bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Bortezomib (Velcade ™) Resistance in a New Set of Human Cell Lines Obtained by Stepwise Selection.
Authors: Cai Jie , Lian Xian Jin , von Roretz Christopher , Shustik Chaim , Gallouzi Imed , Gros Philippe 
Published: October 2019

Abstract
Abstract 2741
Poster Board II-717
Bortezomib (Velcade ™, PS-341, BTZ) is a boronate dipeptide that reversibly inhibits the 26S proteasome, which is essential for the breakdown of ubiquitinated proteins and the regulation of normal cellular homeostasis. The activity of BTZ in treatment of newly diagnosed and refractory/relapsed multiple myeloma may be limited by the development of chemoresistance, the mechanisms of which are poorly understood. To investigate the molecular basis of Bortezomib resistance, BTZ-resistant (BTZr) cell lines were generated by stepwise selection procedures from HeLa, CCRF-CEM, and 4 multiple myeloma cells lines (8226S, U266, H929, and MM.1S), respectively. These BTZ-selected cell lines displayed varying degrees of elevated resistance (2 to 50 fold) to clinically relevant concentrations of BTZ. In addition, while most of the BTZr cells showed cross resistance to several other proteasome inhibitors (PIs), including MG-132 and Epoxomicin, they remained as sensitive to other chemotherapeutic drugs, such as anthracyclines, vinkalkaloids and etoposide, as their parental cells. The proteasome activity profiles are distinct among the cell lines. All parental cell lines displayed varying levels of chymotrypsin-like (CT-L) activity, which is the primary target of BTZ. Most BTZr lines showed markedly decreased CT-L activity, with a few exceptions. Moreover, the observed CT-L activity in all cell lines can be inhibited directly by BTZ and other PIs. In contrast, very low levels of caspase-like or post-glutamyl peptide hydrolyzing (PGPH) proteasome activity were detected in all cell lines. BTZ resistance in HeLa/BTZ cells was closely associated with increased resistance to PI-induced apoptosis, as shown by reduced number of Annexin V-stained cells and by delayed activation/cleavage of apoptosis proteins, such as Caspase-3 and Poly(ADP-ribose) Polymerase (PARP). Furthermore, the resistance to BTZ affected the mechanisms of cell stress responses. As for the parental HeLa cells, HeLa/BTZr cells retained the ability to form, in response to PI treatment, pro-survival foci in the cytoplasm known as stress granules (SGs). However, the drug concentrations required to induce SG formation in HeLa/BTZr cells are much higher (∼4 fold) than those for the parental HeLa, suggesting the development of stress-coping mechanisms in these BTZr cells. Gene expression profiling studies are in progress to identify transcriptomes individually or generally associated with BTZ resistance in these cell lines. Further characterization of these phenotypically similar, yet mechanistically distinct BTZr cell lines may elucidate diverse mechanisms of drug resistance to Bortezomib and other proteasome inhibitors.

Disclosures:
No relevant conflicts of interest to declare.
Targeting Axl Kinase in Hematological Malignancies.
Authors: Gourley Eric S. , Liu Xiao-hui , Hewitt William , Walker Rusty , Warner Steven L. , Vankayalapati Hariprasad , Bearss David J. 
Published: October 2019

Abstract
Abstract 2758
Poster Board II-734
Axl kinase, a member of the TAM family and also known as UFO, ARK, and Tyro7, is a receptor tyrosine kinase implicated in tumorigenesis. Overexpression of Axl is associated with increased cellular transformation, cell survival, proliferation, migration, angiogenesis, and adhesion. The oncogenic potential of Axl was first discovered in chronic myelogenous leukemia (CML) and has been shown to play a role in the development of acute myelogenous leukemia (AML) and myelodysplasia. Binding of Growth Arrest Specific Gene-6 (GAS6), a vitamin K-dependent protein and a known ligand for Axl, leads to subsequent phosphorylation events of downstream effecter molecules such as mitogen-activated protein (MAP) kinase and phosphatidyl-inositol (PI)-3 kinase/AKT pathways which are critical in oncogenic transformation. Furthermore, target validation studies of in vivo cancer models show that inhibition of Axl expression by RNA interference blocked tumor growth in those models. Taken together, this information makes Axl kinase an exciting target for small molecule drug discovery. Effectively utilizing SuperGen's proprietary CLIMB“technology, a series of small molecule inhibitors were rapidly discovered and developed for potency and selectivity against Axl. Lead candidate compounds demonstrate low nano-molar IC50 values in an Axl kinase biochemical assay, and focused selectivity for Axl when screened in kinase panels. Likewise, these compounds showed low and sub micro-molar activity from an anti-proliferative leukemia/lymphoma cell-based panel. Similarly, treatment of leukemia cells with these compounds in combination with known chemotherapeutic agents produced results that implicate combinatorial therapies as potentially beneficial treatments for patients suffering from hematological malignancies. In mechanistic, target validation assays, EC50 data from immunoprecipitated western blots of transiently transfected liquid tumor cells showed nano-molar inhibition of phospho-Axl when detecting with an anti-phospho-tyrosine antibody. A western blot panel of leukemia and lymphoma cell lines showed overexpression of Axl in several of these cell lines, further validating Axl as a potential therapeutic target in hematological cancers. Functional cell-based assays using SuperGen's lead compounds also potently inhibited phospho-AKT (S473). We propose that SuperGen's small molecule inhibitors against Axl kinase represent a new class of compounds with potent and selective activity in hematological malignancies.

Disclosures:
No relevant conflicts of interest to declare.
Inhibition of Cytokine Signaling through Activation of Jak2 Ubiquitination by WP1130.
Authors: Kapuria Vaibhav , Peterson Luke F , Bornmann William , Talpaz Moshe , Donato Nicholas J 
Published: October 2019

Abstract
Abstract 2907
Poster Board II-883
Constitutive activation of Jak2 through mutations or cytokine stimulation leads to abnormal expansion of the myeloid lineage and plays a key role in multiple myeloproliferative disorders. Kinase-specific inhibitors that prevent substrate phosphorylation can provide effective therapy for these disorders. However, this approach is sometimes inadequate as additional mutations and target-specific modifications can alter the kinase in a sufficient fashion to reduce kinase inhibitor affinity. Therefore additional approaches and molecules may be needed to reduce kinase activity through alternate mechanisms. In this report we describe a novel small molecule-mediated approach to block the Jak2/Stat signaling pathway. Using a previously identified small molecule with Jak2 inhibitory activity, we performed structure-activity relationship studies of AG490 derivatives in a cell-based analysis for inhibition of cytokine-stimulated Stat activation. This effort lead to the synthesis of WP1130 which suppressed Jak2 driven Stat activation with nM to μM potency. However, WP1130 did not act as a direct Jak2 kinase inhibitor. Rather, WP1130 treatment induced a rapid, time and dose-dependent trafficking of Jak2 from the detergent soluble to insoluble cellular fraction. We observed a robust induction of protein ubiquitination in WP1130 treated cells which was not directly or indirectly due to proteosome inhibition. Further, WP1130-mediated ubiquitination was unique with respect to other compounds as it did not reduce association of Jak2 with chaperones, did not induce ER stress or increase reactive oxygen species to activate protein ubiquitination. Jak2 was poly-ubiquitinated, primarily with lysine63-linked polymers, after WP1130 treatment and ubiquitination correlated with Jak2 mobilization without its proteolysis. Sub-cellular fractionation and analysis using confocal microscopy suggested rapid trafficking of Jak2 into cellular structures called aggresomes, in complex with Hsp90, Hsp70 and HDAC6. Lysates from WP1130 treated cells had diminished capacity to hydrolyze ubiquitinated substrates, suggesting the involved of reduced deubiquitinase activity as a mediator of Jak2 ubiquitination in WP1130-treated cells. Together, these results suggest that WP1130 enhances a resident ubiquitination cascade that results in Jak2 ubiquitination and its displacement from its peri-membranous, signaling competent sites. The results also suggest that Jak2 is able to undergo K63-linked poly-ubiquitination and may be exploitable as an alternate approach to interrupt Jak/Stat signaling.

Disclosures:
No relevant conflicts of interest to declare.
Procoagulants and Subclinical Atherosclerosis in Young Adults: The CARDIA Study.
Authors: Green David , Foiles Nancy , Chan Cheeling , Kang Joseph , Schreiner Pamela , Liu Kiang 
Published: October 2019

Abstract
Abstract 2973
Poster Board II-948

Background:
Elevated levels of procoagulants (FVII, FVIII, vWF) have been associated with cardiovascular disease, but whether they play a role in atherogenesis is unclear. Study of these factors in young adulthood, prior to clinically manifested disease, and years later when subclinical atherosclerosis has developed, might clarify these associations. The Coronary Artery Risk Development in Young Adults (CARDIA) Study provides an opportunity to examine this issue.

Methods:
Assays of FVII, FVIII, and vWF were performed in 1255 participants ages 23-37 (Year 7,Y7) and repeated at ages 38-50 (Year 20,Y20). Coronary artery calcification (CAC) prevalence and mean intimal-medial thickness (IMT) in the common carotid (CC) and internal carotid (IC) were measured at Y20. Prospective (Y7 clotting factors, Y20 CAC & IMT) and cross-sectional (Y20 clotting factors, Y20 CAC & IMT) analyses were performed. We also grouped participants according to whether they had one or more procoagulants in the highest tertile at Y7, Y20, or both, and evaluated their associations with subclinical disease.

Results:
Y7 levels of procoagulants (%), mean(SD) were: FVII 76(18), FVIII 102(38), and vWF 108(47). At Y20, all had increased by 40% to 55%, and CAC>0 was present in 20% of participants. After adjustment for age, the prospective analyses showed a trend of progressively greater CC thickness from the lowest to the highest tertile of FVII activity in the total group (P=0.007) and in whites (P=0.002) and men (P=0.015). Associations of FVII with IC thickness (0.82 mm to 0.84 mm) and the prevalence of CAC (18.6% to 23%) were weaker (P-trend, 0.1-0.3). Higher FVIII levels were associated with greater IC thickness in the total group, in whites, and in women (highest vs lowest tertile, P<0.05 for each comparison). No associations were seen with vWF. Cross-sectional analyses confirmed the association between FVII and carotid thickening, most strongly with the CC (P=0.002). Most associations were attenuated by multivariable adjustment (BMI, smoking, education, blood pressure, cholesterol, & CRP). Participants with FVII values in the highest tertile at Y7, Y20, or both had a higher prevalence of CAC and greater carotid thickening than those with values in the lowest tertile (P<0.05). Such associations were not observed with FVIII or vWF.

Conclusion:
FVII is a marker but not an independent risk factor for future atherosclerosis in young adults; FVIII is associated with IC thickening, and no associations were observed for vWF.

Disclosures:
No relevant conflicts of interest to declare.
Coating of Tumor Cells by Platelets Confers Expression of Immunoregulatory Molecules Which Impair NK Cell Anti-Tumor Reactivity.
Authors: Placke Theresa , Kopp Hans-Georg , Kanz Lothar , Salih Helmut R 
Published: October 2019

Abstract
Abstract 2993
Poster Board II-970
NK cells play an important role in cancer immunosurveillance and may prevent tumor progression and metastasis due to their ability to mediate direct cellular cytotoxicity and by releasing immunoregulatory cytokines which shape adaptive immune responses. Their reactivity is governed by various activating and inhibitory molecules expressed on target cells and reciprocal interactions with other hematopoietic cells like dendritic cells. Platelets contribute to tumor immune escape, metastasis, and angiogenesis (e.g. Jin et al Nature Med. 2006). In mice, thrombocytopenia inhibits metastasis, and this is reversed by NK cell depletion suggesting that platelets are an important additional player in NK cell-tumor interaction. However, the knowledge regarding the molecular mechanisms by which platelets influence NK cells is fragmentary at best. We found recently that platelet release soluble factors, most notably TGF-β, upon interaction with tumor cells which mediates NK cell silencing through downregulation of the activating immunoreceptor NKG2D (Kopp et al., Cancer Res 2009, in press). However, immunoregulatory molecules residing in the platelet membrane may also modulate NK cell anti-tumor responses. We report here that presence of platelets causes coating of tumor cells, and this markedly reduces NK cell lysis of tumor cells. This is mediated by conferment of “pseudoexpression” of platelet-expressed immunoregulatory molecules to tumor cells which are absent on the tumor cells alone. Among those immunregulatory molecules we identified various ligands for NK cell receptors like MHC class I, GITR ligand or CD62P. To establish the functional significance of tumor cell pseudoexpression of platelet molecules we employed functional analyses of tumor cells and NK cells with or without coating by autologous platelets. The impaired anti-tumor reactivity of NK cells against coated tumor cells was restored by blocking MHC class I on the coating platelets, while isotype control had no effect. Moreover, coating of tumor cells by platelets was validated by ex vivo analyses of primary leukemic cells from patients which also revealed substantial coating by platelets and confered expression of NK cell-modulating molecules. Our data indicate that platelets enable a molecular mimicry of tumor cells, which enables tumor cells to escape NK cell-mediated tumor immunosurveillance.

Disclosures:
No relevant conflicts of interest to declare.
Astrocyte-Plexus Is Formed in the Neovascularization Induced by CD11b+ Cells From An Ischemic Hind-Limb Mouse Model.
Authors: Kim Jeong A. , Hong Oak Kee , Hong Jong Wook , Broxmeyer Hal E. 
Published: October 2019

Abstract
Abstract 3043
Poster Board II-1019
Angiogenic sprouting needs to navigate through tissues to establish a vascular branching pattern. Such angiogenic guidance is mainly inferred by nonrandom angiogenic sprouting in the developing central nervous system (CNS). In mammalian retina, a vascular plexus initially forms, superimposed on a preexisting astrocyte-plexus, and retinal astrocytes begin to express vascular endothelial growth factor (VEGF-A), which stimulates endothelial cells to sprout radially from the optic nerve head into the retinal periphery. However, there has been little investigation of the role of astrocyte-plexus in new vessel formation of other ischemic sites besides retina. We recently found that infiltrated CD11b+ cells in ischemic muscles differentiate into endothelial-like cells in vitro, and that direct-injection of muscle-derived CD11b+ cells enhances recovery of blood perfusion in ischemic hind-limbs of C57BL/6 mice. To study if the astrocytes are related to the neovascularization process in ischemic limbs, infiltrated CD11b+ myeloid-cells in ischemic muscles were isolated 4 days after femoral artery dissection, and then seeded at 0.5×105/cm2 onto Matrigel and cultured in EGM-2 medium. Under these conditions, we tested the following hypotheses: (1) cultured cells initially form an astrocyte-network in vitro, and (2) astrocytes are involved in the formation of vascular structure. Seven days after the cells were embedded in Matrigel, spindle shaped cells grew, spread-out radially from cell clusters to the periphery, and established a mesh-like network expressing glial fibrillary acidic protein (GFAP), a marker for astrocyte. These spindle shaped cells were also positive for pericyte markers: desmin, NG-2, and platelet-derived growth factor receptor (PDGFR)-β. Interestingly, proliferating endothelial cells were closely associated with astrocytes by extension of endothelial cell filopodia on astrocytes. This observation is consistent that astrocyte scaffold guides extension of endothelial cell filopodia. Two weeks after cells were embedded in Matrigel, CD11b+ cells expanded and were layered in the same way to form vessel wall-like structures consisting of hundreds of cells. Spindle shaped GFAP-positive cells gradually expressed smooth muscle-actin at the bottom of the culture plate, migrated to the vessel wall-like structures, and covered the surface of the walls like pericytes. Taken together with the novel finding of astrocyte-like cell differentiation from CD11b+ cells from Matrigel culture, mobilization of CD11b+ myeloid-cells to regions of muscle ischemia would appear to play an important role in neovascularization after ischemic injury.

Disclosures
No relevant conflicts of interest to declare.
Vascular Endothelial-Cadherin Targeted Alpha-Particle Mediated Vascular Killing and Remodeling Augments Subsequent Chemotherapeutic Efficacy.
Authors: Escorcia Freddy E , Henke Erik , McDevitt Michael R. , Villa Carlos , Smith-Jones Peter , Blasberg Ronald , Benezra Robert , Scheinberg David A. 
Published: October 2019

Abstract
Abstract 3054
Poster Board II-1030
Cancer therapies targeting tumor endothelium have shown promising results when combined with chemotherapeutics. Vascular endothelial (VE) cadherin is an endothelial cell specific molecule that is expressed constitutively throughout the vasculature and participates in the formation of adherens junctions between adjacent endothelial cells. The monoclonal antibody E4G10 specifically binds to a cryptic epitope exposed only on the monomeric unengaged form of VE-Cadherin found in neovasculature. We chemically conjugated E4G10 with the cytotoxic, short-range αa-particle emitter, 225Ac, and show that the agent dampens tumor growth as a single agent and this effect is enhanced when given in combination with chemotherapy in a xenograft mouse model. The therapeutic efficacy of the combination therapy is sequence dependent and most pronounced when 225Ac-E4G10 is administered prior to the chemotherapy. Immunohistochemical and immunofluorescence studies for endothelial cells (Meca32), vascular basement membrane (Collagen IV), and pericytes (alpha-smooth muscle actin (alpha-SMA), nerve/glial antigen 2 (NG2) ) showed that the vasculature of 225Ac-E4G10 treated tumors was depleted and the remaining vessels appeared normalized as evidenced by morphological changes, increased pericyte density and coverage. Fluorescence studies with Hoechst 33342 dye i.v. injected showed more homogenous distribution of the dye within 225Ac-E4G10 treated tumors, suggesting improved perfusion, and biodistribution studies with i.v. injected 111In-Diethylene triamine pentaacetic acid (DTPA), a freely diffusible small molecule, showed significantly (p=0.03) increased passive tumor accumulation of 111In-DTPA in 225Ac-E4G10-treated tumors when compared to controls. Quantitative autoradiographic studies on tumor sections also showed both stronger and more homogenous signal distribution in 225Ac-E4G10-treated tumors. These results show that 225Ac-E4G10 treatment leads to ablation and remodeling of the tumor vasculature allowing improved delivery of subsequent small molecules, such as chemotherapeutics, and thereby improves therapeutic outcome.

Disclosures
McDevitt: Actinium Pharma Inc.: Patents & Royalties. Scheinberg:Actinium Pharma Inc.: Patents & Royalties.
The Frequencies of ETV6-RUNX1 Fusion and Hyperdiploidy (>50 chromosomes) in Children with Acute Lymphoblastic Leukemia Are Lower in Far East Than the West..
Authors: Liang Der-Cherng , Shih Lee-Yung , Yang Chao-Ping , Hung Iou-Jih , Liu Hsi-Che , Jaing Tang-Her , Yeh Ting-Chi , Liang Sung-Tzu , Chang Chia-Ling , Lee En-Hui , Liu Jo-Chuan , Yao Hsien-Wen , Chang Wan-Hui 
Published: October 2019

Abstract
Abstract 3064
Poster Board III-1
Both ETV6-RUNX1 (TEL-AML1)fusion and hyperdiploidy (>50 chromosomes) of lymphoblasts are favorable outcome predictors in childhood acute lymphoblastic leukemia (ALL). In 433 children with ALL diagnosed at our hospitals between 1997 and 2007 in Taiwan, the frequency of ETV6-RUNX1 fusion was 15.8%, and the frequency of hyperdiploidy (>50 chromosomes) was 14.1%, both were lower than those of the West. While ETV6-RUNX1 fusion had borderline favorable impact on outcome (p=0.053-0.061), hyperdiploidy showed significant favorable impact on event-free survival (91.1% vs 76.6 %, p= 0.016) in our patients. A meta-analysis from literature enrolled reports in which the case numbers and frequency of ETV6-RUNX1 fusion or hyperdiploidy in childhood ALL were described. It revealed that the frequency of ETV6-RUNX1 fusion in childhood ALL in Far East (Japan, Korea, Hong Kong, Chinese in Singapore, and Taiwan) was 14.2% (127/893, range 10-17%), significantly lower than 21.8% (152/697, range 19-27%) in the West (USA, Germany, Italy, France and Chile) (p < 0.0001). The frequency of hyperdiploidy in Japan and Taiwan was 15.2% (140/921, range 13-20%), significantly lower than 31.6% in the West (977/3,158, range 19-34%) (USA, UK and Germany) (p < 0.0001). So far as we know, there were several articles, including ours, addressing that the frequency of ETV6-RUNX1 fusion in childhood ALL was lower in a Far East country. This is the first meta-analysis to demonstrate that the frequency of ETV6-RUNX1 fusion in childhood ALL in Far East was lower than that in the West. There was no report on that the frequency of hyperdiploidy in Far East was lower than that in the West. This is also the first meta-analysis to demonstrate that the frequency of hyperdiploidy in childhood ALL in Far East is significantly lower than that in the West. The nature of these differences, probably due to racial, needs further study. In Far East, with both a lower frequency of ETV6-RUNX1 fusion, and a lower frequency of hyperdiploidy, it warrants renewed effort to cure a higher proportion of children with ALL.

Disclosures
No relevant conflicts of interest to declare.