartman Caroline , Stonestrom Aaron J , Edwards Christopher R , Gilgenast Thomas , Emerson Daniel , Huang Peng , Werner Michael T. , Jahn Kristen , Keller Cheryl A. , Giardine Belinda , Hardison Ross C. , Raj Arjun , Phillips-Cremins Jennifer E. , Blobel Gerd A. 
Published: November 2019

Abstract
Pharmacologic inhibitors of the bromodomain and extraterminal motif (BET) family of proteins have shown promise in the treatment of hematologic and other malignancies and are being developed for clinical use. However, BET inhibitors do not discriminate between the family members BRD2, BRD3, and BRD4, and thus the mechanistic basis for their therapeutic efficacy is not well understood. In addition, BRD2 and BRD4 are individually required for the activation of genes driven by the erythroid transcription factor GATA1 (Stonestrom et al., Blood 2015), yet how BRD2 in particular contributes to this process has not been studied. We examined BRD2 occupancy genome-wide in erythroid cells and find that BRD2 colocalizes extensively with the architectural/insulator protein CCCTC-binding factor (CTCF). We define a functional hierarchy whereby CTCF is required for BRD2 to occupy co-bound sites, while CTCF binding is BRD2-independent. Using CRISPR/Cas9-based genome editing we identify a boundary element occupied by CTCF and BRD2 that is adjacent to a GATA1-driven enhancer and ensures appropriate transcriptional regulation at the locus. Employing single-molecule RNA FISH we show that either site-specific CTCF disruption or BRD2 depletion leads to increased correlation in mature mRNA levels between the genes flanking this boundary, suggesting that they become inappropriately coregulated. Taken together these findings indicate that BRD2 collaborates with CTCF to constrain the activity of an erythroid enhancer and reveal a potential new role for BET proteins in chromatin domain boundary function.

Disclosures
No relevant conflicts of interest to declare.
FANCA Fine-Tunes Chromosome Segregation By Controlling BUBR1K250 Acetylation at the Kinetochores
Authors: Sater Zahi Abdul , Sharma Richa , Sierra Potchanant Elizabeth , He Ying , Nalepa Grzegorz 
Published: November 2019

Abstract
Fanconi anemia (FA) is an inherited bone marrow failure syndrome associated with genomic instability, high risk of acute myeloid leukemia (AML) and other malignancies. Somatic mutations within the FA/BRCA signaling network occur in AML in the general population, reflecting the importance of FA genes in tumor suppression. While the role of FA signaling in DNA damage repair and replication is well-established, we and others found that the FA network is essential for error-free chromosome segregation during cell division. Both interphase and mitotic errors contribute to the evolution of genomic instability during FA-/- human and murine hematopoiesis in vivo. However, the molecular mechanisms of FA pathway-dependent genome housekeeping during mitosis are incompletely understood.
Through a synthetic lethal kinome-wide shRNA screen in FANCA patient cells, we discovered interphase and mitotic phosphosignaling networks that FANCA-/- cells depend on for survival, including the BUB1-BUBR1 axis of the spindle assembly checkpoint (SAC). BUB1 and BUBR1 are essential SAC kinases that prevent premature anaphase onset and chromosome mis-segregation by inhibiting the APC (anaphase-promoting complex) ubiquitin ligase at the centromeres until all kinetochores achieve correct attachment to the spindle microtubules. Our super-resolution microscopy and biochemistry experiments revealed that FANCA shuttles to kinetochores upon mitotic entry and physically interacts with BUB1 and BUBR1 at the kinetochore-microtubule attachment sites in attachment- and tension-dependent manner. Consistent with impaired SAC, we found that that anaphase onset as well as APC-mediated degradation of cyclin B1, BUBR1 and CDC20 all occur prematurely in FANCA-/- cells. We found that FANCA is essential for BUBR1 lysine 250 (K-250) acetylation at prometaphase kinetochores, and we confirmed that endogenous BUBR1K250 acetylation is disrupted in FANCA-/- primary patient cells using a validated acetyl-specific antibody. BUBR1K250 acetylation event works as a molecular switch in which BUBR1 is converted from a degradation target to a potent inhibitor of the APC ligase. Further, we observed that loss of FANCA disrupts kinetochore recruitment of the BUBR1K250 acetyltransferase PCAF and its upstream regulator, FANCD1/BRCA2.
Our findings establish the first mechanistic connection between FANCA, the canonical SAC tumor suppressor cascade and the FA effector FANCD1/BRCA2. These findings further our understanding of the mechanisms of genomic instability and carcinogenesis resulting from loss of FA signaling. Since impaired BUBR1K250 acetylation causes chromosomal instability and cancer in vivo, our results have a direct translational relevance.
Figure. Figure.

Disclosures
No relevant conflicts of interest to declare.
Low- Molecular Weight Heparins (LMWH) Versus Vitamin K Antagonists (VKAs) in the Treatment and Secondary Prevention of Cancer-Associated Venous Thromboembolism (VTE): A Systematic Review and Meta-Analysis of Randomized Controlled Trials (RCTs)
Authors: Thein Kyaw Zin , Tijani Lukman , Oo Thein H. 
Published: November 2019

Abstract

Introduction: Cancer-associated thrombosis accounts for about 20% of all cases of VTE. VTE increases not only mortality but also morbidity in cancer patients. LMWH is currently recommended over VKAs by major consensus guidelines and it is primarily based on a single, large RCT (CLOT trial) and some smaller studies. However, recently published CATCH trial, which enrolled 900 cancer patients with acute VTE, did not detect a statistically significant reduction in recurrent VTE between tinzaparin and warfarin groups. Hereby, we conducted a systematic review and meta-analysis of RCTs to determine the efficacy and safety of LMWH versus VKA in the treatment and secondary prevention of cancer-associated VTE.
Methods: MEDLINE and EMBASE databases were searched through June 30, 2016. All potential studies and their references were evaluated for any additional relevant studies. The RCTs which compare LMWH vs VKA in the treatment of VTE were incorporated in the analysis. Mantel-Haenszel method was used to calculate the estimated pooled risk ratio (RR), and risk difference (RD) with 95% confidence interval (CI). Fixed effects model was applied.
Results: A total of 2196 cancer patients with acute VTE from 6 RCTs and a subgroup of another 2 RCTs were eligible for analysis. Enoxaparin, dalteparin, tinzaparin and nadroparin were used on LMWH group and warfarin or acenocoumarol were used on VKA group. The treatment duration was from 3 to 6 months. The VTE incidence was 77 (6.832%) in LMWH group vs 128 (11.973%) in VKA group with a RR of 0.572 (95% CI: 0.436 to 0.750, P < 0.001). The absolute RD in VTE was -0.051 (95% CI: -0.076 to -0.027, P < 0.001). Major bleeding (MB) events were reported in 49 (4.757%) patients in LMWH group vs 45 (4.473%) in VKA group according to the analysis of 6 RCTs. The pooled relative risk for MB was statistically non-significant at 1.049 (95% CI: 0.705 to 1.562, P = 0.812). Composite clinically relevant non-major (CRNM) and minor bleeding events were noted in 143 (13.883%) patients in LMWH group and in 163 (16.203%) in VKA group according to the analysis of 6 RCTs. The RR for composite CRNM and minor bleeding was statistically significant at 0.773 (95% CI: 0.630 to 0.948, P 0.014), favoring LMWHs over VKAs.
Conclusions: Our meta-analysis confirms that LMWHs significantly decrease recurrent VTE events compared to VKAs without increasing MB in patients with cancer-associated VTE. LMWH therapy appears to have less CRNM and minor bleeding events compared to VKAs.
Figure Figure.

Disclosures
Oo: Daiichi Sankyo: Research Funding.