NB4 Acute Promyelocytic Leukemia Research Articles

The Transcription Factor PU.1 Controls Alternative Splicing of Cflip By Binding to SRSF2/10 Splice Factors in AML Therapy Responses

cFLIP (CFLAR) is an inactive homolog of caspase 8 that functions downstream of death-receptor mediated apoptosis. CFLAR gene is alternatively spliced and exists in three isoforms: a long isoform (cFLIPL), which partially inhibits CASP8 activity, and two short forms (cFLIPR and cFLIPS) that entirely block caspase 8 activity. Moreover, cFLIPL is crucial for macrophage differentiation. Therefore, the ratio between cFLIPL and cFLIPS/R may regulate myeloid differentiation. The transcription factor PU.1 (SPI1) is necessary for myeloid differentiation as well as myeloid cell survival. PU.1 is expressed at low levels in hematopoietic progenitors and increasing levels of PU.1 promote macrophage and granulocyte differentiation. Additionally, aberrant low PU.1 expression contributes to an immature myeloid phenotype, e.g. seen in acute myeloid leukemia (AML). Interestingly, two studies indicated that high PU.1 protein levels were associated with alternative splicing in a murine erythroleukemia model by either direct binding to splice factors or RNA. Based on these observations we hypothesized that PU.1 controls alternative splicing of cFLIP during myeloid differentiation. In line with above hypothesis, differentiating primary CD34+ hematopoietic progenitors into granulocytes and monocytes or AML cells into granulocytes shifted cFLIP splicing in favor of cFLIPL paralleled by increased PU.1 mRNA and protein levels. Next, we assessed the direct impact of PU.1 protein on splicing during granulocytic differentiation by knocking down PU.1 in NB4 acute promyelocytic leukemia (APL) and MOLM13 AML cells. Silencing PU.1 in NB4 and MOLM13 cells decreased cell death and differentiation during all-trans retinoic acid (ATRA)-mediated granulocytic differentiation. Moreover, knocking down PU.1 reduced cFLIPL/S protein and mRNA ratios during ATRA treatment. Interestingly, increasing the cFLIPL/S ratio by knocking down specifically cFLIPS or overexpressing cFLIPL reduced MOLM13 cell viability during ATRA treatment. Since the canonical function of CFLAR isoforms is the inhibition of extrinsic apoptosis, we next tested if increased cFLIPS levels found in AML cells depleted for PU.1 would show reduced sensitivity to TRAIL treatment. Indeed, TRAIL-induced extrinsic apoptosis via TRAIL receptor was significantly reduced in PU.1 depleted MOLM-13 AML cells. To investigate if PU.1 is directly involved in CFLAR splicing, we analyzed the CFLAR pre-mRNA sequence encompassing the alternative splicing site for PU.1 and splice factor binding motifs. We identified one putative PU.1 and SRSF2 as well as SRSF10 splice factor binding sites in the CFLAR pre-mRNA sequence. Using RNA immunoprecipitation, PU.1 pull-down and proximity ligation assays, we showed that all three proteins bind the CFLAR mRNA and physically interact with each other. Lastly, we asked if PU.1 additionally influences cFLIP alternative splicing via regulating splice factor transcription. Knocking down PU.1 in MOLM13 cells resulted in significantly reduced SRSF2 and SRSF10 mRNA and protein levels. We confirmed PU.1 binding to their promoter using ChIP assays. Accordingly, these splice factors showed altered mRNA expression when comparing mature granulocytes to primary AML cells. Since silencing SRSF2 and SRSF10 switched the CFLARL/S isoform ratio towards the long isoform, we suggest that PU.1-mediated induction of these splice factores represents a negative feedback loop. In conclusion, we provide evidence that PU.1 directly regulates alternative splicing of CFLAR during ATRA-mediated neutrophil differentiation and TRAIL-induced cell death in human AML cells. We propose a model where PU.1 binds to CFLAR pre-mRNA and blocks SRSF2 and SRSF10-mediated splicing ultimatively leading to exon 7 skipping and predominant expression of cFLIPL.

A novel network pharmacology approach for leukaemia differentiation therapy using Mogrify®.

Acute myeloid leukaemia (AML) is a rapidly fatal blood cancer that is characterised by the accumulation of immature myeloid cells in the blood and bone marrow as a result of blocked differentiation. Methods which identify master transcriptional regulators of AML subtype-specific leukaemia cell states and their combinations could be critical for discovering novel differentiation-inducing therapies. In this proof-of-concept study, we demonstrate a novel utility of the Mogrify® algorithm in identifying combinations of transcription factors (TFs) and drugs, which recapitulate granulocytic differentiation of the NB4 acute promyelocytic leukaemia (APL) cell line, using two different approaches. In the first approach, Connectivity Map (CMAP) analysis of these TFs and their target networks outperformed standard approaches, retrieving ATRA as the top hit. We identify dimaprit and mebendazole as a drug combination which induces myeloid differentiation. In the second approach, we show that genetic manipulation of specific Mogrify®-identified TFs (MYC and IRF1) leads to co-operative induction of APL differentiation, as does pharmacological targeting of these TFs using currently available compounds. We also show that loss of IRF1 blunts ATRA-mediated differentiation, and that MYC represses IRF1 expression through recruitment of PML-RARα, the driver fusion oncoprotein in APL, to the IRF1 promoter. Finally, we demonstrate that these drug combinations can also induce differentiation of primary patient-derived APL cells, and highlight the potential of targeting MYC and IRF1 in high-risk APL. Thus, these results suggest that Mogrify® could be used for drug discovery or repositioning in leukaemia differentiation therapy for other subtypes of leukaemia or cancers.

TRAIL Triggers CRAC-Dependent Calcium Influx and Apoptosis through the Recruitment of Autophagy Proteins to Death-Inducing Signaling Complex.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills various cancer cell types, but also leads to the activation of signaling pathways that favor resistance to cell death. Here, we investigated the as yet unknown roles of calcium signaling and autophagy regulatory proteins during TRAIL-induced cell death in leukemia cells. Taking advantage of the Gene Expression Profiling Interactive Analysis (GEPIA) project, we first found that leukemia patients present a unique TRAIL receptor gene expression pattern that may reflect their resistance to TRAIL. The exposure of NB4 acute promyelocytic leukemia cells to TRAIL induces intracellular Ca2+ influx through a calcium release-activated channel (CRAC)-dependent mechanism, leading to an anti-apoptotic response. Mechanistically, we showed that upon TRAIL treatment, two autophagy proteins, ATG7 and p62/SQSTM1, are recruited to the death-inducing signaling complex (DISC) and are essential for TRAIL-induced Ca2+ influx and cell death. Importantly, the treatment of NB4 cells with all-trans retinoic acid (ATRA) led to the upregulation of p62/SQSTM1 and caspase-8 and, when added prior to TRAIL stimulation, significantly enhanced DISC formation and the apoptosis induced by TRAIL. In addition to uncovering new pleiotropic roles for autophagy proteins in controlling the calcium response and apoptosis triggered by TRAIL, our results point to novel therapeutic strategies for sensitizing leukemia cells to TRAIL.

Retinoic Acid Treatment of Human Hematological Malignancies Induces Caspase Dependent and Independent Apoptotic Cell Death

The unprejudiced of this education is to gauge the ability of the retinoic acid to induce apoptotic cell deathin hematological tumors through caspase dependent or independent apoptotic pathway, The cytotoxicityeffects of retinoic acid of different concentrations (400,350,300,250,200,150,100,50,25,12.5 µg\ml) andexposure for all hematological malignancy cell lines (Human non-Hodgkin lymphoma SR and humanmultiple myeloma (COLO 677) and Human Monocytic Leukemia THP1 and Acute promyelocytic leukemiaNB4) have been determined using a microtetrazolium (MTT) assay. Propodeum iodide and alcidine orange(AO/PI) paired discoloration was used to study the ability of retinoic acid to induce apoptosis in the infectedcells and examined under fluorescence microscope and quantified for the percentage of apoptosis induction.Quantitative immunocytochemistry assay was used to study the caspase dependent and independentproteins expression in infected and control cells. Cells treated with Retinoic Acid showed increased celldeath percentage compared to the untreated cells as quantified by MTT assay. AO/PI results revealed thatRetinoic Acid had powerful effect on inducing apoptosis significantly (p<0.001) in human cancer cell linestested, compared to control cell. Immunocytochemistry in Retinoic Acid infected human hematological celllines revealed remarkable increase in expression of caspase 8,9 (dependent pathway) and AIF, ENDOG(independent pathway) induces a significant (p<0.002) as compared untreated cell.This study, which shows the role of the Retinoic Acid in inducing apoptosis through a dependent andindependent pathway in cancer cells, we anticipation these annotations will shanty light on the impendingexploration of retinoic acid in cancer hindrance and rehabilitation

Transcriptional and Metabolic Dissection of ATRA-Induced Granulocytic Differentiation in NB4 Acute Promyelocytic Leukemia Cells.

Acute promyelocytic leukemia (APL) is a hematological disease characterized by a balanced reciprocal translocation that leads to the synthesis of the oncogenic fusion protein PML-RARα. APL is mainly managed by a differentiation therapy based on the administration of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, therapy resistance, differentiation syndrome, and relapses require the development of new low-toxicity therapies based on the induction of blasts differentiation. In keeping with this, we reasoned that a better understanding of the molecular mechanisms pivotal for ATRA-driven differentiation could definitely bolster the identification of new therapeutic strategies in APL patients. We thus performed an in-depth high-throughput transcriptional profile analysis and metabolic characterization of a well-established APL experimental model based on NB4 cells that represent an unevaluable tool to dissect the complex mechanism associated with ATRA-induced granulocytic differentiation. Pathway-reconstruction analysis using genome-wide transcriptional data has allowed us to identify the activation/inhibition of several cancer signaling pathways (e.g., inflammation, immune cell response, DNA repair, and cell proliferation) and master regulators (e.g., transcription factors, epigenetic regulators, and ligand-dependent nuclear receptors). Furthermore, we provide evidence of the regulation of a considerable set of metabolic genes involved in cancer metabolic reprogramming. Consistently, we found that ATRA treatment of NB4 cells drives the activation of aerobic glycolysis pathway and the reduction of OXPHOS-dependent ATP production. Overall, this study represents an important resource in understanding the molecular “portfolio” pivotal for APL differentiation, which can be explored for developing new therapeutic strategies.

Apoptosis-inducing effect of spiroaminopyrimidine analogue in NB4leukemia cells via down-regulation of BIRC5expression.

It has been reported that spiroaminopyrimidine derivatives inhibited the growth and proliferation of various cancer cell lines. In the present study, we evaluated cytotoxic and apoptosis-inducing effects of 2,4-diamino-1,3-diazaspiro[5.5]-9-tert-butyl-2, 4-diene-5-carbonitril (9-tBAP) on NB4acute promyelocytic leukemia (APL) cells. The cells were treated with 10-100µM of 9-tBAP. Cytotoxic activity of the compound was measured using the MTT assay. Apoptosis was investigated by Hoechst 33258staining as well as by Annexin V/PI double staining. The compound under study was found to be highly active cell growth inhibitor with IC50 of 30.0±3.5µM inducing apoptosis in NB4cells. Cell cycle analysis by flow cytometry showed a time-dependent increase in sub-G1 cell population. Real-time polymerase chain reaction analysis revealed that the treatment with the compound down-regulated the BIRC5expression in a time-dependent manner. 9-tBAP displayed potent anti-leukemic activity in vitro thus warranting further investigation.

Autophagy is Required to Regulate Mitochondria Renewal, Cell Attachment, and All-trans–Retinoic Acid–Induced Differentiation in NB4 Acute Promyelocytic Leukemia Cells

All-trans-retinoic acid (ATRA) is a potent inducer of cellular differentiation, growth arrest, and apoptosis as well as a front-line therapy for acute promyelocytic leukemia (APL). The present study provides evidence that induction of autophagy is required for ATRA to induce differentiation of APL (NB4) cells into granulocytes. ATRA treatment causes ~12-fold increase in the number of acidic vesicular organelles and induces marked up-regulation of LC3-II, autophagy-related 5 (ATG5), and Beclin-1. Transmission electron microscopy (TEM) revealed a decrease in mitochondria and ATRA-induced differentiation. To determine the role of autophagy in the differentiation of APL, we knocked down ATG5 in NB4 cells to find that ATRA-induced differentiation is significantly inhibited during ATG5 knock down in cells, indicating the role of autophagy in differentiation of APL. Further experiments revealed restriction of autophagy during ATRA-induced differentiation and inhibition of tissue transglutaminase 2 (TG2) and phospho-focal adhesion kinase (p-FAK), which are known to have roles in differentiation and cell attachment. We examinedexpression of Beclin-1 and B-cell lymphoma-2 (Bcl-2) and levels of mechanistic target of rapamycin (mTOR) after ATRA treatment. ATRA inhibits Bcl-2, up-regulates Beclin-1 expression, and reduces induction of mTOR activation/phosphorylation in NB4 cells. Our results reveal that autophagy has roles in regulation of differentiation, mitochondria elimination, and cell attachment during ATRA-induced APL differentiation.

Hexokinase Proteins Impart Distinct Functions in Myeloid Development and Cell Death

Abstract Many key oncogenic pathways converge to adapt tumor cell metabolism to requirements of cancer cells. Aberrant proliferation that is frequently associated with cancer cells is also linked to an adjustment of metabolism in order to fuel cell growth and division. Cancer cells prefer utilizing glycolysis for energy production and providing essential building blocks for a variety of macromolecules. Hexokinases (HKs) are rate-limiting enzymes that catalyze the first and irreversible step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. Four HK isoforms are expressed in mammalian cells, HK1, HK2, HK3 and HK4 (also known as glucokinase). HKs promote and sustain a concentration gradient that facilitates glucose entry, which ensures the initiation of glucose dependent pathways. In general, HKs have a cytoprotective role that was highlighted by enhanced sensitivity of cancer cells to drugs when HKs were inhibited. Previously we have reported that HK3 was transcriptionally regulated by PU.1 (SPI-1) in myeloid cells. Further, HK3 expression was significantly reduced in patient acute myeloid leukemia (AML) cells, particularly in acute promyelocytic leukemia (APL) cells expressing the PML-RARA oncofusion protein. We now report on the expression and regulatory function of HKs, particularly HK3, during myeloid differentiation and granulocyte associated cell death. First, we analyzed mRNA HK levels in human CD34+ hematopoietic progenitors cells differentiated towards granulocytes or macrophages by qPCR. Interestingly, while HK1 and HK2 levels remain stable during all stages of myeloid differentiation, HK3 mRNA levels significantly increased. The same pattern of HK mRNA expression was seen in NB4 APL and HL60 AML cell lines differentiated towards granulocytes and monocytes using all-trans retinoic acid (ATRA) and vitamin D3, respectively. To determine a specific role for HK1-3 function in myeloid cells, HK1-3 knockdowns (KD) and knockouts (KO) in NB4 and HL60 cell lines, using shRNA or gRNAs (Cas9/CRISPR technology), respectively, were generated. NB4 HK KD AML cells were tested for their differentiation upon ATRA treatment. Knockdown of HKs generally resulted in a decreased differentiation response of about 20% as assessed by the differentiation marker CD11b. We next determined energy metabolism of HK altered KD and KO cells, relative to parental cells, using a seahorse analyzer. While lowering HK1 or HK3 levels in NB4 and HL60 AML cells did not affect glycolytic capacity at steady state, HK2 inhibition significantly reduced steady state glycolytic capacity. In contrast, knocking down or knocking out HK3 resulted in a higher sensitivity to ATRA-induced cell death during differentiation, which was coupled with higher glycolytic capacity. Together, our findings suggest that HK2 has an important role in steady state metabolism of AML cells while HK3 appears to be a metabolic switch for cell survival during myeloid differentiation. Disclosures No relevant conflicts of interest to declare.

RACK1 deficiency synergizes with all-trans retinoic acid to induce apoptosis in human acute promyelocytic leukemia cells.

As a classic differentiation agent, all-trans retinoic acid (ATRA) has been widely used in the treatment of acute promyelocytic leukemia (APL). However, the clinical application of ATRA has strict limitations, for its severe side effects due to the accumulation of peripheral blood leukocytes. The scaffold protein RACK1 (Receptor for activated C kinase 1), which regulates multiple signaling pathways, has been proposed to contribute to the survival of leukemic progenitors. But it remains unclear whether it is also involved in the oncogenic growth of APL. In the present study, we demonstrate that silencing of endogenous RACK1 expression synergized with ATRA to promote the death of NB4 and HL-60 APL cells without effect on cell differentiation induced by ATRA. Interestingly, RACK1 knockdown combined with ATRA treatment mainly induces apoptosis. It is distinct to the necrotic cell death induced by idarubicin in combination with ATRA, a regimen extensively used in the clinic to prevent neutrophil accumulation. Further exploration revealed that the lysosome-autophagy pathway is likely to be responsible for the anti-apoptotic role of RACK1. Taken together, our findings indicate that RACK1 is essential in maintaining the malignant features of APL, and targeting RACK1 may have promising therapeutic implications in the treatment of APL.

Chloroquine exerts antitumor effects on NB4 acute promyelocytic leukemia cells and functions synergistically with arsenic trioxide.

Chloroquine (CQ) has been confirmed to exhibit antitumor effects on different types of cancer cell, but whether it exerts the same effect on acute promyelocytic leukemia (APL) cells remains to be confirmed. In the present study, the effects of various concentrations of CQ on the growth, apoptosis and cell cycle distribution of NB4 cells, as well as the potential mechanisms underlying these effects, were examined. The combined effect of CQ and arsenic trioxide (ATO) on the growth of NB4 cells was also determined. The results of the present study demonstrated that CQ treatment inhibited cell proliferation, and induced mitochondrial pathway apoptosis and S phase arrest in a dose-dependent manner by regulating apoptosis- and cell cycle-related proteins. CQ and ATO had a synergistic effect on the growth inhibition of NB4 cells, which may have been induced through the inhibition of autophagy. In conclusion, the results of the present study indicated that CQ exhibits a cytotoxic effect on NB4 cells and has a synergistic effect when combined with ATO, which thereby improves the curative effect of ATO on APL.

Effect of ATRA and ATO on the expression of tissue factor in NB4 acute promyelocytic leukemia cells and regulatory function of the inflammatory cytokines TNF and IL-1\u03b2

The characteristic hemorrhages of acute promyelocytic leukemia (APL) are caused in part by the high expression of tissue factor (TF) on leukemic cells, which also produce TNF and IL-1β, proinflammatory cytokines known to increase TF in various cell types. Exposure of NB4 cells, an APL cell line, to all-trans retinoic acid (ATRA) or arsenic trioxide (ATO) rapidly and strongly reduced TF mRNA. Both drugs also reduced TNF mRNA, but later, and moreover increased IL-1β mRNA. The effect on procoagulant activity of cells and microparticles, as measured with calibrated automated thrombography, was delayed and only partial at 24 h. TNF and IL-1β inhibition reduced TF mRNA and activity only partially. Inhibition of the inflammatory signaling intermediate p38 reduced TF mRNA by one third but increased TNF and IL-1β mRNA. NF-κB inhibition reduced, within 1 h, TF and TNF mRNA but did not change IL-1β mRNA, and rapidly and markedly reduced cell survival, with procoagulant properties still being present. In conclusion, although we provide evidence that TNF, IL-1β, and their signaling intermediates have a regulatory function on TF expression by NB4 APL cells, the effect of ATRA and ATO on TF can only partially be accounted for by their impact on these cytokines.

Lentivirus‑mediated RIG‑I knockdown relieves cell proliferation inhibition, cell cycle arrest and apoptosis in ATRA‑induced NB4 cells via the AKT‑FOXO3A signaling pathway in vitro.

Retinoic acid inducible gene I (RIG-I) is upregulated during all-trans retinoic acid (ATRA)-induced terminal granulocytic differentiation of NB4 acute promyelocytic leukemia (APL) cells. However, the function and mechanism of RIG-I in NB4 cells remains to be fully elucidated. In the present study, lentivirus-mediated RIG-I-knockdown was used to investigate the proliferation, cell cycle and apoptotic processes of ATRA-induced NB4 cells in vitro using an MTT assay and flow cytometry, respectively. The roles of RIG-I and the AKT-FOXO3A signaling pathway were investigated using western blot analysis. The results showed that the ATRA-induced expression of RIG-I was specifically and effectively knocked down at the mRNA and protein levels by lentivirus mediated RIG-I short hairpin RNA. In addition, silencing of RIG-I reduced the ATRA-induced inhibition of NB4 cell proliferation, cell cycle arrest and apoptosis. Further investigations indicated that with ATRA-induced expression of RIG-I, levels of phosphorylated (p)AKT-Thr308 and pForkhead Box (FOX) O3A-Thr32 were decreased, the expression levels of cell cycle arrest protein p27 and the apoptotic protein, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), directly transcribed by FOXO3A were increased. By contrast, following the knockdown of ATRA-induced expression of RIG-I, the levels of pAKT-Thr308 and pFOXO3A-Thr32 were increased, and the protein expression levels of p27 and TRAIL were decreased. Taken together, these results showed that the knockdown of RIG-I reduced the inhibition of cell proliferation, cell cycle arrest and apoptosis in the ATRA-induced NB4 cells via the AKT-FOXO3A signaling pathway.

Zinc Depletion by TPEN Induces Apoptosis in Human Acute Promyelocytic NB4 Cells

Background/Aims: The effects of zinc signaling on proliferation or apoptosis of leukemia cells remain elusive. In the present study, we used N, N, N’, N’-tetrakis-(2-pyridylmethyl)-ethylene-diamine (TPEN), a membrane-permeable zinc chelator, to evaluate the effect of zinc depletion on survival and apoptosis of NB4 acute promyelocytic leukemia (APL) cells. Methods: The pro-apoptotic effects of TPEN on NB4 cells were examined by flow cytometry, and observed using an optical microscope. Intracellular labile zinc, nitric oxide (NO) or reactive oxygen species (ROS) changes caused by TPEN were measured by flow cytometry. We then explored possible roles of the crosstalk between intracellular labile zinc signaling and nitric oxide signaling in TPEN-triggered apoptosis. Results: we found that TPEN induced apoptosis in NB4 APL cells in a dosage-dependent manner. We further demonstrated that TPEN triggered apoptosis by attenuating intracellular zinc and nitric oxide signaling in NB4 cells. Both exogenous zinc supplement and the nitric donor sodium nitroprusside (SNP) pre-incubation reversed TPEN-mediated inhibition of intracellular NO and Zn²⁺ signaling, and rescued NB4 cells from apoptosis. Conclusion: These results suggest for the first time that crosstalk between zinc signaling and nitric oxide pathway is essential for the survival of NB4 cells. TPEN induces apoptosis in NB4 cells via negatively regulating intracellular NO and Zn²⁺ signaling. Our in vitro data suggest that zinc depletion by TPEN may be a potential therapeutic strategy for APL.

Homoharringtonine production by endophytic fungus isolated from Cephalotaxus hainanensis Li.

Homoharringtonine (HHT), a natural plant alkaloid derived from Cephalotaxus, has demonstrated to have a broad antitumor activity and efficacy in treating human chronic myeloid leukemia. An alternative source is required to substitute for the slow-growing and scarce Cephalotaxus to meet the increasing demand of the drug market. The objective of this study was to screen HHT-producing endophytic fungi from Cephalotaxus hainanensis Li. By screening 213 fungal isolates obtained from the bark parts of Cephalotaxus hainanensis Li, one isolate was found to be capable of biosynthesizing HHT. The fungus was identified as Alternaria tenuissima by morphological characteristics and internal transcribed spacer (ITS) sequence analysis and was named as CH1307. HHT obtained from CH1307 was analyzed through the HPLC and LC-MS/MS and NMR spectroscopy. The extract of the fermentation broth of CH1307 showed antiproliferative activities against K562 (chronic myelocytic leukemia), NB4 (acute promyelocytic leukemia), and HL-60 (promyelocytic leukemia) human cancer cell lines with IC50 values of 67.25±4.26, 65.02±4.75, and 99.23±4.26μg/mL, respectively. The findings suggest that HHT-producing endophytic fungus, Alternaria tenuissima CH1307 might provide a promising source for the research and application of HHT.

Cholinergic activation enhances retinoic acid-induced differentiation in the human NB-4 acute promyelocytic leukemia cell line

The non-neuronal cholinergic system (NNCS) has been shown to play a role in regulating hematopoietic differentiation. We determined the expression of cholinergic components in leukemic cell lines by Western blotting and in normal leukocyte subsets by flow cytometry and found a heterogeneous expression of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), choline transporter (CHT), M3 muscarinic acetylcholine receptor (M3-mAChR) and α7 nicotinic acetylcholine receptor (α7-nAChR). We then evaluated NNCS role in differentiation of human NB-4 acute promyelocytic leukemia cell line and discovered a dramatic induction of M3-mAChR after all-trans retinoic acid (ATRA) treatment (p<0.0001). Adding carbachol which is a cholinergic agonist to the ATRA treatment resulted in an increase of a granulocytic differentiation marker (CD11b) as compared with ATRA treatment alone (p<0.05), indicating that cholinergic activation enhanced ATRA in inducing NB-4 maturation. The combination of carbachol and ATRA treatment for 72h also resulted in decreased viability and increased cleaved caspase-3 expression when compared with ATRA treatment alone (p<0.05). However, this combination did not cause poly (ADP-ribose) polymerase (PARP) cleavage. Overall, we have shown that NB-4 cells expressed M3-mAChR in a differentiation-dependent manner and cholinergic stimulation induced maturation and death of ATRA-induced differentiated NB-4 cells.

Antileukemic effects of piperlongumine and alpha lipoic acid combination on Jurkat, MEC1 and NB4 cells in vitro.

This research indicated to evaluate the effects of piperlongumine (PL), a biologically active alkaloid, and alpha lipoic acid (ALA), a naturally occurring cofactor existed in multienzyme complexes regulating metabolism on leukemia cells. Excessive production of reactive oxygen species (ROS) can lead to oxidative stress, a state that has been observed in several hematopoietic malignancies, including acute and chronic myeloid leukemias. The importance of the association between oxidative stress and malignancy is not currently clear; however, there is evidence that tumor.derived ROS may promote cell survival, migration and metastasis, proliferation and even drug.resistance depending on the origin of the cancer. Increased oxidative stress in leukemic cells may represent a potential therapeutic target, although there are differing opinions on whether therapeutic strategies should aim to antagonize or further promote oxidative stress in leukemic cells. The effects of PL alone (5, 15, 30 μM) and in combination (30 μ M) with ALA (200 μ M) on Jurkat, NB4 and MEC1 leukemia cell lines were investigated through MTT, caspase-3 and cyclooxygenase-2 (COX-2) activities. Inhibition of COX-2 and the induction of caspase.3 cleavage in Nb4 (acute promyelocytic leukemia) cells were found to be significant following PL application and synergistic effects with combination of ALA (inhibition of COX-2 as 23.74% and 3.55-fold increase of caspase-3). PL and ALA may have a potential value as a therapeutic agent for patients with acute promyelocytic leukemia.

The RNA binding proteins RBM38 and DND1 are repressed in AML and have a novel function in APL differentiation

The RNA binding proteins RBM binding motif protein 38 (RBM38) and DEAD END 1 (DND1) selectively stabilize mRNAs by attenuating RNAse activity or protecting them from micro(mi)RNA-mediated cleavage. Furthermore, both proteins can efficiently stabilize the mRNA of the cell cycle inhibitor p21(CIP1). Since acute myeloid leukemia (AML) differentiation requires cell cycle arrest and RBM38 as well as DND1 have antiproliferative functions, we hypothesized that decreased RBM38 and DND1 expression may contribute to the differentiation block seen in this disease. We first quantified RBM38 and DND1 mRNA expression in clinical AML patient samples and CD34(+) progenitor cells and mature granulocytes from healthy donors. We found significantly lower RBM38 and DND1 mRNA levels in AML blasts and CD34(+) progenitor cells as compared to mature neutrophils from healthy donors. Furthermore, the lowest expression of both RBM38 and DND1 mRNA correlated with t(8;21). In addition, neutrophil differentiation of CD34(+) cells in vitro with G-CSF (granulocyte colony stimulating factor) resulted in a significant increase of RBM38 and DND1 mRNA levels. Similarly, neutrophil differentiation of NB4 acute promyelocytic leukemia (APL) cells was associated with a significant induction of RBM38 and DND1 expression. To address the function of RBM38 and DND1 in neutrophil differentiation, we generated two independent NB4RBM38 as well as DND1 knockdown cell lines. Inhibition of both RBM38 and DND1 mRNA significantly attenuated NB4 differentiation and resulted in decreased p21(CIP1) mRNA expression. Our results clearly indicate that expression of the RNA binding proteins RBM38 and DND1 is repressed in primary AML patients, that neutrophil differentiation is dependent on increased expression of both proteins, and that these proteins have a critical role in regulating p21(CIP1) expression during APL differentiation.

RIG-G inhibits the proliferation of NB4 cells and propels ATRA-induced differentiation of APL cells

RIG-G (retinoic acid-induced gene G) was originally identified in ATRA (all-trans retinoic acid)-treated NB4 acute promyelocytic leukemia (APL) cells. It was induced to expression by ATRA along with the differentiation of the cells. However, little is known about its role(s). Here, we established a RIG-G stably expression transformant of NB4 cells. By using the transformant, we showed that expression of RIG-G in NB4 cells not only arrested the cells at G1/G0 transition phase and inhibited their proliferation, but also markedly drive the maturation of NB4 cells in the presence of very low concentration of ATRA (10(-9)mol/L). What's more, by detecting the expression of RIG-G in fresh primary bone marrow mononuclear cells of APL patients in different morbid states, we found high RIG-G expression level in complete remission patients, while low level in untreated or relapsed patients. These results indicated that RIG-G level was high in maturated cells and low in blast cells, and suggested that RIG-G might play a role in the differentiation of bone marrow hemocytes in vivo.

Abstract 510: Inhibition of IRE1α-driven pro-survival pathways is a promising therapeutic application in acute myeloid leukemia

Abstract Survival of cancer cells rely on the unfolded protein response (UPR) to resist stress triggered by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). IRE1α-XBP1 pathway, one of the most important branches of the UPR, is activated in different types of cancer. Upon IRE1α activation, the spliced form of XBP1 (XBP1s) is generated and acts as a transcription factor for many pro-survival genes to prevent cellular death and relieve cellular stress. Blockade of the IRE1α-XBP1 pathway can be considered as a potential anti-cancer strategy. Acute myeloid leukemia (AML) is the most common acute leukemia in adults. We find that XBP1 and XBP1s are up-regulated in AML cell lines and samples from patients. IRE1α RNase inhibitors including STF-083010, HNA and MKC-204 blocked XBP1 mRNA splicing and exhibited modest cytotoxicity in liquid culture against AML cell lines (HNA, mean IC50, 31 µM, n=8) and AML samples from patients (HNA, mean IC50, 35 µM, n=18). Notably, the IRE1α inhibitor HNA caused a significant inhibition (mean IC50, 6 µM, n=6) of clonogenic growth in soft agar of AML cells from patients of different leukemic subtypes. In contrast, HNA had very little toxicity against normal human marrow committed myeloid colony forming cells (mean IC50, 123 µM, n=4). IRE1α inhibition in AML cells induced caspase-dependent apoptosis and G1 cell cycle arrest which was associated with regulation of Bcl-2 family proteins, G1 phase controlling proteins (p21cip1, p27kip1 and Cyclin D1) and Chaperone proteins (CHOP, HERPUD1, DNAJC3, DNAJB9 and EMDM). Absence of Xbp1 (Cre-induced deletion of floxed XBP1) resulted in the myeloid cells becoming resistant to IRE1α inhibitors. Combination of HNA with either bortezomib or As2O3 synergistically inhibited growth of NB4 acute promyelocytic leukemia cells associated with enhanced levels of p-JNK and reduced expression of p-PI3K and p-MAPK. Also, in a dose- and time- dependent manner, inhibition of IRE1α RNase activity increased expression of many miRNAs (especially miR-17, -21, -34a, -96, -125b and -150) in AML cells. Furthermore, HNA treatment inhibited mRNA levels of several miR-34a targeted genes (c-Myc, Cyclin D1 and CDK4). The inhibition of miR-34a conferred cellular resistance to HNA, as measured by significant increase of cell viability. Addition of a miR-34a antagonist restored protein expression levels of c-Myc and Cyclin D1 that had been inhibited by HNA, suggesting a crucial role of miR-34a in the IRE1α-mediated stress response pathway. Taken together, our results strongly suggest that targeting the IRE1α driven pro-survival pathways is an exciting therapeutic approach for the treatment of AML. Note: This abstract was not presented at the meeting. Citation Format: Haibo Sun, Behzad Kharabi Masouleh, Sigal Gery, Qi Cao, Serhan Alkan, Takayuki Ikezoe, Chie Akiba, Ronald Paquette, Wenwen Chien, Carsten Müller-Tidow, Yang Jing, Kharabi Masouleh, Markus Müschen, H. Phillip Koeffler. Inhibition of IRE1α-driven pro-survival pathways is a promising therapeutic application in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 510. doi:10.1158/1538-7445.AM2014-510

Zoledronic acid exerts antitumor effects in NB4 acute promyelocytic leukemia cells by inducing apoptosis and S phase arrest

The aim of this study was to investigate the antitumor effect of zoledronic acid (ZOL) in the NB4 human acute promyelocytic leukemia (APL) cell line and explore the potential mechanism of action of this compound. NB4 cells were exposed to various concentrations (0-200μM) of ZOL. Cell viability was measured by MTS assay. The extent of cell apoptosis and distribution of cells in the different phases of the cell cycle were analyzed with flow cytometry. The expression of apoptosis- and cell cycle-related proteins was assayed by Western blot. The combined effect of ZOL and arsenic trioxide (ATO) on the proliferation of NB4 cells was also determined. The results of this study indicate that ZOL inhibits cell proliferation in a time- and dose-dependent fashion and also induces apoptosis and S phase arrest in a dose-dependent manner. The Western blot analysis confirmed the induction of apoptosis and S phase arrest, revealing that the pro-apoptosis proteins Bax, Puma and activated caspase-9 were upregulated and the anti-apoptosis proteins Bcl-2 and Bcl-xL were downregulated. ZOL at a concentration of 50μM synergized with 0.5μM ATO on the growth inhibition of NB4 cells. Taken together, our data indicate that ZOL exerts a potent antitumor effect on NB4 cells by inducing apoptosis and cell cycle arrest, and that ZOL can synergize with the traditional chemotherapy drug ATO.