bluuebunny/crossref_metadata_2025_split huggingface dataset:
Small Molecule Inhibitor of ITK Disrupts TCR Signaling and Demonstrates Anti-Tumor Efficacy
Authors: Faris Mary , Malyankar Uriel M , Zeng Qingping , Flynn Gary A , Feuer Gerold , Kertesz Nathalie L , Vuga Danka , Lee Sandra , Rosario Darlene , Bot Adrian 
Published: October 2019

Abstract
Abstract 3932
ITK (Interluekin-2 Inducible Tyrosine Kinase) is a member of the TEC family of intracellular protein tyrosine kinases. ITK is highly expressed in T cells and NK cells, with expression detected in mast cells. ITK plays a key role in several aspects of T cell biology, including T cell development, differentiation, migration, proliferation and activation. The function of ITK in immunity and allergy is well documented. T cells from ITK knock out mice show several developmental and functional defects, including defective signal transduction, altered CD4+ to CD8+ T cells ratios, reduced Th2 lineage differentiation, diminished IL4 and IL2 production and reduced T cell proliferation. Importantly ITK deficient mice fail to mount an immune response to infection and show reduced allergic asthma reactions.
In contrast to its well described role in immune function, ITK's function in cancer biology is still emerging. Recent studies had reported enhanced ITK expression and activation of the ITK pathway in several types of leukemias and lymphomas. In addition, the dependence of T cell malignancies on an ITK-regulated pathway, namely the IL2/IL2R (CD25) pathway, has also been observed. Taken together, this information indicates that ITK is a therapeutic target, with applicability in leukemias and lymphomas.
MannKind scientists have developed a series of selective small molecule ITK inhibitors, including the orally available tool compound described within, and evaluated their activity in enzyme, cell-based and in vivo studies. In cellular assays, the compounds showed significant inhibition of the T cell-receptor mediated activation of the ITK pathways and related downstream cytokine production. In addition to inhibiting the phosphorylation of ITK and its downstream mediator, PLCg, our tool compounds inhibited the production of IL2 and expression of CD25 in a dose dependent manner. Importantly, our compound regulated the in vitro growth of tumor T cells but not that of unrelated control cells. In vivo studies revealed that the tool compounds inhibited the growth and progression of patient derived ATL tumors in a xenograft pre-clinical model, and prolonged the survival of treated mice in a dose dependent manner, in addition to regulating cytokine production in vivo. In summary, our team has identified ITK selective compounds with demonstrated on-target and anti-tumor activity in vitro and preclinical T cell tumor models, and validated this pathway relative to T cell malignancies. This effort provides a platform for further compound optimization and evaluation for hematologic malignancies.

Disclosures:
Faris: MannKind Corp: Employment. Malyankar:MannKind Corp: Employment. Zeng:MannKind Corp: Employment. Kertesz:Mannkind Corporation: Employment, Equity Ownership. Vuga:MannKind Corp.: Employment. Rosario:MannKind Corp: Employment. Bot:MannKind Corp: Employment.
The Biocompatibility and Security of Magnetic Nanoparticles Fe3O4-DNR Used In Hematologic Malignancies Therapy
Authors: Chen Bao-An , Wu Wei-wei , Cheng Jian , Wang Jun , Gao Feng , Xu Wen-lin , Gao Chong , Ding Jia-hua , Sun Yun-yu , Song Hui-hui , Bao Wen , Liu Li-jie , Xia Guo-hua , Wang Xue-mei 
Published: October 2019

Abstract
Abstract 3970
The objective of this paper is to study the biocompatibility and security of self-prepared magnetic nanoparticles Fe3O4 (Fe3O4-MNPs) loaded with daunorubicin (DNR), which has the potential application in hematologic malignancies therapy. Hemolysis test was carried out to estimate it s blood toxicity; Fe3O4-MNPs loaded with DNR were intra peritoneally injected into mouse to calculate the LD50; micronucleus (MN) assay was reckoned to identify its genotoxicity; acute toxicity testing was evaluated its influence to mouse hepatic and renal functions. The result of hemolysis rate (HR) of Fe3O4-MNPs loaded with DNR was 2.908%, far less than 5%. Therefore, we concluded that self-prepated Fe3O4-MNPs loaded with DNR nanoparticles had no hemolytic reaction, and they consistent with the requirement of hemolytic test of biomaterials. The LD50 of Fe3O4-MNPs loaded with DNR nanoparticles to the mice was 1009.71mg/kg (relative content of DNR was 10mg/kg) and the 95% confidence interval was 769.11∼1262.40mg/kg, it had no significant difference compared with LD50 of using DNR only, which data was 8.51mg/kg and the 95% confidence interval was 6.48∼10.37mg/kg, and it had wide safety value circumscription. In micronucleus assay, compared Fe3O4-MNPs loaded with DNR nanoparticles experimental groups with negative control group, we found that the result had no significant difference (P > 0. 05) in micronucleus formation rate, while compared experimental groups with positive control group, the result had significant difference (P < 0. 05). The result indicated that Fe3O4 magnetic nanoparticles had no cacogenesis and mutagenesis. Acute toxicity testing showed that mice body weigh of control group, Fe3O4-MNPs loaded with DNR nanoparticles experimental group and isodose DNR group had no significant difference in 24h, 48h, and 72h after intra peritoneally injection; they had normal activity, eating and evacuation; toxic reactions such as instability of gait, convulsion, paralysis and respiratory depression were not been found; the alanine transarninase (ALT), blood urea nitrogen (BUN), and creatinine clearance rate (CCr) of Fe3O4-MNPs loaded with DNR nanoparticles experimental group was 66.0±28.55u/L, 9.06±1.05mmol/L, and 18.03±1.84umol/L, respectively, which had no significant difference compared with control group and isodose DNR group. From the results of our experiment, we could consider that self-prepared Fe3O4-MNPs loaded with DNR nanoparticles is a kind of high biocompatibility and security materials and perhaps is suitable for further application in hematologic malignancies therapy.

Disclosures:
No relevant conflicts of interest to declare.
Curcumin Reverses Resistance to Histone Deacetylase Inhibitors In Cutaneous T-Cell Lymphoma Cells: a Potential Role for NF-κB Signaling
Authors: Zhang Chunlei , Zhang Xiang , Duvic Madeleine 
Published: October 2019

Abstract
Abstract 3973
Histone deacetylase inhibitors (HDACi), including vorinostat (SAHA), depsipeptide (FK228), panobinostat (LBH589), belinostat (PXD101), and entinostat (SNDX275), show in-vitro and clinical activity against cutaneous T-cell lymphoma (CTCL) cell lines and patients' skin lesions [Zhang & Duvic, Expert Rev Dermatol 5: 393–401, 2010]. Vorinostat and depsipeptide were recently approved [Duvic et al, Blood 109: 31-9, 2007; Olsen et al, J Clin Oncol 25: 3109-15, 2007; Piekarz et al, J Clin Oncol 27: 5410-7, 2009], at response rates of 29% and 42%, respectively, but development of resistance remains an important clinical problem. Because we have shown that curcumin, the active ingredient of turmeric, exhibits anti-cancer activity through selective induction of tumor T-cell apoptosis and inhibition of NF-κB signaling in CTCL [Zhang et al, J Invest Dermatol 130: 2110-9, 2010], we now investigated whether curcumin combined with HDACi has synergistic anti-tumor effects in CTCL. HDACi-resistant MJ, HDACi-sensitive HH and HDACi cross-resistant HH/VOR CTCL cells were treated with HDACi (panobinostat, vorinostat, or enlinostat) plus or minus curcumin for up to 48 hrs. Cell viability was examined by the MTS assay and apoptosis by FACS analysis of annexin V/PI binding populations and/or cell cycle distribution. The NF-κB signaling pathway was analyzed by electrophoretic mobility gel shift assay and Western blotting. In MJ and HH cell lines, 5 nM panobinostat induced 1.4% and 11.4% apoptosis and 10 μM curcumin induced 24.5% and 29% apoptosis compared to vehicle controls. Panobinostat combined with curcumin induced 46.9% and 83.4% apoptosis in MJ and HH cell lines, respectively. Of interest, the HDACi cross-resistant HH/VOR CTCL cells were sensitive to curcumin alone and curcumin further enhanced panobinostat-induced apoptosis by 30% in the HH/VOR CTCL cells. Moreover, panobinostat combined with curcumin synergistically suppressed the DNA binding of NF-κB and decreased protein expression of the NF-κB activator RANK and NF-κBp65. Synergism was associated with down-regulation of NF-κB-regulated anti-apoptotic proteins (bcl-2, bcl-xL, and survivin), anti-proliferative proteins (c-myc and cyclooxygenase-2), and pro-invasive protein matrix metalloproteinase-9. Similar synergism was also seen when vorinostat or entinostat was combined with curcumin. These results suggest that HDACi could be combined with curcumin to enhance apoptosis of malignant T-cells through inhibition of NF-κB signaling in CTCL. Curcumin alone and in combination with HDACi may be an attractive strategy for the treatment of HDACi-refractory CTCL patients.

Disclosures:
Zhang: Novartis: Research Funding. Duvic:Novartis: Research Funding.
Indirubin-3'-Monoxime Induces Apoptosis and Autophagy In Acute Lymphoblastic Leukemia Cells and Chronic Myelogenous Leukemia Cells, Exhibits Limited Cytotoxicity In CD34+ Hematopoietic Stem Cells, Lymphocytes and Granulocytes
Authors: Lee Ming-Yang , Liu Yi-Wen , Chen Ming-Ho , Chuang Jing-Jing 
Published: October 2019

Abstract
Abstract 3984
The rates of treatment-related mortality (TRM) and relapse are unacceptably high in adults undergoing antileukemia treatments for acute lymphoblastic leukemia (ALL). So far has no better therapy with low side effects to improve long-term survival in these patients. Indirubin, a Chinese translational anti-chronic myelogenous leukemia (CML) agent, is able to induce cellular apoptosis. However, until now the functional action of IO on ALL remains still unknown. Therefore, here we investigated and compared the cytotoxic efficacy and action of indirubin-3'-monoxime (IO) in JM1 (ALL cell) and K562 (CML cell). ALL and CML cells were treated with a series of IO dose for 24 and 48h, and cell survival was determined by WST-1 assay. ALL and CML cells were shown to be similar susceptible to IO cytotoxicity. In order to clear in which way of cell death induced by IO, we performed analyses for apoptosis, necrosis and autophagy, respectively. After IO treatment, both ALL and CML cells were arrested in the G2/M cell cycle phase. In addition, an increase of sub-G1 proportion was caused. We found also increasing of caspase-3 activation and formation of cleaved PARP in a dose dependent manner. These were associated with the form of apoptosis. However, the caspase inhibitor Z-VAD-FMK only could partially prevent cell death in ALL and CML cells. When further analyzing the necrotic phenomenon through measuring LDH release, the result clearly showed that LDH release was not remarkable after cell treatment with high dose of IO. Besides, we observed surprisingly in Western blot the increasing expression of microtubule-associated protein light chain 3-II (LC3-II), which generally correlates to formation of autophagosomes. Because better antileukemic drug should not induce toxicity in normal blood cells as much as possible, so the cytotoxic effect of IO in CD34+ hematopoietic stem cells, lymphocytes and granulocytes was analyzed. Excitingly, results showed that IO could not affect cell viability of granulocytes, and IO cytotoxicity in lymphocytes was only marginal. If CD34+ hematopoietic stem cells were treated with IO, the rate of cell survival and their ability of differentiation were almost identical in contrast with non-treated control. Apparently, these data indicated that IO possesses the capability to induce apoptosis and autophagy in both CML and ALL cells. The most important is that the IO hardly influences the cell survival and the differentiation of CD34+ hematopoietic stem cells, the cytotoxic effect in granulocytes and lymphocytes is only limited. In conclusion, IO can be considered as a potential agent for clinical anti- ALL treatment.

Disclosures:
No relevant conflicts of interest to declare.
VEGFA Controls Haematopoietic Stem Cell Specification In a Dose-Dependent, Isoform-Specific Manner
Authors: Leung Amy , Ciau-Uitz Aldo , Vyas Paresh , Patient Roger , Porcher Catherine 
Published: October 2019

Abstract
Abstract 406
VEGFA signalling is critical for endothelial and haematopoietic development during embryogenesis. Perturbation of the VEGFA pathway results in defective haemopoietic stem cell (HSC) specification and vascular/arterial formation. Because of the intimate relationship between arterial and blood development, the contribution of VEGFA to HSC specification is unclear. Characterising the exact function of VEGFA is of primary interest for understanding mechanisms regulating haematopoietic specification. Here, we show that morpholino-induced knock-down of the co-repressor and oncoprotein ETO2 in Xenopus embryos leads to a failure of HSC specification via a VEGFA–dependent mechanism, independently from dorsal aorta (DA) formation and arterial specification. Knock-down of ETO2 represses Vegfa levels in somitic tissues along the migratory pathway from the dorsal lateral plate to the midline. This, in turn, leads to absence of Notch1 expression in the hemogenic endothelim of the DA and failure to activate the HSC transcriptional programme. DA formation and arterial specification occur normally, thus uncoupling VEGFA requirements for DA versus HSC formation. Then, we examined expression of the 3 VEGFA isoforms that differ in their physical and biological properties. In Eto2 morphant embryos, there was reduction (∼50%) in mRNA expression of the medium and long Vegfa variants in the somites. Importantly, Vegfa hypomorph embryos phenocopy the Eto2 morphant phenotype; this further demonstrates that reduction in VEGFA levels is sufficient for failure of HSC specification. Furthermore, restoration of medium Vegfa isoform (Vegfa170) levels alone in either Eto2 morphants or Vegfa hypomorph embryos was sufficient to re-establish the HSC programme. In summary, this is the first report of a dose-dependent, isoform-specific function of VEGFA at the onset of the HSC programme, independent from its functions in vessel formation and specification of the DA. We propose that signalling from VEGFA170 in somites instructs the HSC programme of the DA/HSC progenitors as they migrate towards the midline, in a paracrine manner. This VEGFA170 signal is critically required later in the haemogenic endothelium of the newly formed DA to initiate Notch1 expression and trigger the haematopoietic transcriptional programme. This work also reveals for the first time that ETO2 controls the onset of the HSC programme in a non cell-autonomous manner. This work furthers our understanding of the complex interplay between the cell populations controlling key signaling events leading to HSC specification. Manipulation of these signaling cascades may allow us to better develop strategies to produce and expand HSCs for therapeutic and regenerative purposes.

Disclosures:
No relevant conflicts of interest to declare.
High Bcl-2/Mcl-1 Gene Expression Ratio Identifies Molecular Sub-Groups of Multiple Myeloma Patients Extremely Sensitive to ABT-737
Authors: Bodet Linda , Pellat-Deceunynck Catherine , Gomez-Bougie Patricia , Dousset Christelle , Touzeau Cyrille , Le Gouill Steven , Avet-Loiseau Hervé , Bataille Regis , Amiot Martine 
Published: October 2019

Abstract
Abstract 4061
Multiple myeloma (MM) is a plasma cell malignancy presently incurable with heterogeneity in molecular abnormalities and treatment response. While the importance of Mcl-1 in maintaining survival of Human myeloma cell lines (HMCLs) has been well documented we recently identified a sub-group of HMCLs that highly expressed Bcl-2. This finding prompted us to investigate the sensitivity of HMCLs to ABT-737, a cell-permeant compound that binds with high affinity to Bcl-2 and antagonizes its anti-apoptotic function. The effect of ABT-737 on myeloma cell viability was assessed by APO2.7-PE staining on a large panel of HMCLs (n=26) representative of Multiple Myeloma molecular heterogeneity. We found 7 HMCLs highly sensitive to ABT-737 with an EC50 ranging from 7±0.4nM to 150±7.5nM. Myeloma cell death induced by ABT-737 occurred as early as 4 hours after treatment and led to Bax and Bak activation. While all these sensitive cell lines expressed a high level of Bcl-2, only Bcl-2/Mcl-1 gene expression ratio allowed the discrimination of highly ABT-737 sensitive from resistant HMCLs. High sensitivity (EC 50 <150 nM) was correlated to a ratio > 0.4. This result is in agreement with the high affinity of ABT-737 for Bcl-2 in contrast to Mcl-1. Induction of apoptosis may be due to the displacement of Bim, Puma or Bik from their endogenous complexes with Bcl-2, as demonstrated by immunoprecipitation assays. Interestingly, we demonstrated that all sensitive HMCLs harbored a t(11;14) while 77% (7 out of 9) of t(11;14) HMCLs are sensitive to ABT-737. Then, interrogation of the public expression database of MM patients showed that the Bcl-2/Mcl-1 ratio of both t(11;14) (median 0.29, range 0.05–0.71) and hyperdiploid (median 0.24, range 0.04–0.79) patients was significantly higher to all other groups of patients (p<.001). In order to fairly identify MM patients expressing high Bcl-2/Mcl-1 ratio we set up a flow cytometry assay. Preliminary results confirmed that primary myeloma cells with a high Bcl-2/Mcl-1 ratio are also highly sensitive to ABT-737. Altogether, the very high sensitivity of Bcl-2 high Mcl-1weak HMCLs and primary myeloma cells to ABT-737 (similar to the one of CLL) supports the idea of a clinical trial using Bcl-2 antagonists in a selected subset of t(11;14) and hyperdiploid Multiple Myeloma patients.

Disclosures:
Avet-Loiseau: Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees.
A New Xenograft Model for Studying Multiple Myeloma and Drug Response
Authors: Deutsch Varda , Farnoushi Yona , Cipok Michal , Kay Sigi , Naparstek Elizabeth , Goldstein Ronald 
Published: October 2019

Abstract
Abstract 4073
While new treatment options are available, multiple myeloma (MM) still remains an incurable malignancy of plasma cells with a grim prognosis. Practical in vivo models to study human MM may enable a better understanding of the biology of the disease, and better optimization of therapeutic strategies. The best current xenograft model, the immune-deficient NOD/SCID mice, recapitulates MM in vivo, however, the price is very costly and maintenance complex, with >1 month required to establish engraftment. Our goal was to develop a user friendly rapid alternative xenograft system for the preclinical assessment of MM growth and therapy. We recently described this new in-vivo system for studying human leukemia in the pre-immune turkey embryo 1,2. These embryos are inexpensive, require no maintenance, and are easily manipulated experimentally. Described here are the first attempts at application of this novel system to study MM and test therapies. Cell lines ARH-77 and CAG line and fresh patient cells (5 × 106/embryo) were injected IV into turkey egg chorioallantoic membrane veins on embryonic day E11. Engraftment of human cells in hematopoietic organs, bone marrow (BM) and liver was detected 7 days later (E18) by RTPCR, immunohistochemistry and flow cytometry and by circulating free light chain (6-25 mg/L) in the peripheral blood of 100% of the injected cell lines and 50% of patients myelomas. Treatment with Velcade (Bortezomib) or Revlimid IV on E13 (48 hours after MM cell injection), at drug levels that were precalibrated to be non-toxic to the developing embryonic BM, dramatically reduced engraftment, demonstrating the utility of this new model for testing drug activity in vivo. ARH-77 cells, detected by flow cytometry of the embryonic BM cells with anti-human CD19, CD38 and CD138, were inhibited from 8.5% engraftment to 0.72% after a single Velcade treatment, with an 18 fold decrease compared to untreated embryos in the ratio of human to avian cells in BM tissue. determined by Q-RT-PCR analysis of human alpha satellite and avian GAPDH DNA normalized per cell. Very similar results were obtained with Revlimid. The results presented suggest that with further work the turkey embryo model may provide an affordable, rapid and practical xenograft system in vivo for studying the biology of MM, for affordably testing MM therapies, as well for developing a new method for individualized patient screening for response or resistance to particular therapeutic agents.
1. Taizi M, Deutsch VR, Leitner A, Ohana A, Goldstein RS. A novel and rapid in vivo system for testing therapeutics on human leukemias. Exp Hematol. 2006;34:1698-1708.
2. Grinberg I, Reis A, Ohana A, et al. Engraftment of human blood malignancies to the turkey embryo: a robust new in vivo model. Leuk Res. 2009;33:1417-1426.

Disclosures:
No relevant conflicts of interest to declare.
A Novel Anilinoquinazoline Derivative Inhibits Growth of High-Risk Myeloma Cells In Vivo and Regulates Differentiation of Osteoclasts
Authors: Ozaki Yoshie , Naito Yuhei , Yanagawa Hiroshi , Suzuki Yuya , Du Wenlin , Yamada Taketo , Matsuo Koichi , Doi Nobuhide , Iijima Shiro , Matsushita Maiko , Tahara Kayoko , Hattori Yutaka 
Published: October 2019

Abstract
Abstract 4076

Purpose:
Despite recent advances in the use of newly developed drugs, high-risk multiple myeloma (MM) patients harboring del13q, t(4;14) or del17p revealed significantly shorter survival. To overcome the limitation, we have screened forty synthetic anilinoquinazoline (AQ) derivatives, and found a novel compound, Q15, which significantly inhibited the growth of MM cell lines with high-risk chromosomal abnormalities. The purpose of this study is to examine anti-tumor and anti-osteoclastogenic activities of Q15 and to clarify the possibility of development of new drug effective for high-risk MM and bone diseases.

Methods and Results:
Forty AQ derivatives were synthesized and screened for anti-proliferative effect on KMS34 cells. Q15 strongly inhibited growth of t(4;14)-positive KMS34 cells and induced apoptosis in much lower concentration (IC50=78nM) compared with gefitinib (IC50=2500nM), a representative AQ. Q15 also inhibited growth of other MM cell lines harboring high-risk chromosomal abnormalities. It was also found that Q15 did not inhibit intracellular tyrosine phosphorylation induced by EGF, FGF-2, HGF and IL-6, suggesting that Q15 showed anti-tumor activity in a different mechanism from that of gefitinib. In vivo anti-myeloma activity was evaluated by intraperitoneal injection of Q15 into KMS34-bearing lcr/SCID mice. Twenty mg/kg Q15 significantly delayed the tumor growth in these mice. Histopathological examinations revealed apoptosis of MM cells in Q15-treated mice. Growth of colony-forming cells was not suppressed by much higher concentrations (25μ M) of Q15 than IC50, suggesting low hematopoietic toxicity of Q15. In pharmacokinetic study using high-performance liquid chromatography (HPLC), the plasma concentration of Q15 in mice reached a maximum (Cmax=4.5μ M) at 1.5hr after injection, and its half life (T1/2) was 4.5hr. In addition, anti-osteoclastogenic activity was also examined by adding Q15 to M-CSF/RANK ligand-induced osteoclastogenic culture of bone marrow mononuclear cells from C57BL/6JJcl mice. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts was reduced in the presence of Q15.

Conclusion:
Q15, a novel AQ derivative, has anti-MM activity in vivo and is a potentially safe and effective drug for high-risk MM with bone lesions.

Disclosures:
No relevant conflicts of interest to declare.