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1.  Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARα oncoprotein 
Autophagy  2011;7(4):401-411.
PML-RARα oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RARα degradation, which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RARα degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as ATG1, ATG5 and PI3KC3, and by autophagy inhibitors (e.g., 3-methyladenine), blocked PML-RARα degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RARα degradation and myeloid cell differentiation. Moreover, PML-RARα co-immunoprecipitated with the ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RARα degradation and myeloid cell differentiation. The identification of PML-RARα as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL.
doi:10.4161/auto.7.4.14397
PMCID: PMC3127220  PMID: 21187718
autophagy; differentiation; oncoprotein; leukemia; degradation; PML-RARa; p62/SQSTM1
2.  Targeting Expression of the Leukemogenic PML-RARα Fusion Protein by Lentiviral Vector-Mediated Small Interfering RNA Results in Leukemic Cell Differentiation and Apoptosis 
Human Gene Therapy  2011;22(12):1593-1598.
Abstract
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.
Ward and colleagues use lentiviral vector-mediated small interfering RNA to knock down expression of the leukemogenic fusion protein, promyelocytic leukemia–retinoic acid α receptor (PML-RARα). They demonstrate that this approach prevents the development of disease in a mouse model of acute promyelocytic leukemia.
doi:10.1089/hum.2011.079
PMCID: PMC3237690  PMID: 21846246
3.  Altered nuclear co-factor switching in retinoic resistant variants of the PML-RARα oncoprotein of acute promyelocytic leukemia† 
Proteins  2012;80(4):1095-1109.
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 co-repressor and the ACTR nuclear co-activator. The consequences of the mutations on global structure and co-factor interaction functions were assessed quantitatively, providing insights into the basis of agonist resistance. Attenuated co-factor 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.
doi:10.1002/prot.24010
PMCID: PMC3294029  PMID: 22228505
all-trans retinoic acid; coactivator; corepressor; nuclear receptor; transcription regulation; fluorescence anisotropy
4.  Interferon-γ enhances promyelocytic leukemia protein expression in acute promyelocytic cells and cooperates with all-trans-retinoic acid to induce maturation of NB4 and NB4-R1 cells 
In order to investigate the effect and mechanisms of interferon (IFN)-γ in combination with all-trans-retinoic acid (ATRA) on NB4 cells [ATRA-sensitive acute promyelocytic leukemia (APL) cell line] and NB4-R1 cells (ATRA-resistant APL cell line) and to search for a novel approach to solve the problem of ATRA resistance in APL, we initially treated NB4 and NB4-R1 cells with IFN-γ, ATRA and IFN-γ in combination with ATRA, respectively. The cell proliferation was then tested by MTT assay, and the cell differentiation was tested through light microscopy, by NBT test and flow cytometry (FCM). The expression of promyelocytic leukemia (PML) protein was observed by indirect immune fluorescent test. Results showed that ATRA inhibited the growth of NB4 cells, however, it could not inhibit the growth of NB4-R1 cells. IFN-γ inhibited the growth of both NB4 and NB4-R1 cells. Meanwhile, the growth inhibition effect of IFN-γ in combination with ATRA on both NB4 and NB4-R1 cells was significantly stronger than that of any single drug treatment. The results of the NBT reduction test and CD11b antigen detection by FCM indicated that IFN-γ induces the differentiation of NB4 and NB4-R1 cells to some extent. Moreover, the maturation degree of both NB4 and NB4-R1 cells induced by IFN-γ in combination with ATRA was more significant than that of IFN-γ or ATRA alone. After treatment with IFN-γ, the number of fluorescent particles in NB4 and NB4-R1 cell nuclei was higher than those in the control group, which indicated that IFN-γ may induce the expression of PML protein. Together, IFN-γ augments the proliferation inhibition effect of ATRA on NB4 and NB4-R1 cells through enhancing the expression of PML protein. IFN-γ in combination with ATRA not only strengthens the induction differentiation effect of ATRA on NB4 cells, but also can partially induce the maturation of NB4-R1 cells with ATRA resistance.
doi:10.3892/etm.2012.488
PMCID: PMC3438595  PMID: 22969967
acute promyelocytic leukemia; interferon-γ; all-trans-retinoic acid; all-trans-retinoic acid-resistant
5.  Realgar-induced apoptosis and differentiation in all-trans retinoic acid (ATRA)-sensitive NB4 and ATRA-resistant MR2 cells 
International Journal of Oncology  2011;40(4):1089-1096.
Realgar has been used in Western medicine and Chinese traditional medicine since ancient times, and its promising anticancer activity has attracted much attention in recent years, especially for acute promyelocytic leukemia (APL). However, the therapeutic action of realgar treatment for APL remains to be fully elucidated. Cellular cytotoxicity, proliferation, apoptosis and differentiation were comprehensively investigated in realgar-treated cell lines derived from PML-RARα+ APL patient, including the all-trans retinoic acid (ATRA)-sensitive NB4 and ATRA-resistant MR2 cell lines. For analysis of key regulators of apoptosis and differentiation, gene expression profiles were performed in NB4 cells. Realgar was found to induce apoptosis and differentiation in both cell lines, and these effects were exerted simultaneously. Gene expression profiles indicated that genes influenced by realgar treatment were involved in the modulation of signal transduction, translation, transcription, metabolism and the immune response. Given its low toxicity, realgar is a promising alternative reagent for the therapy of APL. Our data contribute to an understanding of the underlying mechanism responsible for the therapeutic effects of realgar in the clinical treatment of APL.
doi:10.3892/ijo.2011.1276
PMCID: PMC3584620  PMID: 22134377
realgar; acute promyelocytic leukemia; apoptosis; differentiation; oligonucleotide microarray
6.  Differentiation Therapy of Acute Myeloid Leukemia 
Cancers  2011;3(2):2402-2420.
Acute Myeloid Leukemia (AML) is a predominant acute leukemia among adults, characterized by accumulation of malignantly transformed immature myeloid precursors. A very attractive way to treat myeloid leukemia, which is now called ‘differentiation therapy’, was proposed as in vitro studies have shown that a variety of agents stimulate differentiation of the cell lines isolated from leukemic patients. One of the differentiation-inducing agents, all-trans retinoic acid (ATRA), which can induce granulocytic differentiation in myeloid leukemic cell lines, has been introduced into clinics to treat patients with acute promyelocytic leukemia (APL) in which a PML-RARA fusion protein is generated by a t(15;17)(q22;q12) chromosomal translocation. Because differentiation therapy using ATRA has significantly improved prognosis for patients with APL, many efforts have been made to find alternative differentiating agents. Since 1,25-dihydroxyvitamin D3 (1,25D) is capable of inducing in vitro monocyte/macrophage differentiation of myeloid leukemic cells, clinical trials have been performed to estimate its potential to treat patients with AML or myelodysplastic syndrome (MDS). Unfortunately therapeutic concentrations of 1,25D can induce potentially fatal systemic hypercalcemia, thus limiting clinical utility of that compound. Attempts to overcome this problem have focused on the synthesis of 1,25D analogs (VDAs) which retain differentiation inducing potential, but lack its hypercalcemic effects. This review aims to discuss current problems and potential solutions in differentiation therapy of AML.
doi:10.3390/cancers3022402
PMCID: PMC3757424  PMID: 24212816
acute myeloid leukemia; targeted therapy; differentiation; all-trans retinoic acid; acute promyelocytic leukemia; 1,25-dihydroxyvitamin D3; vitamin D analogs
7.  Recent advances in the diagnosis and management of childhood acute promyelocytic leukemia 
Korean Journal of Pediatrics  2011;54(3):95-105.
Since the successful introduction of all-trans-retinoic acid (ATRA) and its combination with anthracycline-containing chemotherapy, the prognosis for acute promyelocytic leukemia (APL) has markedly improved. With ATRA and anthracycline-based-chemotherapy, the complete remission rate is greater than 90%, and the long-term survival rate is 70-89%. Moreover, arsenic trioxide (ATO), which was introduced for APL treatment in 1994, resulted in excellent remission rates in relapsed patients with APL, and more recently, several clinical studies have been designed to explore its role in initial therapy either alone or in combination with ATRA. APL is a rare disease in children and is frequently associated with hyperleukocytosis, which is a marker for higher risk of relapse and an increased incidence of microgranular morphology. The frequency of occurrence of the promyelocytic leukemia/retinoic acid receptor-alpha (PML/RARα) isoforms bcr 2 and bcr 3 is higher in children than in adults. Although recent clinical studies have reported comparable long-term survival rates in patients with APL, therapy for APL in children is challenging because of the risk of early death and the potential long-term cardiac toxicity resulting from the need to use high doses of anthracyclines. Additional prospective, randomized, large clinical trials are needed to address several issues in pediatric APL and to possibly minimize or eliminate the need for chemotherapy by combining ATRA and ATO. In this review article, we discuss the molecular pathogenesis, diagnostic progress, and most recent therapeutic advances in the treatment of children with APL.
doi:10.3345/kjp.2011.54.3.95
PMCID: PMC3121002  PMID: 21738538
Acute promyelocytic leukemia; Child; PML-RARA; Tretinoin; Arsenic trioxide; Anthracycline
8.  Retinoid Differentiation Therapy for Common Types of Acute Myeloid Leukemia 
Many cancers arise in a tissue stem cell, and cell differentiation is impaired resulting in an accumulation of immature cells. The introduction of all-trans retinoic acid (ATRA) in 1987 to treat acute promyelocytic leukemia (APL), a rare subtype of acute myeloid leukemia (AML), pioneered a new approach to obtain remission in malignancies by restoring the terminal maturation of leukemia cells resulting in these cells having a limited lifespan. Differentiation therapy also offers the prospect of a less aggressive treatment by virtue of attenuated growth of leukemia cells coupled to limited damage to normal cells. The success of ATRA in differentiation therapy of APL is well known. However, ATRA does not work in non-APL AML. Here we examine some of the molecular pathways towards new retinoid-based differentiation therapy of non-APL AML. Prospects include modulation of the epigenetic status of ATRA-insensitive AML cells, agents that influence intracellular signalling events that are provoked by ATRA, and the use of novel synthetic retinoids.
doi:10.1155/2012/939021
PMCID: PMC3504222  PMID: 23213553
9.  Relapse of Acute Promyelocytic Leukemia with PML-RARα Mutant Subclones Independent of Proximate All-Trans Retinoic Acid Selection Pressure 
Relapse of acute promyelocytic leukemia (APL) following all-trans retinoic acid (ATRA) therapy has been associated with the acquisition of mutations in the high-affinity ATRA binding site in PML-RARα but little information is available about the selection dynamics of the mutation-harboring subclones. In this study, 6/18 patients treated with sequential ATRA and chemotherapy on protocol INT0129 relapsed with complete replacement of the non-mutant pretreatment APL cell population by a PML-RARα mutant subclone. Two patients relapsed in proximity of ATRA treatment, however, in 4 patients there was a 6 to 48 month hiatus between the last ATRA treatment and relapse. The mutant subclones were not detectable in samples tested ≥3 months before relapse at ≥1 in 102 (10−2) sensitivity. In one patient, a functionally-weak mutation was detected at 10−4 sensitivity before therapy but only limited pre-relapse enrichment of the mutant subclone was observed on subsequent ATRA therapy. These results indicate that proximate ATRA selection pressure is frequently not the main determinant for the emergence of strongly dominant PML-RARα mutant subclones and suggest that APL subclones harboring PML-RARα mutations are predisposed to the acquisition of secondary genetic/epigenetic alterations that result in a growth/survival advantage.
doi:10.1038/sj.leu.2404118
PMCID: PMC1410817  PMID: 16437139
Acute promyelocytic leukemia; All-trans retinoic acid; PML-RARα mutations; mutant subclone dynamics; drug selection pressure
10.  Induction of acute lymphocytic leukemia differentiation by maintenance therapy 
Despite extensive study in many malignancies, maintenance therapy has clinically benefited only two diseases: acute lymphocytic leukemia (ALL) and acute promyelocytic leukemia (APL). ALL maintenance therapy utilizes low-dose 6-mercaptopurine (6MP) and methotrexate (MTX), while maintenance in APL primarily consists of all-trans-retinoic acid (ATRA). 6MP and MTX as used in ALL are also now usually added to maintenance ATRA for APL, based on data suggesting an improved disease-free survival. Although the mechanism of action of MTX and 6MP as maintenance is unknown, low-dose cytotoxic agents are potent inducers of differentiation in vitro. Thus, we studied whether maintenance therapy in ALL, like ATRA in APL, may be inducing terminal differentiation of ALL progenitors. The APL cell line NB4, the ALL cell lines REH and RS4;11, and patients' ALL blasts were incubated with ATRA, 6MP, and MTX in vitro. All three drugs inhibited the clonogenic growth of the APL and ALL cell lines without inducing immediate apoptosis, but associated with induction of phenotypic differentiation. The three drugs similarly upregulated lymphoid antigen expression, while decreasing CD34 expression, on patients' ALL blasts. These data suggest that induction of leukemia progenitor differentiation plays an important role in the mechanism of action of maintenance therapy in ALL.
doi:10.1038/sj.leu.2404823
PMCID: PMC2643128  PMID: 17611566
acute lymphocytic leukemia; acute promyelocytic leukemia; maintenance therapy; differentiation
11.  Disseminated Exfoliative Dermatitis Associated with All-Transretinoic Acid in the Treatment of Acute Promyelocytic Leukemia 
Case Reports in Medicine  2012;2012:236174.
Acute promyelocytic leukemia (APL) is a biologically and clinically separate type of acute myeloid leukemia characterized by a translocation involving the retinoic acid receptor-alpha (RARa) locus on chromosome 17, the great majority of which is t(15; 17)(q24.1; q21.1) (Collins (1998), Melnick and Licht (1999), and Grimwade (1999)). Retinoic acid is a critical ligand in the differentiation pathway of multiple tissues, mediated through binding to an RAR. All-trans retinoic acid (ATRA) is a subgroup of the retinoid family, which induces complete remission (CR) in APL by causing differentiation and apoptosis in immature malignant promyelocytes rather than inducing cell death by cytotoxicity (Warrell et al. (1993), Liu et al. (2000), and Cassinat et al. (2001)). ATRA-associated toxicity consisting of headache, fever, weakness, fatigue, dry skin, dermatitis, gastrointestinal disorders, and hypertriglyceridemia has been shown to be mild (Kurzrock et al. (1993)). Herein, we describe a patient with APL that developed an erythematous reaction of the whole body followed by desquamation and exfoliation during ATRA therapy.
doi:10.1155/2012/236174
PMCID: PMC3405663  PMID: 22848226
12.  Regulation of leukemic cell differentiation and retinoid-induced gene expression by statins 
Molecular cancer therapeutics  2009;8(3):615-625.
There is emerging evidence that, beyond their cholesterol lowering properties, statins exhibit important antileukemic effects in vitro and in vivo, but the precise mechanisms by which they generate such responses remain to be determined. We have previously shown that statins promote differentiation of acute promyelocytic leukemia (APL) cells and enhance generation of all-trans-retinoic acid (ATRA)-dependent antileukemic responses. We now provide evidence that statin-dependent leukemic cell differentiation requires engagement and activation of the JNK kinase pathway. In addition, in experiments to define the molecular targets and mediators of statin-induced differentiation we found a remarkable effect of statins on ATRA-dependent gene transcription, evidenced by the selective induction of over 400 genes by the combination of atorvastatin and ATRA. Altogether, our studies identify novel statin molecular targets linked to differentiation, establish that statins modulate ATRA-dependent transcription, and suggest that combined use of statins with retinoids may provide a novel approach to enhance antileukemic responses in APL and possibly other leukemias.
doi:10.1158/1535-7163.MCT-08-1196
PMCID: PMC2681262  PMID: 19240159
statins; atorvastatin; retinoic acid; leukemia
13.  CCAAT/enhancer binding protein ε is a potential retinoid target gene in acute promyelocytic leukemia treatment 
Journal of Clinical Investigation  1999;103(10):1399-1408.
The CCAAT/enhancer binding protein ε (C/EBPε) is a nuclear transcription factor expressed predominantly in myeloid cells and implicated as a potential regulator of myeloid differentiation. We show that it was rapidly induced in the acute promyelocytic leukemia (APL) cell line NB4 during granulocytic differentiation after exposure to retinoic acid (RA). Our data suggest that induction of C/EBPε expression was through the retinoic acid receptor α (RARα) pathway. Reporter gene studies showed that C/EBPε promoter/enhancer activity increased in a retinoid-dependent fashion via the retinoic acid response element (RARE) present in the promoter region of C/EBPε. The RA-induced expression of C/EBPε markedly increased in U937 myelomonoblasts that were induced to express promyelocytic leukemia/RARα (PML/RARα), but not in those induced to express promyelocytic leukemia zinc finger/RARα (PLZF/RARα). In retinoid-resistant APL cell lines, C/EBPε either is not induced or is induced only at very high concentrations of RA (≥10–6 M). In addition, forced expression of C/EBPε in the U937 myelomonoblastic leukemia cells mimicked terminal granulocytic differentiation, including morphologic changes, increased CD11b/CD66b expression, and induction of secondary granule protein expression. Our data strongly suggest that C/EBPε is a downstream target gene responsible for RA-induced granulocytic differentiation of APL cells.
PMCID: PMC408448  PMID: 10330422
14.  Treatment of Acute Promyelocytic Leukemia for Older Patients 
Acute promyelocytic leukemia (APL) represents a remarkable disease in which leukemogenesis is driven by the PML-RARα oncogene and for which targeted treatment with all-trans retinoic acid (ATRA)–based therapy allows substantial chance of cure. APL is seen in a small subset of older patients, with age representing one of the most important prognostic factors for outcome of treatment. Unlike other acute leukemias, the inferior outcomes for APL in older patients relates less to changes in disease biology and more to increased toxicity of ATRA and chemotherapy combination regimens used to induce hematologic and molecular responses. Risk-adapted strategies that use less-toxic agents, such as arsenic trioxide, allow treatment of older patients, with greater efficiency and better chances of cure.
PMCID: PMC3593588  PMID: 21393443
Older patient; all-trans retinoic acid; acute promyelocytic leukemia; arsenic
15.  Retinoic Acid and Arsenic Synergize to Eradicate Leukemic Cells in a Mouse Model of Acute Promyelocytic Leukemia  
The Journal of Experimental Medicine  1999;189(7):1043-1052.
In acute promyelocytic leukemia (APL) patients, retinoic acid (RA) triggers differentiation while arsenic trioxide (arsenic) induces both a partial differentiation and apoptosis. Although their mechanisms of action are believed to be distinct, these two drugs both induce the catabolism of the oncogenic promyelocytic leukemia (PML)/RARα fusion protein. While APL cell lines resistant to one agent are sensitive to the other, the benefit of combining RA and arsenic in cell culture is controversial, and thus far, no data are available in patients. Using syngenic grafts of leukemic blasts from PML/RARα transgenic mice as a model for APL, we demonstrate that arsenic induces apoptosis and modest differentiation, and prolongs mouse survival. Furthermore, combining arsenic with RA accelerates tumor regression through enhanced differentiation and apoptosis. Although RA or arsenic alone only prolongs survival two- to threefold, associating the two drugs leads to tumor clearance after a 9-mo relapse-free period. These studies establishing RA/arsenic synergy in vivo prompt the use of combined arsenic/RA treatments in APL patients and exemplify how mouse models of human leukemia can be used to design or optimize therapies.
PMCID: PMC2193002  PMID: 10190895
differentiation; apoptosis; cancer; clinical trials; transgenics
16.  PIC-1/SUMO-1-Modified PML-Retinoic Acid Receptor α Mediates Arsenic Trioxide-Induced Apoptosis in Acute Promyelocytic Leukemia 
Molecular and Cellular Biology  1999;19(7):5170-5178.
Fusion proteins involving the retinoic acid receptor α (RARα) and PML or PLZF nuclear protein are the genetic markers of acute promyelocytic leukemia (APL). APLs with PML-RARα or PLZF-RARα fusion protein differ only in their response to retinoic acid (RA) treatment: the t(15;17) (PML-RARα-positive) APL blasts are sensitive to RA in vitro, and patients enter disease remission after RA treatment, while those with t(11;17) (PLZF-RARα-positive) APLs do not. Recently it has been shown that complete remission can be achieved upon treatment with arsenic trioxide (As2O3) in PML-RARα-positive APL, even when the patient has relapsed and the disease is RA resistant. This appears to be due to apoptosis induced by As2O3 in the APL blasts by poorly defined mechanisms. Here we report that (i) As2O3 induces apoptosis only in cells expressing the PML-RARα, not the PLZF-RARα, fusion protein; (ii) PML-RARα is partially modified by covalent linkage with a PIC-1/SUMO-1-like protein prior to As2O3 treatment, whereas PLZF-RARα is not; (iii) As2O3 treatment induces a change in the modification pattern of PML-RARα toward highly modified forms; (iv) redistribution of PML nuclear bodies (PML-NBs) upon As2O3 treatment is accompanied by recruitment of PIC-1/SUMO-1 into PML-NBs, probably due to hypermodification of both PML and PML-RARα; (v) As2O3-induced apoptosis is independent of the DNA binding activity located in the RARα portion of the PML-RARα fusion protein; and (vi) the apoptotic process is bcl-2 and caspase 3 independent and is blocked only partially by a global caspase inhibitor. Taken together, these data provide novel insights into the mechanisms involved in As2O3-induced apoptosis in APL and predict that treatment of t(11;17) (PLZF-RARα-positive) APLs with As2O3 will not be successful.
PMCID: PMC84360  PMID: 10373566
17.  Flow cytometric immunobead assay for fast and easy detection of PML–RARA fusion proteins for the diagnosis of acute promyelocytic leukemia 
Leukemia  2012;26(9):1976-1985.
The PML–RARA fusion protein is found in approximately 97% of patients with acute promyelocytic leukemia (APL). APL can be associated with life-threatening bleeding complications when undiagnosed and not treated expeditiously. The PML–RARA fusion protein arrests maturation of myeloid cells at the promyelocytic stage, leading to the accumulation of neoplastic promyelocytes. Complete remission can be obtained by treatment with all-trans-retinoic acid (ATRA) in combination with chemotherapy. Diagnosis of APL is based on the detection of t(15;17) by karyotyping, fluorescence in situ hybridization or PCR. These techniques are laborious and demand specialized laboratories. We developed a fast (performed within 4–5 h) and sensitive (detection of at least 10% malignant cells in normal background) flow cytometric immunobead assay for the detection of PML–RARA fusion proteins in cell lysates using a bead-bound anti-RARA capture antibody and a phycoerythrin-conjugated anti-PML detection antibody. Testing of 163 newly diagnosed patients (including 46 APL cases) with the PML–RARA immunobead assay showed full concordance with the PML–RARA PCR results. As the applied antibodies recognize outer domains of the fusion protein, the assay appeared to work independently of the PML gene break point region. Importantly, the assay can be used in parallel with routine immunophenotyping for fast and easy diagnosis of APL.
doi:10.1038/leu.2012.125
PMCID: PMC3437408  PMID: 22948489
PML–RARA protein; t(15;17); APL; immunobead; flow cytometry
18.  Effects of arsenic trioxide, all-trans-retinoic acid and dexamethasone on NB4 cell line 
Background and the purpose of the study
Experimental and preclinical observations have indicated that combination therapy with all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) may strongly enhance their therapeutic effects in the treatment of acute promyelocytic leukemia (APL). Whilst dexamethasone (Dex) is routinely used for the control of APL- differentiation syndrome, its effect on the pharmacodynamics of ATO is not clear. Therefore, in this study, effects of therapeutic concentrations of ATO, ATRA and Dex and their sequential usages on the proliferation, differentiation and apoptosis in t(15;17)-positive NB4 cells was investigated.
Methods
Cells were treated with therapeutic concentrations of ATO, ATRA and Dex either as single or in combination and cell proliferation was assessed by XTT assay. Expression of CD11b as an indicator of cell differentiation and the percentage of 7-AAD positive cells as a marker of apoptosis were determined by flow cytometry.
Results
ATO, but not ATRA and Dex, decreased proliferation of the cells dose-dependently. Pre-treatment of the cells with any of the drugs did not alter the effects of other drugs on the proliferation. Pre-treatments with Dex blocked the apoptotic effect of ATO (1 µM).
Conclusion
No improvement or antagonistic effects was observed with the pretreatment/ combination of the ATO and ATRA on the differentiation and apoptosis of the cells. It is possible that concomitant usage of Dex with apoptotic doses of ATO in APL patients counteract therapeutic effects of ATO.
PMCID: PMC3304349  PMID: 22615633
ATO; ATRA; Dexamethasone; Combination therapy; Apoptosis.
19.  Inhibition of the LSD1 (KDM1A) demethylase reactivates the all-trans-retinoic acid differentiation pathway in acute myeloid leukemia 
Nature medicine  2012;18(4):605-611.
Acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML), characterized by the t(15;17)-associated PML-RARA fusion, has been successfully treated with therapy utilizing all-trans-retinoic acid (ATRA) to differentiate leukemic blasts. However, among patients with non- APL AML, ATRA-based treatment has not been effective. Here we show that, through epigenetic reprogramming, inhibitors of lysine- specific demethylase 1 (LSD1, also called KDM1A), including tranylcypromine (TCP), unlocked the ATRA-driven therapeutic response in non-APL AML. LSD1 inhibition did not lead to a large-scale increase in histone 3 Lys4 dimethylation (H3K4me2) across the genome, but it did increase H3K4me2 and expression of myeloid-differentiation–associated genes. Notably, treatment with ATRA plus TCP markedly diminished the engraftment of primary human AML cells in vivo in nonobese diabetic (NOD)- severe combined immunodeficient (SCID) mice, suggesting that ATRA in combination with TCP may target leukemia-initiating cells. Furthermore, initiation of ATRA plus TCP treatment 15 d after engraftment of human AML cells in NOD-SCID γ (with interleukin-2 (IL-2) receptor γ chain deficiency) mice also revealed the ATRA plus TCP drug combination to have a potent anti-leukemic effect that was superior to treatment with either drug alone. These data identify LSD1 as a therapeutic target and strongly suggest that it may contribute to AML pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for new combinatorial therapies for AML.
doi:10.1038/nm.2661
PMCID: PMC3539284  PMID: 22406747
20.  UBE1L represses PML/RARα by targeting the PML domain for ISG15ylation 
Molecular cancer therapeutics  2008;7(4):905-914.
Acute promyelocytic leukemia (APL) is characterized by expression of promyelocytic leukemia (PML)/retinoic acid (RA) receptor α (RARα) protein and all-trans-RA-mediated clinical remissions. RA treatment can confer PML/RARα degradation, overcoming dominant-negative effects of this oncogenic protein. The present study uncovered independent retinoid degradation mechanisms, targeting different domains of PML/RARα. RA treatment is known to repress PML/RARα and augment ubiquitin-activating enzyme-E1-like (UBE1L) protein expression in NB4-S1 APL cells. We previously reported RA-induced UBE1L and the IFN-stimulated gene, 15-kDa protein ISG15ylation in APL cells. Whether the ubiquitin-like protein ISG15 directly conjugates with PML/RARα was not explored previously and is examined in this study. Transient transfection experiments with different PML/RARα domains revealed that RA treatment preferentially down-regulated the RARα domain, whereas UBE1L targeted the PML domain for repression. As expected, ubiquitin-specific protease 18 (UBP43/USP18), the ISG15 deconjugase, opposed UBE1L but not RA-dependent PML/RARα degradation. In contrast, the proteasomal inhibitor, N-acetyl-leucinyl-leucinylnorleucinal, inhibited both UBE1L- and RA-mediated PML/RARα degradation. Notably, UBE1L induced ISG15ylation of the PML domain of PML/RARα, causing its repression. These findings confirmed that RA triggers PML/RARα degradation through different domains and distinct mechanisms. Taken together, these findings advance prior work by establishing two pathways converge on the same oncogenic protein to cause its degradation and thereby promote antineoplastic effects. The molecular pharmacologic implications of these findings are discussed.
doi:10.1158/1535-7163.MCT-07-0515
PMCID: PMC2597092  PMID: 18413804
21.  New Role for Granulocyte Colony-Stimulating Factor-Induced Extracellular Signal-Regulated Kinase 1/2 in Histone Modification and Retinoic Acid Receptor α Recruitment to Gene Promoters: Relevance to Acute Promyelocytic Leukemia Cell Differentiation ▿ 
Molecular and Cellular Biology  2011;31(7):1409-1418.
The induction of the granulocytic differentiation of leukemic cells by all-trans retinoic acid (RA) has been a major breakthrough in terms of survival for acute promyelocytic leukemia (APL) patients. Here we highlight the synergism and the underlying novel mechanism between RA and the granulocyte colony-stimulating factor (G-CSF) to restore differentiation of RA-refractory APL blasts. First, we show that in RA-refractory APL cells (UF-1 cell line), PML-RA receptor alpha (RARα) is not released from target promoters in response to RA, resulting in the maintenance of chromatin repression. Consequently, RARα cannot be recruited, and the RA target genes are not activated. We then deciphered how the combination of G-CSF and RA successfully restored the activation of RA target genes to levels achieved in RA-sensitive APL cells. We demonstrate that G-CSF restores RARα recruitment to target gene promoters through the activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and the subsequent derepression of chromatin. Thus, combinatorial activation of cytokines and RARs potentiates transcriptional activity through epigenetic modifications induced by specific signaling pathways.
doi:10.1128/MCB.00756-10
PMCID: PMC3135284  PMID: 21262770
22.  Arsenic Trioxide Is a Potent Inhibitor of the Interaction of SMRT Corepressor with Its Transcription Factor Partners, Including the PML-Retinoic Acid Receptor α Oncoprotein Found in Human Acute Promyelocytic Leukemia 
Molecular and Cellular Biology  2001;21(21):7172-7182.
The SMRT corepressor complex participates in transcriptional repression by a diverse array of vertebrate transcription factors. The ability to recruit SMRT appears to play a crucial role in leukemogenesis by the PML-retinoic acid receptor α (RARα) oncoprotein, an aberrant nuclear hormone receptor implicated in human acute promyelocytic leukemia (APL). Arsenite induces clinical remission of APL through a incompletely understood mechanism. We report here that arsenite is a potent inhibitor of the interaction of SMRT with its transcription factor partners, including PML-RARα. Arsenite operates, in part, through a mitogen-activated protein (MAP) kinase cascade culminating in phosphorylation of the SMRT protein, dissociation of SMRT from its nuclear receptor partners, and a relocalization of SMRT out of the nucleus into the cytoplasm of the cell. Conversely, inhibition of this MAP kinase cascade attenuates the effects of arsenite on APL cells. Our results implicate SMRT as an important biological target for the actions of arsenite in both normal and neoplastic cells.
doi:10.1128/MCB.21.21.7172-7182.2001
PMCID: PMC99892  PMID: 11585900
23.  Blockade of the Ubiquitin Protease UBP43 Destabilizes the Transcription Factor PML/RARα and Inhibits Growth of Acute Promyelocytic Leukemia 
Cancer research  2010;70(23):9875-9885.
New treatments for acute promyelocytic leukemia (APL) are needed. APL cell treatment with all-trans-retinoic acid (RA) degrades the chimeric, dominant negative-acting transcription factor PML/RARα, which is generated by chromosomal translocation. The E1-like ubiquitin-activating enzyme UBE1L associates with interferon stimulated gene ISG15 that binds and represses PML/RARα protein. Ubiquitin protease UBP43/USP18 removes ISG15 from conjugated proteins. In this study, we explored how RA regulates UBP43 expression and the effects of UBP43 on PML/RARα stability and APL growth, apoptosis and differentiation. RA treatment induced UBE1L, ISG15 and UBP43 expression in RA-sensitive but not RA-resistant APL cells. Similar in vivo findings were obtained in a transgenic mouse model of transplantable APL and in the RA response of leukemic cells harvested directly from APL patients. UBP43 knockdown repressed PML/RARα protein levels and inhibited RA-sensitive or RA-resistant APL cell growth by destabilizing the PML domain of PML/RARα. This inhibitory effect promoted apoptosis but did not affect the differentiation response in these APL cells. In contrast, elevation of UBP43 expression stabilized PML/RARα protein and inhibited apoptosis. Taken together, our findings define UBP43 as a novel candidate drug target for APL treatment.
doi:10.1158/0008-5472.CAN-10-1100
PMCID: PMC2999664  PMID: 20935222
UBP43/USP18; UBE1L; ISG15; acute promyelocytic leukemia; and PML/RARα
24.  PML-RARα Initiates Leukemia by Conferring Properties of Self-Renewal to Committed Promyelocytic Progenitors 
Acute promyelocytic leukemia (APL) is characterized by hyperproliferation of promyelocytes, progenitors that are committed to terminal differentiation into granulocytes, making it an ideal disease in which to study the transforming potential of less primitive cell types. We utilized a murine model of APL in which the PML-RARα oncogene is expressed from the endogenous Cathepsin G promoter to test the hypothesis that leukemia stem cell activity resides within the differentiated promyelocyte compartment. We prospectively purified promyelocytes from transgenic mice at various stages of disease and observed that PML-RARα-expressing promyelocytes from young preleukemic mice had acquired properties of self-renewal both in vitro and in vivo. Progression to acute leukemia was associated with an expansion of the promyelocyte compartment at the expense of other stem, progenitor and terminally differentiated populations. Leukemic promyelocytes exhibited properties of self-renewal, and were capable of engendering leukemia in secondary recipient mice. These data indicate that PML-RARα alone can confer properties of self-renewal to committed hematopoietic progenitors prior to the onset of disease. These findings are consistent with the hypothesis that cancer stem cells may arise from committed progenitors that lack stem cell properties, provided that the initiating mutation in cancer progression activates programs that confer properties of self-renewal.
doi:10.1038/leu.2009.63
PMCID: PMC2914549  PMID: 19322209
self-renewal; progenitor; promyelocyte; APL
25.  PML-RARα enhances constitutive autophagic activity through inhibiting the Akt/mTOR pathway 
Autophagy  2011;7(10):1132-1144.
Autophagy is a highly conserved, closely regulated homeostatic cellular activity that allows for the bulk degradation of long-lived proteins and cytoplasmic organelles. Its roles in cancer initiation and progression and in determining the response of tumor cells to anticancer therapy are complicated, and only limited investigation has been conducted on the potential significance of autophagy in the pathogenesis and therapeutic response of acute myeloid leukemia. Here we demonstrate that the inducible or transfected expression of the acute promyelocytic leukemia (APL)-specific PML-RARα, but not PLZF-RARα or NPM-RARα, fusion protein upregulates constitutive autophagy activation in leukemic and nonleukemic cells, as evaluated by hallmarks for autophagy including transmission electron microscopy. The significant increase in autophagic activity is also found in the leukemic cells-infiltrated bone marrow and spleen from PML-RARα-transplanted leukemic mice. The autophagy inhibitor 3-methyladenine significantly abrogates the autophagic events upregulated by PML-RARα, while the autophagic flux assay reveals that the fusion protein induces autophagy by increasing the on-rate of autophagic sequestration. Furthermore, this modulation of autophagy by PML-RARα is possibly mediated by a decreased activation of the Akt/mTOR pathway. Finally, we also show that autophagy contributes to the anti-apoptotic function of the PML-RARα protein. Given the critical role of the PML-RARα oncoprotein in APL pathogenesis, this study suggests an important role of autophagy in the development and treatment of this disease.
doi:10.4161/auto.7.10.16636
PMCID: PMC3210306  PMID: 21673516
autophagy; acute promyelocytic leukemia (APL); PML-RARα; mTOR; apoptosis

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