bluuebunny/crossref_metadata_2025_split huggingface dataset:
5-Azacytidine and Decitabine Induce FAS Re-Expression, Exert Major Proapoptotic Effects, and Cooperate with the FAS Ligand in Producing Apoptosis in Neoplastic Human Mast Cells,
Authors: Ghanim Viviane , Herrmann Harald , Hadzijusufovic Emir , Peter Barbara , Blatt Katharina , Schuch Karina , Cerny-Reiterer Sabine , Mirkina Irina , Heller Gerwin , Zöchbauer-Müller Sabine , Karlic Heidrun , Pickl Winfried F , Valent Peter 
Published: October 2019

Abstract
Abstract 3457
Aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL) are advanced myeloid neoplasms with a poor prognosis. In these patients, neoplastic mast cells (MC) are resistant against most conventional drugs. Demethylating agents reportedly exert beneficial effects in several advanced myelogenous neoplasms, including myelodysplastic syndromes. We examined the effects of two demethylating agents, 5-Azacytidine and 5-Aza-2`Deoxycytidine (Decitabine) on growth and survival (apoptosis) of neoplastic MC and the human MC line HMC-1. Two HMC-1 subclones were used, HMC-1.1 lacking KIT D816V and HMC-1.2 exhibiting KIT D816V. Both demethylating agents were found to induce apoptosis and growth inhibition in HMC-1.1 cells and HMC-1.2 cells in a dose-dependent manner (IC50: 5-Azacytidine: 5–10 μM, Decitabine: 1–5 μM). Interestingly, only Decitabine but not 5-Azacytidine induced a major G2/M cell cycle arrest in HMC-1 cells. Drug-induced apoptosis in HMC-1 cells was accompanied by cleavage and activation of Caspase-8 and Caspase-3 as well as an increased expression of proapoptotic FAS/CD95, whereas no major effects on expression of other surface antigens were seen. We also found that both demethylating agents synergize with the FAS-ligand in inducing apoptosis in neoplastic MC. Methylation-specific PCR and bisulfite genomic sequencing revealed that the FAS-promoter is hypermethylated in HMC-1 cells. In addition, qPCR demonstrated that exposure to 5-Azacytidine or Decitabine leads to re-expression of FAS in neoplastic MC, which was confirmed by flow cytometry. Correspondingly, a FAS-specific siRNA was found to block drug-induced expression of FAS and drug-induced apoptosis in HMC-1 cells. Although other key regulators and tumor suppressor molecules such as p16 were also found to be hypermethylated in HMC-1 cells, no major demethylating effects of 5-Azacytidine or Decitabine were seen. Neither 5-Azacytidine nor Decitabine induced substantial apoptosis or growth arrest in normal human cord blood progenitor-derived MC or in control bone marrow cells. Together, our data show that 5-Azacytidine and Decitabine exert growth-inhibitory and pro-apoptotic effects in neoplastic MC. These effects are mediated through FAS re-expression and are augmented by the FAS ligand. Whether epigenetic drugs produce anti-neoplastic effects in vivo in patients with advanced SM including MCL, remains to be determined in clinical trials.

Disclosures:
Valent: Novartis: Consultancy, Honoraria, Research Funding.
MOZ Is Critical for MOZ/MLL-Fusion-Induced HoxA9/Meis1 Expression and Leukemia Development,
Authors: Katsumoto Takuo , Kitabayashi Issay 
Published: October 2019

Abstract
Abstract 3467
Monocytic leukemia Zinc finger (MOZ), a Myst-type histone acetyltransferase, is involved in chromosome translocations associated with FAB M4/M5 subtypes of acute myeloid leukemia (AML). We have shown that MOZ is critical for hematopoietic cell development and self-renewal of hematopoietic stem cells (HSCs). Recent reports suggest that hematopoietic and leukemic stem cells shares molecular basis for their self-renewal. To clarify the roles of endogenous MOZ in malignant hematopoiesis, we tested MOZ-deficient cells for leukemogenesis by leukemia-associated fusion genes such as MOZ-TIF2, MLL-AF10 and PML-RARa. Hematopoietic stem/progenitor cells (HSPCs), prepared with MOZ+/− or MOZ−/− fetal livers, were stimulated with SCF, IL-3, IL-6 and Osteopontin M for 1 day and infected with retrovirus encoding each fusion genes. Five days after infection, the infected cells were transplanted into recipient mice or cultured in methylcellulose medium with IL-3, SCF and GM-CSF. MOZ+/− HSPCs expressing MOZ-TIF2 or MLL-AF10 fusion gene formed colonies repeatedly and the recipient mice developed AML 2–3 month after transplantation. However, the MOZ−/− HSPCs expressing the MOZ/MLL-fusions showed neither colony-forming activity nor AML-inducing acyivity. On the other hand, when PML-RARa was introduced, both MOZ+/− and MOZ−/− cells showed continuous colony-forming activity. Expression of HoxA9 and Meis1 were high in MOZ+/− cells expressing MOZ/MLL-fusions, but they were very low in MOZ−/− cells expressing MOZ/MLL fusions. We have previously reported that expression of HoxA9 and Meis1 was significantly decreased in HSPCs of MOZ−/− fetal liver. When MOZ−/− HSPCs were stimulated in a condition without IL-6 prior to infection of MOZ-TIF2, the MOZ−/− cells acquired ability to generate colonies serially. In this condition, high HoxA9 expression was induced in the MOZ−/− cells. However, Meis1 expression was low and AML was not induced in recipient mice. These results indicate that endogenous MOZ plays critical roles in MOZ/MLL-fusion-induced AML and in HoxA9 and Meis1 gene expression.

Disclosures:
No relevant conflicts of interest to declare.
Coagulation Profile As Prognostic Indicator in Acute Myeloid Leukemia,
Authors: Gushiken Francisca C. , Vo Yen Dai , Xiao Lianchun , Kadia Tapan , Cortes Jorge E. , Pemmaraju Naveen , Pierce Sherry , Kantarjian Hagop M. , Borthakur Gautam 
Published: October 2019

Abstract
Abstract 3593

Background:
Evidence of deranged coagulation is encountered at presentation in a subset of patients with non-promyelocytic acute myelogenous leukemia (AML). High fibrinogen level at presentation is reported to be predictive of failure to achieve complete remission and of early death in AML (Lancet. 2010 Dec 11;376(9757):2000–8). Broader correlation of abnormal coagulation parameters with additional clinical outcomes in AML is needed.

Objective:
To determine association of coagulation parameters with overall survival (OS), event-free survival (EFS), complete response (CR) as well as CR duration (CRD).

Study design:
We performed a retrospective analysis of prospectively collected data from 869 consecutive patients with acute myeloid leukemia (AML) treated at MD Anderson Cancer Center between the years 2005–2009. Patients with acute promyelocytic leukemia (APL) were excluded. Coagulation parameters were recorded at the time of diagnosis and included prothrombin time (PT), international normalized ratio (INR), activated thromboplastin time (aPTT), fibrinogen level and D-Dimer. Chi-square test, Wilcoxon rank sum test, as well as logistic regression analysis were applied to determine the association of CR with coagulation parameters. The Kaplan-Meier method was used to estimate the probability of survival outcomes and Cox proportional hazards models were fit to determine the association of time to event outcomes (CRD, OS and EFS) with coagulation parameters.

Results:
Univariate logistic regression analysis showed that patients with higher fibrinogen levels had lower CR rates (p=0.02), shorter CRD (p=0.05) and OS (p=0.04). Event free survival was defined as time from presentation to loss of response, failure to respond and death due to any cause. By multivariate logistic regression, high fibrinogen was also associated with shorter EFS (p value = 0.03). PT, aPTT, D-Dimer as continuous variables were not significantly associated with clinical outcomes.

Conclusion:
High fibrinogen level at presentation is associated with poor outcome in patients with acute myeloid leukemia. The potential biological role of high fibrinogen level in resistance to chemotherapy or the possibility of fibrinogen levels being a surrogate marker of heightened inflammatory state needs to be explored.

Disclosures:
No relevant conflicts of interest to declare.
Phase 1/ 2 Study of Sapacitabine and Decitabine Administered Sequentially in Elderly Patients with Newly Diagnosed AML,
Authors: Ravandi Farhad , Faderl Stefan , Cortes Jorge E. , Garcia-Manero Guillermo , Jabbour Elias , Boone Patricia Ann , Kadia Tapan , Borthakur Gautam , Wierda William G. , Wetzler Meir , Venugopal Parameswaran , Chiao Judy , Kantarjian Hagop M. 
Published: October 2019

Abstract
Abstract 3630

Background:
Sapacitabine is a novel nucleoside analogue with a unique ability to induce single-strand DNA breaks after incorporation into DNA, leading to production of double strand DNA breaks and/or G2 cell cycle arrest. It is orally administered and has demonstrated promising activity in patients with acute myeloid leukemia (AML). In AML cell lines, the active metabolite of sapacitabine, CNDAC, is synergistic with hypomethylating agents with the synergy being more apparent if cells are treated with hypomethylating agents first. We are conducting a phase 1/ 2 study to evaluate the safety and efficacy of administering sapacitabine in alternating cycles with decitabine in elderly patients with newly diagnosed AML. Methods: Decitabine 20 mg/m2 was administered intravenously daily × 5 days of a 4-week cycle (odd cycles) alternating with sapacitabine 300 mg po b.i.d. × 3 days/week × 2 weeks of a 4-week cycle (even cycles). After these doses were shown to be safe in the phase 1 part, patients were treated in the Phase 2 part to reach a planned sample size of approximately 24 patients. The primary efficacy endpoint is response rate (CR, CRp, PR, or major HI). The regimen will be considered active if response rate is ≥30%. Eligible patients must be ≥70 years with untreated AML unsuitable for or unwilling to receive standard induction chemotherapy; patients who received hypomethylating agents for prior MDS or MPD are excluded. Results: As of August 2011, 25 patients were treated with the above regimen and 23 had ≥ 60 days of follow-up. Median age is 76 years (range, 72–90). No DLTs were observed. Four patients achieved CR, 3 PR and 2 major HIs in platelets. Median time to response is 2 cycles (range 2– 6). Fifteen patients have received ≥4 cycles of treatment. Three patients died within 60-days and the deaths were unrelated to study drugs by investigator assessment. Common adverse events (regardless of causality) included weakness, anorexia, nausea, constipation, diarrhea, dyspnea, edema, pneumonia, febrile neutropenia, neutropenia, thrombocytopenia, anemia, and hypocalcemia, mostly moderate in intensity. Conclusion: The sequential combination of decitabine and sapacitabine is safe and active in elderly AML.

Disclosures:
Off Label Use: Decitabine for the treatment of elderly AML. Wetzler:Cyclacel Limited: Research Funding. Chiao:Cyclacel Limited: Employment, Equity Ownership.
Oroxylin A Induce Differentiation and G0/G1 Phase Arrest of Acute Myeloid Leukemia (AML)Cells Via Effects on PPARγ/RXRα Heterodimers,
Authors: Hui Hui , Chen Bao-An , Guo Qing-long 
Published: October 2019

Abstract
Abstract 3635

Objective:
It has been shown that oroxylin A, a monoflavonoid extracted from the root of the Chinese herb medicine Scutellaria baicalensis, could effectively inhibit the proliferation of several cancer cell lines. However, little is known about the effect of oroxylin A on leukemic cells. In the current study the effects of oroxylin A were analized on growth, differentiation and cell cycle of myeloid leukemic cell lines.

Methods:
This study used AML cell lines including NB4, U937 and HL60. Expression and distribution of the proteins were analyzed by flow cytometry, western blots and Immunofluoresence assay. The growth inhibition effect of the drug was evaluated by MTT and cell cycle assay. The differentiation of cells was estimated by Giemsa stain and NBT reduction assay.

Results:
The growth of U937, HL60 and NB4 cells incubated with oroxylin A was inhibited in a time- and concentration-dependent manner but no apoptosis effect could be observed. Moreover, oroxylin A induced monocytic differentiation and G0/G1 phase arrest in U937 and HL60 cells, meanwhile, the expresstion of PPARγ, a nuclear receptor correlated with monocytic differentiation, was enhanced specificly and a nuclear accumulation of PPARγ was induced. Effects of differentiation and the upregulation of p21 could be attenuated by GW9662, a specific inhibitor of PPARγ. Oroxylin A dramatically displayed synergistic effect with ATRA or VD3 on growth and differentiation of NB4 or U937 cells respectively. We investigated the phosphorylation of RXRα, a nuclear receptor which assists RARα, PPARγ and VDR to exert differentiation effects through heterodimerize with them, was inhibited especially in nuclear of NB4, U937 and HL60 cells treated with oroxylin A. Activition of MAPK/ERK pathway, which is responsible for phosphorylation of RXRα, was also inhibited by oroxylin A.

Conclusion:
The results showed here demonstrated that oroxylin A could induce growth inhibition, G0/G1 phase arrest and differentiation of U937 and HL60 and show synergistic effects in combination with ATRA in NB4 and with VD3 in U937 cells via potential inhibition effect on phosphorylation of RXRα.

Disclosures:
No relevant conflicts of interest to declare.
Involvement of the Syk-mTOR Pathway in Follicular Lymphoma Cell Invasion and Angiogenesis,
Authors: Bezombes Christine , Fruchon Séverine , Kheirallah Samar , AlSaati Talal , Ysebaert Loic , Laurent Camille , Fournié Jean-Jacques , Laurent Guy 
Published: October 2019

Abstract
Abstract 3656
Follicular lymphoma is the second most common non-Hodgkin's lymphoma. The disease affects the lymph nodes and 50% of patients present with bone marrow infiltration, however the mechanisms involved in dissemination of the disease are not yet known. We previously reported that follicular lymphoma cells display an over-expression of Syk, a tyrosine kinase involved in many cellular processes including cell migration. Therefore we sought to explore its role in the invasive process. Here we show that follicular lymphoma patients display higher MMP-9 and VEGF levels than healthy donors. Moreover, using Syk siRNA and the Syk inhibitor R406, we demonstrate that, in follicular lymphoma cells, Syk is involved in the regulation of MMP-9 and VEGF expression, and that invasion and angiogenesis is mediated through a PI3K-mTOR module. Finally, using a follicular lymphoma xenograft mouse model we observe that R406, the active metabolite of fostamatinib (R788), inhibits MMP-9 expression and angiogenesis in vivo. Altogether, this study provides strong evidence that Syk represents an encouraging therapeutic target in follicular lymphoma and suggests the potential use of fostamatinib as an anti-invasive and anti-angiogenic drug.

Disclosures:
No relevant conflicts of interest to declare.
Incremental Value in Outcome Prediction with Molecular Signatures in Diffuse Large B-Cell Lymphoma,
Authors: Hong Fangxin , Kahl Brad S. , Gray Robert 
Published: October 2019

Abstract
Abstract 3687

INTRODUCTION:
Multiple gene expression-based biomarkers have been identified in diffuse large B-cell lymphoma (DLBCL) that are predictive for survival outcomes. Most studies assess predictive significance based on p-value from multivariate Cox regression; few investigations have evaluated the incremental usefulness of these biomarkers in risk prediction. Using the recently developed concordance measures (e.g., C-statistics) on censored survival data, we assessed the usefulness of two published gene-based risk signatures and compared them to the known clinical prognostic factors; with an overall goal of investigating the added value.

METHOD:
The added value of biomarkers was assessed by C-statistic and the Integrated Discrimination Improvement (IDI). The overall C-statistic is an estimated concordance between prediction and observation (event vs. non-event) - the probability that predicted risk score is higher for subject with earlier time of event. The IDI measures overall improvement in sensitivity and specificity. The signatures we selected are a six-gene predictor by Lossos et al. (2004) and a three-component signature (∼400 genes) by Lenz et al. (2008). We used the Lenz dataset which include 233 patients with DLBCL who received R-CHOP therapy (median follow-up=2.81 yr), and focused on predicting 3-year survival outcome (42% censored). Clinical prognostic factors evaluated are the traditional IPI components (stage, performance status, age, LDH, and number of extra nodal sites).

RESULTS:
The C-statistic was 0.60 and 0.721 for six-gene predictor and three-component signature, suggesting good discrimination ability by molecular signature when used alone. However, the performance is inferior to IPI risk factors, with a C-statistic of 0.733. When integrating gene signatures with IPI risk factors, the C-statistic was increased to 0.744 and 0.762, an improvement of only 0.011 (95% CI, -0.049 to 0.071) and 0.029 (95% CI, -0.033 to 0.091) for six-gene predictor and three-component signature, respectively. Furthermore, assessment by IDI reveals an added value of only 0.011 (95% CI, -0.008 to 0.081) and 0.076 (95% CI, 0.013 to 0.16) for the two molecular signatures. Kaplan-Meier survival curves for the four quartile groups based on the predictor scores confirms the marginal benefit in risk prediction using molecular signatures. (Figure 1).

CONCLUSIONS:
These results indicate that molecular biomarkers are inferior to clinical factors for risk assessment in DLBCL and provide little added value in risk prediction. These calculations suggest we will need to consider more than gene expression to develop highly discriminatory risk prediction models. However, the study of gene expression and clinical outcomes retains considerable potential to enhance understanding of disease mechanisms and uncover new therapeutic targets.

Disclosures:
No relevant conflicts of interest to declare.
Development of a Bruton's Tyrosine Kinase (Btk) Inhibitor, ONO-WG-307: Efficacy in ABC-DLBCL Xenograft Model – Potential Treatment for B-Cell Malignancies,
Authors: Kozaki Ryohei , Yoshizawa Toshio , Tohda Shuji , Yasuhiro Tomoko , Hotta Shingo , Ariza Yuko , Ueda Yoshiko , Narita Masami , Kawabata Kazuhito 
Published: October 2019

Abstract
Abstract 3731

Purpose:
ONO-WG-307 is a small molecule inhibitor that covalently binds to Btk. Signals from B cell receptors (BCR) play a central role in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. BCR signaling is implicated in the survival of malignant B cells and recent studies indicate that targeting Btk, an essential component of the BCR pathway, may be effective in the treatment of B-cell lymphoma. The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis and new therapies, preferably chemo-sparing therapies, or as add-on to existing treatment regimens are required to help treat patients with ABC-DLBCL. Therefore, Btk constitutes an interesting therapeutic target, thus the activity of ONO-WG-307 was evaluated in an ABC-DLBCL xenograft model.

Methods:
Tumor cells (TMD-8) were implanted subcutaneously into female SCID mice. Tumors were allowed to grow to a volume of 100–200 mm3 before the mice were randomized into groups based on tumor size. ONO-WG-307 was administered orally at doses up to 10 mg/kg bid. Tumors were measured two or three times weekly after initiation of treatment, and tumor volumes were determined using the formula volume (=width2xlength)/2. Animals were euthanized when the tumors reached a maximum volume of 2,000 mm3 or after a maximum period of 2 months. In parallel, an exploratory pharmacodynamic marker of Btk inhibition (Phosphorylated-Btk [P-Btk]) was also investigated in vivo.

Results:
Treatment with ONO-WG-307 resulted in a dose-dependent inhibition of tumor growth in a TMD-8 xenograft model. Furthermore, parallel analysis of a pharmacodynamic marker, P-Btk, supported that Btk was inhibited and the level of P-Btk inhibition was correlated with the decreased tumor volumes observed in the TMD-8 model.

Conclusion:
ONO-WG-307 is a highly potent and selective oral Btk inhibitor with evidence of efficacy in the ABC-DLBCL xenograft model, with Btk inhibition further supported using a PD marker. Given the need to treat and overcome disease resistance especially in ABC-DLBCL, the use of a Btk inhibitor is a novel, mechanistic approach to treating B cell malignancies. Additional work is underway, combining ONO-WG-307 with chemotherapy and other targeted agents.

Disclosures:
No relevant conflicts of interest to declare.
Inhibition of AKT Signaling Potently Inhibits the Growth of JAK and MPL-Mutant Cells in the Myeloproliferative Neoplasms In Vitro and In Vivo,
Authors: Khan Irum , Huang Zan , Wen Qiang Jeremy , Koppikar Priya , Levine Ross L , Tefferi Ayalew , Crispino John 
Published: October 2019

Abstract
Abstract 3862
Somatic mutations in JAK2 and MPL are associated with the BCR-ABL negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). While oral JAK2 inhibitors improve peripheral blood counts and splenomegaly, these drugs show varying effects on JAK2 mutant allele burden and do not eliminate the malignant clone in humans or in animal models of MPN. Although much of the research to date has focused on JAK/STAT signaling, other pathways downstream of the class I cytokine receptors, including PI3K/AKT and ERK are also activated in MPNs. Our hypothesis is that persistent activation of these signaling pathways contributes to the progression of myeloproliferative neoplasms (MPNs).
Multiple studies have shown that overexpression of activated JAK2 and MPL mutants (e.g. JAK2 V617F or MPL W515L) in primary murine bone marrow cells causes robust AKT, STAT3, and STAT5, JNK, ERK phosphorylation. To determine the extent to which these signaling pathways are involved in the disease, we cultured JAK2 V617F and MPL W515L expressing clones of the G1ME and 6133 megakaryocytic cell lines with a panel of small molecule kinase inhibitors. We discovered that inhibition of the PI3K/AKT and MAPK/JNK signaling pathways with triciribine and SP600125 respectively, potently suppressed growth of these cells by inducing G1 arrest and apoptosis. In contrast, inhibitors of Ras, ERK, Src family tyrosine kinase, and PKC failed to significantly inhibit proliferation of the JAK2 or MPL mutant expressing cells. Murine bone marrow cells transduced with MPL W515L show a dramatic expansion of megakaryocytes (CFU-MKs) and this expansion was abrogated in the presence of PI3K/AKT inhibitors suggesting a requirement for this pathway in aberrant megakaryocyte expansion.
Since AKT inhibition showed the strongest effect, we assayed the activity of MK-2206, a potent and selective allosteric AKT inhibitor, on multiple models of MPNs. We discovered that MK-2206 induced proliferative arrest and apoptosis accompanied by suppression of PI3K/AKT signaling in G1ME and 6133 cells expressing MPLW515L. MK-2206 also potently inhibited liquid culture growth and colony formation of PMF patient CD34+ cells in vitro. Finally, in preliminary murine transplants experiments with MPLW515L expressing bone marrow progenitors, treatment with MK-2206 led to significant reductions in peripheral blood leukocytosis and extramedullary hematopoiesis. Together, these findings demonstrate that the PI3K/AKT axis represents a rational target for therapy in human myeloproliferative neoplasms.

Disclosures:
No relevant conflicts of interest to declare.