bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Pan-Deacetylase Inhibitor Panobinostat Downregulates HIF-1α and VEGF and, Synergizes with Everolimus In Hodgkin Lymphoma Cell Lines
Authors: Lemoine Manuela , Buglio Daniela , Jona Adam , Derenzini Enrico , Medeiros L. Jeffrey , Berry Donald A , Younes Anas 
Published: October 2019

Abstract
Abstract 2851
Deacetylases (DAC) inhibitors are promising new class of anticancer agents. Panobinostat (LBH589) is a pan-DAC inhibitor with evidence of clinical activity in patients with relapsed classical Hodgkin Lymphoma (HL). However the mechanisms of action of this new drug are not completely understood. The aim of this study was to investigate the mechanisms of antiproliferative effect induced by LBH589 in HL-derived cell lines. Experiments were performed in HL derived-cell lines (HDLM-2, KM-H2 and L-428). Cells were treated with 0.01–1μM of LBH589 alone or in combination with 0.01–1μM of everolimus (RAD001), an inhibitor of the mammalian target of rapamycin (mTOR), for 24–72 hours. Growth inhibition and apoptosis were analyzed in response to treatment using MTS assay, Western blotting and flow cytometric analyses. Effect of panobinostat on a panel of 30 cytokines and chemokines was assayed on cells after incubation of 24 hours using a multiplex assay. Panobinostat demonstrated antiproliferative activity in HL cell lines in a dose-and time-dependant manner with an IC50 ranging between 20 and 40nM at 72 hours. The antiproliferative activity was associated with downregulation of the X-linked inhibitor of apoptosis protein (XIAP), activation of caspase 9 and caspase 3, cleavage of Poly ADP Ribosome polymerase (PARP) and induction of apoptosis. In addition, panobinostat downregulated the level of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1α) a major regulator of tumor cell adaptation to hypoxic stress. Furthermore, panobinostat decreased the level of the vascular endothelial growth factor (VEGF), an HIF-1α target gene, in the cell culture supernatants.The combination of panobinostat and everolimus had synergistic antiproliferative activity in the cells lines. This synergy was due to reciprocal inhibition of negative feedback loops induced by mTOR inhibitor and panobinostat therapy. Collectively, our data demonstrate that panobinostat induces apoptosis in HL cell lines by modulating several survival pathways. Furthermore, the observed synergy between panobinostat and everolimus provides rationale for combining these two active agents in a phase I/II clinical trial in patients with relapsed HL.

Disclosures:
No relevant conflicts of interest to declare.
RNA Interference Targeting Thiopurine Methyl Transferase Gene Alters the Cytotoxic Effect of the 6-Mercaptopurine In Human Leukaemia Cells
Authors: Albertioni Freidoun , Fotoohi Alan , Hashemi Jamileh , Yektaei Elham , Larsson Catharina 
Published: October 2019

Abstract
Abstract 2891
Thiopurines; mercaptopurine (6-MP) and 6-thioguanine (6-TG) are important drugs in treatment of paediatric cancer patients. The activity of these drugs depends on the activity of several common enzymes in the metabolism pathways such as thiopurine methyl transferase (TPMT) and guanine monphosphate synthetase (GMPS). In the present study the efficacy of thiopurines was investigated upon inhibition of TPMT and GMPS gene expression by RNA interference (siRNA). Treatment of the MOLT4 human T-cell leukemia cells with TPMT and GMPS siRNA resulted in decreased mRNA expression as determined by Real-Time PCR by 60% and 70% respectively. When reducing TPMT mRNA, the MOLT-4 cells were 70% less sensitive to 6-MP while the sensitivity to 6-TG was unchanged. When down-regulating GMPS using siRNA the sensitivity was unchanged upon treatment with both drugs. A microarray experiment was conducted using wild type on MOLT-4, 6-MP and 6-TG resistant MOLT-4 variants. The aim was to identify affected genes in response to the resistance induction. The mRNA levels of several nucleoside transporter genes were down-regulated in both thiopurine-resistant sub-lines; concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2). The expression of genes encoding the purine de novo synthesis enzymes was reduced to the same extent in both resistant cell lines as well as the expression of GMPS (>40% in both resistant sub-lines) which indicates defected purine metabolism. The activity of TPMT and other enzymes in metabolism pathway of 6-MP and 6-TG was unchanged in resistant cells. Our results suggest that the contribution of TPMT activity in cytotoxicity of 6-MP is greater than for 6-TG, probably due to inhibition of de novo purine synthesis by methylated nucleotides, whereas TPMT inactivates 6-TG by methylation.

Disclosures:
No relevant conflicts of interest to declare.
Fc-Engineered RANK-Fc Fusion Proteins for Neutralization of Soluble RANKL and Induction of Antibody-Dependent Cellular Cytotoxicity (ADCC) Against Multiple Myeloma
Authors: Schmiedel Benjamin J , Scheible Carolin , Baessler Tina , Wende Constantin M , Wirths Stefan , Azuma Miyuki , Schneider Pascal , Grosse-Hovest Ludger , Kanz Lothar , Salih Helmut R 
Published: October 2019

Abstract
Abstract 3039
Bone resorption is commonly associated with aging, but also with certain cancers. Recent studies identified Receptor Activator of NF-κB (RANK) ligand (RANKL) and its receptors RANK and osteoprotegerin as key regulators of bone remodelling. Multiple myeloma (MM) disrupts the balance within this molecule system towards osteoclastogenesis and bone destruction. Neutralization of RANKL by the monoclonal antibody Denosumab (AMG162) is presently being evaluated for treatment of both non-malignant and malignant osteolysis. We found, in line with previously published data, that primary MM cells (9 of 10) express substantial levels of RANKL at the cell surface and that MM cells directly release RANKL in soluble form (sRANKL). Next we evaluated the possibility to combine neutralization of sRANKL with targeting of MM cells for antibody-dependent cellular cytotoxicity (ADCC) of NK cells utilizing RANK-Ig fusion proteins with modified Fc portions. Compared to wildtype RANK-Fc, our mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (RANK-Fc-ADCC) and abrogated (RANK-Fc-KO) affinity, respectively, to the NK cell FcγRIIIa, but comparable capacity to neutralize RANKL in binding competition and osteoclast formation assays. Analyses with RANKL transfectants and RANKL-negative controls confirmed the high and lacking potential of the RANK-Fc-ADCC and the RANK-Fc-KO to induce NK ADCC, respectively, and ascertained that the RANK-Fc-ADCC specifically induced NK cell lysis of RANKL-expressing but not RANKL-negative target cells. Most notably, in cultures of NK cells with RANKL-expressing primary MM cells RANK-Fc-ADCC potently enhanced NK cell degranulation, cytokine release and MM cells lysis due to enhanced NK reactivity. Thus, our Fc-engineered RANK-Fc-ADCC fusion protein may both neutralize detrimental effects of sRANKL and enhance NK anti-tumor reactivity by targeting RANKL-expressing malignant cells thereby constituting an attractive immunotherapeutic means for treatment of MM.

Disclosures:
No relevant conflicts of interest to declare.
In Vitro and In Vivo Efficacy of CD38 Directed Therapy with Daratumumab In the Treatment of Multiple Myeloma
Authors: Groen Richard W , van der Veer Michael , Hofhuis Frans M , van Kessel Berris , de Weers Michel , Parren Paul W.H.I. , Lokhorst Henk M , Mutis Tuna , Martens Anton 
Published: October 2019

Abstract
Abstract 3058
Daratumumab (DARA) is a fully human antibody against CD38, a membrane-associated antigen, which is considered to be a highly relevant target for the treatment of hematological malignancies such as multiple myeloma (MM) and chronic lymphocytic leukemia (CLL). DARA has the ability to effectively kill target cells by CDC, ADCC and by induction of apoptosis. While CD38 is frequently overexpressed on MM cells, it has a variable degree of expression on the malignant CLL cells. To gain detailed insight into the potential therapeutic power of DARA in tumors with different CD38 expression levels, we now investigated the correlation between the level of CD38 expression and DARA-induced kill by CDC and ADCC. To this end, we first used the human MM cell lines L363 and UM9, expressing low and intermediate CD38 levels, respectively. While both cell lines could be lysed via ADCC, they were not susceptible to killing via CDC. To delineate a possible correlation between CD38 expression and induction of CDC, we lentivirally transduced the CD38 gene into L363 and UM9 cells to generate cell lines ranging in CD38 expression from 50,000 to 800,000 molecules per cell. Hereby we covered the range of CD38 expression found in CLL and MM patients. The L363 and UM9 cell lines were also transduced with the luciferase-GFP marker genes to enable their in vivo quantitative monitoring by bioluminescence imaging (BLI). In CDC assays, up to 95% lysis of CD38-transduced L363 and UM9 cells was achieved and showed an excellent correlation with the level of CD38 expression (r2=0.90). In ADCC assays, however, killing already reached maximum levels at DARA concentrations as low as 1 ng/ml at the lowest CD38 expression levels tested. These in vitro assays indicated that cell killing via CDC is enhanced with increasing CD38 expression levels, while CDC resistant, low CD38-expressing tumors could be targeted by DARA via ADCC. To explore this in an in vivo setting, we used a newly developed MM model in Rag2−/−gc−/− mice and we inoculated the mice with nontransduced, CD38low, UM9 cells, which can be lysed by ADCC but not by CDC. Treatment of the mice with DARA one day after tumor inoculation completely prevented the outgrowth of these UM9 cells; treatment of established UM9 tumours at week 3 resulted in a significant delay in tumor growth. These results confirmed that the CDC resistant UM9 could be readily targeted by DARA in vivo, possibly via ADCC mediated by monocytes in this model. In vivo experiments with variants that express high levels of CD38 are in progress. Taken together, these in vitro and in vivo data underscore the potency of DARA as a novel antibody for CD38-targeted therapy in hematological malignancies, and warrant further exploration in clinical trials.

Disclosures:
de Weers: Genmab bv: Employment. Parren:Genmab: Employment.
NIK Is Involved In the Activation of the Classical and Alternative NF-κB Pathways In Diffuse Large B Cell Lymphoma
Authors: Odqvist Lina , Sánchez-Beato Margarita , Montes-Moreno Santiago , Young Ken H , Acquadro Francesco , Cigudosa Juan , Martinez-Climent Jose A , Sáez Anabel , Sánchez Lydia , Piris Miguel A. 
Published: October 2019

Abstract
Abstract 3099
Deregulated NF-κB activity plays a role in the lymphoma pathogenesis, and has been proposed to constitute a cardinal feature of some subtypes of diffuse large B cell lymphoma (DLBCL). The NF-κB-Inducing Kinase (NIK) is essential for the activation of the alternative NF-κB pathway by inducing the phosphorylation of the NF-κB member p100, which leads to its processing to p52 and its subsequent nuclear translocation. A role for NIK in the classical NF-κB pathway as well has been shown, suggesting NIK as an attractive therapeutic target in lymphomas. Here, we study the frequency and extent of alternative and classical NF-κB activation in diffuse large B cell lymphoma, and the implication of NIK in both pathways. The activation of the classical and alternative NF-κB pathways was present in 28 and 34% of DLBCL cases respectively, as assessed by nuclear expression of p50 (classical pathway) and p52 (alternative pathway) by immunohistochemistry in a series of 301 samples. Activation of both NF-κB pathways was observed in germinal centre B-cell like (GC) and activated B-cell like (ABC) subtypes, with a slight predominance, although not significant, in ABC subtype. In contrast, the levels of p52 and p50 were significantly higher in ABC-DLBCL cell lines than those of GC subtype. The activation of both pathways was mostly overlapped and there was a strong positive correlation between nuclear p52 and p50 (p<0.001). Eighteen % of the cases expressed both p50 and p52 while only 8 and 16% expressed exclusively p50 or p52, respectively. Activation of the alternative NF-κB pathway was strongly associated with Epstein-Barr virus (EBV), since 93% of EBV+ cases expressed nuclear p52 (p<0.001). In our study, no TRAF3 deletions were detected in a panel of 25 DLBCL samples, although absence of TRAF3 was observed in one DLBCL cell line. Since NIK acts as a bottleneck in the activation of the alternative pathway but has also been described to play a role in the classical pathway, we wanted to analyze the effect of the knockdown of NIK on both pathways. Using small interference RNA in two lymphoma cell lines, we observed that the silencing of NIK had an effect on both pathways, decreasing the processing of p100 as well as p105. Taken together, our results show that the activation of NF-κB distinguishes a subset of DLBCL cases, comprising both ABC and GC subtypes, suggest a frequent overlap between the classical and alternative NF-κB pathway in DLBCL, and identify a possible role for NIK in the activation of both pathways.

Disclosures:
No relevant conflicts of interest to declare.
Dysregulation of Micrornas Involved In HTLV-Associated Adult T-Cell Lymphoma
Authors: Beltran Brady , Lin Jianhong , Sokol Lubomir , Zhang Ling , Lwin Tint , Cultrera Jennifer , Quinones Pilar , Morales Domingo , Tannenbaum Victoria , Moscinski Lynn C. , Sotomayor Eduardo M. , Tao Jianguo 
Published: October 2019

Abstract
Abstract 3122
Adult T-cell leukemia/lymphoma (ATL) is an aggressive, fatal malignancy of CD4+ T cells that is etiologically associated with infection by human T-cell leukemia virus-type. ATL has been classified into four main subtypes, indolent smoldering, chronic forms, acute and lymphomatous forms. Most individuals in the chronic stage of ATL eventually undergo progression to a highly aggressive acute stage. Compelling evidence supports a pivotal role for altered tumor suppressor genes involved in leukemogenesis and lymphomagenesis of ATL as well as progression of the disease from the chronic/smoldering to the acute/lymphomatous type. Further investigations are needed as to how the alteration of cell cycle regulatory genes are involved in the aberrant growth of HTLV-infected T cells. The emerging role of microRNAs in tumor development and viral infection has prompted investigations on the role of microRNA in ATL pathogenesis and progression. We therefore performed microRNA microarrays on 16 lymphomatous tissues of ATL patients and normal CD4 lymphocytes of 5 healthy donors. After normalization, we identified a set of aberrantly expressed miRNAs in ATL lymphoma samples when compared with control CD4 lymphocytes including 49 upregulated microRNAs and 89 downregulated microRNAs. MiR-150, miR-24, miR-221, miR-222, miR-142-5p, miR-92b, miR-93, miR-181a, miR-103, miR-107 stands out as highly elevated miRNAs in ATL samples while miR-30b, miR-34a, miR-20b, miR-196b, miR-361-5p, miR-520b/e are found downregulated in ATLL samples. Using gain-and-loss experimentations, we demonstrated miR-181a overexpression decreases, whereas miR-181a inhibition increases a proapoptotic protein, Bim, levels by directly targeting Bim. Furthermore computational analysis, and previous studies revealed that that miR-214, miR-22, miR-93 increased AKT activity by repressing PTEN expression, and miR-221/222 and miR-93 promoted cell proliferation through inhibition of CDKN1B (p27). Overall, this study demonstrated a distinct set of miRNAs that are known to be critical in cellular transformation, representing a novel molecular mechanism and potential therapeutic targets in ATL lymphoma.

Disclosures:
No relevant conflicts of interest to declare.
Low Absolute Lymphocyte Count Predicts Chemotherapy Response and Survival In Peripheral T-Cell Lymphoma, NOS
Authors: Kim Yu Ri , Kim yun Deok , Kim Jin Seok , Cheong June-Won , Kim soo Jeong , Min Yoo-Hong 
Published: October 2019

Abstract
Abstract 3135
Peripheral T cell lymphoma, not otherwise specified (PTCL, NOS) is heterogenous groups of aggressive T-cell lymphoma and treatment outcome is dismal. Lymphopenia is an independent prognostic factor for survival for B-cell lymphoma. The ALC at diagnosis on survival in T-cell lymphoma has not been studied. Thus, we studied the role of ALC at diagnosis on clinical outcome in patients with PTCL, NOS. Between 2001 and 2009, 32 patients with PTCL, NOS reviewed for the study. Median patient age was 57 (range 34–78) years. Median ALC at the time of diagnosis was 1.54 (range 0.41–12.64×109/L). Patients were divided two groups according to ALC count 1.0 ×109/L. Ten patients (31%) had lower ALC at diagnosis. Median follow up duration was 299 days (range 11–2164 days). Overall response rate was 61.5% (16 of 26 patients) and complete response (CR) rate was 42% (11 of 26 patients). Only two patients reached CR in low ALC group.There was no significant difference in overall response rate because of small number of patients. Superior overall survival was observed with an ALC 1.0 × 109/L (N = 22) versus an ALC < 1.0 × 109/L (N=10) (median OS: not reached vs 242 days, OS rates at 5 years, 57% vs 0%, p =0.016, respectively). Multivariate analysis demonstrated ALC to be an independent prognostic indicator for OS (Hazard Ratio 3.5, 95% confidence intervals 1.2–10.2; p<0.019) when compared to the International prognostic index (IPI) and Prognostic Index for PTCLU (PIT). This study suggested that low ALC is an independent prognostic factor for survival in patients with PTCL, NOS.

Disclosures:
No relevant conflicts of interest to declare.
Multiple Mechanisms of Notch-Mediated B-ALL Growth Arrest Via HES1 and PLK1: KIF11, CyclinB and p53
Authors: Kannan Sankaranarayanan , Fang Wendy , Zweidler-McKay Patrick 
Published: October 2019

Abstract
Abstract 3137
Notch signaling inhibits B-ALL growth and survival, while it promotes T-ALL, revealing contrasting cell-specific consequences of Notch signaling in ALL subtypes. We previously reported that HES1 (Hairy and Enhancer of split1) is a Notch pathway member which is sufficient to reproduce these effects in B-ALL.
We have found distinct HES1 protein complexes in B-ALL versus T-ALL, with significantly higher molecular weight complexes found in B-ALL. Through characterization of these complexes, multiple novel HES1 interacting proteins have been identified which regulate HES1 function as well as provide growth inhibiting and pro-apoptotic effects. We previously reported the novel role of HES1:PARP1 complexes in inducing B-ALL apoptosis. This study reports Polo-like kinase1 (PLK1) and Kinesin Family member 11 (KIF11, also known as Eg5) as novel HES1 interacting proteins in B-ALL. PLK1 is highly expressed in B cell leukemias and drives multiple proliferative pathways including activation of KIF11, induction of cyclinB and degradation of p53. KIF11 is also highly expressed in B-ALL and promotes cell cycle progression through enhanced spindle formation, while cyclinB promotes accelerated mitosis.
Through HES1 immunoprecipitation and MALDI-TOF peptide analysis, we identified KIF11 as a potential HES1 interacting protein. Low flow size exclusion chromatography revealed that HES1 and KIF11 co-migrate in high molecular weight complexes. Reciprocal immunoprecipitation (IP) with HES1 and KIF11 antibodies reveal a strong protein-protein interaction in B-ALL cells. PLK1 was also identified in these complexes, revealing a novel interaction of HES1, KIF11 and PLK1. Importantly, induction of Notch signaling via HES1 in B-ALL decreases both PLK1 and KIF11 levels, associated with G1 cell cycle arrest. Furthermore, HES1 expression decreased cyclinB protein levels, but not cyclin A/E, consistent with inhibition of PLK1. Finally, in the same model we observe increased p53 and p21WAF1 protein levels, also contributing to growth arrest.
These data reveal that multiple mechanisms of Notch/HES1-mediated growth arrest may occur via PLK1 in B-ALL. The novel interaction of HES1 with PLK1 and KIF11 and resulting effects on KIF11, cyclinB and p53/p21 pathways may represent important mechanisms of Notch-mediated growth inhibition in B-ALL.

Disclosures:
No relevant conflicts of interest to declare.
A Critical Role of YB-1 In NPMc-Induced Acute Myeloid Leukemia
Authors: Ogawara Yoko , Katsumoto Takuo , Uchiumi Takeshi , Kohno Kimitoshi , Kitabayashi Issay 
Published: October 2019

Abstract
Abstract 3149
Frameshift mutations in Nucleophosmin gene (NPM) are the most frequent abnormality in acute myeloid leukemia (AML), found in approximately 30% of all cases and 50% of patients with normal karyotype (NK) AML. NPM mutations result in an aberrant cytoplasmic localization of NPM protein (NPMc) through a loss of nucleolar localization signal accompanied by acquisition of new nuclear export signal. NPM mutations are heterozygous, so the other wild-type allele is consistently retained. NPMc binds to wild-type NPM through oligomerization domain and impairs its activity by delocalizing to the cytoplasm. It was reported that the NPM-null mice are early embryonic lethal and defective in primary hematopoiesis, suggesting important roles of NPM in early hematopoiesis. However, the molecular mechanism by which NPMc exerts its leukemogenic potential has never been established.
Here we show that ectopic expression of NPMc, but not wild type (WT) NPM, in mouse bone marrow (BM) cells enhanced their colony formation activity in methylcellulose media. Increased expression of HoxA7, 9 and 10 genes were observed in cells expressing NPMc but not in those expressing WT NPM. It has been reported that the expression levels of HOXA genes are upregulated in various types of AML including NPMc+ AML. Since overexpression of HoxA9 immortalizes hematopoietic progenitor cells, our findings suggest that up-regulation of HoxA genes are involved in NPMc-mediated leukemogenesis.
To clarify roles of NPMc in leukemogenesis, we purified the NPM protein complex and identified Y box-binding protein 1 (YB-1) as a binding partner for NPM. YB-1 belongs to the cold shock family and functions in gene transcription and RNA processing. YB-1 strongly bound to WT NPM but not to NPMc. In addition, interaction between YB-1 and NPM was impaired in the presence of NPMc. YB-1-deficient mice were embryonic lethal and their fetal liver were small. YB-1-deficient yolk sac cells showed decreased colony-forming activity, and decreased number of hematopoietic cells were observed when AGM region of YB-1-deficeint embryo were cultured on OP9 cells. Furthermore, expression of Hoxa9 was decreased in fetal liver cells derived from YB-1 knockout mice. To investigate the roles of YB-1 in NPMc-associated leukemogenesis, WT and YB-1-null E14.5 fetal liver cells were infected with retrovirus expressing NPMc. Analyses of colony-forming activity and mRNA expression showed that YB-1 was essential for NPMc-induced increases in colony formation activity as well as in expression of HoxA genes. However, YB-1 was not necessary for colony formation activity induced by other AML-associated fusion genes, such as AML1-MTG8 and MLL-AF10. These data indicate that YB-1 is specifically required for NPMc-induced leukemogenic transformation of hematopoietic cells.

Disclosures:
No relevant conflicts of interest to declare.