ATRA-resistant Acute Promyelocytic Leukemia Research Articles

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.

CAMP protects acute promyelocytic leukemia cells from arsenic trioxide-induced caspase-3 activation and apoptosis

More recently, arsenic trioxide (ATO), was integrated into acute promyelocytic leukemia (APL) treatment, showing high efficacy and tolerability in patients with both ATRA-sensitive and ATRA-resistant APL. ATO could induce apoptosis at relatively high concentrations (0.5 to 2.0 micromol/L) and partial differentiation at low concentrations (0.1 to 0.5 micromol/L) in leukemic promyelocytes. It is known that cAMP agonists enhance low-dose ATO-induced APL cells differentiation. Less well appreciated was the possible interaction between relatively high-doses of ATO and enhanced levels of cAMP in APL cells. Here, we show that elevation of cAMP levels by forskolin inhibited ATO-mediated apoptosis in APL-derived NB4 cells, and this inhibition could be averted by cell permeable cAMP-dependent protein kinase inhibitor (14–22) amide. Inactivating phosphorylation of the proapoptotic protein Bad at Ser118 and phosphorylation of the CREB proto-oncogene at Ser133 were observed upon elevation of cAMP levels in NB4 cells. Phosphorylation of these PKA target proteins is known to promote cell survival in AML cells. The ability of cAMP to endow the APL cells with survival advantage is of particular importance when cAMP agonists may be considered as adjuncts to APL therapy.

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.

Effect of curcumin combined with ATRA on differentiation of ATRA-resistant acute promyelocytic leukemia cells

In order to investigate the effect of curcumin combined with all-trans retinoid acid (ATRA) on differentiation of ATRA-resistant acute promyelocytic leukemia (APL) cells and its molecular mechanism, the NB4-R1, an ATRA-resistant APL cells, was used as a model, counting of NB4-R1 and cell morphologic observation were performed, the effect of curcumin alone or combined with ATRA on proliferation, differentiation of NB4-R1 cells was detected by flow cytometry (FCM), the change of AKT phosphorylation in cell differentiation was detected by Western blot. The results showed that ATRA had no influence on NB4-R1 cell proliferation, but enhanced the inhibitory effect of curcumin on NB4-R1 cell growth; the curcumin or ATRA alone did not affect NB4-R1 differentiation; curcumin combined with ATRA could obviously induce CD11b expression; the cell morphology showed obvious differentiation characteristics. ATRA could promote phosphorylation of AKT in NB4 cells at short time, but not had effect on phosphorylation of AKT in NB4-R1 cells; the curcumin could enhance the phosphorylation of AKT in NB4-1R cells, the curcumin combined with ATRA could further enhance the phosphorylation of AKT. It is concluded that PI3K/AKT pathway inactivation may be one of the factors of drug resistance in APL and curcumin promotes differentiation of NB4-R1 through activating PI3K/AKT pathway.

The Histone Demethylase PHF8 Governs Retinoic Acid Response in Acute Promyelocytic Leukemia

While all-trans retinoic acid (ATRA) treatment in acute promyelocytic leukemia (APL) has been the paradigm of targeted therapy for oncogenic transcription factors, the underlying mechanisms remain largely unknown, and a significant number of patients still relapse and become ATRA resistant. We identified the histone demethylase PHF8 as a coactivator that is specifically recruited by RARα fusions to activate expression of their downstream targets upon ATRA treatment. Forced expression of PHF8 resensitizes ATRA-resistant APL cells, whereas its downregulation confers resistance. ATRA sensitivity depends on the enzymatic activity and phosphorylation status of PHF8, which can be pharmacologically manipulated to resurrect ATRA sensitivity to resistant cells. These findings provide important molecular insights into ATRA response and a promising avenue for overcoming ATRA resistance.

Cyclic AMP can promote APL progression and protect myeloid leukemia cells against anthracycline-induced apoptosis

We show that cyclic AMP (cAMP) elevating agents protect blasts from patients with acute promyelocytic leukemia (APL) against death induced by first-line anti-leukemic anthracyclines like daunorubicin (DNR). The cAMP effect was reproduced in NB4 APL cells, and shown to depend on activation of the generally cytoplasmic cAMP-kinase type I (PKA-I) rather than the perinuclear PKA-II. The protection of both NB4 cells and APL blasts was associated with (inactivating) phosphorylation of PKA site Ser118 of pro-apoptotic Bad and (activating) phosphorylation of PKA site Ser133 of the AML oncogene CREB. Either event would be expected to protect broadly against cell death, and we found cAMP elevation to protect also against 2-deoxyglucose, rotenone, proteasome inhibitor and a BH3-only mimetic. The in vitro findings were mirrored by the findings in NSG mice with orthotopic NB4 cell leukemia. The mice showed more rapid disease progression when given cAMP-increasing agents (prostaglandin E2 analog and theophylline), both with and without DNR chemotherapy. The all-trans retinoic acid (ATRA)-induced terminal APL cell differentiation is a cornerstone in current APL treatment and is enhanced by cAMP. We show also that ATRA-resistant APL cells, believed to be responsible for treatment failure with current ATRA-based treatment protocols, were protected by cAMP against death. This suggests that the beneficial pro-differentiating and non-beneficial pro-survival APL cell effects of cAMP should be weighed against each other. The results suggest also general awareness toward drugs that can affect bone marrow cAMP levels in leukemia patients.

The Diterpenoid Lactone Andrographolide Induces Reactive Oxygen Species (ROS) Dependent Apoptosis in ATRA Sensitive and Resistant APL Cell Lines.

Abstract 2446Andrographolide is a crystalline diterpenoid lactone. It consists of an α-alkylidene- g-butyrolactone moiety and three hydroxyls at C-3, C-14 and C-19, which are responsible for its biological activities. It is the major bioactive ingredient of the medicinal plant Andrographis paniculata and it has been used in Asia for a variety of non-malignant conditions. We previously reported that Andrographolide results in mitochondrial-mediated apoptosis in lymphoma cell lines and fresh malignant cells from patients with lymphoma (Yang et al. Clin Cancer Res 2010:16:4755). Based on the mechanism of action in lymphoma and a prior report in APL (Manikam et al. J Pharm Pharmacol 2009:61:9), we hypothesized that andrographolide may have biological activity in acute promyelocytic leukemia (APL) an that this may be related to reactive oxygen species (ROS). We therefore investigated the effects of andrographolide on cell viability, apoptosis induction, mitochondrial membrane poential and signaling pathways in 3 APL cell lines, the ATRA sensitive line NB4 and the ATRA-resistant lines NB4–007/6 and NB4–306 and 3 samples from patients with APL. Methods:NB4 (ATRA sensitive cell line), NB4–007/6 and NB4–306 (ATRA resistant cell lines) were cultured in RPMI-1640 under standard conditions. Cell viability was measured using the trypan blue or propidium iodide exclusion method. Fresh leukemic cells were obtained from 3 patients after informed consent according to an NU IRB approved protocol. One had ATRA-resistant APL and 2 had de-novo untreated APL. We measured apoptosis by Annexin V-FITC by FACS. We measured mitochondrial membrane potential and cell differentiation by standard techniques. Results:Incubation with increasing concentrations of andrographolide demonstrates loss of cell viability as measured by MTT assay. The IC50 at 48 hours was 6uM for NB4–306, 6.5uM for NB4–007/6 and 9uM for NB4. Apoptosis by Annexin V/FACS demonstrated that at 48 hours there was increasing apoptosis in all 3 cell lines and that the ATRA-resistant cell lines NB4–007/6 and NB4–306 were significantly more sensitive to andrographolide than the ATRA sensitive cell line NB4 (p< 0.025). This was accompanied by PARP and caspase 3-cleavage. There was evidence of decrease in mitochondrial membrane potential, but no effect on differentiation as measured by CD11b expression by flow. We next interrogated signaling pathways and found that in the ATRA resistant line NB4–007/6 there was an increase in phosphorylation of the Forkhead box O transcription factors p-FOXO1 at Thr24 and up-regulation of FasL (which peaked at 6 hours) and p27Kip1. We also demonstrated that andrographolide caused N-acetyl L- cysteine (NAC) reversible down regulation of c-MYC (in the ATRA resistant lines) and p-AKT (T308) (in the ATRA sensitive line) expression. In fresh patient specimens (n=3) there was dose dependent increase in apoptosis at 48 hours (>70% at 10uM, 85% at 20uM). From prior reports and our own data we suspected that the effects of andrographolide were dependent on reactive oxygen species (ROS), and indeed apoptosis was completely inhibited by NAC. Conclusion:Taken together, these data suggest that andrographolide, a novel natural diterpenoid lactone with significant biological activity in cancer, may have activity in patients with ATRA-resistant APL by a mechanism of action that is distinct from ATRA. We believe that these data provide a compelling rationale to add this natural diterpenoid lactone to the clinical trial agenda in APL. Disclosures:No relevant conflicts of interest to declare.

Tanshinone IIA in Acute Promyelocytic Leukemia

IntroductionThe prognosis of acute promyelocytic leukemia (APL) has been significantly improved by the use of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, the utility of these drugs is limited by resistance to ATRA and the side effects of ATO. It has been reported that Tanshinone IIA (Tan IIA), a diterpene quinone isolated from Salvia Miltiorrhiza Bunge, induces apoptosis in the APL cell line, NB4. The effect of Tan IIA on the ATRA-resistant APL cell line (MR2) is unknown. MethodsIn this study, the effects of Tan IIA and ATO, alone and in combination, on MR2 cell apoptosis were investigated using transmission electron microscopy, flow cytom-etry and Western blot analyses. ResultsNuclear changes typical of apoptosis were observed in Tan IIA-treated MR2 cells. Apoptosis was shown to be induced in a dose- and time-dependent manner with the activation of caspase-3 resulting in upregulation of tumor necrosis factor-a expression, activation of caspase-8 and alteration in mitochon-drial transmembrane potential with release of cytochrome c (cyto-c). Tan IIA and ATO acted synergistically on the induction of MR2 cell apoptosis. ConclusionsThese data indicate that Tan IIA may be beneficial in the treatment of ATRA-resistant APL and in combination with ATO for APL therapy in the clinic.

Treatment-influenced associations of PML-RARα mutations, FLT3 mutations, and additional chromosome abnormalities in relapsed acute promyelocytic leukemia

AbstractMutations in the all-trans retinoic acid (ATRA)–targeted ligand binding domain of PML-RARα (PRα/LBD+) have been implicated in the passive selection of ATRA-resistant acute promyelocytic leukemia clones leading to disease relapse. Among 45 relapse patients from the ATRA/chemotherapy arm of intergroup protocol C9710, 18 patients harbored PRα/LBD+ (40%), 7 of whom (39%) relapsed Off-ATRA selection pressure, suggesting a possible active role of PRα/LBD+. Of 41 relapse patients coanalyzed, 15 (37%) had FMS-related tyrosine kinase 3 internal tandem duplication mutations (FLT3-ITD+), which were differentially associated with PRα/LBD+ depending on ATRA treatment status at relapse: positively, On-ATRA; negatively, Off-ATRA. Thirteen of 21 patients (62%) had additional chromosome abnormalities (ACAs); all coanalyzed PRα/LBD mutant patients who relapsed off-ATRA (n = 5) had associated ACA. After relapse Off-ATRA, ACA and FLT3-ITD+ were negatively associated and were oppositely associated with presenting white blood count and PML-RARα type: ACA, low, L-isoform; FLT3-ITD+, high, S-isoform. These exploratory results suggest that differing PRα/LBD+ activities may interact with FLT3-ITD+ or ACA, that FLT3-ITD+ and ACA are associated with different intrinsic disease progression pathways manifest at relapse Off-ATRA, and that these different pathways may be short-circuited by ATRA-selectable defects at relapse On-ATRA. ACA and certain PRα/LBD+ were also associated with reduced postrelapse survival.

Altered nuclear cofactor switching in retinoic‐resistant variants of the PML‐RARα oncoprotein of acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) results from a reciprocal translocation that fuses the gene for the PML tumor suppressor to that encoding the retinoic acid receptor alpha (RARα). The resulting PML-RARα oncogene product interferes with multiple regulatory pathways associated with myeloid differentiation, including normal PML and RARα functions. The standard treatment for APL includes anthracycline-based chemotherapeutic agents plus the RARα agonist all-trans retinoic acid (ATRA). Relapse, which is often accompanied by ATRA resistance, occurs in an appreciable frequency of treated patients. One potential mechanism suggested by model experiments featuring the selection of ATRA-resistant APL cell lines involves ATRA-resistant versions of the PML-RARα oncogene, where the relevant mutations localize to the RARα ligand-binding domain (LBD). Such mutations may act by compromising agonist binding, but other mechanisms are possible. Here, we studied the molecular consequence of ATRA resistance by use of circular dichroism, protease resistance, and fluorescence anisotropy assays employing peptides derived from the NCOR nuclear corepressor and the ACTR nuclear coactivator. The consequences of the mutations on global structure and cofactor interaction functions were assessed quantitatively, providing insights into the basis of agonist resistance. Attenuated cofactor switching and increased protease resistance represent features of the LBDs of ATRA-resistant PML-RARα, and these properties may be recapitulated in the full-length oncoproteins.

Targeting Expression of the Leukemogenic PML-RARα Fusion Protein by Lentiviral Vector-Mediated Small Interfering RNA Results in Leukemic Cell Differentiation and Apoptosis

Acute promyelocytic leukemia (APL) results from a chromosomal translocation that gives rise to the leukemogenic fusion protein PML-RARα (promyelocytic leukemia-retinoic acid α receptor). Differentiation of leukemic cells and complete remission of APL are achieved by treatment of patients with pharmacological doses of all-trans retinoic acid (ATRA), making APL a model disease for differentiation therapy. However, because patients are resistant to further treatment with ATRA on relapse, it is necessary to develop alternative treatment strategies to specifically target APL. We therefore sought to develop a treatment strategy based on lentiviral vector-mediated delivery of small interfering RNA (siRNA) that specifically targets the breakpoint region of PML-RARα. Unlike treatment with ATRA, which resulted in differentiation of leukemic NB4 cells, delivery of siRNA targeting PML-RARα into NB4 cells resulted in both differentiation and apoptosis, consistent with the specific knockdown of PML-RARα. Intraperitoneal injection of NB4 cells transduced with lentiviral vectors delivering PML-RARα-specific siRNA but not control siRNA prevented development of disease in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Taken together, these results indicate that development of PML-RARα-specific siRNA may represent a promising treatment strategy for ATRA-resistant APL.

Treatment with 5-azacytidine accelerates acute promyelocytic leukemia leukemogenesis in a transgenic mouse model.

A key oncogenic force in acute promyelocytic leukemia (APL) is the ability of the promyelocytic leukemia-retinoic acid receptor α (PML-RARA) oncoprotein to recruit transcriptional repressors and DNA methyltransferases at retinoic acid-responsive elements. Pharmacological doses of retinoic acid relieve transcriptional repression inducing terminal differentiation/apoptosis of the leukemic blasts. APL blasts often harbor additional recurrent chromosomal abnormalities, and significantly, APL prevalence is increased in Latino populations. These observations suggest that multiple genetic and environmental/dietary factors are likely implicated in APL. We tested whether dietary or targeted chemopreventive strategies relieving PML-RARA transcriptional repression would be effective in a transgenic mouse model. Surprisingly, we found that 1) treatment with a demethylating agent, 5-azacytidine, results in a striking acceleration of APL; 2) a high fat, low folate/choline-containing diet resulted in a substantial but nonsignificant APL acceleration; and 3) all-trans retinoic acid (ATRA) is ineffective in preventing leukemia and results in ATRA-resistant APL. Our findings have important clinical implications because ATRA is a drug of choice for APL treatment and indicate that global demethylation, whether through dietary manipulations or through the use of a pharmacologic agent such as 5-azacytidine, may have unintended and detrimental consequences in chemopreventive regimens.

Apoptosis induction by (+)α-tocopheryl succinate in the absence or presence of all-trans retinoic acid and arsenic trioxide in NB4, NB4-R2 and primary APL cells

We analyzed the effect of (+)α-tocopheryl succinate (α-TOS) alone or associated with arsenic trioxide (ATO) or all-trans retinoid acid (ATRA) in acute promyelocytic leukemia (APL). α-TOS-induced apoptosis in APL clinical samples and in ATRA-sensitive (NB4) and ATRA-resistant (NB4-R2) APL cell lines. The effective dose 50% (ED-50) was calculated to be 71 and 58 μM, for NB4 and NB4-R2, respectively. α-TOS neither induced nor modified ATRA-induced differentiation of APL cells, and did not affect the proliferation and differentiation of normal CD34 + hematopoietic progenitors in methylcellulose assays. α-TOS exerted a moderate antagonistic effect to ATO-induced apoptosis when treatment was done simultaneously but when α-TOS was added 24 h after ATO, an additive effect was observed. Our results support the concept of α-TOS as an anti-leukemic compound which spares normal hematopoiesis.

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.

PML-RARα inhibitors (ATRA, tamibaroten, arsenic troxide) for acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by generation of the PML-RARalpha fusion gene. PML-RARalpha can homodimerize with another PML-RARalpha, and the hybrid binds the histone-deacetylase recruiting co-repressor complex with higher affinity than the wild-type RARalpha. However, the co-repressor complex is releasable by pharmacological doses of all-trans retinoic acid (ATRA). More than 90% of patients with APL achieve a complete remission (CR) with differentiation therapy consisting of ATRA combined with chemotherapy. A new synthetic retinoid, tamibaroten, showed therapeutic effectiveness in patients with ATRA-resistant APL with increased expression of cellular retinoic acid binding protein (CRABP), and about 60% of patients with relapsed APL achieved a CR. Arsenic trioxide triggers the rapid degradation of PML-RARalpha through the targeting of the PML moieties of the fusion protein and showed a high CR rate in relapsed APL. The combination of ATRA, chemotherapy, and/or new agents improved the long-term survival in patients with APL.

HSP90 inhibitor 17AAG causes apoptosis in ATRA-resistant acute promyelocytic leukemia cells

The effects of a novel heat shock protein inhibitor, 17AAG, on established APL cell lines (NB4 and R1) were analyzed. 17AAG induces apoptosis in APL cell lines both sensitive (NB4) and resistant (R1) to ATRA after 72 h of incubation. Apoptosis occurs by a mechanism different than ATRA-mediated response, as the cells do not undergo differentiation before apoptosis. Analysis of bax and bcl-2 shows that pro-apoptotic (bax) and anti-apoptotic (bcl-2) proteins are decreased in expression after incubation with 17AAG. We believe this data supports potential clinical use of agents that target HSP90 in APL patients failing conventional therapy.

Designed ATRA analogue active against ATRA-resistant acute promyelocytic leukemia cells having a single nucleotide substitution in their retinoic acid receptor

All- trans retinoic acid (ATRA) induces the differentiation of acute promyelocytic leukemia (APL) cells into neutrophils. UF-1 cells were established from an ATRA-resistant APL patient, and were previously shown to possess a single amino acid (or nucleotide) substitution, Arg276Trp, in their ATRA receptor. In the present research, we designed several ATRA derivatives having a hydrophobic alkyl ketone moiety instead of the negatively charged carboxylic acid moiety. Among them the ethyl ketone derivative, Et-ketone ATRA, was shown to induce the differentiation of UF-1 cells when assessed in terms of intracellular ROS production. It also induced the formation of PML NBs and expression of CD11b antigen marker and p21, transcriptional targets of RARα. Et-ketone ATRA did not induce these phenotypic changes in wild-type APL NB4 cells. Furthermore, we found that Et-ketone ATRA induced apoptosis selectively in UF-1 cells, i.e., not in other leukemic cells. The induction of apoptosis was shown to be partly due to the up-regulation of Bax protein. Thus, Et-ketone ATRA selectively induced differentiation and apoptosis in ATRA-resistant APL UF-1 cells, and is likely to be useful for the clinical treatment of the Arg276Trp-type of ATRA-resistant APL.

Two Patients with All-trans Retinoic Acid-Resistant Acute Promyelocytic Leukemia Treated Successfully with Gemtuzumab Ozogamicin as a Single Agent

Acute promyelocytic leukemia (APL) cells express a considerable level of CD33, which is the target of gemtuzumab ozogamicin (GO), and a significantly lower level of P-glycoprotein (P-gp). Therefore, GO is predicted to be a successful treatment for APL. In this article, we report on the GO treatment of 2 patients with APL, who had fully relapsed after induction therapy with all-trans retinoic acid (ATRA) following chemotherapy. Both patients had relapsed 3 times and were resistant to reinduction therapy with ATRA. GO (9 mg/m2) was administered on days 1 and 15. After GO treatment, both patients achieved complete hematologic and molecular remission. GO may be another promising agent for the treatment of ATRA-resistant relapsed APL when given as salvage chemotherapy.

All Trans Retinoic Acid (atRA) Differentiation Markers in Normal and Retinoid-Resistant Acute Promyelocytic Leukemia Cells Revealed Induction of atRA Metabolism as Relevant Prognostic of APL Sensitivity to Therapy.

It is now well established that all trans Retinoic Acid (atRA), administered at pharmacological doses to Acute Promyelocytic Leukemia (APL) patients, provides the first example of therapy by differentiation. Clinical remission is often transient as resistance develops. Because mechanisms of installation are still unclear, gene expression changes during APL cells differentiation were identified by Serial Analysis of Gene Expression (SAGE). Construction of proliferative and 48 hours atRA-treated NB4 cells libraries allowed us to identify a set of new transcriptional markers.Expression profiles of atRA response were performed on NB4, two atRA-resistant cell lines (NB4-LR2 and UF1) and APL blasts by real time PCR analyze on Microfluidic Card. We choose a hundred genes from multiple functional classes since there are several potential mechanisms for atRA resistance: target gene expression, transcription regulation, atRA metabolism, proteasome pathways≡ Reliability between SAGE and Microfluidic technologies is high since 95% of significantly modulated SAGE transcripts (p<0.01) show the same modulation by PCR. Analyzes provide valuable markers of the granulocytic phenotype (ICAM3, S100A9, CYP4F3, TMSB10), relevant and new atRA target genes (HIC1, ID2). Moreover, SAGE highlights chromatin remodeling factors, histone deacetylase (HDAC11) or coactivator (NCOA3) which may play a crucial role in the differentiation process. Finally, we insulate markers that correlate with resistance (CEBPA, CRABP2, NDRG1, CYP26).Transcriptome studies were conducted onto blast of patients with distinct long-term sensitivity, established by correlation to the in vitro differentiation rate (Cassinat, B. et al. Blood, 2001). As a result, all patients show transcriptional response to retinoid. However, once blast differentiation reached, induction of atRA-response element genes in high sensitive blasts is reduced. In opposition, transcripts expression of low sensitive patients is still high, revealing a delay in differentiation establishment. Conversely, expression of cytochrome p450 CYP26, involved in the atRA catabolism, is maintained in highly sensitive blast whereas no modulation is observed in low sensitive blast. Because promoter analysis reveals Homeobox response element, involvement of HOX factors found in SAGE librairies was investigated. In cell lines, HOX factors cooperate with retinoid receptors to increase CYP26 transcription. Furthermore, high-pressure chromatography shows a switch between atRA and its metabolites, 6 hours after atRA addition but only in sensitive cell line.To conclude, CYP26 is a relevant marker of resistance prognostic. Since metabolites show similar efficiency for cell growth inhibition and differentiation than atRA, their implication in resistance installation is investigated.

Retinoid/arsenic combination therapy of promyelocytic leukemia: induction of telomerase-dependent cell death

Acute promyelocytic leukemia (APL) is efficiently treated with a cell differentiation inducer, all-trans retinoic acid (ATRA). However, a significant percentage of patients still develop resistance to this treatment. Recently, arsenic trioxide (As2O3), alone or in combination with ATRA, has been identified as an alternative therapy in patients with both ATRA-sensitive and ATRA-resistant APL. Previous investigations restricted the mechanism of this synergism to the modulation and/or degradation of PML-RARalpha oncoprotein through distinct pathways. In this study, using several ATRA maturation-resistant APL cell lines, we demonstrate in vitro that the success of ATRA/As2O3 treatment in APL pathology can be explained, at least in part, by a synergistic effect of these two drugs in triggering downregulation of telomerase efficient enough to cause telomere shortening and subsequent cell death. Such long-term low-dose combinatorial therapy strategies, developed also to avoid acute side effects, reinforce the notion that the antitelomerase strategy, based on a combination of active agents, should now be considered and evaluated not only in APL but also in other malignancies.