bluuebunny/crossref_metadata_2025_split huggingface dataset:
Azacitidine/Decitabine Synergism with the NEDD8-Activating Enzyme Inhibitor MLN4924 in Pre-Clinical AML Models
Authors: Smith Peter G , Traore Tary , Grossman Steve , Narayanan Usha , Carew Jennifer S , Lublinksky Anya , Kuranda Michael , Milhollen Michael 
Published: October 2019

Abstract
Abstract 578
MLN4924 is an investigational small molecule inhibitor of NEDD8-activating enzyme that has shown clinical activity in a Phase I clinical trial in Acute Myelogenous Leukemia (AML). To identify potential combination partners of MLN4924 we performed a high-throughput viability screen in AML cells with 40 approved and investigational agents. In vitro characterization of AML cell lines revealed two distinct cell cycle phenotypes suggesting alternate mechanism of action following MLN4924 inhibition of NAE. One group demonstrated moderate S-phase accumulation with greater than 4N DNA content consistent with DNA-rereplication as a result of CDT1 dysregulation. The second group demonstrated distinct and rapid accumulation of subG1 cells without S-phase accumulation or DNA re-replication suggesting induction of apoptosis and cell death. These observations led us to choose two cells lines representative of each mechanism to understand potential for synergy in AML cells. Two hypomethylating agents were included in the screen (decitabine and azacitidine) and were found to be synergistic with MLN4924 by Combination Index and Blending Synergy Analysis. These data were confirmed with a second NAE inhibitor that is structurally dissimilar to MLN4924. The combination of azacitidine and MLN4924 were shown to result in significantly increased DNA-damage and cell death compared to single agent alone as measured by Western Blotting and FACS analysis of cell cycle distributions. In vivo studies were performed in HL-60 and THP-1 xenografts using MLN4924 on a clinically relevant dosing schedule twice weekly. Single agent azacitidine at its Maximum Tolerated Dose (MTD) had minimal activity in the HL-60 model and was combined with a sub-optimal dose of MLN4924 that when combined induced complete and sustained tumor regressions. The mechanism for the apparent synthetic lethality in this in vivo model is currently under evaluation; however it is supported by a dramatic elevation in DNA damage and cleaved caspase-3 in vivo in the combination arm. A second xenograft model (THP-1) that was also insensitive to single agent azacitidine treatment underwent complete and sustained tumor regressions when combined with MLN4924. Thus MLN4924 and azacitidine can combine to produce synergistic antitumor activity in pre-clinical models of AML. Coupled with their non-overlapping clinical toxicities these data suggest the potential for future combination studies in clinical trials.

Disclosures:
Smith: Millennium Pharmaceuticals: Employment. Traore:Millennium Pharmaceuticals: Employment. Grossman:Millennium Pharmaceuticals: Employment. Narayanan:Millennium Pharmaceuticals: Employment. Carew:Millennium Pharmaceuticals: Research Funding. Lublinksky:Millennium Pharmaceuticals: Employment. Kuranda:Millennium Pharmaceuticals: Employment. Milhollen:Millennium Pharmaceuticals: Employment.
Inactivation of Polycomb Repressive Complex 2 Components in Myeloproliferative and Myelodysplastic/Myeloproliferative Neoplasms
Authors: Cross Nicholas C.P. , Score Joannah , Hidalgo-Curtis Claire , Jones Amy V , Winkelmann Nils , Skinner Alison , Ward Daniel , Zoi Katerina , Ernst Thomas , Stegelmann Frank , Dohner Konstanze , Chase Andrew J 
Published: October 2019

Abstract
Abstract 617
Single nucleotide polymorphism (SNP) arrays have revealed regions of acquired uniparental disomy (aUPD) as recurrent events in hematological malignancies. Some of these regions are associated with the acquisition of somatic mutations in specific genes but the presumptive targets of many stretches of aUPD remain to be identified. We and others recently identified EZH2 as a target for 7q aUPD in myeloid malignancies, specifically myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) and the overlap myelodysplastic/myeloproliferative neoplasms (MDS/MPN). EZH2 is the catalytic component of the polycomb repressive complex 2 (PRC2), a highly conserved histone H3 lysine 27 methyltransferase that regulates the expression of developmental genes. We reasoned that the other two core PRC2 components SUZ12 and EED may also be mutational targets in these diseases, as well as associated factors such as Jarid2. We identified SUZ12 mutations in 1 of 2 cases with MDS/MPN with 17q aUPD and 2 of 2 myelofibrosis cases with focal 17q11 deletions. All three were missense mutations affecting the highly conserved VEFS domain, necessary for the interaction between SUZ12 and EZH2 and thought to help stabilize the PRC2 complex. Analysis of a further 146 MDS/MPN cases revealed an additional VEFS domain mutant, yielding a total mutation frequency of 2/148 (1.4%). We did not find mutations of Jarid2 or EED in association with aUPD for chromosome 6p or 11q, respectively, however screening additional cases identified missense mutations in EED (1/87; 1%) and Jarid2 (3/148; 2%). All three SUZ12 mutations tested and the EED mutation reduced PRC2 histone methyltransferase (HMT) activity as assessed by an in vitro baculovirus expression assay, thus demonstrating that PRC2 function may be compromised in myeloid disorders by mutation of distinct genes. SUZ12 is located at 17q11.2 close to NF1, a gene that encodes a negative regulator of Ras signalling that has been strongly implicated in the pathogenesis of myeloid neoplasms. Of the cases with SUZ12 mutations, one with del(17q11.2) had mutations of both NF1 and SUZ12. Both of these mutations were clearly inactivating: a frameshift in exon 5 of NF1 and a D605V change in SUZ12 that greatly reduced PRC2 HMT activity. In summary, we have shown that inactivation of the PRC2 can occur through mutation of any of the 3 core components, and that inactivating mutations of EZH2, SUZ12 or EED are seen in 15% of cases of MDS/MPN. These findings emphasize the importance of PRC2 function in hemopoiesis and suggest that deregulation of the epigenetic control of gene expression plays an important role in the development of myeloid disorders.

Disclosures:
No relevant conflicts of interest to declare.
Blocking the ZZ Domain of Sequestosome 1/p62 Suppress the Enhancement of Myeloma Cell Growth and Osteoclast Formation by Marrow Stromal Cells
Authors: Teramachi Jumpei , Myint Kyaw Ze Yar , Feng Rentian , Xie Xiangqun , Windle Jolene J. , Roodman David , Kurihara Noriyoshi 
Published: October 2019

Abstract
Abstract 888
The marrow microenvironment enhances both tumor growth and bone destruction in multiple myeloma (MM) through MM cell-induced activation of multiple signaling pathways in bone marrow stromal cells (BMSC) by TNFα. We reported that sequestosome-1 (p62) acts as a signaling hub for NF-kB, MAPK and PI3K activation in BMSC of MM patients and enhances MM growth and osteoclast (OCL) formation. p62 is composed of 5 domains that are involved in protein–protein interactions required for formation of these signaling complexes, but which domain of p62 mediates increase MM growth and OCL formation is unclear. Therefore, deletion constructs of p62 that lacked each of the 5 domains (PB1, ZZ, p38, TBS or UBA) were transfected into a p62−/− stromal cell line. We found that the ZZ domains mediated BMSC enhancement of MM cell growth, IL-6 production, VCAM-1 expression and OCL formation. Using virtual modeling of the ZZ domain, we identified 6 candidate p62-ZZ inhibitory molecules and tested them for their capacity to block enhanced MM cell growth, OCL formation, IL-6 production, and VCAM-1 expression on BMSC induced by TNFα.
When MM1.S, RPMI8266, ANBL6 MM cell-lines or CD138+ primary MM cells were cultured in the absence of BMSC with p62-ZZ inhibitor #3 (10mM), this inhibitor directly induced cell death. The p62-ZZ inhibitor-induced cell death was characterized by an increase in reactive oxygen species (ROS) production by inhibiting NF-kB activation, and apoptotic cell death by triggering the activation of caspases 3, 7, and 9. This inhibitor had an IC50 of 4.6mM for MM1.S survival. In contrast, CFU-Blast formation by human CD34+ cells was not inhibited by p62-ZZ inhibitor #3 (10mM). p62-ZZ inhibitor #3 (10mM) treatment of human OCL precursors derived from CFU-GM, inhibited OCL formation by blocking precursor proliferation. To examine the specificity of the p62-ZZ inhibitor #3, we tested its effects on OCL formation by CD11b+ mononuclear cells from wild type (WT) and p62−/− mice cultured with TNFα for 7 days. p62-ZZ inhibitor #3 blocked WT OCL formation but did not block p62−/− OCL formation. The p62-ZZ inhibitor also blocked VCAM-1 expression and IL-6 production by normal and MM patient stromal cells induced by TNFα compared to vehicle. Importantly, the inhibitor (10mM) did not block stromal cell proliferation. Further, the inhibitor blocked TNFα induced PKCζ phosphorylation in stromal cells and MM1.S when the cells were pretreated with the p62-ZZ inhibitor for 3 hours. These results demonstrate that p62-ZZ inhibitor #3 specifically blocks both stromal cells independent and dependent MM cell growth and OCL formation but does not affect hematopoietic or stromal cell growth. These results support p62 as a potential novel therapeutic target for MM.

Disclosures:
Roodman: Amgen: Consultancy; Millennium: Consultancy.
Cyclin A2 Plays a Critical Role in Proliferation of Lymphoid Progenitors
Authors: Kohno Kentaro , Iwasaki Hiromi , Iino Tadafumi , Mizuno Shin-ichi , Sicinski Peter , Akashi Koichi 
Published: October 2019

Abstract
Abstract 914
Cell cycle regulators could be differentially used among self–renewing stem cells, rapidly expanding progenitor cells, and terminally differentiated cells those clonally replicate. Cyclin A is a regulatory subunit for cyclin dependent kinase (Cdk) 1 and Cdk2, and it drives S phase progression as well as transition to G2/M phase in cell cycle. We have previously reported that cyclin A2 is not required for fibroblast replication but it is indispensable in maintenance of self-renewing stem cells, including embryonic stem cells and hematopoietic stem cells (HSCs) (Cell 138 2009). The question is whether cyclin A2 plays a role in proliferation of hematopoietic progenitors downstream of the HSC. Here, we further assessed the requirement of cyclin A2 in non-self-renewing hematopoietic progenitors. Quantitative RT-PCR analysis showed that cyclin A2 was expressed in hematopoietic progenitor cells as well as stem cells, and its expression level is highest in lymphoid-committed progenitor stages of both T and B cell lineages. Thus, in order to test the role of cylin A2 in early lymphopoiesis, we crossed cyclin A2 floxed mice with Rag1-Cre knock-in mice. Because recombination activating gene (RAG)-1 is essential for generation of pre-BCRs and pre-TCRs that are critical for expansion of B and T lymphoid progenitor cells, respectively, we hypothesized that the requirement of Cyclin A2 in early lymphopoiesis can be assessed in this system. As we expected, the Rag1-Cre cyclin A2 floxed/floxed mice were viable, and have normal numbers of HSCs and myeloid progenitors. They, however, displayed severe reduction of mature T and B cell numbers that were only 1/100 - 1/10 of wild-type controls. The number of common lymphoid progenitor was unchanged, but there were severely reduced preB cells in bone marrow and T cell progenitors from CD4-CD8- double negative stage in thymus. Furthermore, cell cycle analysis shows that the Cyclin A2 disrupted progenitors are unable to progress from S to G2/M phase, and in vitro culture clearly showed that those progenitors are unable to proliferate and resulted in apoptosis. These findings clearly demonstrate that cyclin A2 is indispensable not only for self-renewing HSCs, but also for proliferation of T and B cell progenitors.

Disclosures:
No relevant conflicts of interest to declare.
Activation of Vascular Cells by Microparticles and Other Danger Signals Via the CD36 Scavenger Receptor
Authors: Silverstein Roy L. 
Published: October 2019

Abstract SCI-12
Clinically significant thrombotic events are common complications of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes mellitus, cancer, and chronic autoimmune rheumatologic disorders. Many of these potentially life-threatening thrombi occur in the arterial circulation and likely relate to inappropriate platelet activation and/or vessel wall injury. CD36 is a type B scavenger receptor expressed on platelets, monocytes, microvascular endothelial cells (MVEC), and vascular smooth muscle cells (VSMC) that recognizes and responds to endogenous and exogenous “danger signals” including advanced glycated proteins (AGE), microbial cell wall components, and oxidized phospholipids that are found on the surface of oxidized LDL (oxLDL) and cell-derived microparticles (MP). Mouse FeCl3 thrombosis model systems revealed that genetic deletion of cd36 rescued the prothrombotic phenotypes associated with hyperlipidemia, oxidant stress, or diabetes/hyperglycemia. Interestingly, compared to wild type mice, fewer MP were incorporated into thrombi formed in cd36 null mice after FeCl3-induced vascular injury. Cd36 deficiency also provides partial protection from atherosclerosis in experimental mouse models. On a cellular level, the prothrombotic, pro-atherogenic, and anti-angiogenic activities of CD36 relate to ligand-dependent alterations of platelet, macrophage, and vessel wall cellular functions. For example, CD36 binding to MP, AGE, or oxLDL “sensitizes” platelets to activation by low doses of agonists, such as ADP. In macrophages, CD36 interactions with oxLDL leads to lipid internalization, foam cell formation, and inhibition of migration. In the vessel wall CD36 promotes VSMC oxidant stress and MVEC apoptosis. Mechanistically, CD36 transmits intracellular signals in response to ligand engagement that involve recruitment of specific Src family kinases to its short cytoplasmic tail, with subsequent activation of specific MAP kinase pathways and Vav family guanine nucleotide exchange factors. These signaling pathways remain incompletely understood, but impact many important cellular processes, including cytoskeletal dynamics, cell polarity, transcription of antioxidant genes controlled by the transcription factor Nrf2, and activation of NADPH oxidases. Specific components of CD36 signaling pathways depend on the cellular context and in some cases involve engagement of co-receptors, including integrins, TLRs, and tetraspanins. Recent targeted genome-wide association studies suggest that cd36 polymorphisms associate with CD36 expression levels on platelets and monocytes as well as with risk of thrombotic, metabolic, and atherosclerotic disorders. Targeting CD36 may therefore provide novel approaches to many important human vascular diseases.

Disclosures:
No relevant conflicts of interest to declare.
The Role of Activated Protein C in Cancer
Authors: Spek C. Arnold , Arruda Valder 
Published: October 2019

Abstract
Abstract SCI-18
Activated protein C (APC) is a natural anticoagulant that blocks the amplification of the coagulation cascade via inactivation of factors Va and VIIIa. The APC/PC pathway is initiated by complex formation of thrombin, thrombomodulin (TM), and the endothelial protein C receptor (EPCR) allowing the conversion of zymogen protein C into its activated form. Based on the well-accepted view that coagulation contributes to cancer progression and that anticoagulant treatment may benefit some cancer patient groups, it has been hypothesized that the natural anticoagulant protein C pathway may also play a role in cancer progression. Interestingly, it has recently been shown that endogenous APC limits experimental metastasis of B16 melanoma cells in mouse lungs. Notably, an APC-blocking antibody dramatically increased the number of experimental lung metastasis although not due to diminished anticoagulant activity of APC but largely due to reduced APC-driven S1P1-mediated VE-cadherin-dependent vascular barrier enhancement. In line with these findings repeated administration of recombinant human APC as well as transgenic overexpression of EPCR also diminished experimental metastasis of B16 melanoma cells. It may thus be tempting to speculate that recombinant APC could be a novel treatment strategy to limit cancer progression. However, APC has however a short half-life, needs intravenous administration, and is associated with severe bleeding complications, complicating the potential clinical application of these findings. In contrast to APC, zymogen PC has a longer half-life and is associated with significantly less bleeding complications. To prove or refute the hypothesis that zymogen PC may be an attractive alternative treatment option for APC in cancer patients, we recently compared the effect of continuous overexpression of murine APC or zymogen PC in the liver by viral-mediated gene transfer in experimental metastasis. Interestingly, both APC and zymogen PC overexpression was highly effective in limiting experimental metastasis. An APC variant (APC-5A) with reduced anticoagulant but normal signaling properties did not limit experimental metastasis, whereas the protective effect of zymogen PC remained even in the absence of protease activated receptor-1 (PAR-1), which is the main mediator of APCs cytoprotective effect. Zymogen PC may thus be a novel therapeutic target to limit cancer progression. In conclusion, the natural anticoagulant APC pathway may play an important role in limiting cancer cell extravasation and interventions seeking to modulate the PC system may ultimately benefit the cancer patient. The challenge is however to confirm these findings in alternative preclinical cancer models and eventually to translate our findings into a clinical setting.

Disclosures:
No relevant conflicts of interest to declare.
Genetics of Graft-Versus-Host Disease
Authors: Petersdorf Effie W. 
Published: October 2019

Abstract
Abstract SCI-49
The HLA barrier remains the primary roadblock to hematopoietic cell transplantation from alternative donors for the treatment of blood disorders. Currently over 18 million unrelated donors are represented by registries worldwide and they serve as a critically important resource for patients in need of a transplant. The basis for the selection of unrelated donors has evolved with advances in HLA typing technology. The demonstration that serologically identical HLA phenotypes have DNA-defined allelic variants that can provoke graft-versus-host reactions has served as the basis for the current criteria for the selection of donors. Although donor HLA matching lowers morbidity and mortality from graft-versus-host disease (GVHD), matching does not guarantee that the patient will not experience life-threatening GVHD and require prolonged immunosuppression after transplantation. Furthermore, the risks of acute and chronic GVHD associated with transplantation from HLA mismatched donors has lead to a reluctance to use mismatched donors for some patients. In 2011, the unmet need is two-fold. First, the vast majority of patients in need of a transplant have no HLA matched unrelated donor. To permit these patients the opportunity for a life-saving transplant, more information on the rules that govern permissible donor-recipient HLA mismatches is needed. Second, information is needed on the extent of non-HLA genetic variation that resides within the major histocompatibility complex (MHC) region and the manner in which such variation contributes to the transplantation barrier. Several research strategies have been applied to identify HLA mismatch combinations that can be used safely, including but not limited to analysis of individual amino acid residues that define the peptide binding repertoire of HLA class I and II alleles and antigens, and computational approaches that relate the sequence to structure of HLA molecules. The availability of a dense map of over 36,000 single nucleotide polymorphisms and complete sequence information for common HLA haplotypes has recently provided new information on the extent of human genetic variation and its organization on haplotypes. These data serve as a rich resource for mapping novel MHC resident variation associated with GVHD risk and transplant outcome. New information is emerging on the diversity of the MHC among transplant patient-donor pairs, the organization of simple and complex genetic variation relative to the classical HLA loci, and the putative regions within the MHC that are amenable to fine mapping. Future investigation of the genetic basis of GVHD will be enhanced with more complete information on MHC region variation in diverse human populations, haplotype content, and robust tools for both querying and analyzing complex variation.

Disclosures:
No relevant conflicts of interest to declare.
Targeted ShRNA Screening Identified Critical Role of Pleckstrin-2 in Erythropoiesis
Authors: Keerthivasan Ganesan , Yang Jing , Wong Piu , Doench John , Root David E. , Ji Peng 
Published: October 2019

Abstract
Abstract 3199
Mammalian erythropoiesis is globally regulated by erythropoietin (Epo). Epo binds to its receptor on the cell surface of erythroid precursor; induces a series of downstream pathways that promote cell differentiation and inhibit apoptosis. Recent genome wide transcriptional profile study demonstrated that over 500 genes are up-regulated during erythropoiesis. Many of these genes encode erythroid specific proteins that play well-known functions in red cells. However, the functions of the most other genes in the erythroid cells are still unknown. To identify novel genes in erythropoiesis, we infected mouse fetal liver erythroblasts with lentiviruses containing mammalian shRNA knockdown library that selectively includes the most highly upregulated 100 genes with unknown functions in erythroid cells. The infected cells were cultured in two different conditions for the characterization of early and late stage erythropoiesis using a high throughput flow cytometry based analysis. With these methods, we identified 33 novel genes that regulate cell differentiation or apoptosis in early stage erythropoisis; 20 genes play important roles in late stage erythropoiesis including enucleation. Significantly, there is an overlap of 16 genes that function in both early and late stage erythropoiesis. We focused on pleckstrin-2, which is specifically and abundantly expressed in erythroid cells, to further characterize its detailed functions in red cell development. We found that knockdown of pleckstrin-2 leads to dramatic apoptosis in early stage erythropoiesis. Knockdown of pleckstrin-2 in late stage erythropoiesis blocks enucleation with no apparent effects on cell differentiation, proliferation or apoptosis. We further discovered that pleckstrin-2 deficiency in early and late erythroblasts disrupts normal actin cytoskeleton as evidenced by super-resolution immunofluorescence microscope. To elucidate the detailed mechanisms of the functions of pleckstrin-2 in different stages of erythropoiesis, we performed proteomic studies and identified candidate proteins that interact with pleckstrin-2 that may contribute to the phenotypes of apoptosis and enucleation defects. In summary, our study identified pleckstrin-2 as a critical regulator of mammalian erythropoiesis and proved the significance of large-scale shRNA screening in the discovery of novel genes in development.

Disclosures:
No relevant conflicts of interest to declare.