Resistant Acute Promyelocytic Leukemia Research Articles

Restoration of CCAAT enhancer binding protein α P42 induces myeloid differentiation and overcomes all-trans retinoic acid resistance in human acute promyelocytic leukemia NB4-R1 cells.

All-trans retinoic acid (ATRA) is one of the first line agents in differentiation therapy for acute promyelocytic leukemia (APL). However, drug resistance is a major problem influencing the efficacy of ATRA. Identification of mechanisms of ATRA resistance are urgenly needed. In the present study, we found that expression of C/EBPα, an important transcription factor for myeloid differentiation, was significantly suppressed in ATRA resistant APL cell line NB4-R1 compared with ATRA sensitive NB4 cells. Moreover, two forms of C/EBPα were unequally suppressed in NB4-R1 cells. Suppression of the full-length form P42 was more pronounced than the truncated form P30. Inhibition of PI3K/Akt/mTOR pathway was also observed in NB4-R1 cells. Moreover, C/EBPα expression was reduced by PI3K inhibitor LY294002 and mTOR inhibitor RAD001 in NB4 cells, suggesting that inactivation of the PI3K/Akt/mTOR pathway was responsible for C/EBPα suppression in APL cells. We restored C/EBPα P42 and P30 by lentivirus vectors in NB4-R1 cells, respectively, and found C/EBPα P42, but not P30, could increase CD11b, CD14, G-CSFR and GM-CSFR expression, which indicated the occurrence of myeloid differentiation. Further upregulating of CD11b expression and differential morphological changes were found in NB4-R1 cells with restored C/EBPα P42 after ATRA treatment. However, CD11b expression and differential morphological changes could not be induced by ATRA in NB4-R1 cells infected with P30 expressing or control vector. Thus, we inferred that ATRA sensitivity of NB4-R1 cells was enhanced by restoration of C/EBPα P42. In addition, we used histone deacetylase inhibitor trichostatin (TSA) to restore C/EBPα expression in NB4-R1 cells. Similar enhancement of myeloid differentiation and cell growth arrest were detected. Together, the present study demonstrated that suppression of C/EBPα P42 induced by PI3K/Akt/mTOR inhibition impaired the differentiation and ATRA sensitivity of APL cells. Restoring C/EBPα P42 is an attractive approach for differentiation therapy in ATRA resistant APL.

Proteasome Activity Is Dispensable for the Degradation of PML-RARα: Efficacy of Bortezomib Along with Arsenic Trioxide in the Treatment of Arsenic Sensitive and Resistant Acute Promyelocytic Leukemia

Degradation of PML-RARA upon treatment with ATO is predominantly mediated by the proteasome complex. Reports suggest that in relapsed APL patients who were treated upfront with ATO, mutations in the B2 domain of PML in PML-RARA gene are involved in resistance to ATO. These mutations predict a poor clinical outcome in spite of subsequent combination of ATO with chemotherapy (NEJM 2014). We had previously reported that Bortezomib (Bo) was able to synergize with ATO by inducing apoptosis through increased levels of ROS and up regulation of the UPR pathway (Blood. 2012;120, 3552) and we had also noted PML-RARA degradation and nuclear body formation with this combination. We further evaluated the mechanism of degradation of PML-RARA when ATO was combined with Bo and the effect of this combination on resistant cell lines, a mouse model and in relapsed patients.We generated in-house ATO resistant NB4 cell lines (NB4EV-ASR1, ASR2 and ASR3). In NB4EV-ASR1 we confirmed the presence of A216V mutation in the PML B2 domain (previously reported to be involved in ATO resistance) while the other 2 clones did not have this or any other mutation in PML-RARA. We noted an increase in the baseline proteasomal activity in all the resistant cell lines when compared to naïve NB4 cells (n=3; data not shown). The combination of ATO and Bo induced a significant apoptosis in all the resistant cells similar to naïve NB4 cells (Figure 1A: n=4; Combination Index = 0.02).The mechanism of inducing apoptosis in the resistant cell lines was similar to naïve NB4 cells, as previously reported by us, and involved an increased level of ROS, decreased mitochondrial membrane potential, induction of UPR and activation of caspase-3 (Figure 1B).We next evaluated PML-RARA degradation in NB4 naive cells treated with a combination of ATO+Bo. At 24 hours, there was an evidence of induction in autophagy as shown by LC3II formation using western blot technique which increased at 48 hours; this time point coincides with time at which maximum PML-RARA degradation occurred (Figure1C). Similar results were seen in the resistant cell lines (with and without mutation A216V). Blocking autophagy by 3-methyl adenine showed a partial inhibition in the degradation of PML-RARA. We have also observed that there is an accumulation of p62 (ubiquitin binding protein) at 24 hours and this was degraded by 48 hours suggests that accumulated ubiquitinated products were cleared by autophagy via p62 (Figure1D). In a co-immunoprecipitation experiment, p62 and LC3II proteins precipitated along with PML-RARA (figure 1E). Knock down of p62 transcript by siRNA followed by ATO+Bo treatment showed an accumulation of PML-RARA in the treated cells in comparison to the scrambled and control.In an APL transplanted mice model, combination of ATO and Bo prolonged the life span of the mice as illustrated in Figure1F. In this group there was a significant decrease in the leukemia burden evidenced by decreased leukemic cells in bone marrow, peripheral blood and spleen by flow cytometry, RQ-PCR and decreased spleen size on day +20. A reduction in the LIC was demonstrated by secondary transplants. We also observed that transplantation of bone marrow cells from the long term surviving mice post ATO+Bo therapy did not induce leukemia (Figure1G) and no transcripts of PML-RARA were detected in the recipients.A phase II clinical study combining Bo with ATO and chemotherapy has been initiated for patients with relapsed APL (NCT01950611). In this ongoing study 11 patients have been enrolled. The median age was 32 years. 7 were males. All patients achieved hematological remission and the median time to complete molecular remission was 42 days (one patient still on induction therapy). The addition of Bo was well tolerated. None of the cases had evidence of significant neuropathy, worsening of coagulopathy, IC bleed or a differentiation syndrome. Long term follow up is awaited to comment on the efficacy of this combination.In conclusion, the mechanism of ATO+Bo synergy is multi-factorial and appears to be predominantly due to increase in ROS activity and up regulation of UPR pathway leading to apoptosis. In spite of proteasomal inhibition by addition of Bo with ATO, PML-RARA continues to be degraded and this is mediated by up-regulation of autophagy pathway. ATO+Bo synergy was further confirmed in a pre-clinical model. This combination is also effective in ATO resistant cell lines with high levels of synergism. [Display omitted] DisclosuresOff Label Use: Bortezomib in the treatment of acute promyelocytic leukemia.

Arsenic Trioxide Resistance: More to It Than Mutations in PML-RARα

There has been a recent concern of arsenic trioxide (ATO) resistance in patients with acute promyelocytic leukemia (APL) treated with ATO as upfront therapy. The focus of ATO resistance has centred on mutations in PML-RARA gene (Blood 2011, NEJM 2014). NB4 cells in our laboratory were exposed to serial increasing concentrations of ATO. We subsequently generated 3 ATO resistant clones, NB4EV-ASR1, ASR2 and ASR3. In addition we have also evaluated an established ATRA resistant APL cell line UF1 (Gift from Dr. Chomienne. C).In a viability assay, we observed that these cell lines are resistant to ATO and had an IC50 above 2µm (Table1). These resistant cell lines also had a higher IC50 to other therapeutic drugs such as Daunorubicin and Cytosine arabinoside and a reduced differentiation effect on exposure to ATRA (summarized in Table 1). To identify the differences between the naïve cells and the resistant cells, we did whole exome sequencing (NGS) by Iontorrent and found that only NB4 EV-ASR1 clone had ATO resistance causing mutation (A216V) in the PML B2 domain (VAF=91.7%) while the other two cell lines (NB4EV-ASR2 and ASR3) did not have a mutation in PML-RARA. Next, we did an expression array to find the differential regulated genes between the naïve cell line and the parent resistant cell line from where all the 3 cell lines had been derived. We found that 1490 genes were differentially regulated (> 2 fold). The pathways significantly enriched for differentially expressed genes were cell survival, ABC transporters, Glutathione synthesis, Ubiquitin- proteasome degradation system and signalling pathways like PI3-AKT and PTEN.Validating the micro-array data, we found that there is an increased expression of ABC transporters such as MRP4, AQP9 (n=3; Figure1A) which are known to efflux ATO from the cells and a decreased expression of ABCA1 (known to efflux glutathione). The up regulation of these transporters also correlated with decreased levels of intracellular ATO (IC-ATO; measured using AAS, see Figure 1B for details) in the resistant cell lines. We also noted that there is a varying reduction in the basal reactive oxygen species levels and a varying increase in the amount of basal reduced glutathione (GSH) levels in the resistant cell lines (n=3, Table 1). We have noted that adding Buthionine sulphoximine (BSO - GSH inhibitor) along with ATO was able to restore the sensitivity of ATO in the resistant cells lines, however there was significant variation in the sensitivity of ATO among the cell lines when treated with the same concentration of BSO (Figure 1C).At the transcript levels we did not find any difference in expression of PML-RARA but at the protein level we noted a significant reduction in the levels of PML-RARA in the resistant cell lines (Figure 1D). We also observed an increase in the proteasome activity in the resistant cell lines compared to naïve cells (data not shown). In an immunofluorescence assay probing for PML, we found an absence of micro-speckled pattern in the resistant cell lines and UF1 cell lines compared to naive cells (Figure 1D).In conclusion, we have observed that in addition to PML-RARA mutations, variations in the Redox system, ABC transporters, intracellular ATO concentration and anti-apoptosis pathways are likely to be altered in ATO resistance. It is likely that ATO resistance is multi-factorial and that the dominant mechanism can vary between different resistant cell lines and potentially the same variation could be seen in relapsed patients. Importantly in the presence of ATO resistance there was also a decrease in sensitivity to other conventional agents used to treat APL. Novel agents and strategies based on these observations are required to address the issue of ATO resistance in patients with relapsed APL.Abstract 3554. TableCharacteristic featuresNB4 naïveNB4EV-AsR1NB4EV-AsR2NB4EV-AsR3UF1Sensitivity to ATO (IC 50 -µM)0.93.093.442.884.1Differentiation with ATRA exposure (1uM for 72hrs) (n=4)(CD11b% expression) (mean±SD)49.2±7.340.1±3.012.4±2.530.5±2.60.57±0.23Sensitivity to other chemotherapy drugs (IC50) (n=3)a) Daunorubicin(µM)b)Cytosine arabinoside (µM)0.14 8.30.22 16.50.19 4.70.2 13.10.18 NAMRP4 expression (Fold difference)14.23.84.2NAReactive oxygen species (ROS) levels (MFI Fold difference normalized to NB4 cells) (n=3)10.740.860.680.3Glutathionine levels measured by flowcytometry (MFI Fold difference normalized to NB4 cells) (n=3)11.371.451.390.5 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Preclinical Studies of the IAP Antagonist Debio 1143 in Combination with Cytarabine or Doxorubicin in a Mouse Model of AML

Background: Relapse and drug resistance remains the major cause of therapy failure in Acute Myeloid Leukemia (AML). Inhibitors of apoptosis proteins (IAPs) are a family of proteins that suppress proapoptotic signaling and represent attractive targets to overcome chemotherapy resistance. Debio 1143 (a.k.a. AT-406) is an orally-active IAP antagonist able to promote apoptosis alone and/or in combination with chemotherapy. We recently reported the results of a Phase 1 study of Debio 1143 in combination with daunorubicin and cytarabine (araC) in poor-risk AML patients (DiPersio et al. J Clin Oncol 32:5s, 2014, a7029). Although Debio 1143 showed good tolerability and could be safely combined with chemotherapy, 62% (8/13) of patients with complete remissions relapsed within the study period. Here, we used a preclinical AML model to begin examining the effects of BM stroma on the efficacy of Debio 1143 combined with araC or doxorubicin (dox).Methods: We generated murine acute promyelocytic leukemia (APL) cell lines that are resistant to araC and dox both in vitro and in vivo by chronically exposing APL cells to increasing concentrations of drug in vitro. This APL model was previously generated by knocking in the human PML-RARa cDNA into the cathepsin G locus (Westervelt et al. Blood, 2003). We used WST-1 cell proliferation assays and bioluminescence imaging (BLI) to evaluate the efficacy of Debio 1143 to induce apoptosis alone and in combination with araC or dox in vitro in chemotherapy sensitive and resistant APL cells in the presence and absence of M2-10B4 stromal cells. Dox or araC was provided for (a) a 6h period initiated 24h before Debio 1143 (i.e., with an intervening 18h washout of the chemotherapy), (b) simultaneously with Debio 1143, or (c) 6h after Debio 1143. To assess Debio 1143 in vivo, we injected APL cells into syngeneic mice. In our first experiment, mice (n=5/cohort) were treated with PBS, araC (500 mg/kg, s.c.), dox (50 mg/kg, s.c.), Debio 1143 (100 mg/kg, p.o.) or their combination on days 5, 6, 12, and 13 after APL injection. In our second experiment, mice (n=8/cohort) were treated with PBS, araC (200 mg/kg, s.c.), dox (10 mg/kg, s.c.), Debio 1143 (100 mg/kg, p.o.) or their combination on days 3 to 7 and 10 to 16 after APL injection. In both experiments, Debio 1143 was given 2 hours prior to chemotherapy.Results: Figure 1 shows that coculture of APL cells with stromal cells protects them from araC-induced killing (Figure 1A) and that our araC-resistant cells are indeed resistant to araC (Figure 1B). Treatment with Debio 1143 alone has no effect on parental or araC-resistant APL survival in the presence or absence of stroma cells. However, Debio 1143 in combination with araC killed the parental APL cells and overcame the stroma cell-mediated chemotherapy protection (Figure 1A). Although combination therapy partially inhibited araC-resistant APL cell proliferation in the absence of stroma, it had no effect when the cells were cultured in the presence of stroma (Figure 1B). We obtained similar results with dox and dox-resistant cells (data not shown). This in vitro data with the parental APL cell line was confirmed in vivo. Combination therapy with chemotherapy (araC or dox) and Debio 1143 for 4 days (EXP#1) or 10 days (EXP#2) significantly prolonged the overall survival of mice by 4 to 7 days compared to single agent therapy (Debio 1143, araC, or dox) in this aggressive tumor model (Table 1). Preliminary in vitro data suggests that pretreatment of parental or araC-resistant APL cells with chemotherapy (araC or dox) enhances their killing by Debio 1143 compared to simultaneous treatment.Conclusions: Debio 1143 combined with chemotherapy inhibits parental and chemotherapy-resistant APL growth in vitro and in vivo. Future mechanistic studies are required to determine how Debio 1143 can overcome stroma-mediated resistance. [Display omitted] [Display omitted] DisclosuresRettig:Debiopharm International SA: Research Funding. Vuagniaux:Debiopharm International SA: Employment. Zanna:Debiopharm International SA: Employment. DiPersio:Debiopharm International SA: Research Funding.

Abstract 2291: Arsenic trioxide induces cell cycle arrest, apoptosis and MAPKinase signaling cascade in acute promyelocytic leukemia cells

Abstract Arsenic trioxide (ATO) has recently been successfully used in the treatment of all-trans retinoic acid resistant relapsing acute promyelocytic leukemia (APL) patients both alone or combination with other drugs. Its use as induction and consolidation therapy has resulted in complete remission rate of both de novo and relapsed APL patients. However, the detailed molecular mechanisms of its therapeutic action are poorly known. We have used human leukemia (HL-60) cells as a model to elucidate the anti-cancer properties of arsenic trioxide. We hypothesized that ATO arrests cell cycle progression of HL-60 cells at G1 phase and leading to cell death by intrinsic pathway of apoptotic signaling. To test the hypothesis, we used western blotting, confocal imaging and spectrofluoremetric techniques to identify detailed cellular and molecular mechanisms of ATO action in HL-60 cells. We found that the expression levels of pro-apoptotic molecules (Bax) and cytochrome C were up-regulated, while that of anti-apoptotic Bcl2 protein was down-regulated at 4 and 6mg/mL ATO doses significantly. We also found that ATO stimulates signaling molecules like P38, JNK in a dose dependent manner in HL-60 cells. Oxidative stress and related imbalance of pro- and anti-apoptotic molecules lead to change of mitochondrial membrane potential and opening of the mitochondrial membrane transition pores. Our confocal imaging data shows a clear translocation of Bax from cytosol to mitochondria and of cytochrome c from mitochondria to cytosol in ATO-treated cells. A dose-dependent decrease/drop-down in mitochondrial membrane potential was also observed in ATO-treated cells, as evidenced by JC-1 leveling and spectrofluoremetric potential recording. On the basis of these findings, we conclude that mitochondrial pathway of apoptosis plays a key role in ATO pharmacology and should be further assessed as a drug target in APL chemotherapy. Citation Format: Sanjay Kumar, Clement Yedjou, Paul B. Tchounwou. Arsenic trioxide induces cell cycle arrest, apoptosis and MAPKinase signaling cascade in acute promyelocytic leukemia cells. [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 2291. doi:10.1158/1538-7445.AM2014-2291

Evaluation of meisoindigo, an indirubin derivative: in vitro antileukemic activity and in vivo pharmacokinetics.

Meisoindigo has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia(CML) in China since the 1980s. In the present study, the invitro antileukemic activity of meisoindigo was investigated in acute promyelocytic leukemia (APL) cells, acute myeloid leukemia(AML) cells, and myelomonocytic leukemia cells (NB4, NB4.007/6, HL-60 and U937) comprising both retinoic acid-sensitive and retinoic acid-resistant cells. We found that meisoindigo effectively inhibited the growth and/or proliferation of these four cell types at µM levels. The effects of meisoindigo in these cells are related to its proliferation inhibition and apoptosis induction, and are independent of cell cycle arrest, indicating that meisoindigo could be possible in the treatment of APL, AML and retinoic acid resistant APL. The invivo pharmacokinetics of meisoindigo and its major circulatory metabolites in rat plasma were then investigated by a newly developed and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The profiles of plasma concentration versus time were plotted and the relevant pharmacokinetic parameters were calculated for meisoindigo and its reductive metabolites. The plasma concentrations of meisoindigo after oral administration were much lower than the invitro IC50s determined in the leukemic cells. The contradicting poor pharmacokinetic characteristics and the established clinical efficacy of meisoindigo could indicate the presence of active metabolites invivo.

Combined staurosporine and retinoic acid induces differentiation in retinoic acid resistant acute promyelocytic leukemia cell lines.

All-trans retinoic acid (ATRA) resistance has been a critical problem in acute promyelocytic leukemia (APL) relapsed patients. In ATRA resistant APL cell lines NB4-R1 and NB4-R2, the combination of staurosporine and ATRA synergized to trigger differentiation accompanied by significantly enhanced protein level of CCAAT/enhancer binding protein ε (C/EBPε) and C/EBPβ as well as the phosphorylation of mitogen-activated protein (MEK) and extracellular signal-regulated kinase (ERK). Furthermore, attenuation of the MEK activation blocked not only the differentiation but also the increased protein level of C/EBPε and C/EBPβ. Taken together, we concluded that the combination of ATRA and staurosporine could overcome differentiation block via MEK/ERK signaling pathway in ATRA-resistant APL cell lines.

Inhibition Of Nuclear Transport Modulator Xpo1/CRM1 For The Treatment Of Acute Myeloid Leukemia (AML)

BackgroundExportin 1 (XPO1) also called CRM1, is a widely expressed nuclear export protein, transporting a variety of molecules including tumor suppressor proteins and cell cycle regulators. Targeted inhibition of XPO1 is a new strategy to restore multiple cell death pathways in various malignant diseases. SINEs are novel, orally available, small molecule Selective Inhibitors of Nuclear Export (SINE) that specifically bind to XPO1 and inhibit its function. MethodsWe used WST-1 cell proliferation assays, flow cytometry, and bioluminescence imaging to evaluate the efficacy of multiple SINEs to induce apoptosis alone and in combination with cytarabine (AraC) or doxorubicin in vitro in chemotherapy sensitive and resistant murine acute promyelocytic leukemia (APL) cells. This murine model of APL was previously generated by knocking in the human PML-RARa cDNA into the 5' regulatory sequence of the cathepsin G locus (Westervelt et al. Blood, 2003). The abnormal co-expression of the myeloid surface antigen Gr1 and the early hematopoietic markers CD34 and CD117 identify leukemic blasts. These Gr1+CD34+CD117+ APL cells partially retain the ability to terminally differentiate toward mature granulocytes (mimicking more traditional AML models) and can be adoptively transferred to secondary recipients, which develop a rapidly fatal leukemia within 3 weeks after tumor inoculation. To assess the safety and efficacy of SINEs in vivo, we injected cryopreserved APL cells intravenously via the tail vein into unconditioned genetically compatible C57BL/6 recipients and treated leukemic and non-leukemic mice (n=15/cohort) with 15 mg/kg of the oral clinical staged SINE KPT-330 (currently in Phase 1 studies in patients with solid tumors and hematological malignancies) alone or in combination with 200 mg/kg cytarabine every other day for a total of 2 weeks. Peripheral blood was obtained weekly from mice for complete blood counts and flow cytometry to screen for development of APL. ResultsThe first generation SINE, KPT214, inhibited the proliferation of murine APL cell lines in a dose and time dependent manner with IC50 values ranging from of 95 nM to 750 nM. IC50 values decreased 2.4-fold (KPT-185) and 3.5-fold (KPT-249) with subsequent generations of the SINEs. Consistent with the WST-1 results, Annexin V/7-aminoactinomycin D flow cytometry showed a significant increase of APL apoptosis within 6 hours of KPT-249 application. Minimal toxicity against normal murine lymphocytes was observed with SINEs even up to doses of 500 nM. Additional WST-1 assays using AraC-resistant and doxorubicin-resistant APL cell lines demonstrated cell death of both chemotherapy-resistant cell lines at levels comparable to the parental chemosensitive APL cell lines. Combination therapy with low dose KPT-330 and AraC showed additive effects on inhibition of cell proliferation in vitro. This additive effect of KPT-330 and chemotherapy on APL killing was maintained in vivo. As shown in Figure 1, treatment with AraC or KPT-330 alone significantly prolonged the survival of leukemic mice from a median survival of 24 days (APL + vehicle) to 33 days or 39 days, respectively (P < 0.0001). Encouragingly, combination therapy with AraC + KPT-330 further prolonged survival compared to monotherapy (P < 0.0001), with some mice being cured of the disease. Similar in vivo studies with the AraC-resistant and doxorubicin-resistant APL cells are just being initiated. [Display omitted] ConclusionsOur data suggests that the addition of a CRM1 inhibitor to a chemotherapy regimen offers a promising avenue for treatment of AML. Disclosures:Shacham:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. McCauley:Karyopharm Therapeutics, Inc: Employment, Equity Ownership.

Oridonin Upregulates All-Trans Retinoic Acid Receptor Alpha and Induces Differentiation Of NB4 Cells Though NF-Kb Pathway

Nuclear retinoic acid receptors (RARs) function as ligand-dependent transcriptional regulators, often heterodimerized with retinoid X receptors. They play critical roles in a variety of biological processes, including development, differentiation and homeostasis. However, it is still not very clear about the mechanism of RARα catabolism. Almost all cases of acute promyelocytic leukemia (APL) express oncogenic fusion protein involving the RARα due to chromosome translocations. Despite the great success of ATRA therapy, it is still important to search for a variety of promising compounds to enhance the pharmacologic activity of ATRA and alleviate its adverse effects. And some of these compounds may be useful as a probe to investigate the mechanism of RARα catabolism.Our previous studies showed that oridonin (Ori), a diterpenoid isolated from Rabdosia rubescens, could have an ability to accumulate RARα, enhance ATRA-induced differentiation in ATRA-sensitive as well as -resistant APL cells, and induce apoptosis which is initiated by reactive oxygen species (ROS) production. Based on those discoveries, we supposed that inhibition of RARα degradation may be responsible for these serial actions. Herein, we used Ori as a probe to explore how Ori stabilized RARα and its potent mechanisms. Firstly, our results showed that Ori upregulated RARa in a dose- and time- dependent manner in NB4 cells while anti-oxidant agent N-acetyl-L-cysteine (NAC) addition completely blocked this effect, indicating that RARa was stabilized via Ori-induced intracellular ROS formation. Secondly, we found H2O2, which could increase ROS level also, upregulated RARa in a dose- and time- dependent manner. More of interest, we found that both Ori and H2O2 upregulated RARa through NF-kB-signaling, proved by the accumulate evidence that either NF-κB pathway inhibitor or knockdown of P65 or inhibition IkBa phosphorylation could block the RARa stabilization. Whereas, P65 overexpression or knockdown of IkBa or some NF-κB pathway activating agents like tumor necrosis factor alpha (TNFa) and lipopolysaccharide ( LPS) could increase the RARa level.Collectively, these findings suggested that Ori is an extraordinary tool to enhance the pathophysiological and potential therapeutic roles of RARα expression though NF-kB pathway. Moreover, an approach combining ATRA with Ori warrants further investigation as a therapeutic strategy for APL patients. Disclosures:No relevant conflicts of interest to declare.

Effect of flavonoids of puerarin on the proliferation and apoptosis of retinoic acid resistant acute promyelocytic leukemia cell line NB4-R1 cells

Effect of flavonoids of puerarin on the proliferation and apoptosis of retinoic acid resistant acute promyelocytic leukemia cell line NB4-R1 cells

Natural products-based chemical biology research on leukemic cell differentiation

Natural products are structurally diverse and novel, and they often possess unique biological activities. Therefore, natural compounds can serve as an important tool for chemical biology research and a great resource of innovative drugs and lead compounds. Based on the discovery that natural compound adenanthin induces differentiation of ATRA-sensitive and resistant acute promyelocytic leukemia (APL) cells in vivo and in vitro as well as prolongs the survival of APL leukemic mice. Recently we apply adenanthin as a chemical probe to identify its potential targets by cell extract-based pull-down assay and find that the adenanthin specifically targets peroxiredoxin I/II proteins and thus induces H2O2 accumulation, and the signal axsis involved in adenanthin-induced differentiation is explored as well. This minireview attempts to summarize these results and give prospect to its future development.

Lithium Treatment Potentiates Both in Vitro and in Vivo Retinoic Acid Efficacy in APL.

AbstractAbstract 2614We previously demonstrated that although retinoic acid (RA) has targeted efficacy in Acute Promyelocytic Leukemia (APL), heterogeneity exists leading to the appearance of un-targeted clones at the time of relapse. Characterization of these clones is not yet fully unraveled though we and others have previously highlighted the roles of RARα mutations, pharmacogenomics or APL miRNome. We recently identified that the ERK1/2 pathway synergized with RA to restore the transcriptional activity of RA in resistant APL cells, thus restoring RA induced differentiation (Cassinat et al. Mol Cell Biol 2011). These results suggest that targeting interconnected signaling pathways could optimize differentiation therapy efficacy. To this effect, we studied known signaling pathway activators or inhibitors that could potentiate with RA and identified Lithium chloride (LiCl).Treatment of the ATRA sensitive-APL NB4 cell line with LiCl (25mM) decreases proliferation and increases apoptosis (25% and 40% of Annexin V-positive cells at day 1 and 2 respectively) with evidence of caspase 3 cleavage at day 2. Because NB4 cells fully differentiated with RA alone we were unable to observe any synergy when combined with LiCl. Treatment of the RA-resistant APL UF-1 cell line with RA or LiCl alone does not induce differentiation. Combination of RA+LiCl restores differentiation after 3 days of culture (65% CD11b positive and 55% NBT test positive cells). Similar results were obtained with different GSK3 inhibitors, suggesting that the LiCL effects were in part linked to its well characterized GSK3 inhibitory activity. Interestingly, we noted that LiCl treatment induces rapid phosphorylation of ERK1/2 and pretreatment with the MEK/ERK1/2 inhibitor UO126 fully abolished the differentiation induced by the RA+LiCl combination. The combination restores in UF-1 the expression of RA target genes (such as RARα2) to the same levels obtained in NB4 cells treated by RA alone. The level of luciferase activity of an RA responsive element reporter gene was increased with the RA+LiCl combination compared to RA alone. Both target gene expression and luciferase activiy were abolished after inhibition of the MEK/ERK1/2 pathway. Thus, increase in differentiation of UF-1 cells by RA+LiCl is linked to increased RA transcriptional activation. Similar studies in fresh APL patient cells confirmed both the increase in differentiation and level of RA target gene expression and their inhibition by UO126. Finally, to translate these findings in vivo, we used the APL-transplantable mouse model. Plasma lithium levels in treated mice were measured between 0.6 and 1.05 mmol/l, levels reached in humans. When LiCl was combined with RA we repeatedly observed a pronounced survival advantage compared to mice treated by RA alone as evaluated by Kaplan Meier analysis.In this work we demonstrate that LiCl, a well tolerated agent in humans, has the potential, when combined with RA, to restore RA induced transcriptional activation and differentiation in RA resistant APL cells. Furthermore, this combination also increases RA efficacy in an in vivo APL mouse model. Disclosures:Off Label Use: Lithium is a mood modulator administered for bipolar disorders.

Inhibition of mitotic kinase Aurora suppresses Akt-1 activation and induces apoptotic cell death in all-trans retinoid acid-resistant acute promyelocytic leukemia cells

BackgroundAurora kinase ensures accurate chromosome segregation during cell cycle, maintaining genetic integrity in cell division. VX-680, a small-molecule Aurora kinase inhibitor, interferes with mitotic entry and formation of bipolar spindles. Here, we evaluated VX-680 as a potential agent for treatment of all-trans retinoid acid (ATRA)-resistant acute promyelocytic leukemia (APL) in vitro.MethodsCD11b expression was utilized to assess cell differentiation by flow cytometry. Immunofluorescence staining was conducted to analyze formation of cell monopolar spindle. Cell proliferation was evaluated by MTT assay. Sub-G1 population and Annexin V/PI staining were used to measure cell apoptosis. Hoechst 33342 staining was applied for identifying morphological changes in nucleus of apoptotic cell. Aurora-A (Aur-A) activation and the signaling pathways involved in apoptosis were detected by Western blot. JC-1 probe was employed to measure mitochondrial depolarization.ResultsVX-680 inhibited Aur-A by reducing autophosphorylation at the activation site, Thr288, accompanied by producing monopolar mitotic spindles in APL cell line NB4-R2 that was resistant to ATRA. In addition, we found that VX-680 inhibited cell proliferation as assessed by MTT assay. Flow cytometry showed that VX-680 led to apoptotic cell death in both dose- and time-dependent manners by either Sub-G1 or Annexin V/PI analysis. Hoechst 33342 staining represented typical apoptotic cells with nuclear fragmentation in VX-680 treated cells. Importantly, VX-680 inhibition of Aurora kinase suppressed Akt-1 activation and induced mitochondrial depolarization, which eventually resulted in apoptosis by activation of caspase pathway, as indicated by increasing proteolytic cleavage of procaspase-3 and poly ADP ribose polymerase (PARP) in NB4-R2 cells.ConclusionsOur study suggested potential clinical use of mitotic Aurora kinase inhibitor in targeting ATRA-resistant leukemic cells.

Development of real-time quantitative polymerase chain reaction assays to track treatment response in retinoid resistant acute promyelocytic leukemia.

Molecular detection of minimal residual disease (MRD) has become established to assess remission status and guide therapy in patients with ProMyelocytic Leukemia–RARA+ acute promyelocytic leukemia (APL). However, there are few data on tracking disease response in patients with rarer retinoid resistant subtypes of APL, characterized by PLZF–RARA and STAT5b–RARA. Despite their rarity (<1% of APL) we identified 6 cases (PLZF–RARA, n = 5; STAT5b–RARA, n = 1), established the respective breakpoint junction regions and designed reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) assays to detect leukemic transcripts. The relative level of fusion gene expression in diagnostic samples was comparable to that observed in t(15;17) – associated APL, affording assay sensitivities of ∼1 in 104−105. Serial samples were available from two PLZF–RARA APL patients. One showed persistent polymerase chain reaction positivity, predicting subsequent relapse, and remains in CR2, ∼11 years post-autograft. The other, achieved molecular remission (CRm) with combination chemotherapy, remaining in CR1 at 6 years. The STAT5b–RARA patient failed to achieve CRm following frontline combination chemotherapy and ultimately proceeded to allogeneic transplant on the basis of a steadily rising fusion transcript level. These data highlight the potential of RT-qPCR detection of MRD to facilitate development of more individualized approaches to the management of rarer molecularly defined subsets of acute leukemia.

Comparative proteome study of apoptosis induced by As 4S 4 in retinoid acid resistant human acute promyelocytic leukemia NB4-R1 cells

Tetra-arsenic tetra-sulfide (As 4S 4), with improved toxicity profiles relative to arsenic trioxide, is the essential component of the new oral arsenic formulation which is highly effective and safe in the treatment of both newly diagnosed acute promyelocytic leukemia (APL) and more importantly relapsed/refractory APL. Although it is investigated that the therapeutic action of As 4S 4 is closely associated with its induced cells apoptosis, the definitive systematic molecular mechanism of action of As 4S 4 in APL therapy is still remained unknown. In this study, a serial of assays in vitro about the cytotoxicity of As 4S 4 and cellular apoptotic evidences were done, then a proteomic investigation with the high-resolution two-dimensional electrophoresis system and mass spectrometry were performed to obtain for the first time systematic identification and characterization of the global proteome of apoptosis induced by As 4S 4 in retinoic acid (RA)-resistant cells. Among them, expressional and functional regulations of target proteins SET, RPP2 and PHB might be the potential novel effective therapeutic strategies for RA-resistant APL. This study will not only facilitate to understand the signal transduction of apoptosis of RA-resistant APL cells induced by As 4S 4 as a whole, but also is important to screen for drug targets as a new therapeutic strategy for hematopoietic malignant tumors.

Apoptosis inducing and differentiation enhancement effect of oridonin on the all‐trans‐retinoic acid‐sensitive and ‐resistant acute promyelocytic leukemia cells

We investigated the effects of oridonin (Ori), a diterpenoid isolated from Rabdosia rubescens, on apoptosis and differentiation of all-trans-retinoic acid (ATRA)-sensitive (NB4) and ATRA-resistant (NB4-R1) cells. The results showed that reactive oxygen species initiates Ori-induced apoptosis. In addition, we found that neither Ori nor ATRA (10 nM) alone induced marked cell differentiation, while co-treatment of these two compounds can induce differentiation of NB4 and NB4-R1 cells which was accompanied by increased RARalpha, C/EBPepsilon or C/EBPbeta. This is the first report to show that RARalpha could be accumulated by Ori which may be useful as a probe to investigate the mechanism of RARalpha catabolism. These results suggest that Ori is a potential candidate for acute promyelocytic leukemia cancer therapy.

Modulation of p53, c-fos, RARE, cyclin A, and cyclin D1 expression in human leukemia (HL-60) cells exposed to arsenic trioxide.

Arsenic trioxide (As(2)O(3)) has recently been successfully used to treat all trans retinoic acid (ATRA) resistant relapsing acute promyelocytic leukemia. However, its molecular mechanisms of action are poorly understood. In the present study, we used the human leukemia (HL-60) cell line as a test model to study the cellular and molecular mechanisms of anti-cancer properties of As(2)O(3). We hypothesized that As(2)O(3)-induced expression of stress genes and related proteins may play a role in the cellular and molecular events leading to cell cycle modulation in leukemic cells. To test this hypothesis, we performed Western blot analysis to assess the expression of specific cellular response proteins including p53, c-fos, RARE, Cyclin A, and Cyclin D1. Densitometric analysis was performed to determine the relative abundance of these proteins. Western Blot and densitometric analyses demonstrated a strong dose-response relationship with regard to p53 and RARE expression within the dose-range of 0-8 microg/ml. Expression of c-fos was slightly up-regulated at 2 microg/ml, and down-regulated within the dose-range of 4-8 microg/ml. A statistically significant down-regulation of this protein was detected at the 6 and 8 microg/ml dose levels. No statistically significant differences (p > 0.05) in Cyclin D1 expression was found between As(2)O(3)-treated cells and the control. Cyclin A expression in As(2)O(3)-treated HL-60 cells was up-regulated at 6 microg/ml, suggesting that it is required for S phase and passage through G(2) phase in cell cycle progression. Taken together, these results indicate that As(2)O(3) has the potential to induce cell cycle arrest through activation of the 53-kDa tumor suppressor protein and repression of the c-fos transcription factor. Up-regulation of RARE by As(2)O(3) indicates that its cytotoxicity may be mediated through interaction/binding with the retinoic acid receptor, and subsequent inhibition of growth and differentiation.

Adenanthin Upregulates c/ebp β Expression with Induction of Differentiation of the ATRA-Sensitive and -Resistant leukemic Cells.

Application of all-trans retinoic acid (ATRA) has significantly improved the outcome of patients with acute promyelocytic leukemia (APL), a unique subtype of acute myeloid leukemia (AML) characterized with the reciprocal translocation t(15;17) that results in the expression of leukemia-promoting promyelocytic leukemia-retinoic acid receptor-α (PML-RARα) protein. However, most patients usually relapsed in a short period and followed by generation of retinoid resistance. Therefore, it is imperative to explore agents to overcome the resistance. In this work, we found that adenanthin, a diterpenoid compound extracted from medicinal herbs, could induce monocytic differentiation of ATRA-sensitive APL-derived NB4 cells. Especially, the differentiation-inducing effect could also be seen in the agent-treated ATRA-resistant NB4-LR1, NB4-LR2 cells, as evidenced by the morphology and the increased expression of cell surface differentiation antigens CD11b and CD14. Based on this discovery, we also found that adenanthin significantly upregulated the expression of CCAAT enhancer–binding protein beta (C/EBPβ), but not the C/EBPα and C/EBPε at its transcription and protein levels, which was accompanied by the activation of c-Jun N-terminal kinases signal pathway. On the other hand, silence of C/EBPβ by specific small-interfering RNA abrogated the adenanthin induced differentiation in NB4 cells. All these data proposed the role of C/EBPβ in adenanthin induced differentiation. Furthermore, we investigated the potential combinational effects of the natural compound with other known differentiation-inducing agents including ATRA and 1, 25-dihydroxy vitamin D3. The results demonstrated that adenanthin strikingly enhanced ATRA (10nM) induced granulocyte differentiation and 1, 25-dihydroxy vitamin D3 (1nM) induced monocytic differentiation. These results suggest that adenanthin may be a potential antileukemia agent for the ATRA-sensitive and -resistant APL and deserves to be further investigated in the future.

RARα-PLZF overcomes PLZF-mediated repression of CRABPI , contributing to retinoid resistance in t(11;17) acute promyelocytic leukemia

Leukemia-associated chimeric oncoproteins often act as transcriptional repressors, targeting promoters of master genes involved in hematopoiesis. We show that CRABPI (encoding cellular retinoic acid binding protein I) is a target of PLZF, which is fused to RARalpha by the t(11;17)(q23;q21) translocation associated with retinoic acid (RA)-resistant acute promyelocytic leukemia (APL). PLZF represses the CRABPI locus through propagation of chromatin condensation from a remote intronic binding element culminating in silencing of the promoter. Although the canonical, PLZF-RARalpha oncoprotein has no impact on PLZF-mediated repression, the reciprocal translocation product RARalpha-PLZF binds to this remote binding site, recruiting p300, inducing promoter hypomethylation and CRABPI gene up-regulation. In line with these observations, RA-resistant murine PLZF/RARalpha+RARalpha/PLZF APL blasts express much higher levels of CRABPI than standard RA-sensitive PML/RARalpha APL. RARalpha-PLZF confers RA resistance to a retinoid-sensitive acute myeloid leukemia (AML) cell line in a CRABPI-dependent fashion. This study supports an active role for PLZF and RARalpha-PLZF in leukemogenesis, identifies up-regulation of CRABPI as a mechanism contributing to retinoid resistance, and reveals the ability of the reciprocal fusion gene products to mediate distinct epigenetic effects contributing to the leukemic phenotype.

Effects of Celecoxib on Human Acute Promyelocytic Leukemia Cell Line and Its Mechanism.

Acute promyelocytic leukemia (APL) is a special subtype of acute myelogenous leukemia (AML) which is characterized for a specific translocation between chromosome 15 and 17 [t(15;17)] and the expression of PML/RARα fusion gene. Celecoxib, one of the specific inhibitors of cyclooxygenase-2 (COX-2), has been reported to induce anti-neoplastic activity on many solid human tumor cell lines in recent years. In our study, ATRA resistant APL cell line MR2 cells were used to investigate the effects of celecoxib on hematological malignancy. MR2 cells were treated with celecoxib at different concentration (0, 20, 40, 80, 120 and 160μmol/L). The proliferation of MR2 cells was observed by MTT assay and apoptosis was detected by DNA fragmentation analysis and flow cytometry using Annexin V-FITC/PI staining. Western blot was used to detect the change of caspase-8, -9, -3 and PARP in MR2 cells. The expression of mRNA of fusion gene PML/RARα, COX-2 and survivin, bcl-2/bax, CIAP1 and CIAP2 was assessed by reverse transcription polymerase chain reaction (RT-PCR). Cell cycle analyzed by flow cytometry with PI staining and western blot was used to detect the expression of cell-cycle-regulating proteins. The telomerase activity of MR2 cells was analyzed by PCR-ELISA. The expression of hTERT mRNA and c-myc mRNA was assessed by RT-PCR. MR2 cells viability in presence of celecoxib decreased markedly in a dose- and time- dependent manner. After treated with celecoxib (20-160μmol/L) for 12-48h, the proliferation of MR2 cells were inhibited significantly, in comparison with the control group (P<0.01). 50% growth inhibition (IC50) at 24h and 48h was 80.93μmol/L and 71.72μmol/L, respectively. A DNA ladder pattern of internucleosomal fragmentation was observed. The translocation of phosphatidylserine at the outer surface of the cell plasma membrane could be induced by celecoxib and its level increased following the augmentation of the drug concentration. MR2 cells exposure to 40-160μmol/L celecoxib for 24h caused 9.59%, 24.00%, 36.10% apoptotic cells, which was more than that of the untreated group 2.84% (P<0.01). The expression of survivin mRNA decreased dramatically, while no significant change with PML/RARα and COX-2. Treatment with celecoxib for 24h resulted in the activation of caspase-3 and caspase-9, cleavage of PARP. 40-160μmol/L celecoxib led to cell cycle arrest in G1/S phase, and CyclinD1 and CyclinE decreased, accompanied with up-regulation of P21waf/cip1, P27KIP, P16INK4a. celecoxib could inhibit the telomerase activity of APL cell line, and the inhibition was dose- and time- dependent. The expression of hTERT mRNA and c-myc mRNA were down-regulated by celecoxib in dose- dependent manner. These results indicated that celecoxib could inhibit MR2 cells proliferation by inducing apoptosis, cell cycle arrest and suppression of telomerase activity.