bluuebunny/crossref_metadata_2025_split huggingface dataset:
Combined Carfilzomib and Selective PI3Kδ Inhibition (TGR1202) Results In Enhanced Myeloma Cell Apoptosis
Authors: Torre Claire , Gu Yanyan , Boise Lawrence H. , Lonial Sagar 
Published: October 2019

Abstract

Introduction
The PI3K signaling pathway plays a vital role in regulating cell growth, proliferation and survival in multiple myeloma (MM) as well as in many other cancers. TGR-1202, an isoform-specific PI3Kδ inhibitor, with efficacy in preclinical models of hematologic malignancies, is currently in Phase I clinical development. In multiple myeloma, PI3K signaling appears to be very important for many extracellular signals, yet inhibition with -pan PI3K inhibitors have not exhibited significant activity. However, literature reports indicate that there are several MM cell lines that express PI3K -δ, and do appear to have differential sensitivity to specific isoform inhibition as opposed to pan PI3K inhibition. In this report, we sought to evaluate the effects of TGR-1202 alone and in combination with the proteasome inhibitor carfilzomib, with the intent of further understanding the mechanism of action and evaluating the impact of the combination.

Methods
Human myeloma cell lines (MM.1S, MY5, RPMI8226, U266, KMS11, ARH-77, OPM1, OPM2, LP1, JJN3 and L363) were treated with TGR-1202 alone, carfilzomib alone, or with the combination of TGR-1202 and carfilzomib. Annexin V/PI staining and Western blot were used to identify the cellular and molecular sequelae of the combination.

Result
10 µM TGR-1202 alone did not cause significant cell death in the MM cell lines tested at 48 hours. When cells were treated with the combination of TGR-1202 and 3 nM carfilzomib, we observed enhanced apoptosis in all of the tested cell lines. In the U266 cell line, 3 nM carfilzomib and 10 µM TGR-1202 induced 16% and 14% cell apoptosis respectively. In the combination treatment, apoptosis increased to 75%. To explore the molecular mechanisms underlying the combination, we used a Western blot assay to evaluate the impact of the combination on the mTOR signaling pathway, a known reciprocal feedback loop when PI3K is blocked. TGR-1202 alone did not have an obvious effect on the mTOR signaling pathway, yet combining TGR-1202 with carfilzomib, significantly inhibited phospho-mTOR, suggesting total pathway blockade.

Conclusion
The combination of TGR-1202 with carfilzomib induces synergistic apoptosis in MM cell lines. Preliminary data suggests that this occurs through blockade of the entire reciprocal feedback loop of mTOR activation. Additional data from primary patient samples and underlying mechanisms will be presented at the meeting. These findings support the rationale for future clinical studies of TGR-1202, a selective PI3K-δ inhibitor in combination with the proteasome inhibitor carfilzomib.

Disclosures:
Boise: Onyx: Consultancy. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy.
A Systematic Review Of The Role Of Erythropoietin In The Pathophysiology Of Anemia In Elderly Patients
Authors: Sriram Swetha , Xenocostas Anargyros , Lazo-Langner Alejandro 
Published: October 2019

Abstract

Introduction
Anemia is a significant issue in the geriatric population, having an impact on morbidity, mortality and quality of life. However, identifying the etiology of anemia is not possible in ∼30% of patients. Postulated mechanisms of anemia of unknown etiology (AUE) include a blunted response to erythropoietin (EPO) or inadequate EPO production in response to anemia. The latter mechanism is of particular interest since it might be amenable to pharmacological intervention. Therefore, in order to explore the relationship between EPO levels and hemoglobin in elderly individuals with AUE, we conducted a systematic review of observational studies.

Methods
We searched Medline, EMBASE, Web of Science, Biosis Previews and Dissertations and Theses using the terms erythropoietin, anemia, elderly and diagnosis as MeSH subject headings. Additional relevant articles were identified by hand searching the meeting abstracts of the European Hematology Association (2006 - 2012) and the American Society of Hematology (2004 - 2012). For inclusion into the final review, studies needed to report data on EPO levels in elderly individuals diagnosed with anemia of unknown etiology. No meta-analysis was conducted due to the heterogeneity of the retrieved studies.

Results
The search identified 4277 potentially relevant citations, of which 31 studies were reviewed in full and 7 cohort studies (2 retrospective, 5 prospective) were included in the final review. The included studies involved 2534 participants ( 1). In general, studies found: 1) lower EPO levels in AUE compared to iron deficiency anemia and other forms of anemia; 2) a lack of correlation between EPO levels and the severity of anemia; and 3) EPO levels in AUE that are in general higher than in non-anemic patients.

Conclusion
Our findings suggest that EPO levels are generally elevated in elderly individuals with AUE, but remain inappropriately low, particularly when compared to anemia of other etiologies suggesting either a relative EPO deficiency, an abnormal EPO response or an abnormal erythroid cell response to EPO. Further research is required to elucidate the mechanisms involved, and the value of pharmacological interventions.

Disclosures:
No relevant conflicts of interest to declare.
H3K9me3-Governed Senescence In Tumor Development and Lymphoma Treatment
Authors: Kase Julia , Dörr Jan , Yu Yong , Milanovic Maja , Ji Sujuan , Spiesicke-Wegener Sandra , Lohneis Philipp , Dörken Bernd , Lee Soyoung , Schmitt Clemens A. 
Published: October 2019

Abstract

Introduction
Premature senescence, a terminal cell-cycle arrest condition of viable cells in response to acute cellular stresses such as oncogenic activation or DNA-damaging anticancer therapy, is characterized by S-phase entry-controlling histone H3 lysine 9 trimethylation (H3K9me3). Previously, we reported an essential, tumor-suppressive role for the H3K9 histone methyltransferase Suv39h1 in oncogene-induced senescence (OIS) as a barrier to lymphoma development in vivo (Braig-M et al., Nature, 2005). In the current study, we focused, in addition to Suv39h1, on the H3K9-active demethylases LSD1 and JMJD2C in both OIS and therapy-induced senescence (TIS).

Methods
Human diploid fibroblasts (HDFs) and mouse embryo fibroblasts (MEFs) were stably transfected with H-RasG12V to induce OIS. S-phase-promoting E2F target gene control by H3K9me3 was analyzed by chromatin immunoprecipitation. Transformation was assessed by anchorage-independent colony formation in vitro and tumor development in nude mice. To establish TIS, primary Eµ-myc transgenic mouse lymphoma cells, retrovirally transduced with bcl2 to block apoptosis, were exposed to adriamycin in vitro or cyclophosphamide in vivo. Senescence was analyzed by staining for senescence-associated b-galactosidase activity (SA-b-gal), Ki67 and BrdU incorporation. Lymphoma formation and treatment in vivo were monitored by luciferase and GFP imaging, SA-b-gal/Ki67 staining in situ, and overall survival was assessed by Kaplan Meier analysis.

Results
H3K9-active demethylases – like overexpression of a dysfunctional H3R9 mutant – blocked cellular senescence, and permitted direct transformation under oncogenic Ras. In Myc-driven lymphomas, either loss of Suv39h1 or overexpression of LSD1 or JMJD2C cancelled TIS in vitro and in vivo. Notably, H3K9me3-impaired lymphomas resembled control lymphomas in their proliferation rate and sensitivity to drug-induced apoptosis, but displayed significantly shorter progression-free and overall survival after chemotherapy. Extended data sets on LSD1 and JMJD2C expression in human diffuse large B-cell lymphoma samples and their correlation to treatment outcome will be presented at the meeting.

Conclusion
The data underscore the essential albeit dynamic role of the H3K9me3 mark in OIS and TIS, and unveil the oncogenic potential of H3K9 demethylases, thereby providing a mechanistic basis for JMJD2C- or LSD1-targeting strategies in lymphoma therapy.

Disclosures:
No relevant conflicts of interest to declare.
DUSP4 Stabilizes FOXP3 Expression In Human Regulatory T Cells
Authors: Sugimoto Naoshi , Liu Yong-Jun 
Published: October 2019

Abstract

Background
Naturally occurring regulatory T cells (Treg) are a subset of CD4+T lymphocytes derived from the thymus. Treg suppress immune responses through multiple mechanisms and are involved in inhibiting the development of autoimmunity. Treg constitutively express the master regulatory gene FOXP3, which is necessary to maintain their phenotype and functions. Human blood circulating FOXP3+ Treg are composed of naïve and activated subsets. CD45RA+CD45RO- naïve Treg enter the blood from the thymus and become CD45RA-CD45RO+ activated Treg after activation. Activated Treg show higher suppressive capacity than naïve Treg.
We have previously compared the gene expression profiles of the four human blood CD4+T cell subsets: naïve T helper (Th) cells, memory Th cells, naïve Treg, and activated Treg. We found that some memory Th cell signature genes (those that are highly expressed in memory Th cells compared to naïve Th cells) were also strongly expressed in activated Treg. DUSP4 (dual specificity phosphatase 4) was one such gene. The DUSP family molecules are known to act as inhibitors of T cell activation signaling, primarily by dephosphorylating the activated MAP kinases. In this study, we hypothesized that DUSP4 might be associated with the phenotype and the functions of human activated Treg.

Experimental Procedures and Results
Human Treg cells were sorted from the peripheral blood of healthy donors and infected with shRNA virus targeting DUSP4 upon stimulation with anti-CD3 and anti-CD28 antibodies. The phenotype and the functions of DUSP4 shRNA-transfected Treg cells were analyzed by real-time quantitative PCR, flow cytometry, and in vitro suppression assay. Strikingly, shRNA knockdown of DUSP4 in Treg resulted in more than 90% reduction in the FOXP3 mRNA level. FOXP3 protein down-regulation was observed as well by intracellular FOXP3 staining. Furthermore, the in vitro suppression capacity of DUSP4 shRNA-transfected Treg decreased in proportion to the FOXP3 level. The decreased FOXP3 expression by DUSP4 knockdown was considered to be due to increased activity of ERK1/2 MAP kinase, which has significant roles in T cell activation. We are currently testing whether MAP kinase inhibitors and MAP kinase genes knockdown modify FOXP3 expression by Treg.

Conclusion
Our study suggests that DUSP4 plays a fundamental role in maintaining the phenotype and the suppressive functions of human activated Treg. DUSP4 and MAP kinases may become novel targets to control Treg function in autoimmunity, allergy, allo-immunity, and cancer immunotherapy.

Disclosures:
No relevant conflicts of interest to declare.
Generation and Functional Properties Of FVIII-Specific Human T Regulatory Cells: A Novel Approach Using Hemophilia A patient’s T Cell Receptors
Authors: Kim Yongchan , Su Yan , Rossi Robert J. , Ettinger Ruth A. , Pratt Kathleen P. , Scott David William , Shevach Ethan 
Published: October 2019

Abstract
Antibody responses (inhibitors) against human factor VIII (FVIII) can obviate successful therapeutic efficacy of FVIII replacement therapy in hemophilia A patients. Novel efforts are necessary to overcome this problem: we propose that specific T regulatory cells (Tregs) can be used to prevent and reverse inhibitor formation. Tregs are the most important T subset for the maintenance of self-tolerance and homeostasis of our immune system. Clinical applications using Tregs are now considered a next-generation cellular therapy in a variety of autoimmune and inflammatory immune disorders such as type 1 diabetes, GVHD, and transplantation, and potentially as a treatment for antibody-mediated diseases such as hemophilia inhibitor formation. However, therapy with polyclonal Tregs is non-specific and a potential drawback is that it could be generally immunosuppressive. Therapy with antigen-specific Tregs (CARTs) would be highly preferable, as these have proved more potent in animal models of autoimmune diseases. We demonstrate herein the generation and functional properties of human T regulatory cells that specifically recognize FVIII. Tregs were generated that expressed the same T cell receptor (TCR) as that found on an HLA-DRB1*01:01-restricted T cell clone isolated from a hemophilia subject with a high-titer inhibitor. The T effector clone was isolated by MHC (DR0101) tetramer staining of this subject’s CD4 cells followed by single-cell sorting and expansion in cell culture. Nucleotide/amino acid sequences were identified to design retroviral FVIII-specific TCR constructs. To produce FVIII-specific human Foxp3+ Tregs, we transduced both expanded T effector cells and Tregs with a TCR expressed on an effector CD4 clone isolated from an inhibitor subject; these T cells recognize the same HLA-DRB1*01:01 restricted epitope as the “parent” effector T cell clone. Specific reactivity of the transduced cells bearing the designed TCR was validated by staining of the transduced T effector and Treg cells by DR0101 tetramers loaded with the same peptide as that recognized by the original effector T cell clone. Transduced effector T cells were expanded by stimulation with the expected FVIII peptide in vitro. Moreover, activation by this peptide in the presence of oligonucleotides mediated functional expansion and stabilization of transduced Tregs in ex vivo culture. Importantly, the expanded transduced Tregs were immunosuppressive only when stimulated by FVIII, even when FVIII-specific effector T cells were present in large excess. (Supported by NIH grant RO1 HL061883-15 to DWS and unrestricted research funding from Bayer, Pfizer and CSL Behring to KPP, and the intramural program of the NIAID to EMS).

Disclosures:
Pratt: Bayer: Research Funding; Pfizer: Research Funding; CSL Behring: Research Funding.
The Clinical Utility Of Bone Turnover Markers In The Evaluation Of Bone Disease In Patients With Haemophilia A and B
Authors: Anagnostis Panagiotis , Vakalopoulou Sofia , Timoleon-Achilleas Vyzantiadis , Charizopoulou Maria , Mandala Eudokia M. , Agapidou Alexandra , Vassiliki Violaki , Vasilia Garipidou 
Published: October 2019

Abstract

Aim
Hemophilia A and B has been associated with increased prevalence of low bone mineral density (BMD). However, the utility of bone turnover markers (BTM) is unknown. The aim of this study was to evaluate bone metabolism in men with hemophilia and investigate associations between BTM and bone disease.

Methods
Serum N- (NTX-I), C-terminal telopeptide of type I collagen (CTX-I) and tartrate-resistant acid phosphatase band-5b (TRAP-5b), as bone resorption markers, and osteocalcin (OC) and bone-specific alkaline phosphatase (b-ALP), as bone formation markers, were assessed.

Results
Seventy men (mean age 45.4±14.7 years) with hemophilia A (n=59) or B (n=11) were studied. Patients with low BMD had significantly higher b-ALP concentrations compared with those with normal BMD, without any differences in the other BTM. NTX-I and CTX-I concentrations were negatively associated with estradiol concentrations and hip BMD and positively with HIV infection, number of affected joints and arthropathy scores. B-ALP and OC concentrations were negatively associated with hip BMD, severity of hemophilia and fracture history, and positively with the number of affected joints and testosterone concentrations.
In men with low BMD, NTX-I, TRAP-5b and b-ALP concentrations were positively correlated with the number of affected joints, whereas TRAP-5b concentrations were positively associated with arthropathy scores and negatively with the level of physical activity.

Conclusion
Increased bone metabolism exists in men with hemophilia and low BMD. Arthropathy, low physical activity and low estradiol concentrations are associated with increased bone resorption and may contribute to the pathogenesis of low BMD in these patients.

Disclosures:
No relevant conflicts of interest to declare.
MiR-150 Inhibits MLL-AF9 Associated Leukemia By Suppressing Leukemic Stem Cells
Authors: Cheruku Patali S , Bousquet Marina , Zhang Guoqing , Ge Guangtao , Ying Wei , Meng Cong , Shie Andrew , Wang Stephanie , Wong Piu , Wang Gang , Safe Stephen , Zhou Beiyan 
Published: October 2019

Abstract
Leukemic stem cells (LSCs) are derived from hematopoietic stem or progenitor cells and often share gene expression patterns and specific pathways. Characterization and mechanistic studies of LSCs are critical as they are responsible for the initiation and potential relapse of leukemias, however the overall framework, including epigenetic regulation, is not yet clear. We previously identified microRNA-150 (miR-150) as a critical regulator of mixed lineage leukemia (MLL) -associated leukemias by targeting oncogenes. Our additional results suggest that miR-150 can inhibit LSC survival and disease initiating capacity by suppressing more than 30% of “stem cell signature genes,” hence altering multiple cancer pathways and/or stem cell identities. MLL-AF9 cells derived from miR-150 deficient hematopoietic stem/progenitor cells displayed significant proliferating advantage and enhanced leukemic colony formation. Whereas, with ectopic miR-150 expression, the MLL-AF9 associated LSC population (defined as Lin-ckit+sca1- cells) was significantly decreased in culture. This is further confirmed by decreased blast leukemic colony formation in vitro. Furthermore, restoration of miR-150 levels in transformed MLL-AF9 cells, which often display loss of miR-150 expression in AML patients with MLL-fusion protein expressing, completely blocked the myeloid leukemia development in a transplantation mouse model. Gene profiling analysis demonstrated that an increased level of miR-150 expression down regulates 30 of 114 stem cell signature genes by more than 1.5 fold, partially mediated by the suppressive effects of miR-150 on CBL, c-Myb and Egr2 oncogenes. In conclusion, our results suggest that miR-150 is a potent MLL-AF9 leukemic inhibitor that may act by suppressing the survival and leukemic initiating potency of MLL-AF9 LSCs.

Disclosures:
No relevant conflicts of interest to declare.
Induction Of Apoptosis By The Dual PI3K/Mtor Inhibitor NVP-BEZ235 Is Impaired By a Profound G1/G0 Arrest In Acute Leukemia Which Can Be Overcome By Combination Approaches
Authors: Kampa-Schittenhelm Kerstin M , Akmut Figen , Illing Barbara , Schittenhelm Marcus M 
Published: October 2019

Abstract

Introduction
Constitutive phosphorylation of AKT is frequently found in acute leukemia. In a proportion of patients, activation of the PI3K/AKT pathway can be linked to gain-of-function tyrosine kinase mutations. We have previously shown that TKI only incompletely inactivate AKT signaling. PI3K/AKT pathway inhibitors are attractive for targeting this pathway. However, most inhibitors lead to G1/G0 arrest. Likely for this reason, response rates to PI3K inhibitor treatment were moderate in various neoplasms. We here demonstrate that the dual PI3K-MTORC1/2 inhibitor NVP-BEZ235, which is currently under clinical investigation in relapsed acute leukemia, mediates a profound G1/G0 arrest in cells harboring leukemia-driving FLT3 ITD or BCR-ABL1 mutations, impairing induction of apoptosis. Furthermore, combination with TKI or chemotherapy overcomes cellular resistance.

Methods
Proliferation and apoptosis assays were performed in leukemia cell lines, an isogenic Ba/F3 cell model harboring mutant-KIT, FLT3 or ABL1 isoforms and primary leukemic cells as well as healthy donor cells. Immunoblots were performed to study effects on AKT signaling pathways including downstream effectors of autophagy (p-ULK1), proliferation, cell cycle (p70S6Kinases, cyclinD, pRB) and apoptosis (caspases). Isobolograms were created to compute combination indices.

Results
NVP-BEZ235 profoundly inhibited phosphorylation of AKT and p70S6K, leading to a potent antiproliferative effect in the low nanomolar range in most cell lines as well as patient blasts. However, no meaningful induction of apoptosis was observed. Instead, a sustained G1/G0 cell cycle arrest with dephosphorylation of RB and upregulation of cyclinD, induction of protective autophagy pathways (via ULK1) and absence of cleaved caspase 3 was detected. An isogenic Ba/F3 model confirmed the low proapoptotic efficacy especially for FLT3 ITD and BCR-ABL1. In an attempt to overcome NVP-BEZ235 induced cell cycle arrest, we co-treated leukemic cells with specific TK inhibitors or chemotherapeutics such as daunorubicin in a fixed dose-dilution ratio. Combination indices revealed additive to superadditive proapoptotic effects for all tested combinations.

Conclusion
We provide evidence that the antileukemic activity of NVP-BEZ235 in acute leukemia with mutations in tyrosine kinases is abrogated due to induction of profound G1/G0 cell cycle arrest which can be overcome by combination with TKI or chemotherapy.

Disclosures:
No relevant conflicts of interest to declare.
Combination Of Regulatory T Cells and Rapamycin As Treatment For Experimental Chronic Graft-Versus-Host Disease
Authors: Belle Ludovic , Ehx Gregory , Somja Joan , Binsfeld Marilene , Hannon Muriel , Caers Jo , Fransolet Gilles , Beguin Yves , Humblet Stephanie , Baron Frederic 
Published: October 2019

Background
Chronic graft-versus-host disease (cGvHD) is one of the main complications of allogeneic hematopoietic cell transplantation (allo-HCT). Donor CD4+ conventional T cells (Tconv) as well as regulatory T cells (Treg) are the key-players in its pathogenesis. Rapamycin (sirolimus, rapa) is a mTor inhibitor that can suppress activation and proliferation of Tconv without inhibiting Treg.

Aims
To assess the impact of a combined treatment with Tregand rapa on experimental cGvHD.

Methods
Lethally irradiated BALB/c mice were injected with 10x106 bone marrow cells and 70x106 splenocytes from B10.D2 donor mice. Mice were divided in four groups on day -1 and treatments were started on day 20 with either PBS, rapa 1 mg/kg/Day, Treg 1.106 cells (D+20), or rapa 1 mg/kg/Day + Treg 1.106 cells (D+20). Treg (CD4+ CD25+ cells) were purified from spleen cells from donor B10.D2 mice using the CD4+ CD25+ regulatory T cell isolation kit (Miltenyi Biotec, GmbH, Germany). Treg purity (defined as CD4+ FoxP3+ cells) was ≥ 70% of total cells and > 92% of CD4+T cells.
The severity of sclerodermatous cGvHD was assessed with the following clinical scoring system. Briefly, animals were individually scored every 3 days for five parameters: weight loss (1, 10-20%; 2, > 20%), posture (1, kyphosis only at rest; 2, severe kyphosis when the animal moved), activity (1, moderate activity impairment; 2, no move unless stimulated), skin (1, erythema or scaling tail; 2, open lesion on the body surface) and hair loss (1, > 1 cm2; 2, > 2 cm2). Mice which reached a score of 8 were estimated to have terminal GvHD and were sacrificed. Terminal GvHD-free survivals between the 4 groups were compared using the Log-rank test.

Results
The effects of the treatments were evaluated 7 days after starting the treatments (D+27 post-transplantation). Numbers of total and central memory and effector memory CD4+ T cells/µL were significantly (p<0.05) decreased in rapa-, Treg-, and Treg + rapa- treated mice compared to PBS mice, while numbers of naïve CD4+ T cells/µL were significantly (p<0.05) decreased in Treg-, and Treg + rapa- treated mice. Further, proliferation of CD4+ T cells (assessed by Ki67 expression) was significantly decreased in rapa- (p<0.05) and Treg + rapa- treated mice (p<0.05). In addition, counts and proliferation of CD8+ T cells/µL were significantly (p<0.05) lower in rapa- and Treg+ rapa- treated mice compared to PBS mice.
Importantly, terminal-GvHD-free survival was significantly shorter in PBS mice than in Treg (P=0.03), rapa (P=0.04), and Treg+ rapa mice (P=0.02).

Conclusion
Our results showed that Treg and rapa administration improved cGvHD in this model of cGvHD.

Disclosures:
No relevant conflicts of interest to declare.