Ara-C Treatment Research Articles

Autophagy Is an Important Event for Low Dose Cytarabine Treatment in Acute Myeloid Leukemia U937 Cell Line

ObjectiveTo investigate the mechanisms underlying the low dose cytarabine induced autophagy and differentiation in human acute myeloid leukemia (AML) cell lines in vitro.IntroductionCytarabine (Ara-C) is a fundamental drug used in the treatment of AML for years, but its specific mechanism of action is still not well documented. Autophagy, characterized by the formation of double-membrane vesicles called phagosomes, is a general process for degradation of cellular proteins and cytoplasmic organelles in many physiological and pathophysiological conditions. Recent studies have established the role of autophagy in cell differentiation and chemotherapy effects. Here we sought to determine the importance of autophagy in the AML treatment with especially low dose cytarabine (LD-Ara-C).MethodsCell viability after Ara-C treatment was evaluated using the Cell and Particle Counter. The expression of LC3, p62, Beclin 1 and Bcl-2 was detected by Western blot analysis, so as the Akt-mTOR pathway. Ultrastructural features in cells with or without Ara-C treatment were observed through transmission electron microscope (TEM).Cell morphology was determined by Wright-Giemsa staining. Flow cytometry was performed to examine cell differentiation and apoptosis in the presence or absence of 3-MA, a PI3K inhibitor, or CQ, a lysosomal inhibitor. A beclin1-specific short hairpin RNA (shRNA) was transfected into cells using lentivirus to obtain beclin1 silencing.ResultsThe AML cell lines U937 and HEL were treated with increasing concentrations of Ara-C range from 10nM to 500nM for 24/48h and then analyzed for cell viability, which showed the growth inhibition rates in a time and dose-dependent manner. The cytotoxic effects of Ara-C on U937 cells were also studied by flow cytometry analysis of Annexin V/PI strained samples. Cell apoptosis and death were not detected when exposed to low concentration (10-50nM) while about 50% proliferation inhibition rate exists. To test whether autophagy was involved in the action of LD-Ara-C on leukemia cells, we detected microtubule-associated protein light chain 3 (LC3) conversion and sequestosome1 (p62) decrease by immunobloting. Treatment with LD-Ara-C (50nM) induced a time-dependent increase in the expression of LC3-Ⅱ and beclin1, but degradation of p62 in both U937 and HEL cells. Besides, vacuoles surrounded by a double membrane and containing cytoplasmic contents, a characteristic of autophagosomes appeared after 24h treatment, further suggesting an autophagic response caused by LD-Ara-C. Meanwhile, deregulation of Akt-mTOR pathway was also detected. LD-Ara-C induced increase of CD11b expression and morphologic changes of partial differentiation within 4 days in U937 revealed differentiation as another cellular effect of Ara-C in AML cells. When cultured in the presence of autophagy inhibitor, 3-MA or CQ, LD-Ara-C induced cell autophagy and differentiation were reversed, and cell growth inhibition was also attenuated. The similar phenomenon could also be seen when we down-regulated the expression of beclin1, an essential protein for the mammalian autophagy cascade, by shRNA transfection in U937 cells. Taken together, LD-Ara-C-induced autophagy appeared to play an important role in the following cell differentiation and death in AML cells.ConclusionOur data demonstrate that LD-Ara-C plays the therapeutic effect in human acute myeloid leukemia cells by triggering both autophagy and differentiation. Downreguation of Akt-mTOR pathway is involved in these processes. We suggest that cytarabine-induced autophagy is not a pro-survival mechanism, but accounts for its antineoplastic effects, which provides the first evidence for the potential of autophagy as a novel approach for AML treatment with LD-Ara-C, however the underlying mechanisms remain to be well elucidated. DisclosuresNo relevant conflicts of interest to declare.

Activated Fibroblasts Protect Acute Myeloid Leukemia Cells from Chemotherapy

Introduction: It has become clear that cancer is not only just a disease of the genes, but the tumor microenvironment (TME) plays an important role in cancer progression (Borriello L & DeClerck YA, 2014). In leukemia, the bone marrow plays a special role in environment-mediated drug resistance as it is not only a sanctuary protecting tumor cells from cytotoxic drugs, but also a source of many stromal cells that colonize primary tumors and contribute to the pre-metastatic niche (Borriello L & DeClerck YA, 2014; Meads MB, et al, 2008). As we all know, there exists two distinct BM niches in this significant microenvironment: ‘osteoblastic (endosteal)’ and ‘vascular’ niches (Tabe Y & Konopleva M, 2014; Konopleva M, et al, 2009). Besides these two, scholars transferred to focus on the stromal in bone marrow，which may have importance effects on leukemic cells (Wu S, et al, 2005; Campana D, et al, 2005). Shriram V. Nath et al. found that there was a negative correlation between reticulin fiber density (RFD) at diagnosis in childhood ALL and white blood cell count in peripheral blood. Similarly, there was a negative correlation between RFD and the percentage of blast cells in blood. They speculated fiber in the bone marrow leading to poor prognosis in acute lymphoblastic leukemia (ALL) via anchoring leukemia cells in bone marrow stromal (Nath SV, et al. 2011). But the role of fibers and its mainly producing cells fibroblasts has not been previously studied in acute myeloid leukemia (AML). Therefore, we retrospectively investigate the bone marrow biopsies of primary AML patients, to analyze the relationship between the RFD and prognosis in adult AML, and to identify and quantitative analyze protective effects of the fibroblasts on leukemia cells from chemotherapy in vitro.Methods: Makers of activated fibroblasts were stained by immunohistochemistry on bone marrow biopsies; The RFD were evaluated based on the grid point method of computer; Obtained activated fibroblasts by induced BM-MSC of primary AML with human recombinant TGFβ1.(4) Analyzed protective effects of activated fibroblasts by co-cultured with leukemia cells such as THP-1/K562, futher explored the mechanism via SB431542, a specific inhibitor of TGFβ signaling pathway.Result:RFD in primary AML was significant higher than control. Patients with higher RFD indicated poorer prognosis; However, collagenI coated plates showed no effects on survival rate of the leukemic cells. Here, we demonstrated that the general makers of activated fibroblasts: FSP1, α-SMA and FAP in primary AML were significant higher than control, implied that there existed amount of activated fibroblasts in patients; Activated fibroblast protect both THP-1 and K562 from apoptosis with treatment of Ara-c, and arrested them in phase of G0/G1. Moreover, this protect effects of activated fibroblast will be cancelled with SB431542.Conclusion:Activated fibroblasts and fibrous tissue were both proliferated abnormally in primary AML, and the later was significantly associated with relapse-free survival and overall survival of patients, indicating that RFD could be used as an important factor of poor prognosis and should guide clinical intervention. In vitro induced activated fibroblasts could be able to protect leukemia cells from chemotherapy. The possible mechanisms might be that the soluble cytokines TGFβ1, but not collagen I, which were secreted by activated fibroblasts, contributed to leukemia protection. In addition to this, activated fibroblasts could also change cell cycle of tumor cells, make them more arrested in G0/G1 phase, thus decreased the their chemotherapy sensitivity. DisclosuresNo relevant conflicts of interest to declare.

Ablation of Autophagy-Related E1 Ligase Atg7 Primes Acute Myelogenous Leukemia Cells to Apoptosis, Sensitizes to Chemotherapy and Overcomes Stroma Mediated Protection

Background: Except for the 'good risk’ groups identified by current cytogenetic and molecular criteria, patients with acute myelogenous leukemia (AML) largely relapse after attaining initial remissions, and outcomes of patients with early relapse is uniformly poor, highlighting the need for strategies to overcome resistance to agents used in frontline therapy. Autophagy is an ancestral adaptive mechanism to stress and can be triggered by exposure to chemotherapy. Autophagy is also believed to play an important role in tumor protection by the microenvironment. Autophagy inhibition increases chemosensitivity in several experimental models of solid tumors. The role of autophagy is not well studied in AML, but the hypoxic bone marrow niche is expected to induce autophagy in AML cells and render them chemo-resistant. Our reverse phase protein array (RPPA) analysis of over 500 newly diagnosed AML patient samples indicated that abnormal expression of several autophagy proteins in leukemic blasts (LKB1, BECLIN 1 and ATG7) is associated with poor prognosis in AML (Borthakur, G et al. ASH 2011# 2513), suggesting a therapeutic role for autophagy suppression. Atg7 is a key molecule in autophagy vesicle elongation with a role in two essential ubiquitin-like reactions (LC3 lipidation and Atg 5/12 conjugation), and its knockdown is expected to inhibit autophagy globally.Aim: To determine the effects of autophagy inhibition on chemosensitivity of AML cells and on stroma induced resistance.Results: We semi-quantitatively assessed ATG7 expression in AML cell lines (THP1, OCI-AML2 and 3, HL-60, MOLT-4, MOLM 13 and 16) by Western blot. HL-60 and MOLM13 cells had the lowest expression of Atg7 and were most sensitive to treatment with ara-C and idarubicin while OCI-AML3 and THP-1 had highest levels of Atg7 and were most resistant to treatment (p= .001-.004). In addition, high expression of Atg7 is associated with high levels of anti-apoptotic Mcl-1 in these lines.As available autophagy inhibitors are non-specific, we used lentiviral shRNA to knock down Atg7 and study the role of autophagy. Atg7 knockdown cells (ATG7-KD) were treated with ara-C and idarubicin for 48-96 hours. At all time points, apoptosis was significantly higher in ATG7-KD cells compared to cells transduced with a non-silencing scrambled control (ATG7-Scr) at 96 hrs: 39.6±2.4 vs 22.4±1.3, p=.002 for ara-C 2 µM and 47.9±2.6 vs 34.7±2.3, p=.0004 for idarubicin. We co-cultured OCI-AML3 (ATG7-KD and -Scr) cells with normal bone marrow derived mesenchymal stromal cells (MSCs) to mimic the bone marrow micro-environment. Sensitivity to ara-C and idarubicin was reduced by co-culture for both cell types, but ATG7-KD cells remained more sensitive than were ATG7-Scr cells.Western blot analysis confirmed increased p62 and decreased LC3 lipidation in ATG7-KD cells (LC3 II/I ratio= 0.15 vs 0.7 in ATG7-Scr), indicating a block in autophagy, and increased caspase 3 cleavage, indicating apoptosis. The higher expression of pro-apoptotic NOXA and BAX, and lower expression of anti-apoptotic MCL-1 and BCL-2, in ATG7-KD cells compare to ATG7-Scr indicating mitochondrial pathway apoptosis. Gene expression profiling on Illumina HT12 arrays was used to study OCI-AML3 ATG7-KD and -Scr cells at baseline and after 24 and 48 hr of araC 0.5 µM. Gene set enrichment analysis (GSEA) showed significantly lower baseline expression of interferon response genes and JAK-STAT pathway genes in OCI-AML3 ATG7-KD cells. Changes with araC treatment were largely similar, but the response to araC was different between ATG7-KD and –Scr cells for a small number of genes. In-vivo chemo-sensitivity experiments are in progress.Conclusion: Autophagy inhibition by genetic silencing of ATG7 increases chemosensitivity of AML cells, even in the presence of bone marrow stromal cells. ATG7-KD cells appear to be more primed for apoptosis compared to their controls. Concomitant inhibition of ATG7, a potentially drugable E1 ligase, appears to be a valid strategy to enhance sensitivity to front-line treatment agents in AML. DisclosuresNo relevant conflicts of interest to declare.

Development of an Axl/Mer Dual Inhibitor, ONO-9330547: Promising Single Agent Activity in an Acute Myeloid Leukemia (AML) Model

Purpose: Axl and Mer belong to the TAM (Tyro3, Axl, Mer) family of receptor tyrosine kinases and play a role in regulating cell proliferation, survival, migration and cytokine production. Axl/Mer are over-expressed in various types of haematological and solid tumour cancers and appear to play a key role in epithelial-to-mesenchymal transitions (EMTs), known to be important in tumour metastases and drug resistance. AML is a clonal disease of haematopoietic progenitors characterised by acquired heterogeneous genetic changes that alter cell proliferation and differentiation and Axl is an independent prognostic marker and therapeutic target in AML. Recent studies revealed that AML cells induce expression and secretion of the Axl ligand growth arrest specific gene 6 (Gas6) by bone marrow derived stromal cells (BMDSCs). Gas6 mediates proliferation, survival and chemoresistance of Axl-expressing AML cells, giving rise to a chemoprotective tumour niche environment. Standard chemo-treatment for AML affects not only AML cells but healthy cells which cause severe side effects. In addition, only 40-45% of patients <65 years reach long-term survival with current treatment options and only 10% of patients >65 can be cured. Therefore, there is still a high, unmet medical need for new therapies. ONO-9330547 is a small molecule inhibitor that binds to Axl and Mer kinase with a potency (IC50) of 2.2 and 0.4 nM respectively, therefore, the anti-tumour activity of ONO-9330547 in AML cells was evaluated.Methods: AML cell lines were implanted subcutaneously into female SCID mice. Randomization of mice occurred when mean tumour volume was 100-200 mm3. ONO-9330547 was administered orally at doses up to 3 mg/kg twice daily (BD) for 24 days. Tumour volumes were measured twice a week after initiation of treatment, and tumor volumes were determined using the formula volume (=width2xlength)/2. Animals were euthanized when the tumours reached a maximum volume of 2,000 mm3. Phosphorylation of Axl (P-Axl) and Mer (P-Mer) were detected by Western blotting or Flow cytometry.Results: In AML cell lines assays, ONO-9330547 strongly inhibited both P-Axl and P-Mer with an IC50in the nM range, which resulted in growth inhibitions and inductions of apoptosis. In the MV-4-11 xenograft model, tumour growth inhibition at the final treatment day was 40% in the 0.3 mg/kg BD group and 78% in the 1 mg/kg BD and 100% in the 3 mg/kg BD groups respectively (All treatment groups: P<0.001 vs Vehicle). Specifically, the treatment with ONO-9330547 1 or 3 mg/kg BD resulted in higher and sustained P-Axl inhibition throughout the study, and treatment of 3 mg kg BD resulted in complete remission in all treated mice. Interestingly, up-regulation of Axl and Mer were observed in AML cell lines after the treatment of doxorubicin or Ara-C. The combination of ONO-9330547 with Ara-C resulted in significant anti-tumour effect compared with respective monotherapy.Conclusion: ONO-9330547 is a highly potent dual Axl/Mer inhibitor with evidence of efficacy in AML cells. Moreover, up-regulation of Axl/Mer after chemotherapy indicates the potential combination of ONO-9330547 with standard therapy to overcome treatment resistance in AML. Additional work to investigate and clarify the involvement of Axl/Mer in other types of leukemia are currently underway. DisclosuresYasuhiro:Ono Pharmaceutical Co., Ltd.: Employment. Yoshizawa:Ono Pharmaceutical Co., Ltd.: Employment. Fujikawa:ONO Pharmaceutical Co., Ltd.: Employment. Tanaka:ONO Pharmaceutical Co., Ltd.: Employment. Koike:ONO Pharmaceutical Co., Ltd.: Employment. Kawabata:Ono Pharmaceutical Co., Ltd.: Employment.

Pre-pubertal exposure of cytarabine-induced testicular atrophy, impaired spermatogenesis and germ cell DNA damage in SD rats

Context: Cytarabine (Ara-C) is an effective chemotherapeutic drug for the treatment of acute leukaemias. It inhibits the DNA synthesis and repair, thereby causes cytotoxicity in the proliferating cells.Objective: This study was aimed to investigate the effects of pre-pubertal exposure of Ara-C on testesticular development in juvenile SD rats and their function at puberty.Materials and methods: Ara-C was injected at the doses of 50, 100 and 200 mg/kg/day from postnatal day (PND) 29–42 (14 days) by intraperitoneal (i.p.) route. Half of the animals were sacrificed on PND 43 and remaining on PND 70. End points of the evaluation included gross pathological examination, histomorphometric analysis, sperm count and sperm head morphology, cell proliferation and DNA damage as well as apoptosis analysis.Results: Ara-C treatment significantly decreased food and water intake, weight gain, testes and epididymis weight and increased histological alterations in the seminiferous tubule. Furthermore, Ara-C treatment significantly decreased the PCNA-positive cells and sperm count in a dose-dependent manner. Ara-C treatment also increased the DNA damage and apoptosis in testes and sperm as evident from the comet and TUNEL assays results.Discussion: The present study results clearly indicated that Ara-C treatment impaired spermatogenesis and adversely affects the testicular development and its function in rats by reducing the germ cell proliferation and the inducing DNA damage and apoptosis.

Clinical Efficacy of Mitoxantrone and Ara-C with or without Etoposide Salvage Chemotherapy in Adult Patients with Relapsed or Refractory Acute Lymphoblastic Leukemia: Retrospective Multicenter Study of the Korean Adult ALL Working Party

Mitoxantrone is a conventional agent for relapsed or refractory acute lymphoblastic leukemia (ALL). However, an effective combination with other drugs and a feasible dosage has not been identified. A retrospective study of 46 patients with relapsed or refractory ALL was conducted to determine the efficacy of mitoxantrone and Ara-C treatment with (MEC) and or without etoposide (MC). Twenty-seven and 19 patients received MC and MEC chemotherapy, respectively. Twenty-two (48%) patients showed overall response [complete response (CR), 33%; CR with incomplete platelet recovery (CRp), 15%], and 10 of 22 responders received allogeneic stem cell transplantation (SCT). Median overall survival (OS) was 6.2 months (95% confidence interval, 3.41-9.0). Thirteen (48%) patients in the MC group and 9 (47%) in the MEC group achieved CR/CRp (p = 0.96). Treatment-related mortalities in the MC and MEC groups were 3 (11%) and 4 (21%), respectively (p = 0.36). However, the MEC group frequently presented with grade 3 or higher bacteremia/candidemia (p = 0.013). No difference in OS was observed between the two groups (p = 0.769). In conclusion, salvage therapy consisting of mitoxantrone and Ara-C without etoposide appeared to be an effective bridge therapy to allogeneic SCT for patients with refractory or relapsed ALL.

An intragenic long noncoding RNA interacts epigenetically with the RUNX1 promoter and enhancer chromatin DNA in hematopoietic malignancies.

RUNX1, a master regulator of hematopoiesis, is the most commonly perturbed target of chromosomal abnormalities in hematopoietic malignancies. The t(8;21) translocation is found in 30-40% of cases of acute myeloid leukemia (AML). Recent whole-exome sequencing also reveals mutations and deletions of RUNX1 in some solid tumors. We describe a RUNX1-intragenic long noncoding RNA RUNXOR that is transcribed as unspliced transcript from an upstream overlapping promoter. RUNXOR was upregulated in AML samples and in response to Ara-C treatment in vitro. RUNXOR utilizes its 3'-terminal fragment to directly interact with the RUNX1 promoter and enhancers and participates in the orchestration of an intrachromosomal loop. The 3' region of RUNXOR also participates in long-range interchromosomal interactions with chromatin regions that are involved in multiple RUNX1 translocations. These data suggest that RUNXOR noncoding RNA may function as a previously unidentified candidate component that is involved in chromosomal translocation in hematopoietic malignancies.

Chromosome Breakage Induced by the Genotoxic Agents Mitomycin C and Cytosine arabinoside is Concentration and p53 Dependent

The p53 tumor suppressor protein plays an essential role in cellular integrity and inactivation of the TP53 gene by mutation is the most frequent alteration in human cancer. As loss of p53 function is associated with increased genetic instability, it is important in genotoxicity testing to explore the role of p53 competency. In vitro model systems for genotoxicity testing are sometimes prone to misleading positive results; some of this loss of predictivity may be caused by p53 inactivation in some cell models. To explore whether impaired p53 function plays a role in mutation sensitivity, TK6 cells (p53 competent) and NH32 cells (p53 deficient) were treated with two known genotoxicants, mitomycin C (MMC) and cytosine arabinoside (araC). Chromosomal damage was assessed in the low dose region by an automated micronucleus system and p53 activity was investigated by gene and protein expression analysis. Cell cycle progression studies were also assessed. Low levels of micronucleus and p53 induction were observed in TK6 cells treated with MMC. On the other hand, higher levels of micronucleus and p53 induction were shown in TK6 cells treated with araC and a G1/S arrest was observed after araC treatment. p53 deficient NH32 cells showed an increased sensitivity of micronucleus(MN) induction after araC treatment compared with TK6 cells and less of an active G1/S phase checkpoint. Thus, impaired p53 function sensitizes cells to genotoxicants and plays a central role in the DNA damage response. This data has clear importance for safety assessment of genotoxicity and shows how crucial p53 competence is.

Comparative study of autophagy inhibition by 3MA and CQ on Cytarabine-induced death of leukaemia cells

As the molecular mechanisms of Cytarabine,one of the most important drugs used in the leukaemia’s treatment, are only partially understood and the role of autophagy on leukaemia development and treatment is only recently being investigated, in this study, by using Chloroquine (CQ) and 3-methyladenine (3MA) as autophagy inhibitors, we aim to evaluate the contribution of an autophagic mechanism to Cytarabine (AraC)-induced death of HL60 leukaemia cells. Trypan blue exclusion and AnnexinV/PI assays were used to evaluate HL60 cell death under AraC treatment in the presence or absence of 3MA and CQ. Western blotting and immunofluorescence experiments were performed to show the involvement of apoptosis and autophagy protein expressions. Phenotypic characterization of HL60-treated cells was performed by using immunophenotyping. Clonogenic assays were applied to analyse clonal function of HL60-treated cells. We observed that although autophagy inhibition by 3MA, but not CQ, increased the death of HL60 AraC cells after 24 h of treatment, no significant differences between AraC and AraC + 3MA-treated groups were observed by using clonogenic assay. In addition, increased number of immature (CD34(+)/CD38(−)Lin(−/low)) HL60 cells was found in AraC and AraC-3MA groups when compared with control untreated cells. Although AraC anti-leukaemia effects could be potentiated by 3MA autophagy inhibition after 24 h of exposure, leukaemia cell resistance, the main causes of treatment failure, is also promoted by autophagy initial stage impairment by 3MA, denoting the complex role of autophagy in leukaemia cells’ response to chemotherapy.

Progenitor Cell Dynamics in the Newt Telencephalon during Homeostasis and Neuronal Regeneration

SummaryThe adult newt brain has a marked neurogenic potential and is highly regenerative. Ventricular, radial glia-like ependymoglia cells give rise to neurons both during normal homeostasis and after injury, but subpopulations among ependymoglia cells have not been defined. We show here that a substantial portion of GFAP+ ependymoglia cells in the proliferative hot spots of the telencephalon has transit-amplifying characteristics. In contrast, proliferating ependymoglia cells, which are scattered along the ventricular wall, have stem cell features in terms of label retention and insensitivity to AraC treatment. Ablation of neurons remodels the proliferation dynamics and leads to de novo formation of regions displaying features of neurogenic niches, such as the appearance of cells with transit-amplifying features and proliferating neuroblasts. The results have implication both for our understanding of the evolutionary diversification of radial glia cells as well as the processes regulating neurogenesis and regeneration in the adult vertebrate brain.

Outcome and prognostic factors in patients with mantle-cell lymphoma relapsing after autologous stem-cell transplantation: a retrospective study of the European Group for Blood and Marrow Transplantation (EBMT)

Autologous stem-cell transplantation (autoSCT) is considered a standard treatment of non-frail patients with mantle cell lymphoma (MCL), but little is known about outcome of MCL patients relapsing after autoSCT. We therefore sought to analyse the outcome after autoSCT failure and the efficacy of a rescue stem-cell transplantation (SCT) in this setting. Patients with MCL were eligible if they had relapsed after autoSCT performed between 2000 and 2009. A total of 1054 patients could be identified in the EBMT registry. By contacting the transplant centres, a full dataset could be retrieved for 360 patients. Median overall survival (OS) after relapse of the whole study group was 19 months. A long (>12 months) interval between autoSCT and relapse [P < 0.001, hazard ratio (HR) 0.62], primary refractory disease (P < 0.02, HR 1.92), prior high-dose ARA-C treatment (P = 0.04, HR 1.43), and the year of relapse (P = 0.02, HR 0.92) significantly influenced OS from relapse in multivariate analysis. Eighty patients (22%) received a rescue allogeneic SCT (alloSCT). Relapse incidence, non-relapse mortality, and OS 2 years after alloSCT was 33% [confidence interval (95% CI 21% to 45%)], 30% (95% CI 19% to 42%), and 46% (95% CI 33% to 59%), respectively. Remission duration after autoSCT was the only variable significantly affecting the outcome of salvage alloSCT. In contrast, rescue autoSCT was not associated with long-term disease control. However, individual patients survived long term even without salvage transplantation. MCL recurrence within 1 year after autoSCT has an extremely dismal outcome, while the prognosis of patients with longer remission durations after autoSCT is significantly better. AlloSCT may offer the possibility of durable survival when performed for patients with a remission duration of more than 12 months after first autoSCT, but the favourable effect of a salvage alloSCT in this setting needs further validation.

Progress of the study on cytosine arabinoside in the treatment of acute leukemia in children

Cytosine arabinoside (Ara-C) is one of the key chemotherapy drugs comprising all stages combined protocols during long-term chemotherapy for children with acute leukemias, which demonstrated its efficacy and safety. Along with the progress of basic and clinical researches, in Ara-C clinical dose and treatment, prevention of adverse reactions, relationship between the drug metabolism and long-term clinical results, and new nucleoside drugs have achieved remarkable results. This paper on the above fields of research, progress and trends, briefly summarized as follows. Key words: Cytosine arabinoside; Leukemia; Metabolic key enzyme; Long-term efficacy; Adverse reaction

Effect of cytosine arabinoside on cerebellar neurofilaments during development: A sexual dimorphism

Previous reports suggest that the resistance of neuronal cytoskeleton to drug toxicity may vary with age and gender. The aim of the present study was to assess the impact of cytosine arabinoside (AraC) treatment on neurofilament (NF) levels and phosphorylation status in the developing cerebellum of male, female and testosterone propionate (1.25 mg/rat)-androgenized female rats. AraC (200 mg/kg bw) was administered from postnatal day (PND) 14–16 and changes in the level and phosphorylation of NFs were detected at PND 16 by Western blot analysis. The drug had no effect in male pups, while it increased the non-phosphorylated NF subunits of medium and low molecular weight in females. Androgenization of females prevented the AraC-induced increase in NF subunits. The levels of estrogen receptor beta (ER-β), known to mediate neuroprotective actions of estrogens in the brain, were significantly higher in the developing female cerebellum, as compared to males and androgenized females.These data show that the neurofilament cytoskeleton in the developing rat cerebellum exhibits resistance to AraC that appears sexually dimorphic. In young males the resistance is exemplified by a lack of responsiveness, whereas in juvenile females it is presented by an androgenization-sensitive NF upregulation.

HERG Potassium Channel Blockage by Scorpion Toxin BmKKx2 Enhances Erythroid Differentiation of Human Leukemia Cells K562

BackgroundThe hERG potassium channel can modulate the proliferation of the chronic myelogenous leukemic K562 cells, and its role in the erythroid differentiation of K562 cells still remains unclear.Principal FindingsThe hERG potassium channel blockage by a new 36-residue scorpion toxin BmKKx2, a potent hERG channel blocker with IC50 of 6.7±1.7 nM, enhanced the erythroid differentiation of K562 cells. The mean values of GPA (CD235a) fluorescence intensity in the group of K562 cells pretreated by the toxin for 24 h and followed by cytosine arabinoside (Ara-C) treatment for 72 h were about 2-fold stronger than those of K562 cells induced by Ara-C alone. Such unique role of hERG potassium channel was also supported by the evidence that the effect of the toxin BmKKx2 on cell differentiation was nullified in hERG-deficient cell lines. During the K562 cell differentiation, BmKKx2 could also suppress the expression of hERG channels at both mRNA and protein levels. Besides the function of differentiation enhancement, BmKKx2 was also found to promote the differentiation-dependent apoptosis during the differentiation process of K562 cells. In addition, the blockage of hERG potassium channel by toxin BmKKx2 was able to decrease the intracellular Ca2+ concentration during the K562 cell differentiation, providing an insight into the mechanism of hERG potassium channel regulating this cellular process. Conclusions/SignificanceOur results revealed scorpion toxin BmKKx2 could enhance the erythroid differentiation of leukemic K562 cells via inhibiting hERG potassium channel currents. These findings would not only accelerate the functional research of hERG channel in different leukemic cells, but also present the prospects of natural scorpion toxins as anti-leukemic drugs.

Cytosine Arabinoside Chemotherapy Does Not Enrich For Leukemic Stem Cells In Xenotransplantation Model Of Human Acute Myeloid Leukemia

Despite a high rate of complete remission after treatment with conventional genotoxic agents, the overall survival of patients with acute myeloid leukemia (AML) is poor due to frequent relapses caused by the chemoresistance of rare leukemic stem cells (LSCs, also called Scid-Leukemia Initiating Cells). This unfavorable situation leads to a strong need to characterize those cells in order to target them with new specific therapies. Using a robust immunodeficient mouse model (NOD/LtSz-scid IL2Rγchainnull or NSG), we have previously shown that these LSCs were rare and not restricted to the CD34+CD38- immature compartment. This phenotypical heterogeneity of LSCs suggests that pharmacological targeting of LSC will not work if solely based on their cell surface markers. A better understanding of the mechanisms underlying the in vivo chemoresistance is required for the development of innovative targeted therapies. Aracytine (Ara-C, a pyrimidine analog), the most clinically used chemotherapeutic agents for AML patients, inhibits DNA synthesis and, therefore, targets and kills cycling AML cells in S phase of the cell cycle. Based on this mechanism of action, we hypothesized that Ara-C treatment will spare and enrich quiescent LSCs in vivo.We analyzed the response to Ara-C and residual disease in NSG mice engrafted with primary AML cells from 13 patients in two clinical centers (University of Pennsylvania, Philadelphia, USA and Purpan Hospital, Toulouse, France). A sub-lethal treatment of 60 mg/kg Ara-C given daily for five days induced a 5- to 50- fold reduction of peripheral blood blasts and total tumor burden in spleen and bone marrow in all patients tested. For 5 patients, we observed relapse within 4 to 6 weeks post-chemotherapy. Surprisingly, residual viable cells after Ara-C treatment showed no significant enrichment in quiescent cells and CD34+CD38- cells for the majority of primary samples tested (12 and 10 out of 13 total tested, respectively). Of note, the largest fraction (70-90%) of leukemic cells is in G0/G1 phase (including 0.5-20% in G0) in untreated engrafted mice. Moreover, we observed no significant changes in cell cycle profile of residual leukemic cells during the time course of the disease progression for 3 out of 4 patients. Finally, we assessed the frequency of LSCs in Ara-C-treated and control mice using transplantation and limiting dilution analysis in secondary recipients. Interestingly, we observed that Ara-C treatment did not increase the frequency of SL-ICs in residual cells, suggesting that blasts and LSC were equally sensitive to Ara-C in vivo.Our results show that sub-lethal regimen of Ara-C does not lead to enrichment of LSCs and induces cell death of both leukemic bulk and stem/progenitor cells independently of their cell cycle status probably through another in vivo mechanism such as apoptosis, autophagy or necroptosis. This study also suggests that further characterization of chemoresistant leukemic cells beyond phenotype and cell cycle status must rely on more functional properties in order to better elucidate the molecular basis of resistance in AML. Disclosures:Perry:MERCK: Employment. Carroll:Leukemia and Lymphoma Society: Research Funding. Sarry:AFFICHEM SA: Membership on an entity's Board of Directors or advisory committees.

Epigenetic Deregulation In Relapsed Acute Myeloid Leukemia

Treatment failure in Acute Myeloid Leukemia (AML) is attributed in many cases to relapsed disease. Relapsed AML is a fundamental clinical challenge since most patients have poor clinical outcomes. The exact biological basis of AML relapse remains unclear. Genetic clonal evolution is widely believed to underlie the emergence of chemotherapy resistant clones. However, only limited, predominantly non-overlapping, somatic mutations and copy number aberrations were found to occur upon AML relapse. Furthermore, in a subset of cases, no relapse specific somatic mutations or copy number aberrations were identified. This suggests a role for other mechanisms in relapsed AML. We hypothesize that epigenetic plasticity and deregulation contributes to the pathogenesis of relapse in AML.To explore this notion, we performed a genome scale epigenetic and genetic analysis of thirty-nine paired diagnosis and relapsed AML human patient samples using exome capture, RNA-seq and ERRBS for DNA methylation sequencing. Exome capture was performed on each patient's germline DNA as well. Exome capture revealed only a limited number of known recurrent somatic mutations acquired upon disease relapse, in agreement with previous reports. In contrast, upon disease relapse we identified thousands of statistically significant changes in cytosine methylation patterns. Globally, the majority of patients (85%) displayed striking predominance of DNA hypermethylation (p= 1.00433e-05, binomial test for equality of proportions) upon disease relapse. Notably a smaller set of patients displayed the opposite epigenetic phenotype with prominent loss of cytosine methylation. While differential methylation in the hypermethylated group of patients localized predominantly to CpG islands, the majority of differential methylation in the hypomethylated group localized to regions lacking both CpG islands and shores. In spite of these two distinct overall cytosine methylation patterns, the majority of differentially methylated cytosines are located in intergenic regions in all cases, and a subset of promoters were hypermethylated in almost all patients at relapse. A pathway analysis indicated that the commonly hypermethylated gene promoters at relapse are involved in the Hedghog, Wnt and calcium signaling pathways (p<0.05, modified Fisher Exact test). Integration of these findings with mutational and transcriptional profiles is underway.In order to determine whether epigenetic events linked to AML relapse could be modeled experimentally we performed a pilot study of a human AML xenograft in immunocompromised mice. Engrafted mice were treated with Ara-C at a clinically relevant dose (60mg/Kg; n=2) or vehicle alone (n=3) for five consecutive days. Human AML cells were collected at various timepoints including 28 days after Ara-C treatment where the AML had frankly relapsed in mice. Cytosine methylation profiles obtained through ERRBS revealed predominantly hypermethylated cytosines when compared to the xenotransplanted diagnostic sample (72% hypermethylated versus 28% hypomethylated). Remarkably, there was a strong overlap with gene promoters that are also aberrantly methylated in relapsed AML patients (p<0.01, hypergeometric test), including members of the Wnt signaling pathway.We conclude that there are epigenetically distinct forms of relapsed AML. Nonetheless, there is convergent epigenetic regulation of specific gene pathways that may contribute to relapsed AML pathogenesis and xenotransplanted AML mice can serve as experimental models for further study. Finally, the genomic distribution of reprogrammed methylation suggests a role for epigenetic plasticity at distal regulatory elements. Whereas it remains unclear whether these changes represent clonal selection, their extensive and dynamic range suggest that exposure to chemotherapy may alter the fidelity of mechanisms that control cytosine methylation distribution thus permitting widespread and distant epigenetic reprogramming and contributing to disease relapse. Disclosures:No relevant conflicts of interest to declare.

Rituximab Maintenance Therapy After Autologous Stem Cell Transplantation Prolongs Progression Free Survival In Patients With Mantle Cell Lymphoma

BackgroundIt was recently shown that rituximab maintenance therapy prolongs progression free (PFS) and overall survival (OS) in patients with mantle cell lymphoma (MCL) after conventional chemotherapy treatment. However, it is not clear if rituximab maintenance can further improve outcome of MCL patients after autologous stem cell transplantation (autoSCT). MethodsAll patients with MCL who underwent an autoSCT at the University Hospital of Heidelberg from 2000 onwards were selected for this analysis. MCL patients who have received rituximab maintenance therapy after autoSCT in a prospective phase-2 trial were compared with regard to PFS and OS with MCL patients who were transplanted during the same time period but did not receive rituximab maintenance therapy by multivariate cox regression analysis correcting for known confounders. For the comparison of rituximab maintenance therapy with the control group, rituximab maintenance therapy was considered as a time dependent event in order to avoid that early progression before start of rituximab maintenance therapy may compromise results. ResultsA total of 72 patients met the inclusion criteria. Twenty-two patients participated in a phase-2 trial and received rituximab maintenance therapy for two years. Median age was 60 years (30-74). Prior to autoSCT all patients received rituximab and 45 patients were treated with high dose cytarabine (HD-ARA-C). AutoSCT was performed after first line treatment in 51 patients. A complete response (CR) before autoSCT was achieved by 27 patients. Patients with and without rituximab maintenance therapy did not significantly differ with regard to age, upfront autoSCT, CR rates and HD-ARA-C treatment prior to autoSCT. However, there was a trend that patients who received rituximab maintenance therapy were transplanted more recently (p=0.06).Median observation time after autoSCT was 56 months. Two-year PFS and OS of the control group were 65% and 80%, respectively, as compared to 90% and 88%, respectively, of the rituximab maintenance group. By univariate analysis rituximab maintenance therapy was associated with significantly better PFS (HR 0.21, p=0.014; Figure 1A) but so far not OS (Figure 1B). Multivariate adjustment for age (HR per year 0.98; p=0.79), year of transplant (HR per calendar year 1.0; p=0.96), achievement of CR prior autoSCT (HR 1.59; p=0.26), upfront autoSCT (HR 0.81; p=0.80) and high dose ARA-C treatment (HR 0.69; p=0.63) confirmed the beneficial impact of rituximab maintenance therapy (p=0.02 HR 0.23). [Display omitted] SummaryRituximab may prolong PFS in MCL patients who have undergone autoSCT. Prospective studies are highly warranted to confirm our preliminary finding. Disclosures:Ho:Sanofi-Genzyme: Research Funding, travel grant Other.

Enriched environment increases neurogenesis and improves social memory persistence in socially isolated adult mice

Social memory consists of the information necessary to identify and recognize cospecifics and is essential to many forms of social interaction. Social memory persistence is strongly modulated by the animal's experiences. We have shown in previous studies that social isolation (SI) in adulthood impairs social memory persistence and that an enriched environment (EE) prevents this impairment. However, the mechanisms involved in the effects of SI and EE on social memory persistence remain unknown. We hypothesized that the mechanism by which SI and EE affect social memory persistence is through their modulation of neurogenesis. To investigate this hypothesis, adult mice were submitted to 7 days of one of the following conditions: group-housing in a standard (GH) or enriched environment (GH+EE); social isolation in standard (SI) or enriched environment (SI+EE). We observed an increase in the number of newborn neurons in the dentate gyrus of the hippocampus (DG) and glomerular layer of the olfactory bulb (OB) in both GH+EE and SI+EE mice. However, this increase of newborn neurons in the granule cell layer of the OB was restricted to the GH+EE group. Furthermore, both SI and SI+EE groups showed less neurogenesis in the mitral layer of the OB. Interestingly, the performance of the SI mice in the buried food-finding task was inferior to that of the GH mice. To further analyze whether increased neurogenesis is in fact the mechanism by which the EE improves social memory persistence in SI mice, we administered the mitotic inhibitor AraC or saline directly into the lateral ventricles of the SI+EE mice. We found that the AraC treatment decreased cell proliferation in both the DG and OB, and impaired social memory persistence in the SI+EE mice. Taken together, our results strongly suggest that neurogenesis is what supports social memory persistence in socially isolated mice.

Investigation and verification of a bioluminescent biosensor for the quantitation of ara-CTP generation: A biomarker for cytosine arabinoside sensitivity in acute myeloid leukaemia

A novel whole cell bacterial biosensor, which emits light in response to the active metabolite of cytosine arabinoside (ara-C, cytarabine), ara-CTP, has been investigated and verified. The biosensor has been formulated as an ex vivo assay, designed for peripheral blood or bone marrow cells, which can produce a clinical result within a working day. The nucleoside analogue ara-C is a key agent for treatment of acute myeloid leukaemia (AML); treatment decisions are made rapidly with AML, patients often receiving same-day commencement of chemotherapy. Currently no rapid predictive test is available to select appropriate therapy for patients prior to treatment. Experiments were designed to determine optimal assay conditions using leukaemic cell lines. We observed a significant increase (~15 fold) in bioluminescence signal compared to control after 8-h incubation of the biosensor with ara-C. This corresponded to a >2-log increase in light output per bacterial cell. Interestingly, bioluminescence conferred a survival advantage to the bacteria following ara-C treatment. The assay is sensitive (lower limit of quantitation of 0.05µM), selective, accurate (≤15% RE) and precise (≤15% coefficient of variation) over a linear concentration range of ara-CTP (0.05–0.5µM), and detection is independent of reaction volume. Recovery of added standard was tested using ex vivo patient leukaemic cells (n=5). Stability studies on lyophilized bacterial biosensor were performed to ensure maintenance of performance over 12 months. The biosensor assay could be invaluable to the clinician, assisting with treatment selection, and potentially mitigating the risks of resistance and toxicity observed with this drug.

ARC Is Regulated by MAPK and PI3K and Confers Drug Resistance and Survival Advantage to AML in vitro and in vivo

Abstract Abstract 893 ARC (Apoptosis repressor with caspase recruitment domain) is a unique antiapoptotic protein that has been shown to suppress the activation of both intrinsic and extrinsic apoptosis. We previously reported that ARC is one of the most potent adverse prognostic factors in AML and that high ARC protein expression predicted shorter survival and poor clinical outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report how ARC is regulated and its role in inhibition of AML apoptosis and in cell survival. We provide evidence that ARC expression is regulated by MAPK and PI3K signaling. Inhibition of MAPK and PI3K pathways decreased ARC mRNA and protein levels in AML cells. ARC expression in AML cells is upregulated in co-cultures with bone marrow-derived mesenchymal stromal cells (MSCs) and the upregulation is suppressed in the presence of MAPK or PI3K inhibitors. To investigate the role of ARC in apoptosis resistance in AML, we generated stable ARC overexpressing (O/E) KG-1 and stable ARC knock down (K/D) OCI-AML3 and Molm13 cells and treated them with Ara-C and agents selectively inducing intrinsic (ABT-737) or extrinsic (TRAIL) apoptosis. We found that ARC O/E cells are more resistant and ARC K/D cells more sensitive to Ara-C, ABT-737, and TRAIL-induced apoptosis: EC50s of Ara-C, ABT-737, or TRAIL treatment at 48 hours for ARC O/E KG-1 and control cells were 1.5 ± 0.1 μM vs. 83.5 ± 4.6 nM, 2.2 ± 0.2 μM vs. 60.2 ± 3.1 nM, or 0.97 ± 0.03 μg/mL vs. 0.17 ± 0.08 μg/mL, respectively and for ARC K/D OCI-AML3 and control cells were 0.33 ± 0.02 μM vs. 3.4 ± 0.2 μM, 0.24 ± 0.01 μM vs. 1.3 ± 0.1 μM, or 0.13 ± 0.09 μg/mL vs. 0.36 ± 0.03 μg/mL, respectively. Bone marrow microenvironment is known to play critical roles in AML disease progression and in protecting leukemia cells from various therapeutic agent-induced apoptosis. Leukemia cells were co-cultured with MSCs in vitro study to mimic the in vivo condition. ARC was found to be highly expressed in MSCs and stable ARC K/D MSCs were generated. AML cell lines and primary patient samples were co-cultured with ARC K/D or control MSCs and treated with Ara-C, ABT-737, or TRAIL. Interestingly, ARC K/D MSCs lost their protective activity for leukemia cells treated with these agents. EC50s for OCI-AML3 cells co-cultured with ARC K/D or control MSCs for 48 hours treated with Ara-C, ABT-737, or TRAIL were 1.0 ± 0.04 μM vs. 4.5 ± 0.2 μM, 0.15 ± 0.06 μM vs. 0.53 ± 0.02 μM, or 1.4 ± 0.8 μg/mL vs. 8.1 ± 0.3 μg/mL, respectively. In addition, ARC O/E KG-1 cells grew faster and ARC K/D OCI-AML3 and Molm13 cells and ARC K/D MSCs grew slower than their respective controls. We then injected KG-1 cells into mice and found that NOD-SCID mice harboring ARC O/E KG-1 had significantly shorter survival than mice injected with the vector control KG-1 (median 84 vs. 111 days) as shown in the figure. Download : Download high-res image (24KB) Download : Download full-size image Collectively, results demonstrate that ARC plays critical roles in AML. ARC is regulated by MSCs through various signaling pathways in AML cells, protects leukemia cells from apoptosis induced by chemotherapy and by agents selectively inducing intrinsic and extrinsic apoptosis. ARC regulates leukemia cell growth in vitro and in vivo. The results suggest that ARC is a potential target for AML therapy. In addition, targeting ARC in MSCs suppresses microenvironmental protection of AML cells. Disclosures: No relevant conflicts of interest to declare.