bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Polymorphism In the 3'UTR Region of ABCB1 Is Associated with Increased Allele Activity with Corresponding Increases In P-Glycoprotein Expression and Function
Authors: Bajuaifer Nada , Grundy Martin , Hills Robert , Russell Nigel H. , Pallis Monica , Seedhouse Claire 
Published: October 2019

Abstract
Abstract 3990
Multidrug resistance is one of the major prognostic factors in acute myeloid leukemia (AML). Expression of p-glycoprotein (p-gp), a member of the ABC transporter family and encoded by the ABCB1 gene, is associated with failure to respond to chemotherapy. Control of MDR1 expression is multifactorial and to-date emphasis has been on the 5'-promoter region of the gene. Evidence is now accumulating regarding a role for the 3'-untranslated region (3' UTR) of genes in controlling expression. We therefore studied a single nucleotide polymorphism (SNP) in the 3'UTR of ABCB1 (rs3842) and determined its effect on p-gp expression.
Samples were available on 450 AML patients who had been entered into the UK LRF AML14 and NCRN AML15 trials. The distribution of the rs3482 A/G SNP was determined using the SNaPshot method; 10% of samples had their genotype confirmed by sequencing. P-gp protein expression and function were analysed by flow cytometry of CD45-gated cells; expression using MRK-16 and function by assessing the modulation of R123 accumulation by PSC833. To determine the activity of the two polymorphic alleles the 3'UTR of ABCB1 was cloned into a modified pGL3 luciferase vector and the normalised luciferase activity was assessed for both the A and G allele.
The distribution of the rs3482 SNP in our AML patient cohort was 71% AA, 27% AG and 1.5% GG. These results are comparable with the published prevalence of the SNP in European populations. Approximately 60% of AML patients do not express p-gp so we therefore focused upon the 40% of patients who expressed p-gp. We found that in the p-gp-expressing patients a higher p-gp protein and function was associated with patients with the wild–type homozygous AA genotype when compared to the GA+GG genotypes. Furthermore when the ABCB1 3'UTR was cloned downstream of the firefly luciferase gene and transfected into HL-60 cells the ‘A’ construct demonstrated significantly increased activity when compared to the G construct (normalized ratio (firefly luciferase/renilla luciferase): A 7.7, G 1.9; n=4, p<0.05).
Outcome data was available on 414 of the intensively treated trial patients. Whilst the ABCB1 3'UTR polymorphism had no impact on overall survival or relapse risk there was a trend towards an increased relapse free survival in patients with the wildtype AA genotype (RFS AA 37%; AG+GG 28%, p=0.08). This may correspond to higher p-gp expression in immunocompetent cells post-remission.
In conclusion this work demonstrates that the ABCB1 rs3482 3'UTR SNP is functionally significant with the A allele associated with higher activity and a concomitant increase in p-gp expression and function in p-gp expressing patient samples.

Disclosures:
No relevant conflicts of interest to declare.
Profiling of Multiple Gene Mutations In Myelodysplastic Syndromes Using High-Throughput Resequenceing Combined with Barcode Labeling
Authors: Nagata Yasunobu , Sanada Masashi , Yoshida Kenichi , Nakaya Takeo , Matsubara Aiko , Obara Naoshi , Ishiyama Ken , Miyawaki Shuichi , Takita Jyunko , Chiba Shigeru , Shih Lee-Yung , Koeffler H.Phillip , Ogawa Seishi 
Published: October 2019

Abstract
Abstract 4011
Myelodysplastic syndromes (MDS) are a highly heterogeneous group of myeloid neoplasms characterized by ineffective hematopoiesis and a predisposition to acute myeloid leukemia, where a model of multisteponcogenesis has been implicated in their pathogenesis. On the other hand, recent advances in cancer genome analysis disclosed a number of gene mutations involved in the development of MDS, including mutations of RAS, RUNX1, CEBPA, TET2, CBL, EZH2 and TP53, where multiple gene mutations frequently harbor in a single case. However, the entire profiles of these multiple gene mutations with their relationship with WHO classification, chromosomal alterations, and clinical pictures have not been explored in a large series of MDS cases. Screening possible gene mutations in dozens of candidate genes in a large number of samples using Sanger sequencing were a time-consuming and labor-intensive task. So in order to overcome this issue and to obtain comprehensive registries of gene mutations in known candidate genes in a total of 170 cases with MDS and related disorders, we performed high throughput mutation analyses of more than 80 candidate genes using Genome Solexa-based next-generation resequencing technology combined with target gene capture and barcode labeling of individual samples. Briefly, each fragmented genomic DNA was frist amplified by single-primer polymerase-chain reactions (PCR), from which target sequences were concentrated using the SureSelect-system® (Agilent). Captured targets were primed with 6-base barcode sequences to discriminate the sample, which were subjected to high-thoughput resequencing using Genome Analizer®(Illumina). All 170 cases were already analyzed by Affymetrix SNP arrays, and their mutation status regarding RUNX1, p53, NRAS and KRAS, c-CBL and TET2 had been determined by Sanger sequencing, and thus were considered to an ideal sample set for this study, in which genome-wide copy numbers were characterized in detail and the known mutations works as a control to measure the performance of the barcode resequencing. Targeted 80 genes consisted of exons with total length of ∼500Kb, and included genes which were known to be mutated in MDS and related disorders, and other candidate targets of mutations. We were able to analyze up to 80 samples per 1 run and efficiently detected mutations in targeted genes by the high average coverage obtained from these sequences. On average 80% of targeted regions were covered with >20 depths of reading. In this meeting, we will present the result of our large-scale mutation study in MDS and related disorders and discuss the genetic basis of MDS in terms of multiple gene mutations as well as copy number alterations.

Disclosures:
No relevant conflicts of interest to declare.
Promoter Methylation and Decreased Expression of Mir-124 In Myelodysplastic Syndromes
Authors: Meng Yuesheng , Xia Qiao , Hu Jun 
Published: October 2019

Abstract
Abstract 4014
The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal disorders of hematopoietic stem/progenitor cells. Although demethylating agents such azacytidine and decitabine have been widely used to treat MDS, the underlying molecular mechanisms remain obscure. Abnormalities of microRNAs (miRNA) have been recently associated with hematological malignancies including MDS. The miR-124 was initially demonstrated to modulate neurogenesis. It was recently shown that EVI1-induced methylation and silencing of miR-124 were present in murine MDS cells. In the retrospective study we evaluated methylation status and expression levels of miR-124 in fifteen MDS patients (subtypes included RCUD, RCMD, RAEB-1, RAEB-2 and CMML). Genomic DNA samples were modified with bisulfite and methylation at three promoter regions of miR-124 was examined with methylation-specific real time quantitative polymerase chain reaction analysis (MQPCR). In general, we observed an increased methylation levels of miR-124 in MDS patients than that in normal bone marrow (NBM, P<0.01). In accordance with this, marked depression of miR-124 was seen in six patients when compared with NBM (more than 2 times lower), as determined with quantitative reverse-transcriptive PCR assay. Moreover, there were higher degrees of promoter methylation in cases with depressed miR-124 than that in remaining cases. A negative correlation between the expression and methylation levels was statistically significant (R= -0.498, P<0.01). The change of miR-124 was not directly related to short-term clinical response or prognosis, possibly due to limited size of the sample. However, the miR-124 amount returned to basal levels in two cases (RCMD and CMML subtypes respectively) after low-dose decitabine therapy and DNA methylation of all three loci disappeared. Continued work is underway to accumulate more cases and make long-term clinical follow-up. In conclusion, this primary work suggested a possible role of the methylation-mediated silencing of miR-124 in the pathogenesis or disease progression of MDS.

Disclosures:
No relevant conflicts of interest to declare.
Novel PI3K Inhibitor Compound A Induces Myeloma Cell Apoptosis and Shows Synergistic Cytotoxicity with Dexamethasone In Multiple Myeloma
Authors: Zheng Yuhuan , Yang Jing , Zhang Liang , Qian Jianfei , Matthews Jairo , Wang Michael , Kwak Larry , Yi Qing 
Published: October 2019

Abstract
Abstract 4080
Phosphatidylinositol 3-kinase (PI3K) plays a central role in cell metabolism. PI3K is activated by growth factors, cytokines, and other stimulatory factors in association with their receptors. Activated PI3K in turn initiates signaling transduction to Akt-mTOR and leads to regulation of cell growth, proliferation, and apoptosis. Dysregulation of the pathway is widely seen in different types of human cancers, including multiple myeloma (MM). Therefore, PI3K-Akt inhibition is expected to exert broad anti-MM activity. Compound A (CA) is a novel pan-PI3K inhibitor, developed by Novartis Oncology. This compound has shown significant cell growth inhibition and induction of apoptosis in a variety of tumor cell lines. CA is currently being investigated in Phase I clinical trials in solid tumor patients. In this study, we investigated the in vitro and in vivo anti-MM activity of CA. Our findings showed that CA induces apoptosis in MM cell lines, ARP1, ARK, MM.1S, MM.1R, CAG and U266, and primary MM cells in both a time-dependent and a dose-dependent manner in vitro. Western blot analysis indicated activation of caspases after CA exposure. The presence of MM bone marrow stromal cells (BMSCs) or addition of IL-6, the growth cytokine for MM, did not attenuate CA-induced MM cell apoptosis. More importantly, CA only showed limited cytotoxicity toward normal lymphocytes or non-tumoric BMSCs. Results from mechanistic studies showed that CA treatment results in cell cycle arrest in G1 phase by upregulating cell cycle repressor p27 (Kip1) and downregulating cyclin D1. CA treatment also caused decreased anti-apoptotic XIAP expression, and increased cytotoxic small isoform of Bim, BimS expression, both of which may contribute to CA-induced cell apoptosis. In addition to its effect in vitro, CA showed potent anti-MM activity in vivo in an established MM model in SCID mice. CA treatment repressed tumor growth and prolonged the survival of tumor-bearing mice. To test the synergistic/addictive effect of CA with other MM chemotherapeutics, we combined CA with melphalan, dexamethasone, lenalidomide, or bortezomib to treat MM cells. Our results showed that low doses of CA and dexamethasone, either of which alone has only limited cytotoxicity, exhibited synergistic anti-MM activity in dexamethasone-sensitive cell lines ARP1 and MM.1S, but not in dexamethasone-resistant cell MM.1R. Western blot analysis suggested that CA and dexamethasone combined treatment in MM.1S results in accumulation of the cytotoxic BimS. Increased BimS expression may cause the synergistic effect of CA and dexamethasone. Thus, our findings suggest CA alone or together with dexamethasone may be a promising treatment for MM.

Disclosures:
No relevant conflicts of interest to declare.
Long-Term Remission Maintenance on Weekly Imatinib Dosage In Patients with FIP1L1-PDGFRA-Positive Chronic Eosinophilic Leukaemia
Authors: Helbig Grzegorz , Hus Marek , Moskwa Andrzej , Zawilska Krystyna , Molendowicz-Portala Lucyna , Świderska Alina , Urbanowicz Alina , &dot;Zuk Ewa , Raźny Małgorzata , Rodzaj Marek , Seferyńska Ilona , Całbecka Małgorzata , Wichary Ryszard , Kyrcz-Krzemien Slawomira 
Published: October 2019

Abstract
Abstract 4097
A small subset of patients with hypereosinophilic syndrome (HES) presents an interstitial deletion in chromosome 4q12, which leads to the expression of an imatinib -responsive fusion gene- called FIP1L1-PDGFRA (F/P). These patients have chronic eosinophilic leukemia (CEL). Here, we treated twenty five F/P-positive CEL patients (22 male, 2 female; median age of 50 years) with imatinib using initial daily doses ranging from 100 – 400 mg. At diagnosis a median peripheral blood eosinophilia and eosinophil marrow infiltration were 12×109/L (range 2.5–40.8) and 39% (range 7–80), respectively. Splenomagaly was the most frequent clinical manifestation in this patient subgroup. All imatinib-treated patients achieved clinical and molecular response. A complete haematological remission (CHR) was demonstrated after median of 13 days (range 3–90) whereas molecular response (MR) was confirmed after median of 9 months (range 3–24). In a remission maintenance phase, imatinib doses were de-escalated and they were following: 100mg once weekly (n=11), 100mg twice weekly (n=6), 100mg daily (n=5), 200mg once weekly (n=2) and 400mg once weekly (n=1). Plasma imatinib level was measured 24 hours after the last drug intake in 7 patients treated in once weekly schedule and it remained extremely low, ranging between 44–167 ng/ml. Molecular studies performed at the same time points confirmed molecular remission. With a median follow-up of 40 months all patients remained in CHR and FIP1L1-PDGFRA expression was undetectable in all treated patients. These data indicate that even very low imatinib doses are highly effective in remission maintenance of patients with F/P-positive CEL.

Disclosures:
No relevant conflicts of interest to declare.
UCH-L1, a Potent Oncogene That Drives the Development of B-Cell Lymphoma, Promotes Akt Signaling by Regulating Mtor Complex Assembly
Authors: Hussain Sajjad , Galardy Paul J. 
Published: October 2019

Abstract
Abstract 4182
De-ubiquitinating enzymes (DUBs) are important regulators of many cellular processes through their ability to reverse the modifications installed by ubiquitin ligases. Several members of the DUB family are implicated in human cancers, where they may act as oncogenes or tumor suppressors. There is little direct in vivo evidence, however, for their roles in cancer. We recently reported that the DUB UCH-L1 is a potent oncogene that drives the development of spontaneous B-cell lymphoma in transgenic mice, and is required for the survival of malignant B-cells in vitro. The mechanism of these effects involves the strong induction of Akt signaling that is observed in culture and in lymphoid tissues of Uchl1 transgenic mice. Akt is phosphorylated on two distinct sites by the kinases PDK1 and mTOR, with both phosphorylation events required for maximal signaling. The activity of mTOR towards its substrates is dependent on the binding partners to which it is bound. When mTOR binds to raptor, it forms mTORC1, an important regulator of nutrient sensitive protein translation, whereas when complexed with rictor and SIN1, it forms mTORC2, a critical regulator of Akt signaling. It is unclear how the cell regulates the formation of mTORC1/2. Here we show a novel mechanism by which UCH-L1 regulates mTOR-dependent Akt signaling. By co-immunoprecipitating mTOR and its binding partners, we find that UCH-L1 expression promotes the binding of mTOR with rictor at the expense of raptor, without changing the level of either protein. These data suggest a novel ubiquitin-dependent regulatory step in mTOR complex assembly. In addition, UCH-L1 expression leads to a reduction in the levels of the Akt antagonist phosphatase PHLPP1 in vitro and in vivo. This decline is completely dependent on UCH-L1 DUB activity as expression of a catalytic mutant does not affect levels of PHLPP1. We find that the mechanism of this decline is not due to proteasome degradation, nor transcriptional control. We therefore hypothesize that the UCH-L1 associated shift in cellular mTOR activities leads to reduced mTORC1-dependent translation of PHLPP1. To test this, we inhibited mTORC1 activity by depleting raptor using shRNA and found a dramatic decline in PHLPP1 levels, whereas depletion of rictor had no effect on PHLPP1. We conclude that UCH-L1 boosts Akt signaling through a novel two-step mechanism by which it promotes the generation of mTORC2, reduces the levels of mTORC1, and thereby impairs the translation of PHLPP1. These data have important implications for understanding the roles of ubiquitin, mTOR, and Akt, in lymphoma biology and may lead to more intelligent use of next generation mTOR inhibitors in the treatment of lymphoma.

Disclosures:
No relevant conflicts of interest to declare.
The Ubiquitination-Dependent Interaction of p210 BCR/ABL with β-Catenin Contributes to Chronic Myeloid Leukemogenesis
Authors: Chen Ru , Gwen Mahon , Tala Ilona , Whitehead Ian 
Published: October 2019

Abstract
Abstract 4186
Chronic myelogenous leukemia (CML) is a malignant hematopoietic stem cell disorder that is invariably associated with the kinase activity of BCR/ABL. Although the ABL-encoded kinase activity is essential for disease progression, several studies have shown that BCR encoded sequences are also necessary for BCR/ABL-mediated leukemogenesis. In this current study we have identified a ubiquitin binding domain (UBD) in p210 BCR/ABL which is present in all fusion variants of BCR/ABL (p190, p210 and p230). This UBD does not conform to any known consensus sequence, and is unique to BCR and BCR/ABL. Deletion of the UBD does not impair the auto- or trans-kinase activity of p210 BCR/ABL, nor does it impair the interaction between p210 BCR/ABL and GRB2, or the ability of p210 BCR/ABL to activate ERK1/2. A mutation at residue tyr-177 of p210 BCR/ABL also does not impair the interaction with ubiquitin suggesting that the GRB2 and ubiquitin binding sites are adjacent, but separable. β-catenin has been identified as a binding partner for BCR and BCR/ABL, and the docking site has been mapped to a region of BCR/ABL that contains the UBD. Over-expression of p210 BCR/ABL, but not the ubiquitin binding mutant, leads to an accumulation of β-catenin that is phosphorylated on the serine residues that normally trigger ubiquitin-mediated turnover. This difference cannot be attributed to a difference in the activation status of GSK-3β. Treatment with an E1 inhibitor impairs the interaction between β-catenin and p210 BCR/ABL suggesting that the interaction is ubiquitination-dependent. Consistent with this possibility, previous studies have identified a glycine residue in β-catenin that is required for the BCR interaction. Based on these observations we propose a model in which p210 BCR/ABL may influence the Wnt signaling pathway by binding ubiquitinated β-catenin, and stabilizing it against degradation. In a murine bone marrow transplantation model for CML, mice transplanted with hematopoietic cells that express the ubiquitin binding mutant have significantly increased life spans compared to mice transplanted with p210 BCR-ABL expressing cells. This delayed disease progression may be due to a decrease in levels of β-catenin and less granulocyte macrophage progenitors (GMPs), which are thought to function as leukemic stem cells in the blast phase of CML. The role of ubiquitin-mediated b-catenin binding in p210 BCR/ABL-mediated leukemogenesis is currently being explored.

Disclosures:
No relevant conflicts of interest to declare.
Intrinsic Requirement of MicroRNA In Hox-Based Leukemia Initiating Cell Maintenance
Authors: Chaubey Aditya , Velu Chinavenmeni Subramani , Horman Shane , Jegga Anil , Guzman Monica L. , Zeleznik-Le Nancy , Jordan Craig T. , Carroll Martin , Gebelein Brian , Grimes H. Leighton 
Published: October 2019

Abstract
Abstract 4192
Mouse models of MLL-Af9 leukemia have been exploited to roughly determine leukemia initiating cell (LIC) characteristics and biology. Each colony formed by MLL-Af9 leukemic splenocytes is capable of initiating leukemia upon transplantation. MLL-fusion oncoproteins signal through elevated expression of HoxA9, Meis and Pbx proteins. The HoxA9 transcription factor is of critical interest in human AML because it is; 1) directly affected by chromosomal translocations, 2) upregulated by leukemia oncoproteins, and 3) the level of HoxA9 in cytogenetically normal human AML predicts outcome. However, the direct transcriptional targets of endogenous HoxA9 that mediate transformation remain largely unknown. The Growth factor independent-1 (Gfi1) transcriptional repressor is known to induce granulopoiesis and inhibit myeloid progenitor proliferation. GFI1 is mutated in patients with severe congenital neutropenia (SCN). SCN patients are at increased risk for AML. We have shown that Hox and Gfi1 orthologs antagonize each other during Drosophila anterior posterior patterning, and that in mammalian myeloid progenitors Gfi1 directly represses the expression of Hoxa9, Pbx1 and Meis1. Moreover, Gfi1 regulates the expression of miR-21 and miR-196b, and forced expression of these miR blocks G-CSF instructed granulopoiesis. Here we demonstrate that microRNA genes are the targets of endogenous HoxA9 versus Gfi1 antagonism and that this antagonism is relevant in the context of human leukemia. Moreover, we also show that miR-196b and miR-21 are activated by Hox-signaling leukemia oncoproteins. Next, in both murine leukemia models and primary human AML samples, antagomir-mediated inhibition of microRNA function specifically disrupts colony replating potential of Lin- bone marrow cells transformed by HoxA9, Nup98-HoxA9 and MLL-Af9, as well as bona fide MLL-Af9 leukemias. In contrast, cells transformed by AML-ETO (which does not signal through HoxA9) are not affected. In vivo, antagomir treatment blocked MLL-Af9-initiated leukemia (but not AML-ETO leukemia) lethality. Finally, limiting dilution analyses demonstrate that antagomirs inhibit Hox-based transformation by targeting the LIC. Antagomir treatment induces the re-expression of a histone lysine demethylase, downregulation of gene expression associated with maintenance of MLL-Af9 leukemia and associated H3K4me3 marks on these genes. Our data establish microRNA genes as functional downstream targets of endogenous HoxA9, and implicate epigenetic signaling as critical client/mediators of Hox-based leukemia oncoproteins in LIC maintenance.

Disclosures:
Carroll: Sanofi Aventis Corporation: Research Funding; Kyowa Hakko Kirin Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding.