bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Functional Analysis of Variants Associated with Therapy-Induced Second Malignancies After Hodgkin Lymphoma Identified by a Genome-Wide Scan
Authors: Best Timothy , Skol Andrew D , Gamazon Eric , Onel Kenan 
Published: October 2019

Abstract
Abstract 3892
Survivors of Hodgkin lymphoma (HL) are susceptible to radiation-induced second malignant neoplasms (SMNs). In a genome-wide association study (GWAS) of patients treated for HL who did or did not develop SMNs, we identified and validated two SMN-associated single nucleotide polymorphisms (SNPs) at 6q21, intergenic between PRDM1 and ATG5 [rs4946728: P = 1.04×10-9, OR = 3.21 (95% CI = 2.37–6.42), and rs1040411: P = 4.24×10-8, OR = 2.43 (95% CI = 1.76–3.34)]. Recently, it was demonstrated that disease-associated SNPs are more likely to be expression quantitative trait loci (eQTLs), SNPs that regulate gene expression, than are randomly chosen SNPs matched for their population allele frequencies. Indeed, we found that the 1000 SNPs most associated with SMNs are significantly enriched for eQTLs (P = 0.01). Exploring the processes regulated by SMN-associated SNPs can inform the mechanism by which SMNs result in patients treated with radiation therapy. As an initial investigation of the effect of these SNPs on gene expression, we studied the effect of the validated 6q21 haplotype (comprised of rs4946728 and rs1040411) on global gene expression in HapMap lymphoblastoid cell lines (LCLs). Gene set enrichment analysis of genes differentially expressed (log2>0.05) between cell lines carrying either the risk or protective haplotype revealed that carriage of the risk-associated haplotype was associated with increased expression of transmembrane proteins (enrichment P = 2.1×10-13) and immune response proteins (enrichment P = 1.2×10-6). Because the 6q21 haplotype is in close physical proximity to ATG5 and PRDM1, we investigated its functional consequence on expression of these genes. We discovered the risk-associated haplotype was significantly associated with lower levels of PRDM1 mRNA (P = 0.04) but not ATG5 mRNA. As exposure to radiation is the primary etiologic factor for SMNs, we assessed the effect of the risk haplotype on protein levels of PRDM1 and ATG5 in six LCLs (three with the risk haplotype and three with the protective haplotype) following 10Gy of gamma irradiation (IR). PRDM1 protein levels were significantly lower in LCLs carrying the risk-associated haplotype in the absence of IR. In all lines, PRDM1 levels increased following radiation exposure, but this effect was significantly attenuated in presence of the risk haplotype. In sum, these data suggest that SNPs associated with SMNs following HL are enriched for SNPs that regulate gene expression. We demonstrate that the validated risk alleles at 6q21 are associated with differences in PRDM1 mRNA and protein levels and response to radiation. These observations suggest a model in which PRDM1 may be a key regulator of the radiation-response that protects against the emergence of SMNs.

Disclosures:
No relevant conflicts of interest to declare.
The Oncogenic Mir-17-92 MicroRNA Cluster Is Inhibited by EGR2 During Macrophage Differentiation Via JARID1b-Mediated Histone 3 Lysine 4 Demethylation
Authors: Pospisil Vit , Vargova Karin , Kokavec Juraj , Rybarova Jana , Savvulidi Filipp , Necas Emanuel , Laslo Peter , Zavadil Jiri , Stopka Tomas 
Published: October 2019

Abstract
Abstract 390
The miR-17-92 cluster (Oncomir1) encodes seven related microRNAs associated with cell proliferation, apoptosis and development and is overexpressed in number of malignancies including myeloid leukemias. The miR-17-92 cluster is highly expressed in myeloid progenitors, while it becomes downregulated upon the onset of macrophage differentiation. Conversely, sustained expression of miR-17-92 is associated with a differentiation blockade (Fontana 2007). Here we report a novel mechanism of the regulation of miR-17-92 cluster within differentiating myeloid progenitors. During macrophage differentiation, the myeloid transcription factor PU.1 transcriptionally induces the secondary determinant Early growth factor 2 (Egr2). Subsequently, Egr2 binds to the miR-17-92 cluster promoter and recruits histone demethylase Jarid1b resulting in histone H3 lysine K4 (H3K4) demethylation within the CpG island upstream miR-17-92 cluster leading to the repression of miR-17-92 expression. Reporter assays using deletion constructs of the miR-17-92 promoter region revealed that Egr2 is required for targeting the Jarid1b onto critical minimal region within CpG island of upstream miR-17-92 locus and its decreased H3K4 methylation. In addition, functional assays identified the 3'UTR of Egr2 as the target of multiple miRNAs of the miR-17-92 cluster, indicating existence of a mutual regulation between miR-17-92 cluster and Egr2, putatively involved in macrophage differentiation characterized by a bistable state, where Egr2 negatively regulates miR-17-92 cluster in differentiating cells and, in turn, miR-17-92 cluster negatively regulates Egr2 in highly proliferating progenitor cells to achieve homeostatic regulation. Ectopic expression of miR-17-92 cluster within myeloid progenitors blocked macrophage differentiation indicating that leukemogenesis may involve miR-17-92 mediated differentiation blockade. To determine if the newly identified negative regulatory mechanism of miR-17-92 is involved in leukemogenesis, we tested peripheral blood mononuclear cells isolated from acute myeloid leukemia (AML) patients (N=27). 14 of 27 AML patients exhibited significantly downregulated transcriptional factors PU.1 and Egr2 and high levels of the miR-17-92 cluster expression. Ectopic expression of Egr2 within AML blast cells (N=2) led to decreased levels of miR-17-92 and restored the expression of p21 and BIM, two established miR-17-92 targets downregulated in AML. We conclude that PU.1 represses miR-17-92 cluster during macrophage differentiation by Egr2 mediated recruitment of the Jarid1b demethylase and that dysregulation in the miR-17-92 repression mechanism may contribute to the pathogenesis of AML. (Grants # IGA 10310-3, MSMT 2B06077, 0021620806, LC06044, SVV-2010-254260507).

Disclosures:
No relevant conflicts of interest to declare.
CXCR7 Regulates Retention of Splenic Marginal Zone B Cells and Availability of SDF-1/CXCL12
Authors: Wang Hongsheng , Shin Dong Mi , Qi Chenfeng , Morse Herbert 
Published: October 2019

Abstract
Abstract 3902
CXCR7 is a newly discovered receptor for the chemokines I-TAC/CXCL11 and SDF-1/CXCL12. Overexpression of CXCR7 in certain tumors has been associated with increased activities of adhesion, invasion and survival. CXCR7 has thus been investigated as a potential chemotherapeutic target in the treatment of metastatic cancers. Our analyses of murine B cell lymphomas revealed that marginal zone B (MZB) cell lymphomas expressed higher levels of CXCR7 than other types of lymphomas. This prompted us to investigate the expression and function of CXCR7 in normal B cells. In this report, we demonstrate that normal MZB cells expressed the highest level of CXCR7 among all B cell subsets. This pattern of expression was consistent with gene profiling studies using cDNA microarrays. Injection of mice with CCX754 or CCX771, a specific blocker of CXCR7, resulted in a significant reduction of MZB cells in the spleen. Immunohistological analyses revealed disrupted integrity and reduced size of the MZ in spleens of CCX754-treated mice. In addition, CCX754 significantly blocked internalization of CXCR7 resulting in an increase of CXCR7 expression on MZB cells but not follicular B cells. This indicates that CXCR7 constantly removes its ligands from the extracellular environment. Taking together, our data suggest that CXCR7 controls CXCL12 availability influencing MZB cell retention in the spleen.

Disclosures:
No relevant conflicts of interest to declare.
The Medicinal and Antitumor Mushroom, Agaricus Blazei Murill, Activates NF-κB Via TLR2 but Not TLR4 In Monocytic Cells, and Stimulates Monocyte-Derived Dendritic Cells (MDDC) to Increased Cell Surface Marker Expression and Cytokine Production, and May Thus Have Adjuvant Effect In MDDC Cancer Vaccines
Authors: Hetland Geir , Tryggestad Anne Merete Aaland , Espevik Terje , Ryan Liv , Tangen Jon-Magnus , Johnson Egil , Kvalheim Gunnar 
Published: October 2019

Abstract
Abstract 3904
Agaricus bM (AbM) is an edible Basidiomycetes mushroom from Brazilian rain forest that is used in traditional medicine against cancer and other diseases. It is rich in immunomodulating substances like β-glucans and is shown to inhibit tumors and adjuvate hepatitis B virus DNA vaccine in mouse models. Previously, we have found that an AbM-based (82%) extract, AndoSan™, additionally containing Basidiomycetes mushrooms Hericium erinaceum and Grifola frondosa, also protected against gram positive and gram-negative sepsis and allergy in mice. Now, to examine the mechanisms behind AbM's biological effects and to see whether it is beneficial as adjuvant in DC cancer vaccines, cellular surface markers on stimulated MDDC were studied by flow cytometry and cytokine production by a 17-plex Luminex system. Transcription factor NF-kB activation, which is linked to cytokine production, cell cyclus regulation and more, was then determined by a luciferase assay system and western blotting as translocation of NF-kB from cytosol to nucleus in AbM stimulated promonocytic THP-1 cells and in HEK293 cells, transfected with CD14/MD2 and TLR2 or TLR4. In both cell lines, AbM induced NF-kB activation via TLR2, but inhibited such activation induced via TLR4. After 24h incubation of MDDC with AndoSan™, there was down-regulated CD11c, de novo CD69 and enhanced CD86 expression on the cells, as well as increased levels in the culture supernatants of cytokines TNFα, IL-1β, IL-2, IL-6, IL-8, MIP-1β, IFNγ and G-SCF. Moreover, when this mixed mushroom extract was added as adjuvant to a DC melanoma vaccine, there was increased proliferation (H3-thymidine incorporation) of cancer-peptide-specific T cells. We conclude that in monocytes and probably also MDDC, AbM activates transcription factor NF-kB via TLR2 stimulation but inhibits such activation induced via the LPS receptor, TLR4. In MDDC the AbM-based extract AndoSan™ induced differential expression of cell surface markers for cell adhesion, activation and antigen presentation, and increased production of a leukocyte growth factor and proinflammatory, chemotactic and some Th1-type but not Th2-type cytokines in vitro. This may explain some of the known antitumor properties of AbM and its effect against infection and allergy. Since preliminary data suggest that the AbM-based extract, AndoSan™, may also have positive adjuvant effect in MDDC-based cancer vaccine, it will be tested further ex vivo and in clinical trials with cancer patients subjected to such DC vaccines.

Disclosures:
Hetland: Immunpharma AS: Patents & Royalties, Research Funding. Tangen:Immunopharma AS: Research Funding. Johnson:Immunopharma AS: Patents & Royalties.
Incompletely Activated CD4+ T Cells Induce Granzyme B+ Regulatory B Cells In An Interleukin 21-Dependent Manner
Authors: Jahrsdoerfer Bernd , Dahlke Karen , Hagn Magdalena , Sontheimer Kai , Beyer Thamara , Lunov Oleg , Fabricius Dorit , Tron Kyrylo , Nienhaus G. Ulrich , Simmet Thomas 
Published: October 2019

Abstract
Abstract 3905
Immune regulation is central for the development of an efficient cellular immune response. Both Treg cells and plasmacytoid DC can suppress T cell proliferation in a granzyme B (GzmB)-dependent and perforin-independent manner. In the present study we found that, depending on stimulation with interleukin (IL-) 21, B cells (BC) can also express GzmB and effectively suppress T cell proliferation. GzmB expression in BC is enhanced by BC receptor engagement, and is suppressed by CD40 ligation. Since CD4+ T cells are a main source of IL-21, we tested whether they can induce GzmB in BC. We found that incompletely activated CD4+ T cells, but not fully activated T cells induce GzmB in co-cultured BC. Using confocal microscopy, we showed that BC-derived GzmB is enzymatically active and that GzmB+ BC transfer GzmB to CD4+ T cells. Furthermore, GzmB+ BC decreased CD4+ T cell expression of the TCR-zeta chain, a GzmB target, which is required for T cell proliferation. Our results suggest BC may regulate cellular adaptive immune responses by Treg cell-like mechanisms. Inhibition of BC-derived GzmB may represent a novel strategy to induce more effective and comprehensive cellular immune responses.

Disclosures:
No relevant conflicts of interest to declare.
Activation of Acute Lymphocytic Leukemia Blasts with CpG ODN, IL-4 and CD40 Ligand Facilitates Enhanced Anti-Leukemic CTL Responses
Authors: Fabricius Dorit , Breckerbohm Lucia , Queudeville Manon , Vollmer Angelika , Eckhoff Sarah M. , Pfeil Johanna , Strauss Gudrun , Fulda Simone , Jahrsdoerfer Bernd , Meyer Lueder H , Debatin Klaus-Michael 
Published: October 2019

Abstract
Abstract 3907
Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Although the vast majority of patients initially respond to up-front chemotherapy, relapses occur in approximately 20% of cases and have a poor prognosis. Thus, novel therapeutic strategies are required to treat minimal residual disease and improve long-term survival. B cell precursor (BCP)-ALL cells express low levels of costimulatory and antigen-presenting molecules and therefore are poorly recognized by the immune system. Previous reports show that CpG oligodeoxynucleotides (CpG) can induce immunogenicity of non-Hodgkin's lymphomas including B-CLL, and that CpG may decrease BCP-ALL tumor burden in a murine xenograft model. In the present study we investigated the effect of various combinations of known potent B cell stimulators including CpG, Interleukin (IL)-2 family cytokines and CD40 ligand (CD40L) on the immunogenicity of both primary BCP-ALL cells and a series of established BCP-ALL cell lines. Using combinations of CpG, IL-4 and CD40L we were able to enhance expression of CD40, CD54, CD86 and MHC class II on BCP-ALL cells, resulting in an increased capacity of BCP-ALL cells to induce proliferative T cell responses and to generate cytotoxic T cells (CTLs). Of note, these CTLs exhibited significantly enhanced anti-leukemic cytotoxicity not only towards treated but also towards untreated BCP-ALL cells, with similar effects found in both allogeneic and syngeneic settings. The strongest effects were observed after exposure of BCP-ALL cells to a combination of CpG, IL-4 and CD40L, whereas untreated control BCP-ALL cells induced minimal T-cell proliferation and cytotoxicity only. Importantly, CpG in combination with IL-4 and CD40L was significantly more effective than CpG alone. Our results demonstrate that combined treatment with CpG, IL-2 family cytokines and CD40L is more efficient than CpG alone in inducing an immunogenic phenotype in BCP-ALL cells. Further studies using a humanized leukemia mouse model may open novel therapeutic approaches in the management of BCP-ALL, for example by using adoptive T cell transfer after in-vitro stimulation of autologous T cells with activated BCP-ALL cells.

Disclosures:
No relevant conflicts of interest to declare.
AT9283, a Novel Pan-Aurora/JAK-2 Kinase Inhibitor Suppresses Tumor Growth In Aggressive B-Cell Non-Hodgkin's Lymphoma
Authors: Mahadevan Daruka , Liu Xiaobing , Persky Daniel Oscar , Miller Thomas P. , Squires Matthew S , Qi Wenqing 
Published: October 2019

Abstract
Abstract 3930
Aurora kinases (A and B) are oncogenic serine/threonine kinases that play a central role in regulating the mitotic phase of the eukaryotic cell cycle. Auroras are over-expressed in numerous tumors including aggressive B-cell non-Hodgkin's lymphomas (B-NHL) and are considered validated oncology targets. AT9283 a pan-Aurora/JAK-2 inhibitor has undergone early phase trials in acute and chronic myeloid leukemia with promising anti-tumor activity. Hence, we hypothesized that 1) targeting mitosis with AT9283 would be effective in promoting apoptosis in aggressive B-NHL cell lines, and 2) addition of a microtubule targeting agent activating the spindle assembly checkpoint would lead to enhanced anti-tumor activity in a mouse xenograft model of mantle cell lymphoma (MCL).
AT9283 inhibited Aurora A and B activity as demonstrated by reducing phosphorylation of Aurora A and histone H3 as well as inhibition of cell proliferation (IC50 of 0.02–1.6 μM) in a variety of aggressive B-NHL cell lines (diffuse large B-cell lymphoma, MCL and transformed follicular lymphoma). B-NHL cells treated with AT9283 exhibited endoreduplication confirming the mechanism of action of an Aurora kinase inhibitor. Treatment of B-NHL cells with AT9283 induced apoptosis demonstrated by flow cytometry and PARP-cleavage in a dose and time dependent manner. Aurora A over-expression has been demonstrated to override the spindle assembly checkpoint (SAC) and result in resistance to microtubule targeting agent (e.g. taxols and vinca alkaloids) induced apoptosis. At very low doses (5-50 nM) apoptosis was almost doubled in the combination (30%) compared to AT9283 or docetaxel alone (15%) compared to controls. A mouse xenograft MCL model (Granta-519) demonstrated that AT9283 (10 and 15 mg/kg) or docetaxel (10 mg/kg) alone had modest anti-tumor activity (10-20% tumor growth inhibition). However, AT9283 plus docetaxel demonstrated a statistically significant tumor growth inhibition of >60% over control. The body weights of all mice in all cohorts did not change significantly (within 10%) during the study and mice appeared to tolerate treatment(s) well. Together, our results suggest that AT9283 plus docetaxel may represent a novel therapeutic strategy in aggressive B-cell NHL and warrant early phase clinical trial evaluation [Funded by the Lymphoma SPORE, P50 CA130805501A1].

Disclosures:
No relevant conflicts of interest to declare.
Identification of AML and MDS Patients Who Could Potentially Benefit From Temozolomide Therapy: Proportion of Patients with Low Methyl Guanine Methyltransferase (MGMT) Expression Level
Authors: Brandwein Joseph , Kassis Jeannine , Leber Brian , Howson-Jan Kang , Minden Mark D. , Greaves Kim , Pouliot Jean-Francois 
Published: October 2019

Abstract
Abstract 3985

Introduction:
Temozolomide is an imidazotetrazine derivative of dacarbazine that functions as an alkylating agent. Temozolomide anti-tumor activity is attributed to methylation of DNA, leading to the formation of O6-methylguanine that eventually leads to apoptosis. Resistance to temozolomide may largely be conferred by O6-methylguanine DNA methyltransferase (MGMT) over-expression. The level of MGMT expression will determine the ability of a cell to withstand damage from an alkylating chemotherapeutic agent. Brandwein et al. (Leukemia 2007;21:821-4) previously showed that the complete response rate to temozolomide therapy in patients with acute myeloid leukemia (AML) was higher when MGMT expression was low.

Methods:
In this prospective multicenter study we evaluated MGMT expression by Western blot in bone marrow or peripheral blood from older patients with previously untreated AML and high risk myelodysplastic syndrome (MDS). Expression was characterized by comparison with b-actin and results were expressed as an MGMT:b-actin ratio: Negative: < 0.2; Weak: 0.2 – 0.25; and Strong:> 0.25 MGMT promoter methylation was also evaluated using methylation-specific PCR. Both protein expression and promoter methylation were tested in a central lab.

Results:
Data were obtained for 152 patients; median age was 76 years old (range 46–94 years). A majority (63%) were male. Primary diagnosis was AML (84%) and MDS (16%). MGMT expression level could be determined in 128 of the 152 patients tested (84%). Expression ratios were: Negative (41%), Weak (11%), and Strong (48%). MGMT promoter methylation could be detected in 16% of cases in patients with either Negative or Weak expression ratio and in 2% of patients with Strong expression ratio. A majority of patients with promoter methylation (82%) had low MGMT expression.

Discussion:
One-half of patients with AML/MDS had either a Negative or Weak MGMT expression level, and would therefore be potential candidates for temozolomide therapy. These low levels could be explained by promoter methylation in a minority of cases. Thus, low level of MGMT expression in AML/MDS may be due to direct alteration of the MGMT gene or modulation of expression at the post-transcriptional level.

Disclosures:
Brandwein: Merck: Honoraria, Research Funding. Off Label Use: temozolomide for acute myeloid leukemia. Kassis:Merck: Research Funding. Leber:Merck: Research Funding. Howson-Jan:Merck: Research Funding. Minden:Merck: Research Funding. Greaves:Schering-Plough/Merck: Employment. Pouliot:Schering-Plough/Merck: Employment.
Biomarkers In Cell Death of Imatinib-Resistant Ph-Leukemia Cells During Treatment with mTOR Inhibitor, Everolimus
Authors: Minami Miho , Minami Yosuke , Kuwatsuka Yachiyo , Kiyoi Hitoshi , Naoe Tomoki 
Published: October 2019

Abstract
Abstract 3988
Aberrant activation of mammalian target of rapamycin (mTOR) signaling pathway has been reported in hematological malignancies including leukemia initiating cells. Although rapamycin and its analogs have proven effective as anticancer agents, the mechanism of action and the solid biomarkers of response have not been fully elucidated. We investigated detailed biomarkers during the cell death of imatinib (IM)-resistant Ph-positive (Ph+) leukemia cells due to quiescence or mutations at the ABL-kinase domain after treatment with mTOR inhibitor, everolimus (Eve, RAD001). Ph+ leukemic NOD/SCID/IL2rγnull (NOG) mice cells were long co-cultured with S17 stromal cells and treated with IM and Eve. While slow-cycling (Hoechst 33342low/Pyronin Ylow) CD34+ cells were insensitive to IM in spite of BCR-ABL-dephosphorylation, combination treatment with IM and Eve induced substantial cell death including the CD34+ population. In Baf3/p210T315I cells, IM-resistant Ph+ leukemia cell line harboring T315I-mutation, Eve also induced cell death with low IC50 values in PI-exclusion assays. In murine model cutaneously injected with Baf3/p210T315I cells, in vivo-treatment with Eve decreased tumor formation. In these systems during treatment with Eve, we did not observe evident dephosphorylations of BCR-ABL, mTOR itself and 4EBP1, but rapid S6K-dephosphorylation with lower doses and decreased expression of MCL-1. Furthermore, the feedback-loop effects such as reversely increased phosphorylations of AKT (Ser473) and FOXO1/3a were also detected during the cell death. We are now investigating more efficient strategies using inhibitors screening kit and also planning to examine new generation of mTOR inhibitors to overcome the IM-resistance due to quiescence or T315I-mutation.

Disclosures:
Naoe: Kyowa-Kirin: Research Funding; Novartis: Research Funding; Janssen: Research Funding.