bluuebunny/crossref_metadata_2025_split huggingface dataset:
High-Resolution Comparative Genomic Hybridization of Mirna Genes In Therapy-Related AML Identifies a Somatic Deletion of MiR-223
Authors: Ramsingh Giridharan , Matson Angela , Walter Matthew J. , Link Daniel C. 
Published: October 2019

Abstract
Abstract 2759
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and diverse cellular processes. Expression profiling of miRNAs has identified dysregulated miRNAs in many cancers, including acute myeloid leukemia (AML). However, the mechanism for altered miRNA gene expression and the frequency of miRNA gene mutations in AML is largely unknown. We performed next-generation sequencing and high-resolution comparative genomic hybridization (CGH) to determine the frequency of miRNA gene mutations in 30 patients with therapy-related AML (t-AML). The sequencing is completed, and final analysis is underway. Herein, we report the results of the CGH. We designed custom CGH arrays for all 835 miRNA genes in miRBase (version 14) and 44 genes involved in miRNA processing. Average probe spacing was 30 bp for miRNA genes and 80 bp for miRNA processing genes and 10 kb flanking regions were interrogated for all genes. In each case, genomic DNA from leukemic blasts was compared with DNA from a skin biopsy from that patient. The median age of the 30 patients with t-AML was 49.2 years (25-77) with M:F ratio 13:17. The mean blast % was 81% (31-100). Consistent with previous reports, many of these patients had an abnormal karyotype with abnormalities of chromosome 5 and 7, 13% and 17% respectively. As expected, CGH analysis confirmed copy number alterations already identified by cytogenetics. In addition, we identified a single t-AML sample (from a male patient) that carried a small (435 kb) hemizygous deletion of miR-223 on chromosome × that was not apparent by cytogenetics. Quantitative PCR of genomic DNA confirmed the loss of the miR-223 gene, and real time RT-PCR demonstrated loss of miR-223 expression. Of note, miR-223 has been implicated in the regulation of granulopoiesis, and mice lacking miR-223 display a myeloproliferative phenotype (Johnnidis, Nature 2008). We screened an additional 27 AML patients for miR-223 expression and identified 3 other samples with miR-223 expression 2 standard deviations below the normal (based on CD34+ cells from healthy donors). No copy number alterations in miR-223 were detected in these patients, suggesting epigenetic silencing of miR-223. Consistent with this possibility, one of these patients carried a t(8;21), which has been shown to epigenetically silence miR-223 (Fazi, Cancer Cell 2007). The mechanism by which miR-223 is silenced in the other two AML samples is under investigation. In summary, cytogenetically silent deletions of miRNA genes are uncommon in t-AML. Loss of miR-223 expression can occur through somatic mutation or epigenetic silencing and is likely to contribute to leukemic transformation in a subset of patients with AML.

Disclosures:
No relevant conflicts of interest to declare.
Lack of IL-7 Receptor Alpha Chain (CD127) Expression In T Cells Is a Hallmark of T-Cell Immunodeficiency In Schimke Immuno-Osseous Dysplasia (SIOD)
Authors: Sanyal Mrinmoy , Dionis Kira Y , Baradaran-Heravi Alireza , Dekel Benjamin , Bökenkamp Arend , Boerkoel Cornelius F. , Lewis David B. 
Published: October 2019

Abstract
Abstract 2767
Schimke immuno-osseous dysplasia (SIOD) is an autosomal recessive, incompletely penetrant, childhood disorder associated with biallelic loss-of-function mutations of SMARCAL1 (swi/snf-related matrix-associated actin-dependent regulator of chromatin, subfamily-a-like-1) gene. The SMARCAL1 gene encodes for the DNA annealing helicase although it is unknown why this impairment results in skeletal dysplasia, renal dysfunction, and T-cell lymphopenia. The representation of T-cell subsets in SIOD patients is further characterized by a high proportion of memory (CD45RA−CD45RO+) T cells, however, the etiology of T-cell immunodeficiency has not been elucidated. Here, we demonstrate that T cells in individuals affected by SIOD express markedly reduced surface levels of IL-7 receptor alpha chain (CD127) as compared to their unaffected sibling or normal individuals. In contrast, the alpha-chain component of the IL-2 receptor (CD25) was expressed normally. This reduction in IL-7 receptor alpha chain was observed in all T-cell subsets including naïve T cells. This suggests that the origin of the IL-7 receptor alpha chain deficiency is likely intrathymic, rather than occurring after emigration to the periphery. In addition, T cells from SIOD patients were less responsive to stimulation with IL-7, indicating a loss of functional receptor. From these observations, we propose the lack of functional IL-7 receptor expression in T cells, and possibly their earlier progenitors, may have restricted T-cell development in SIOD patients. The mechanism for reduced IL-7 receptor expression is currently under investigation.

Disclosures:
No relevant conflicts of interest to declare.
Characterization of the Immunoregulatory Function of Human TCRαβ+ CD4-CD8- Double-Negative T Cells
Authors: Voelkl Simon , Gary Regina , Mackensen Andreas 
Published: October 2019

Abstract
Abstract 2776
Regulatory T (Treg) cells have been shown to be involved in downregulating immune responses in autoimmunity, transplant rejection, and graft-versus-host disease. Moreover, the important role of Treg cells in tumor progression has also been extensively investigated and Treg-mediated immune suppression has emerged as a crucial mechanism of tumor evasion. Recently, a novel subset of TCRαβ+ CD4−CD8− (double-negative, DN) T cells has been characterized to specifically suppress immune responses in mice. Here we demonstrate that human DN T cells belong to the family of inducible Treg cells with potent suppressor activity towards CD4+ and CD8+ T-cell responses. Resting DN T cells failed to suppress responder cells, whereas antigen presenting cell (APC)-stimulated DN T-cells and freshly isolated CD4+CD25+ Treg cells revealed a strong suppressive activity. Of importance, when more potent stimulators such as allogeneic dendritic cells were used for activation of responder T cells, CD4+CD25+ Treg cells were unable to mediate any suppressor function, while APC-primed DN T cells were still able to suppress. The suppressive activity of DN T cells is neither mediated indirectly by modulation of APCs nor by competition for T-cell growth factors. Furthermore, DN T-cell mediated suppression towards responder T cells requires cell-cell contact and is TCR dependent. Based on the importance of Treg cells in tumor immunity, we determined the frequency of circulating DN T cells in patients with malignant diseases. Of interest, the DN T-cell pool revealed an increased frequency in cancer patients compared to healthy controls, indicating that these suppressor cells might limit tumor-specific T-cell responses and thereby impair tumor immunosurveillance. Taken together, our results demonstrate that human DN T cells are a new subset of inducible Treg cells exerting a very potent suppressive activity towards cellular immune responses. Further understanding of the mechanisms involved in human DN T-cell suppression may have important implications for novel immunotherapies.

Disclosures:
No relevant conflicts of interest to declare.
Resveratrol Induces Cell Growth Arrest and Cell Death of Adult T-Cell Leukemia Cells Via Deacetylation of STAT3 by SIRT1
Authors: Ito Shigeki , Oyake Tatsuo , Murai Kazunori , Ishida Yoji 
Published: October 2019

Abstract
Abstract 2844
Adult T-cell leukemia (ATL) is an aggressive malignancy of peripheral T cells infected with human T-cell leukemia virus type 1 (HTLV-1). The prognosis of aggressive ATL patients remains poor because of its resistance to conventional chemotherapy. Therefore, new therapeutic targets should be explored. We recently reported that signal transducers, and activators of transcription 3 (STAT3)/survivin pathway is critical for cell proliferation and survival of ATL cells (Ito S. et al. Leuk Res. 2010. 34:352-357). Thus, STAT3 may be one of the therapeutic targets in ATL. Recent report suggested that acetylation in the SH2 domain of STAT3 is an important regulatory modification that influences protein-protein interaction and transcriptional regulation. Although STAT3 has been shown to interact with p300 acetyltransferase, the mechanism of deacetylation of STAT3 remains unknown. It has been recently shown that fasting-activated longevity protein sirtuin 1 (SIRT1) deacetylase/STAT3 pathway in the regulation of gluconeogenesis. We hypothesized that STAT3 deacetylation by SIRT1 may influence the cell growth and survival of ATL cells. In this study, we investigated the effects of resveratrol, an activator of SIRT1 deacetylase, on HTLV-1 infected T-cell lines, KUT1 and MT2 cells. We found that resveratrol significantly suppressed cell proliferation in a dose- and time-dependent manner by trypan blue exclusion method (P<0.01, ED50=50mM). Resveratrol also induced apoptosis in these cells by flow cytometric analysis with AnnexinV/propidium iodide staining (P<0.01). We next examined intracellular signaling in resveratrol-treated cells. Immunoblot analysis showed the suppression of constitutive phosphorylation of STAT3 at Tyr705 and IkBα at Ser32 but not JAK3 and STAT5 of both cells 24 hours after the treatment of resveratrol. Interestingly, resveratrol reduced acetylation of STAT3 at Lys685. We also observed the cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP) in resveratrol-treated cells, indicating that resveratrol induces caspase-dependent apoptosis in these cells. To better understand the mechanism by which resveratrol induces STAT3 deacetylation, we examined the relationship between SIRT1 and STAT3. SIRT1 protein expression level was elevated after the treatment of resveratrol. Immunoprecipitation assay revealed the physical interaction of STAT3 with SIRT1 in MT2 cells, suggesting that the mechanism of action of resveratrol involves the deacetylation of STAT3 through the interaction of STAT3 with SIRT1.
Our data indicate that resveratrol presents a potent anti-leukemia effect in part via the deacetylation of STAT3 in HTLV-1 infected cells. These mechanistic findings suggest that resveratrol may have a potential in the treatment of ATL.

Disclosures:
No relevant conflicts of interest to declare.
The Novel Bispecific Antibody [(CD20)2xCD16] Efficiently Triggers Lysis of Neoplastic B Cells
Authors: Glorius Pia , Baerenwaldt Anne , Kellner Christian , Staudinger Matthias , Parren Paul W.H.I. , van De Winkel Jan G. J. , Humpe Andreas , Valerius Thomas , Repp Roland , Nimmerjahn Falk , Gramatzki Martin , Peipp Matthias 
Published: October 2019

Abstract
Abstract 2846
Although the CD20 antibody rituximab considerably improved the treatment of B-cell neoplasias, novel optimized CD20-targeting strategies may still offer therapeutic gains. Analysis of Fc receptor (FcR) polymorphisms of rituximab-treated patients and animal studies demonstrated the crucial role for activating FcRs and effector cell recruitment. In the current study a novel CD20-directed bispecific antibody (bsab) [(CD20)2×CD16] in the tribody format was evaluated to enhance antibody dependent cellular cytotoxicity (ADCC). The heterodimeric molecule forms a CD16-directed F(ab) fragment with a CD20-directed scFv c-terminally fused to both peptide chains. [(CD20)2×CD16] was expressed in HEK-293T cells and purified using affinity chromatography. Specific binding to CD20- or FcγRIIIa- (CD16a) positive cells was demonstrated by flow cytometry. No significant difference in binding to both CD16a alloforms (CD16a-F158 and CD16a-V158) was observed. In chromium release assays [(CD20)2×CD16] mediated specific lysis of different burkitt's lymphoma cell lines in a dose-dependent manner even at low picomolar concentrations. Potency of target cell lysis was improved compared to rituximab (10-fold) irrespective of the effector cell donors' CD16a V/F-polymorphism status. In addition, the bsab triggered lysis of primary tumor cells from 12/12 lymphoma patients (CLL; MCL; MZL). [(CD20)2×CD16] triggered significantly stronger target cell lysis than rituximab or the parental CD20 antibody. Importantly, specific lysis of primary tumor cells was also observed using autologous NK cells as effector cells, suggesting that [(CD20)2×CD16] is capable in triggering tumor cell lysis in the presence of a strong MHC I inhibitory signal. Interestingly, when B-cell depletion was analyzed in whole blood, significantly higher depletion rates were obtained with the bsab compared to rituximab (75% vs 25%). Depletion of B cells was accompanied by activation of NK cells, as evidenced by CD69 expression on NK cells. Finally, NOD-SCID mice were transplanted with CD34-positive cord blood cells to reconstitute a humanized hematopoietic system. Distribution and numbers of engrafted human B cells and human CD16-positive effector cells were analyzed by flow cytometry. CD16 was expressed on splenic monocytes and NK cells. After daily injection of [(CD20)2×CD16] for three days a significant reduction of B cells was observed. Thus, the CD16-directed bispecific antibody [(CD20)2×CD16] may extend the therapeutic range and represents a promising candidate for further clinical development.

Disclosures:
Parren: Genmab: Employment. van De Winkel:Genmab: Employment.
Bortezomib Induces Caspase-9-Mediated Apoptosis through Activation of the BH3-Only Proteins Noxa, Bik and Puma In Both ALK-Positive and ALK-Negative Anaplastic Large Cell Lymphoma Cells
Authors: Cillessen Saskia AGM , Hijmering Nathalie J , Moesbergen Laura M , Ossenkoppele Gert J. , Oudejans Joost J , Meijer Chris JLM 
Published: October 2019

Abstract
Abstract 2847
Anaplastic large cell lymphoma (ALCL) is a CD30 positive T-cell lymphoma that can be divided into a systemic and a primary cutaneous type. Systemic ALCL can be further divided into an anaplastic lymphoma kinase (ALK) expressing type and an ALK-negative type. Despite intensive treatment regimens, the disease will be fatal in 20–30% of the systemic ALK-positive and 50–70% of the systemic ALK-negative ALCL patients. A recent study in primary ALCL samples has demonstrated an increased expression of a fraction of NF-κB target genes, suggesting upregulation of NF-κB activity in ALCL tumor cells. NF-κB activity can be inhibited by the proteasome inhibitor bortezomib resulting in induction of apoptosis. In this study, we therefore investigated if bortezomib can induce apoptosis of cultured lymphoma cells of three systemic ALK-positive and three ALK-negative ALCL patients and seven ALCL cell lines and we examined the mechanisms by which bortezomib induced cytotoxicity in these ALCL cells.
Treatment with bortezomib resulted in induction of apoptosis in all ALK-positive and ALK-negative ALCL patient samples and ALCL cell lines tested, when we compared the percentage cell death with the non-neoplastic CD4- and CD8-positive PBMC and tonsil T-cells from healthy donors. The lethal dose (LD50) varied between 54nM and more than 100nM after 24 hours and varied between 21nM and 52nM after 48 hours of exposure. ALK-negative ALCL cases were more sensitive to bortezomib and showed significant lower LD50 values than ALK-positive ALCL cells. We show that bortezomib-induced cell death in ALK-positive and ALK-negative ALCL is dependent on caspase-9 and/or caspase-8 mediated apoptosis and that bortezomib induces depolarization of the mitochondrial membrane. mRNA-expression and protein analysis revealed clearly upregulation of the BH3-only proteins Noxa, Bik and Puma, resulting in Bak and Bax release from the anti-apoptotic proteins Mcl-1 and Bcl-2. We also demonstrated that ALCL cells relatively resistant to bortezomib were characterized by high expression of Bcl-2A1, suggesting the possibility of pre-defining patients most likely to benefit from bortezomib therapy.
Our preclinical data support the therapeutic application of bortezomib as potential drug in the treatment of ALCL, especially ALK-negative ALCL patients to improve their prognosis.

Disclosures:
No relevant conflicts of interest to declare.
Preclinical Evaluation of A Potent 2nd Generation Small-Molecule Hsp90 Inhibitor STA-9090 In Hematological Cell Lines
Authors: Ying Weiwen , Proia David , He Suqin , Sang Jim , Foley Kevin , Du Zhenjian , Blackman Ronald k , Wada Yumiko , Sun Lijun , Koya Keizo 
Published: October 2019

Abstract
Abstract 2899
STA-9090 is a potent, second generation, small-molecule Hsp90 inhibitor, with a chemical structure unrelated to the first-generation, ansamycin family of Hsp90 inhibitors. In preclinical in vitro and in vivo studies, STA-9090 has shown potency up to 100 times greater than the first-generation Hsp90 inhibitors against a wide range of solid and hematological cancer types including those resistant to imatinib, sunitinib, erlotinib, and dasatinib. STA-9090 is currently being evaluated two Phase 1 and four Phase 2 trials (non-small cell lung, GIST, colon, and gastric) in solid tumor cancers; and two trials in hematologic cancers. Additional Phase 2 trials in several other indications are planned for 2H 2010.
Inhibition of Hsp90 by STA-9090 results in the destabilization of a broad range of oncogenic kinases often overexpressed or mutated in hematological cancers. For example, the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressed in the anaplastic large cell lymphoma (ALCL) cell line Karpas 299, is degraded rapidly in the presence of STA-9090 in vitro, resulting in the loss of viability. Similar results were shown in other NPM-ALK driven ALCL cells including SU-DHL-1 and SR-786 with IC50 less than 20 nM. Stability of other kinases common to hematological malignancies, such as Bcr-Abl, FLT3 and c-Kit, were also shown to be highly sensitive to STA-9090, resulting in potent cell death of cell lines addicted to signaling by these kinases.
In vivo, STA-9090 was highly effective in a subcutaneous xenograft model of diffuse large B-cell lymphoma SU-DHL-4 with resulting %T/C values of 26, 4, -90 and -93 when dosed at 25, 50, 75 and 100 mg twice per week, respectively. Importantly, 75 and 100 mg/kg STA-9090 dosed 2 times per week for a total of 3 weeks (150 and 200 mg/kg weekly) resulted in 25% and 50% of the animals in each group being free of tumors by the end of the study, respectively. MV4-11, an AML (FLT3ITD) cell line, turned out to be one of the most sensitive xenograft models to STA-9090 treatment. STA-9090 at 100 mg/kg or125mg/kg once weekly was highly efficacious with 37.5% of mice achieving tumor free with acceptable toxicity at the end of the 3-week treatment period.
In conclusion, STA-9090 exhibits preferable biological profiles both in vitro and in vivo in treating hematological malignances. Clinical studies for using STA-9090 both once weekly and twice weekly are ongoing.

Disclosures:
Ying: Synta Pharmaceuticals: Employment. Proia:Synta Pharmaceuticals: Employment. He:Synta Pharmaceur: Employment. Sang:Synta Pharmaceuticals: Employment. Foley:Synta Pharmaceuticals: former employee. Du:Synta Pharmaceuticals: former employee. Blackman:Synta Pharmaceuticals: Employment. Wada:Synta Pharmaceuticals: Employment. Sun:Synta Pharmaceuticals: Employment. Koya:Synta Pharmaceuticals: Employment.
PDE4 Inhibitors Enhance DNA Damage-Induced Apoptosis In CLL
Authors: Meyers John , Lerner Adam 
Published: October 2019

Abstract
Abstract 2902
Treatment of primary chronic lymphocytic leukemia (CLL) cells with PDE4 inhibitors induces apoptosis while comparable treatment of primary human T and B cells from healthy donors does not. PDE4 inhibitor treatment also augments glucocorticoid-induced apoptosis in CLL cells. We now report that PDE4 inhibitor treatment enhances CLL apoptosis induced by DNA damage. Using drug combination analysis, treatment with etoposide and rolipram resulted in a greater than additive apoptotic effect than predicted by a Loewe additivity model at many dose combinations. In order to directly address the role of DNA damage and rule out effects on drug uptake and efflux, we next examined the ability of PDE4 inhibitors to modulate gamma-irradiation-induced apoptosis in CLL. While most patients demonstrated in vitro sensitivity to rolipram and gamma irradiation, a small subset of patients, including those known to be p53 mutant, that lacked a substantial response to ionizing radiation also lacked sensitivity to rolipram (p < 0.01). Those patients with intact sensitivity to radiation, demonstrated at least an additive, or greater-than-additive at some doses, effect on apoptosis when PDE4 inhibitor treatment was combined with gamma irradiation treatment (p < 0.05 at 1 Gy and 20 mM rolipram). As judged by comet assays, gamma radiation-induced DNA double stranded breaks were repaired within one hour of induction regardless of exposure to rolipram. As judged by Western analysis and flow cytometry, the kinetics of gamma irradiation-induced H2AX phosphorylation, a marker for DNA damage, were not altered by addition of rolipram in a manner that correlated with apoptosis. Finally, rolipram treatment did not further augment levels of p53, p21cip, or phospho-Chk2. These results suggest that PDE4 inhibitors did not alter double strand DNA break repair nor alter upstream radiation-induced p53-mediated signaling events. As PDE4 inhibitors are known to induce CLL apoptosis through a mitochondrial pathway, our current studies are focused on identifying the mechanism by which DNA damage and PDE4 inhibitor signaling pathways converge at the mitochondrion to enhance apoptosis. Regardless of the precise mechanism by which these supra-additive apoptotic effects occur, our study adds credence to the concept that PDE4 inhibitors may prove to be important sensitizing adjuvants in the treatment of human B cell malignancies.

Disclosures:
No relevant conflicts of interest to declare.