bluuebunny/crossref_metadata_2025_split huggingface dataset:
Combination Of Inhibitors Against DNA Methyltransferases and Histone Deacetylases Synergistically Suppresses Cell Viability In Acute Myeloid Cells In a p53-Dependent Manner
Authors: Gopalakrishnapillai Anilkumar , Barwe Sonali P , Kolb E. Anders 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) accounts for 18% of childhood leukemia diagnoses. Although, the 5-year survival rate for children with AML is estimated at 64%, novel therapeutic options are required to improve the outcome for certain subsets of AML that are refractory. The contribution of epigenetic modifiers to the pathogenesis of AML in children is becoming evident following the discovery of mutations in patients, which affect DNA and/or histone methylation. Although DNA methyltransferase (DNMT) inhibitors have been approved in AML, their efficacy as single agents has been limited. This could be due to the crosstalk between DNA methylation and histone modifications that together regulate gene expression. This raises the possibility that optimal re-expression of silenced tumor suppressor genes in AML requires treatment with both DNMT and histone deacetylase (HDAC) inhibitors.
We evaluated the effect of a combination of DNA methyltransferase inhibitor azacytidine with HDAC inhibitors panobinostat or romidepsin on the viability of three pediatric AML cell lines- MV4;11 (expressing wild-type p53), THP-1 (with no detectable p53 mRNA) and AML-193 (with mutated p53). We observed that azacytidine as a single-agent only moderately reduced cell viability of MV4;11 cells (IC50 – 7 uM), and it failed to induce cell death in THP-1 and AML-193 cells. However, azacytidine in combination with panobinostat and romidepsin was more effective at reducing cell viability in MV4;11 and THP-1 cells (with a combination index of 0.5 to 0.6). AML-193 cells were almost insensitive to cell death induced by the combination treatment. This suggests that p53 is required for induction of cell death by azacytidine and panobinostat/romidepsin combination. Although THP-1 cells have no detectable p53 expression, other studies have reported the upregulation of p53 following stimulation with nitric oxide or 7-oxysterols. The effect on cell viability in THP-1 cells is likely due to the reexpression of silenced p53 in these cells. Taken together, our data suggests that combining azacytidine with panobinostat/romidepsin induces synergistic cell death in a p53-dependent manner.

Disclosures:
No relevant conflicts of interest to declare.
The O-Linked N-Acetylglucosamine Transferase Modifies HOXA9 and Inhibits HOXA9-Driven Immortalization
Authors: M Rual Jean-Francois , Hess Jay L. , Xu Tao , Collins Cailin , Zhang Honglai , Ha Kimberly , Park Sung Soo , Bronstein Joel , Wang Jingya 
Published: October 2019

Abstract
The homeodomain-containing transcription factor HOXA9 is a core element of the HOXA9 enhanceosome, a critical DNA-protein complex that regulates hematopoietic stem cell self-renewal during hematopoiesis. Several genetic mutations observed in acute myeloid leukemia (AML) patients, including MLL translocations, are associated with aberrant up regulation of HOXA9, thus disrupting the hematopoietic balance towards leukemogenesis. While analyses of HOXA9 and cofactors have uncovered fundamental aspects of the mechanisms through which these proteins mediate their functions, questions remain. For example, what molecular mechanisms contribute to switching HOXA9 enhanceosomes off during myeloid differentiation? Could these mechanisms be targeted for the therapeutic benefit of leukemia patients? Characterization of the molecular interactions in which HOXA9 enhanceosome proteins are involved should shed light on the mechanisms that govern these proteins during both normal hematopoiesis and leukemogenesis. We recently discovered that HOXA9 interacts physically with OGT, the only O-linked N-acetyl glucosamine transferase in humans. We also demonstrated that HOXA9 is O-GlcNAcylated by OGT. Investigation of the functional relevance of this interaction to HOXA9-driven leukemogenesis is currently under way using interaction- and O-GlcNAcylation-deficient alleles of HOXA9 in a colony formation assay. Our preliminary results suggest that OGT inhibits HOXA9’s ability to transform primary bone marrow cells, thus suggesting OGT is a potential tumor suppressor of HOXA9-driven leukemogenesis. Current efforts focus on further dissecting the molecular interplay occurring between HOXA9 and OGT on chromatin, its impact on the regulation of HOXA9 targets and its role in HOXA9-driven leukemogenesis. Work is also under way to identify factors involved in the OGT-mediated regulation of HOXA9 enhanceosomes.

Disclosures:
No relevant conflicts of interest to declare.
Improved Modifications Of Nitric Oxide Donating Aspirin (NO-ASA): Getting a Grip On TP53 Mutated High-Risk Chronic Lymphocytic Leukemia (CLL)
Authors: Krallman Sylvia , Poll-Wolbeck Simon Jonas , Flamme Hanna , Krueger Mark , Berkessel Albrecht , Hallek Michael , Kreuzer Karl-Anton , med. Dr. 
Published: October 2019

Abstract

Introduction
Chronic lymphocytic leukemia (CLL) shows a conspicuous inhomogeniety in the clinical course among patient subgroups with pronounced genetic aberrations. Some patients have a rapidly fatal disease despite therapy, which is accompanied by the incidence of deletion of chromosome 17p and mutation of TP53. Searching for new therapeutic options we investigate the cytotoxic effects and mechanistically opportunities of NO-ASA on CLL cells.

Methods
Primary cells were isolated from peripheral blood of CLL patients or healthy individuals. Primary CLL cells with or without TP53 mutation, as well as PBMCs and B-cells of healthy volunteers were tested in a luminometric assay (ATP content) after 24 h NO-ASA incubation. Caspase-dependency was determined in a luminometric assay after 6 h (n=5) as well as in immunoblot-analysis (Caspase 3, PARP; n=3) after 24 h. Influence on Btk- and NF-κB pathways was investigated after 3 h via immunoblot-analysis. Additional, expressions of NF-κB target genes (BCl-2, MCl-1 and CFLAR) were investigated via qRT-PCR after 4 h.

Results
NO-ASA effectively reduced ATP content in CLL cells from a mixed patient population (LD50 4.34 µM). Primary CLL cells from TP53 mutated patients (n=5) showed a slightly increased LD50 of 25.56 µM, which was still significantly lower than for healthy PBMCs (LD50 63.72 µM, n=5). In addition a concentration-dependent activation of the caspase cascade could be shown compared to untreated control (p<0.001) via luminometric assay as well as a visual apoptosis induction via western blot analysis. Phosphorylation of the NF-κB p65 subunit at Ser 536 was decreased by NO-ASA to ≈58% (n=3), while NO-ASA had no impact on BtK-Phosphorylation status (n=3). The relative expression of BCl-2 and CFLAR is significantly reduced (p<0.05, n=3) by NO-ASA, although MCl-2 was overexpressed (p< 0.001) under treatment’s influence.

Conclusion
NO-ASA shows potent reduction of ATP content in primary CLL cells. TP53 mutated patients are about three fold more sensitive towards NO-ASA treatment as PBMCs derived from healthy individuals. The cytotoxic effect depends on caspase-activation and might be influenced by inhibition of NFkB-pathway without impact on BtK-pathway. Overexpression of MCl-2 could present an escape-mechanism. Hence, NO-ASA is worth further evaluation as a treatment for poor prognosis patients unresponsive to conventional CLL treatment regimens.

Disclosures:
Hallek: Janssen: Research Funding; Gilead: Research Funding; Roche: Research Funding. Kreuzer:Roche: Honoraria; Mundipharma: Honoraria.
Synergistic Cytotoxic Effect Of The PARP Inhibitor ABT888 and Busulfan In Acute Leukemia Cell Lines
Authors: Patel Pritesh R. , Mahmud Dolores , Rondelli Damiano 
Published: October 2019

Abstract
The alkylator Busulfan (Bu) is used in conditioning regimens for acute leukemia prior to allogeneic stem cell transplant. Multiple DNA repair mechanisms including mismatch repair and base excision repair have been implicated in resistance to Bu. The enzyme PARP is central to base excision repair. We hypothesized that treatment of acute leukemia cell lines with both ABT888 (Veliparib), an inhibitor of PARP 1 and 2, and Bu would lead to synergistic cell kill and that this effect is maximal in mismatch repair deficient cells.
Two mismatch repair proficient cell lines (K562 and HL60) and 2 mismatch repair deficient cell lines (NB4 and REH) were treated with ABT888 alone, Bu alone or a combination of both. In single drug experiments, doses of drug treatment ranged from 0-400mcg/ml. In combination experiments a fixed dose of ABT888 of 1.25mcg/ml was utilized with Bu doses varying from 0-200mcg/ml. This dose of ABT888 was chosen as it approximated to patient blood levels in clinical trials. After 24 hours of treatment, cells were washed and resuspended in fresh medium. Proliferation of cells was measured by standard 3H-thymidine uptake assay at 48 hours. Sigmoidal dose response curves and GI50 values were then calculated. In addition, cells were tested for apoptosis by flow cytometry using activated caspase 3 and annexin/ PI staining at 24 and 48 hours after treatment.
All 4 cell lines were found to be resistant to single agent ABT888. Despite mismatch repair deficiency in REH cells, therapeutic doses of ABT888 did not cause significant decreases in proliferation. The effect of ABT888 was, as expected, much less evident in the mismatch repair proficient K562 cells. These cells were also relatively resistant to single agent Bu in vitro. The combination of Bu and ABT888 was synergistic in all cell lines with GI50 (micromoles/ml) for Bu decreasing from 67.8 to 45.7 in K562, from 23.3 to 8.0 in HL60, from 46.6 to 36.1 in NB4 and from 34.4 to 17.0 in REH cells. The Combination Index was <1 in all cell lines indicating synergy. Dose Reduction Index, indicating the factor by which the dose of Bu can be decreased to achieve the same treatment effect size, ranged from 1.45 to 3.1. The synergistic effect was greatest in the mismatch repair deficient cell line REH (Combination Index 0.53, Dose Reduction Index 3.1). As expected, the synergistic effect observed did not correlate with increased apoptotic death of leukemic cells implying an alternative mechanism for the derease in proliferation.
To our knowledge, this is the first study to show synergy of a clinically available PARP inhibitor with Bu. We believe this data warrants further study with the potential clinical application of increasing the antileukemic effect of stem cell transplantation conditioned with Bu containing preparative regimens.

Disclosures:
No relevant conflicts of interest to declare.
Influence Of Expression Levels Of ATP-Binding-Cassette Transporters (ABC-B1, ABC-G2) On Survival Outcome In Adults Patients With Acute Lymphoblastic Leukemia
Authors: Delgado Carolina Balderas , Garcia Alvaro Cabrera , Carrillo Irma Olarte , Fuller Etta Rozen , Ramos Peñafiel Christian Omar , Jaloma Juan Collazo , Castellanos Sinco Humberto Baldemar 
Published: October 2019

Abstract
Drug resistance is a major obstacle in the successful treatment and an important cause of death in acute leukemia. One of the well-known genes responsable for drug resistance are ABC-B1 and ABC-G2. The association of transcript levels with survival outcome has not systematically been investigated in mexican adults with acute lymphoblastic leukemia patients. We therefore applied real-time RT-PCR technology to study the association between the ABC-B1 and ABC-G2 genes with overall survival in mexican adults leukemia patients. A total of 36 patients were analyzed at diagnosis and followed for 25 months to assess their development and survival. The 57% were female, mean age at diagnosis was 33.5 years (19-78 years), 60% of patients were positive for gene expression ABC-B1 and 78% for ABC-G2. When performing quantitative analysis of ABC-B1 expression, those with high levels (> 1.0) had worse clinical behavior (P = 0.007) and death (P = <.001). We found no statistical significance with ABC-G2. The data suggest that higher expression levels of ABC-B1 gene contributes to the poor outcome of adult patients with acute lymphoblastic leukemia.

Disclosures:
No relevant conflicts of interest to declare.
Cellular Intrinsic Mechanism Affecting The Outcome Of AML Treatment With Ara-C In a Syngeneic Mouse Model
Authors: Chen Zheng , Tan Dongming , Zhao Wenjun , Su Guangsong , Zheng Yanwen , He Chao , Diers Miechaleen D , Mao Zhengwei , Singleton Timothy , Largaespada David A. , Yin Bin 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) is a deadly form of hematologic malignancy. Refractoriness to treatment in AML represents a common reason for treatment failure. However, the underlying biological mechanisms are not clear. Mouse models of types of leukemia have proved valuable in the identification of mechanisms responsible for leukemogenesis and in the evaluation of efficacy of therapeutics. In this work, we established and characterized a mouse model of AML, by transplantation of BXH-2 derived myeloid leukemic cells into syngeneic mice. The recipient mice developed leukemia shortly following transplantation, with the presence of blast cells in peripheral blood and infiltrated organs. We then developed an efficacious Ara-C-based regimen for the treatment of AML in mice, which was comprised of two courses of ten daily intraperitoneal injections of 50 mg/kg of Ara-C with a 5-day break. Using this immunocompetent mouse model, we proved that leukemic cell load was correlated with the survival of leukemia mice. We also demonstrated that the sensitivity of leukemia cells to Ara-C could significantly affect the survival of AML mice. To examine the difference between sensitive and resistant leukemic cells at the molecular level, genome-wide expression in these cells was profiled, revealing that overall 366 and 212 genes became upreglated or downregulated, respectively, in the resistant cells. Many of these genes are involved in the regulation of cell cycle, cellular proliferation, and apoptosis. Among them, the differential expression levels of Dnmt3L, Plau, Bcl2, CD72, Uba7 and Pdrg1 genes are further validated by quantitative PCR. These genes are known to be related with cellular differentiation and apoptosis pathway. Interestingly, the Ara-C resistant leukemic cells retained sensitivity to an inhibitor of anti-apoptosis proteins, ABT-737, and the treatment with this inhibitor prolonged life span of mice engrafted with resistant leukemic cells. These results suggest that leukemic load and leukemic cell intrinsic resistance can affect the outcome of AML treatment with Ara-C. Incorporation of apoptosis inhibitors, such as ABT-737, into traditional cytotoxic regimens merits consideration for the treatment of a subset of, especially, Ara-C insensitive, patients with AML.

Disclosures:
Largaespada: Discovery Genomics, Inc: Consultancy, Share Holder Other; NeoClone Biotechnology, Inc: Consultancy, Share Holder, Share Holder Other.
Bifunctional Immunoactive siRNAs as an Approach to Personalized AML Therapy
Authors: Meinl Hanna , Zeitlhöfler Marcus , Kocabey Samet , Liedl Tim , Hiddemann Wolfgang , Subklewe Marion , Endres Stefan , Rothenfußer Simon , Lichtenegger Felix S 
Published: October 2019

Abstract
The prognosis of acute myeloid leukemia (AML) is poor due to frequent relapse after initial remission. The development of new approaches to postremission therapy for elimination of minimal residual disease remains a major scientific and clinical challenge. We strive to combine two different innovative therapeutic concepts to develop a new specific and personalized treatment for AML. siRNAs are used to knock down either a gene that drives leukemogenesis due to genetic alterations in specific cases of AML (e.g., FLT3, NPM1) or a gene that is essential for the survival of the leukemic cells (e.g., BRD4, MCL1, PLK1). By adding a triphosphate modification to the 5’ end, the siRNA molecules additionally become ligands for the cytosolic pattern recognition receptor RIG-I (retinoic acid inducible gene I). Its activation mimics viral infection and leads to the production of inflammatory cytokines and induction of apoptosis in the target cell. We expect these bifunctional molecules to result in a decrease of viable AML cells and in the induction of an immune response similar to an active immunization.
This concept was successfully tested in vitro for several target genes in AML cell lines. We could demonstrate that the specific gene knockdown leads to inhibited proliferation, increased apoptosis and higher sensitivity to chemotherapeutic agents. Activation of RIG-I by triphosphate-modified RNA additionally stimulated an inflammatory response by the leukemic cells and increased the apoptosis rate.
A major hurdle for all siRNA-based anti-cancer strategies is the specific delivery of the RNA into tumor cells. In vivo liposomal transfection of siRNA molecules has been used in various tumor models, but generally results in ineffective and unspecific delivery. We are testing DNA-based nanoparticles coupled with molecules that target receptors specific for or overexpressed on AML cells. By coupling bifunctional siRNA molecules to these nanoparticles, they should be efficiently and selectively transported into the cytosol of AML cells.
Proof-of-concept in vivo studies in AML mouse models are in preparation. The long-term goal of this project is the development of a set of bifunctional siRNA molecules for the individualized treatment of AML.

Disclosures:
No relevant conflicts of interest to declare.
Locally Directed Therapy In Limited Stage Mantle Cell Lymphoma
Authors: Dalia Samir , Chavez Julio C , Bello Celeste M. , Chervenick Paul A. , Sokol Lubomir , Tao Jianguo , Sotomayor Eduardo M. , Shah Bijal D. 
Published: October 2019

Abstract

Introduction
Mantle-cell lymphoma (MCL) is a rare B-cell malignancy that usually presents in stage III-IV disease (classical) with poor outcomes even with aggressive therapy. Rarely, patients present with localized disease and the role of localized or systemic therapy remains a clinical choice.

Purpose
To assess outcomes in our patients with early stage MCL with a focus on treatment approaches.

Methods
Patients with early stage (Ann Arbor Stage I or II) between 1992 and 2012 were identified through our MCL database. Demographic , treatment, and outcomes data was collected for each patient. Cox regression was used to identify risk factors for progression and survival. Kaplan-Meier (KM) method was used to estimate median time to progression (TTP) and overall survival (OS), and comparisons were estimated using the log rank test. A p-value of <0.05 was considered significant.

Results
From our database of 300 patients with MCL, 34 were identified to have stage I or II disease (Table 1). 7 received local therapy, consisting of either radiation or resection. 20 received systemic therapy, including 1 who received only rituximab. 7 received a combination of local therapy and systemic therapy, including 2 who received rituximab with radiation.
With a median followup of 73.7 months, TTP was 22.8 months and OS was 83.8 months. MIPI could be determined for 56% of the cohort, however, was neither prognostic for TTP or OS. WBC was available for 74% of the cohort, and was prognostic for OS only [HR 1.46 (95%CI 1.02-2.08, p-value=0.04)]. None of the remaining MIPI covariates were prognostic. Ki67 was reported for only 8 patients limiting analysis as a continuous variable. Blastoid/Pleomorphic histology, alternatively, was prognostic for OS [HR 24.5 (95%CI 1.22-25.72, p-value=0.03)], but not TTP. Locally directed therapy was not associated with a decrease in OS or TTP, with some patients showing no evidence of relapse with extended followup (figure 1).

Conclusions
In our series patients with early stage MCL demonstrated a longer TTP and OS than those with extensive stage disease. The MIPI failed to stratify patients, likely related to small sample size. Aggressive histology may confer a worse prognosis, though, it remains unclear whether this can be abrogated by more intensive management, as all with blastoid/pleomorphic histology received some form of systemic therapy. Notably, as described by others, locally directed therapy did not compromise outcomes, with some showing extended TTP and OS.

Disclosures:
No relevant conflicts of interest to declare.
Cytogenetic Aberrations Characterize Mantle Cell Lymphoma With Bone Marrow Involvement and Provide The Most Important Prognostic Information
Authors: Yi Shuhua , Li Zengjun , An Gang , Li Chengwen , Zou Dehui , Qi Junyuan , Peihong Zhang , Qiu Lugui 
Published: October 2019

Abstract

Introduction
The clinical characteristics and cytogenetic aberrations of mantle cell lymphoma (MCL) is well described in the West, but not in China, and also not mentioned in patients with bone marrow involvement (BMI). The aim of this study was to characterize the clinical and cytogenetic feature of Chinese patients with MCL.

Methods
During the period July 2003 through November 2012, 50 MCL patients with BMI were diagnosed at our Hospital. Cytogenetic aberrations were detected by FISH on bone marrow cells, using a panel probes including Rb1, TP53, ATM and c-MYC.

Results
The median age of the 50 patients was 55.5 years at diagnosis, with 38 male patients (76%). Eighteen patients had B symptom, 36 patients with splenomegaly, while 4 patients had hepatomegaly. Twenty-four of the forty-five patients had elevated serum LDH. According to MIPI system, 13 patients (27.7%) were classified into medium risk group, while 34 patients (72.3%) in the high risk prognosis group. In aspect of the cytogenetic aberrations, eleven of forty-two patients (26.2%) had Rb-1 deletion, 7/39 patients (17.9%) with ATM deletion, 16/42 patients (38.1%) with TP53 deletion, while 15/37 patients (40.5%) had c-MYC abnormality, including amplification and translocation.
With a median follow-up of 21.5 months, the median estimated progression-free survival (PFS) was 15 months (95% CI 8.6-21.4), and the median estimated overall survival (OS) was 27 months (95% CI 17.5-36.5). Using the Kaplan-Meier method, the MIPI high risk group, deletion of Rb-1, ATM, TP53 and c-MYC abnormality were the adverse prognostic factors for PFS, while deletion of Rb-1, ATM, TP53 and c-MYC abnormality predicted the worse OS in the univariate analysis. All other clinical characteristics did not significantly influence the PFS and OS(p>0.05). TP53 deletion and c-MYC abnormality were the independent prognostic factors for both of PFS and OS in the multivariate analysis.

Conclusions
The outcome of MCL with BMI was poor. TP53 deletion and c-MYC abnormality were common in MCL with BMI and represented the worst factors for survival.

Disclosures:
No relevant conflicts of interest to declare.