Retinoic Acid-resistant Acute Promyelocytic Leukemia Research Articles

Mechanical phenotyping reveals unique biomechanical responses in retinoic acid-resistant acute promyelocytic leukemia

SummaryAll-trans retinoic acid (ATRA) is an essential therapy in the treatment of acute promyelocytic leukemia (APL), but nearly 20% of patients with APL are resistant to ATRA. As there are no biomarkers for ATRA resistance that yet exist, we investigated whether cell mechanics could be associated with this pathological phenotype. Using mechano-node-pore sensing, a single-cell mechanical phenotyping platform, and patient-derived APL cell lines, we discovered that ATRA-resistant APL cells are less mechanically pliable. By investigating how different subcellular components of APL cells contribute to whole-cell mechanical phenotype, we determined that nuclear mechanics strongly influence an APL cell’s mechanical response. Moreover, decondensing chromatin with trichostatin A is especially effective in softening ATRA-resistant APL cells. RNA-seq allowed us to compare the transcriptomic differences between ATRA-resistant and ATRA-responsive APL cells and highlighted gene expression changes that could be associated with mechanical changes. Overall, we have demonstrated the potential of “physical” biomarkers in identifying APL resistance.

Single Cell Analyses Reveal a Non-Canonical EZH2 Activity As a Main Driver of Retinoic Acid Resistance in PLZF/Rara Leukemia

Resistance to treatment is due to the heterogeneity of the tumor which contains a subset of cancer cells that escape treatment and are responsible for the relapse. Acute Promyelocytic Leukemia (APL), the M3 subtype of AML, is a good model to illustrate these problematics. Indeed, APL driven by oncogenic fusion proteins such PML/RARA t(15;17) or PLZF/RARA t(11;17) behave differently to differentiation therapeutics. Both APLs differentiate in vivo upon Retinoic Acid (RA) treatment; however, while PML/RARA APL patients exhibit partial or complete remission, PLZF/RARA APL patients remain clinically resistant. In the present study we aim to decipher the transcriptional and epigenetic networks that is linked to t(11;17) APL resistance towards RA.We took advantage of the PLZF/RARA RA resistant murine APL model to catch relapse-initiating cell features and their vulnerabilities. By developing an integrative single-cell multi-omics analysis (scRNA-seq and scATAC-seq), we uncovered transcriptional and chromatin heterogeneity of the PLZF/RARA APL blasts. We highlighted a subset of cells insensitive to RA-induced differentiation with a strong DNA repair signature (“Rep” cluster) and exhibiting a fine tuned transcriptional network targeting the histone methyltransferase Ezh2. To validate the function of Ezh2 in APL physiology, we combined epigenomic studies with RA-treated and non-RA-treated bone marrow transplantation experiments. We revealed high Ezh2 activity that marks the relapse of RA-treated APL. However, targeting Ezh2 methyltransferase activity was not sufficient to achieve disease cure and, suggests an independent methyltransferase Ezh2 activity linked to RA resistance.These findings demonstrate the power of single-cell multi-omics integration to highlight path to sensitize therapy-resistant leukemia cells. In addition, our study uncovers a dual role of Ezh2 in APL and suggests that targeting non-canonical Ezh2 activity could be a new promising therapeutic approach for RA resistant APL. DisclosuresIwama: Nissan Chemical Corporation: Research Funding.

Dual origin of relapses in retinoic-acid resistant acute promyelocytic leukemia

Retinoic acid (RA) and arsenic target the t(15;17)(q24;q21) PML/RARA driver of acute promyelocytic leukemia (APL), their combination now curing over 95% patients. We report exome sequencing of 64 matched samples collected from patients at initial diagnosis, during remission, and following relapse after historical combined RA-chemotherapy treatments. A first subgroup presents a high incidence of additional oncogenic mutations disrupting key epigenetic or transcriptional regulators (primarily WT1) or activating MAPK signaling at diagnosis. Relapses retain these cooperating oncogenes and exhibit additional oncogenic alterations and/or mutations impeding therapy response (RARA, NT5C2). The second group primarily exhibits FLT3 activation at diagnosis, which is lost upon relapse together with most other passenger mutations, implying that these relapses derive from ancestral pre-leukemic PML/RARA-expressing cells that survived RA/chemotherapy. Accordingly, clonogenic activity of PML/RARA-immortalized progenitors ex vivo is only transiently affected by RA, but selectively abrogated by arsenic. Our studies stress the role of cooperating oncogenes in direct relapses and suggest that targeting pre-leukemic cells by arsenic contributes to its clinical efficacy.

Tetra-arsenic tetra-sulfide induces cell cycle arrest and apoptosis in retinoic acid-resistant acute promyelocytic leukemia cells.

Previous studies have shown that the therapeutic action of tetra-arsenic tetra-sulfide (As4S4) is effective for acute promyelocytic leukemia. However, the molecular mechanism of the action of As4S4 in retinoic acid-resistant acute promyelocytic leukemia (APL) therapy remains unclear. In the present study, the signaling of the cytotoxic effects induced by As4S4 on retinoic acid-resistant APL NB4-R1 cells was investigated. A time-dependent increase in cell death and DNA cleavage was observed following As4S4 treatment. Changes in B-cell lymphoma 2 and Bax accompanied by the activation of caspase-3 and cleavage of poly ADP-ribose polymerase were observed as actions of As4S4. As4S4 induced an accumulation of NB4-R1 cells in the S and G2/M phases, as detected by flow cytometry. Therefore, the present results suggest that As4S4-mediated apoptosis in NB4-R1 cells involves a mitochondria-dependent pathway.

Apoptosis of retinoic acid-resistant APL NB4-R1 cells induced by As4S4 and its molecular mechanism

This study was to investigate the apoptosis-inducing effect of As(4)S(4) on the retinoic acid-resistant acute promyelocytic leukemia (APL) NB4-R1 cells and its potential mechanisms. The leukemia cell line NB4-R1 was cultured in vitro and divided into control group and treatment group. The apoptosis rate and cell cycle were detected by flow cytometry. The apoptotic DNA fragments were analyzed by agarose gel electrophoresis. The changes of BCL-2, BAX and Caspase-3 were determined by Western blot. After NB4-R1 cells were treated with As(4)S(4)(25 µmol/L) for 0 h, 24 h, 48 h, the percentage of early apoptotic cells was obviously raised from 0% to 24.49% and 47.41%, the percentage of late apoptotic cells were elevated from 0.08% to 14.72% and 20.70%. Compared with control group, the DNA degradation revealed a characteristic DNA ladder during agarose gel electrophoresis after treatment for 24 h. The drug significantly induced an accumulation of the S phase cell population from 31.85% of the untreated cells to 42.53% and 55.12% treated with the different time whereas the NB4-R1 cells in G0/G1 phase decreased from 57.30% to 37.56% and 28.51%. As(4)S(4) could decrease the expression of BCL-2 and increase the level of BAX. Pro-caspase-3 could be cleaved into small active fragments under the apoptotic stimulation. As(4)S(4) can efficiently induce NB4-R1 cell apoptosis, which may be related with the down-regulation of BCL-2 and the up-regulation of BAX, as well as the activation of Caspase-3.

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.

Matrine Cooperates with All-Trans Retinoic Acid on Differentiation Induction of All-Trans Retinoic Acid-Resistant Acute Promyelocytic Leukemia Cells (NB4-LR1): Possible Mechanisms

Retinoic acid resistance results in refractory disease, and recovery in acute promyelocytic leukemia remains a challenge in clinical practice, with no ideal chemotherapeutic drug currently available. Here we report on the effect of an active compound of Sophora flavescens called matrine (0.1 mmol/L) combined with all-trans retinoic acid (1 µmol/L) in alleviating retinoic acid resistance in acute promyelocytic leukemia-derived NB4-LR1 cells by differentiation induction, as can be seen by an induced morphology change, increased CD11b expression, and nitro blue tetrazolium reduction activity, and a decreased expression of the promyelocytic leukemia-retinoic acid receptor α fusion gene and protein product. We further explored the probable mechanism of how matrine promotes the recovery of differentiation ability in NB4-LR1 cells when exposed to all-trans retinoic acid. We observed that the combination of all-trans retinoic acid and matrine can increase the level of cyclic adenosine monophosphate and protein kinase A activity, reduce telomerase activity, and downregulate the protein expression of topoisomerase II beta in NB4-LR1 cells. The results of this study suggest the possible clinical utility of matrine in the treatment of retinoic acid-resistant acute promyelocytic leukemia.

Silencing of the human SET gene in vitro with lentivirus-mediated RNA interference

In our previous study, SET was identified as one of the differentially expressed proteins that was associated with tetra-arsenic tetra-sulfide (As4S4)-induced NB4-R1 [retinoic acid-resistant acute promyelocytic leukemia (APL) cell line] apoptosis. However, the mechanism through which SET regulates pathways during this process remains unclear. The aim of this study was to construct lentivirus-mediated short hairpin RNA (shRNA) against SET and investigate the effect of SET on As4S4-induced retinoic acid-resistant APL cell apoptosis. In the present study, 4different oligonucleotides targeting the human SET gene were synthesized and cloned into the eukaryotic expression plasmid pGCSIL-GFP. The recombinant vectors were introduced into NB4-R1 cells. The silencing efficiency was measured by real-time quantitative PCR (RT-qPCR) and western blotting. Our results showed that the 4recombinant RNA interference (RNAi) vectors were constructed successfully. Fluorescence microscopy demonstrated that infection efficiency ranged from 70to90%. Infection with the 4different RNAi vectors significantly knocked down the expression of SET by 52.8, 69.1, 48.9 and 90.3% at the mRNA level, and 92.5, 96.3, 91.7 and 98.4% at the protein level, respectively. We attempt to clarify the mechanism of As4S4 treatment on retinoic acid-resistant APL.

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.

Lentiviral vector-mediated RNA interference targeted against prohibitin inhibits apoptosis of the retinoic acid-resistant acute promyelocytic leukemia cell line NB4-R1

To investigate the possibility of prohibitin (PHB) inhibition by lentiviral vector-mediated RNA interference (RNAi) and its influence on cell apoptosis in the retinoic acid-resistant acute promyelocytic leukemia cell line NB4-R1, a lentiviral vector encoding a short hairpin RNA (shRNA) targeted against PHB (pGCSIL-GFP-PHB) was constructed and transfected into the packaging cells 293T, and the viral supernatant was collected to transfect NB4-R1 cells. Quantitative real-time fluorescent PCR and western blotting were used to detect the expression levels of PHB. Flow cytometry and detection of enzymatic activity of caspase-3 by western blotting were employed to examine cell apoptosis. Our results provide evidence that the lentiviral vector pGCSIL-GFP-PHB was constructed successfully, and the PHB mRNA and the protein expression inhibitory rates were 90.3 and 95.8%, respectively. When compared to the control group, the activity of caspase-3 decreased significantly, which showed a 57.3% downregulation, and the apoptosis rate was reduced by 44.6% (P<0.05). In conclusion, downregulation of the PHB gene may inhibit apoptosis of NB4-R1 cells, and it is speculated that this was at least partly due to the downregulation of caspase-3, and PHB may be a novel target for gene therapy for retinoic acid-resistant acute promyelocytic leukemia.

Targeting PKCδ-mediated topoisomerase IIβ overexpression subverts the differentiation block in a retinoic acid-resistant APL cell line

Retinoic acid (RA) relieves the maturation block in t(15:17) acute promyelocytic leukemia (APL), leading to granulocytic differentiation. However, RA treatment alone invariably results in RA resistance, both in vivo and in vitro. RA-resistant cell lines have been shown to serve as useful models for elucidation of mechanisms of resistance. Previously, we identified topoisomerase II beta (TOP2B) as a novel mediator of RA-resistance in APL cell lines. In this study, we show that both TOP2B protein stability and activity are regulated by a member of the protein kinase C (PRKC) family, PRKC delta (PRKCD). Co-treatment with a pharmacologic inhibitor of PRKCD and RA resulted in the induction of an RA responsive reporter construct, as well as the endogenous RA target genes, CEBPE, CYP26A1 and RIG-I. Furthermore, the co-treatment overcame the differentiation block in RA-resistant cells, as assessed by morphological analysis, restoration of promyelocytic leukemia nuclear bodies, induction of CD11c cell surface expression and an increase in nitro-blue-tetrazolium reduction. Cumulatively, our data suggest a model whereby inhibition of PRKCD decreases TOP2B protein levels, leading to a loss of TOP2B-mediated repressive effects on RA-induced transcription and granulocytic differentiation.

The Combination of 9-cis Retinoic Acid Plus MEK Inhibitor Induces Apoptosis Via Dephosphorylation of Retinoid X Receptor α in HL-60R Cells.

Resistance against retinoic acid (RA) is a serious problem of differentiation-induction therapy for acute promyelocytic leukemia (APL) in clinical practice. RA exerts its biological activities primarily through the nuclear receptor dimmer, consisting of retinoic acid receptor (RAR) and retinoid X receptor (RXR). Both receptors consist of three subtypes (α, β and γ) and form heterodimeric RAR/RXR and homodimeric RXR/RXR complexes. 9-cis RA, which is a high-affinity ligand for RXR but also binds to RAR, induces cell differentiation in wild type HL-60 human myeloid leukemia cells. However, in HL-60R cells, the RA-resistant subclone of HL-60, 9-cis RA can not induce cell differentiation and apoptosis. We recently reported that malfunction of RXRα due to posttranslational modification by phosphorylation to be associated with carcinogenesis of hepatocellular carcinoma (HCC). Phosphorylated form of RXRα (p-RXRα) at serine 260 by Ras/MAPK is resistant to ubiquitin/proteasome-mediated degradation and the accumulation of p-RXRα interferes with the function of remaining normal RXRα in a dominant negative manner, thereby promoting growth of HCC cells. We also found that in the presence of MEK inhibitor PD98059, 9-cis RA can induce the degradation of p-RXRα and thus restoring the function of this receptor in RXRα-phosphorylated human HCC cells. Based on the results as described above, we initiated this study to examine whether 9-cis RA can exert growth inhibitory effects on RA-resistant HL-60R cells when combined with MEK inhibitor, with focusing on the inhibition of expression of p-RXRα protein. We found that RXRα protein was originally expressed in both HL-60 and HL-60R cells, and that the expression level of RXRα protein was inhibited by about 60% and 20%, respectively, when those cells were treated with 0.5 μM 9-cis RA. Not only total RXRα protein, but also the level of p-RXRα protein was constitutively expressed in both HL-60 and HL-60R cells, and was significantly decreased in HL-60 cells by treatment with 9-cis RA alone. On the other hand, in HL-60R cells, 9-cis RA alone nor 20 μM PD98059 alone did not cause a down regulation of these proteins. However, when HL-60R cells were treated with the combination of 9-cis RA plus PD98059, the expression level of p-RXRα protein was markedly decreased. Moreover, the combination of these agents induced apoptosis in HL-60R cells, whereas similar effect was not obtained when the cells were treated with either agent alone. The combined treatment of these agents also cooperatively inhibited the growth of HL-60 R cells. The present findings suggest that the accumulation of p-RXRα might impair the function of normal RXRα as a master regulator of nuclear receptors and, thus, contributing to the resistance to RA in HL-60R cells. Combination of 9-cis RA plus MEK inhibitor might be an effective regimen for patients with RA resistant APL.

ATR, PML, and CHK2 Play a Role in Arsenic Trioxide-induced Apoptosis

Arsenic trioxide (ATO) is a potent anti-leukemic chemotherapeutic agent for acute promyelocytic leukemia (APL) that results from a t (15, 17) chromosomal translocation that produces PML-RARalpha, a fusion protein between a tumor suppressor PML and the retinoic acid receptor RARalpha. APL patients are initially treated with retinoic acid, but most develop resistance and relapse. In contrast, ATO induces prolonged remissions even in the relapsed cases. However, the molecular mechanisms by which ATO kills the leukemic cells are not fully understood. We find that ATO induces apoptosis, at least in part, by activating proapoptotic kinase Chk2. ATO does this by stimulating ATR (ataxia telangiectasia mutated and Rad3-related) kinase, a Chk2-activating kinase. In conjunction, ATO degrades PML-RARalpha, resulting in the restoration of PML, which is required for autophosphorylation and full activation of Chk2. As a result, the p53-dependent apoptosis pathway is activated. Based on this, we propose that a pathway composed of ATR, PML, Chk2, and p53 plays a role in ATO-mediated apoptosis, a notion that is consistent with the observation that Chk2 is genetically intact and mutations in the p53 gene are extremely rare in 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.

19-Nor-1,25(OH)2D2 (a Novel, Noncalcemic Vitamin D Analogue), Combined with Arsenic Trioxide, Has Potent Antitumor Activity against Myeloid Leukemia

Recently, we reported that a novel, noncalcemic vitamin D analogue (19-nor-1,25(OH)2D2; paricalcitol) had anticancer activity. In this study, we explored if paricalcitol enhanced anticancer effects of other clinically useful drugs in vitro against a large variety of cancer cells. Paricalcitol, when combined with As2O3, showed a markedly enhanced antiproliferative effect against acute myeloid leukemia (AML) cells. This combination induced monocytic differentiation of NB-4 acute promyelocytic leukemia (APL) cells and HL-60 AML cells and caused both to undergo apoptosis associated with down-regulation of Bcl-2 and Bcl-x(L). Paricalcitol induced monocytic differentiation of U937 AML cells, which was partially blocked by inducing expression of APL-related PML-retinoic acid receptor alpha (RARalpha) chimeric protein in the U937 cells containing a Zn2+-inducible expression vector coding for this fusion protein (PR9 cells). Exposure to As2O3 decreased levels of PML-RARalpha in PR9 cells, and the combination of paricalcitol and As2O3 enhanced their monocytic differentiation in parallel with the As2O3-mediated decrease of PML-RARalpha. Furthermore, As2O3 increased the transcriptional activity of paricalcitol probably by increasing intracellular levels of paricalcitol by decreasing the function of the mitochondrial enzyme 25-hydroxyvitamin D3-24-hydroxylase, which functions to metabolize the active vitamin D in cells. In summary, the combination of paricalcitol and As2O3 potently decreased growth and induced differentiation and apoptosis of AML cells. This probably occurred by As2O3 decreasing levels of both the repressive PML-RARalpha fusion protein and the vitamin D metabolizing protein, 25-hydroxyvitamin D3-24-hydroxylase, resulting in increased activity of paricalcitol. The combination of both of these Food and Drug Administration-approved drugs should be considered for treatment of all-trans retinoic acid-resistant APL patients as well as those with other types of AML.

Combination of retinoic acid and tumor necrosis factor overcomes the maturation block in a variety of retinoic acid-resistant acute promyelocytic leukemia cells

AbstractRetinoic acid (RA) overcomes the maturation block in t(15:17) acute promyelocytic leukemia (APL), leading to granulocytic differentiation. Patients receiving RA alone invariably develop RA resistance. RA-resistant cells can serve as useful models for the development of treatments for both APL and other leukemias. Previously, we showed that RA and tumor necrosis factor (TNF) promote monocytic differentiation of the APL cell line NB4 and U937 monoblastic cells. Here, we report that combining TNF with RA leads to maturation of several RA-resistant APL cells along a monocytic pathway, whereas UF-1, a patient-derived RA-resistant cell line, showed characteristics of granulocytic differentiation. We found distinct differences in gene regulation between UF-1 cells and cells showing monocytic differentiation. Although IRF-7 was up-regulated by TNF and RA in all cells tested, expression of c-jun and PU.1 correlated with monocytic differentiation. Furthermore, synergistic induction of PU.1 DNA binding and macrophage colony-stimulating factor receptor (m-CSF-1R) mRNA was observed only in cells differentiating into monocytes. Using neutralizing antibodies against m-CSF-1R or its ligand, we found that inhibiting this pathway strongly reduced CD14 expression in response to RA and TNF, suggesting that this pathway is essential for their synergy in RA-resistant leukemia cells. (Blood. 2004;104:3335-3342)

Induction of differentiation of retinoic acid-resistant acute promyelocytic leukemia cells by the combination of all- trans retinoic acid and granulocyte colony-stimulating factor

An acute promyelocytic leukemia (APL) cell line with natural resistance to all- trans retinoic acid (ATRA), UF-1, was induced to differentiate into mature granulocyte when treated with the combination of ATRA and granulocyte colony-stimulating factor (G-CSF), while neither of them alone was capable of inducing the differentiation effectively. Continuous presence of both agents was required for the maximal differentiation-inductive effect. Neither proliferation arrest nor induction of apoptosis preceded the differentiation. Differentiated phenotype was accompanied by growth arrest, however, not by increased apoptosis. It was assumed that cellular events at the downstream of the signaling pathways of ATRA and G-CSF cooperatively played pivotal roles in the differentiation–induction.

Response to histone deacetylase inhibition of novel PML/RARα mutants detected in retinoic acid–resistant APL cells

Resistance to all-trans retinoic acid (ATRA) remains a clinical problem in the treatment of acute promyelocytic leukemia (APL) and provides a model for the development of novel therapies. Molecular alterations in the ligand-binding domain (LBD) of the PML/RARalpha fusion gene that characterizes APL constitute one mechanism of acquired resistance to ATRA. We identified missense mutations in PML/RARalpha from an additional ATRA-resistant patient at relapse and in a novel ATRA-resistant cell line, NB4-MRA1. These cause altered binding to ligand and transcriptional coregulators, leading to a dominant-negative block of transcription. These mutations are in regions of the LBD that appear to be mutational hot spots occurring repeatedly in ATRA-resistant APL patient cells. We evaluated whether histone deacetylase (HDAC) inhibition could overcome the effects of these mutations on ATRA-induced gene expression. Cotreatment with ATRA and TSA restored RARbeta gene expression in NB4-MRA1 cells, whose PML/RARalpha mutation is in helix 12 of the LBD, but not in an APL cell line harboring the patient-derived PML/RARalpha mutation, which was between helix 5 and 6. Furthermore, ATRA combined with TSA increases histone 4 acetylation on the RARbeta promoter only in NB4-MRA1 cells. Consistent with these results, the combined treatment induces differentiation of NB4-MRA1 only. Thus, the ability of an HDAC inhibitor to restore ATRA sensitivity in resistant cells may depend on their specific molecular defects. The variety of PML/RARalpha mutations arising in ATRA-resistant patients begins to explain how APL patients in relapse may differ in response to transcription therapy with HDAC inhibitors.

A novel retinoic acid-resistant acute promyelocytic leukemia model in vitro and in vivo (review).

Differentiation-inducing therapy by all-trans retinoic acid (RA) is now a standard therapy in patients with acute promyelocytic leukemia (APL). Nearly all patients achieve complete remission by the treatment of all-trans RA, however, clinical remissions are usually of brief duration, and these patients often develop RA-resistant disease. The mechanisms of RA-resistance in APL cells are poorly understood and most clinical approaches have not been successful in overcoming RA-resistance. We have recently established a novel APL cell line (UF-1) with RA-resistant features. In addition, we have established human GM-CSF-producing transgenic (hGMTg) SCID mice system. UF-1 cells were inoculated either intraperitoneally or subcutaneously into hGMTg SCID mice and made the first RA-resistant murine APL model. These RA-resistant APL model systems in vitro and in vivo may be useful for investigating the molecular studies on the block of leukemic cell differentiation and as means to investigate the mechanisms of RA-resistance. Moreover, this murine model system will be important for developing novel therapeutic strategies in RA-resistant APL.

1,25-Dihydroxyvitamin D3 Induces Differentiation of a Retinoic Acid–Resistant Acute Promyelocytic Leukemia Cell Line (UF-1) Associated With Expression of p21WAF1/CIP1 and p27KIP1

Retinoic acid (RA) resistance is a serious problem for patients with acute promyelocytic leukemia (APL) who are receiving all-transRA. However, the mechanisms and strategies to overcome RA resistance by APL cells are still unclear. The biologic effects of RA are mediated by two distinct families of transcriptional factors: RA receptors (RARs) and retinoid X receptors (RXRs). RXRs heterodimerize with 1,25-dihydroxyvitamin D3[1,25(OH)2D3] receptor (VDR), enabling their efficient transcriptional activation. The cyclin-dependent kinase (cdk) inhibitor p21WAF1/CIP1 has a vitamin D3–responsive element (VDRE) in its promoter, and 1,25(OH)2D3 enhances the expression of p21WAF1/CIP1 and induces differentiation of selected myeloid leukemic cell lines. We have recently established a novel APL cell line (UF-1) with features of RA resistance. 1,25(OH)2D3 can induce growth inhibition and G1 arrest of UF-1 cells, resulting in differentiation of these cells toward granulocytes. This 1,25(OH)2D3-induced G1 arrest is enhanced by all-trans RA. Also, 1,25(OH)2D3 (10−10 to 10−7 mol/L) in combination with RA markedly inhibits cellular proliferation in a dose- and time-dependent manner. Associated with these findings, the levels of p21WAF1/CIP1 and p27KIP1 mRNA and protein increased in these cells. Northern blot analysis showed that p21WAF1/CIP1 and p27KIP1 mRNA and protein increased in these cells. Northern blot analysis showed that p21WAF1/CIP1 and p27KIP1 transcripts were induced after 6 hours’ exposure to 1,25(OH)2D3 and then decreased to basal levels over 48 hours. Western blot experiments showed that p21WAF1/CIP1 protein levels increased and became detectable after 12 hours of 1,25(OH)2D3treatment and induction of p27KIP1 protein was much more gradual and sustained in UF-1 cells. Interestingly, the combination of 1,25(OH)2D3 and RA markedly enhanced the levels of p27KIP1 transcript and protein as compared with levels induced by 1,25(OH)2D3 alone. In addition, exogenous p27KIP1 expression can enhance the level of CD11b antigen in myeloid leukemic cells. In contrast, RA alone can induce G1 arrest of UF-1 cells; however, it did not result in an increase of p21WAF1/CIP1 and p27KIP1transcript and protein expression in RA-resistant cells. Taken together, we conclude that 1,25(OH)2D3 induces increased expression of cdk inhibitors, which mediates a G1 arrest, and this may be associated with differentiation of RA-resistant UF-1 cells toward mature granulocytes.