All-trans Retinoic Acid Differentiation Research Articles

The transcriptional corepressor CBFA2T3 inhibits all-trans-retinoic acid–induced myeloid gene expression and differentiation in acute myeloid leukemia

CBFA2/RUNX1 partner transcriptional co-repressor 3 (CBFA2T3, also known as MTG16 or ETO2) is a myeloid translocation gene family protein that functions as a master transcriptional corepressor in hematopoiesis. Recently, it has been shown that CBFA2T3 maintains leukemia stem cell gene expression and promotes relapse in acute myeloid leukemia (AML). However, a role for CBFA2T3 in myeloid differentiation of AML has not been reported. Here, we show that CBFA2T3 represses retinoic acid receptor (RAR) target gene expression and inhibits all-trans-retinoic acid (ATRA)-induced myeloid differentiation of AML cells. ChIP-Seq revealed that CBFA2T3 targets the RARα/RXRα cistrome in U937 AML cells, predominantly at myeloid-specific enhancers associated with terminal differentiation. CRISPR/Cas9-mediated abrogation of CBFA2T3 resulted in spontaneous and ATRA-induced activation of myeloid-specific genes in a manner correlated with myeloid differentiation. Importantly, these effects were reversed by CBFA2T3 re-expression. Mechanistic studies showed that CBFA2T3 inhibits RAR target gene transcription by acting at an early step to regulate histone acetyltransferase recruitment, histone acetylation, and chromatin accessibility at RARα target sites, independently of the downstream, RAR-mediated steps of transcription. Finally, we validated the inhibitory effect of CBFA2T3 on RAR in multiple AML subtypes and patient samples. To our knowledge, this is the first study to show that CBFA2T3 down-regulation is both necessary and sufficient for enhancing ATRA-induced myeloid gene expression and differentiation of AML cells. Our findings suggest that CBFA2T3 can serve as a potential target for enhancing AML responsiveness to ATRA differentiation therapies.

Depletion of the Long Non-Coding RNA MALAT1 primes Chronic Myelomonocytic Leukemia (CMML) for Differentiation Therapy with All-Trans retinoic Acid (ATRA) through the Transcription Factor CREB

Improvements in antisense technology have now enabled clinically relevant therapeutic credentialing of the noncoding genome. MALAT1 is a long non-coding RNA that, among other functions, is thought to serve as a nuclear scaffold for splicing and transcription factors. MALAT1 expression is associated with inferior prognosis across solid tumors and its depletion impairs proliferation and metastasis in preclinical solid tumor models. We found that elevated MALAT1 levels are independently associated with inferior overall survival in patients with CMML. Further, RNA-sequencing of primary CMML monocytes identified MALAT1 as the fourth most over-expressed transcript compared to controls. Therefore, we explored the biologic relevance and therapeutic candidacy of MALAT1 across several human and murine models of CMML. First, we crossed NRASQ61R/+Mx1-cre driven mice, which display a CMML-like phenotype, to MALAT1KO/KOmice. Although MALAT1KO/KOmice did not have abnormalities in complete blood counts, immunophenotyping of the hematopoietic stem cell (HSC) compartment identified statistically significantly lower numbers of HSC compared to wild type (WT) controls and a non-significant decrease in NRASQ61R/+/MALAT1KO/KOcompared to NRASQ61R/+alone. This decrease in HSC was not a result of impaired self-renewal as no differences were observed in these models after in vivo competitive transplant experiments. Therefore, we reasoned that MALAT1 expression may be controlling HSC differentiation. To test this, we transformed bone marrow cells from these models with an estrogen-regulated (ER) Hoxb8 construct enabling cells to maintain an HSC state until ER is withdrawn and myeloid differentiation is induced. ER-Hoxb8NRASQ61R/+/MALAT1KO/KOcells had increased basal levels of Gr-1 compared to ER-Hoxb8 transformed NRASQ61R/+alone that was dramatically enhanced upon ER withdrawal suggesting that MALAT1 depletion regulates myeloid differentiation. These findings were validated by assessment of morphology, transcriptome, and in vivo immunophenotyping of bone marrow and spleen cells. Further, moribund NRASQ61R/+/MALAT1KO/KOmice displayed a reduction in organomegaly typically associated with leukemic burden. We validated this in human monocytic leukemia by generating MALAT1 depleted THP-1 isogeneic cell lines using the CRISPR/Cas9 system. MALAT1 depleted THP-1 cells (MKO) had greater terminal differentiation according to immunophenotypic markers and morphology that was greatly enhanced when treated with phorbol myristate acetate. Last, MKO orthotopic xenografts demonstrated inferior human leukemia engraftment and decreased spleen and liver weights, and heterotopic xenografts exhibited reduced tumor volume, collectively suggesting diminished leukemic burden. Because ATRA has been clinically tested in CMML with minimal effects, we next explored whether MALAT1 depletion could potentiate ATRA differentiation in CMML. First, we treated MKO cells with ATRA and observed a large induction of myeloid differentiation by marker expression and morphologic assessment compared to isogenic controls. This was validated by NRASQ61R/+/MALAT1KO/KOmice demonstrating that ATRA more robustly induced differentiation compared to vehicle which was not seen in NRASQ61R/+/MALAT1+/+mice. Next, we tested MALAT1 antisense oligonucleotides (ASOs) currently under clinical development in THP-1 cells +/- ATRA and demonstrated both an increase in myeloid differentiation and apoptosis compared to ATRA alone. To test this therapeutic strategy in primary CMML specimens, we generated CMML patient-derived xenografts (n=30 mice) and treated each with ASO, ATRA, the combination, or controls and identified a more robust reduction in human HSC engraftment with the combination. To explore the mechanistic basis for these findings, we performed RNA-sequencing of MALAT1-depleted or control cells and identified that CREB target genes were differentially expressed. Basal protein levels of p-CREB were also decreased in MKO cells and were further reduced in the nucleus of MKO by western and microscopy. Lastly, overexpression of WT or constitutively active CREB but not its dominant negative rescued the differentiation effect seen in ATRA treated MKO cells. Taken together, MALAT1 is a novel, CREB-dependent regulator of myeloid differentiation and its depletion potentiates ATRA therapy. Disclosures Cluzeau: Menarini: Consultancy; Jazz Pharma: Consultancy; Abbvie: Consultancy. Komrokji:celgene: Consultancy; pfizer: Consultancy; DSI: Consultancy; JAZZ: Speakers Bureau; Novartis: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy. MacLeod:Ionis Pharmaceuticals: Employment. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Epling-Burnette:Forma Therapeutics: Research Funding; Celgene Corporation: Patents & Royalties, Research Funding; Incyte Corporation: Research Funding.

Abstract 3064: Epigenetic manipulation can sensitize AML cells to differentiate with ATRA

Abstract Human acute myeloid leukemia (AML), the most common type of acute leukemia, has approximately eight subtypes, many of which have poor prognosis. Most of these subtypes are associated with specific, recurrent chromosome translocations. These translocations result in fusion genes, which encode oncoproteins that block differentiation and promote proliferation of immature cells. The Myeloid Lymphoid Leukemia gene (MLL) is frequently involved in these translocations, and is considered a driver of the AML. Differentiation promoting drugs, such as all-trans-retinoic acid (ATRA) are an attractive alternative to cytotoxic chemotherapy, but few types of AML, other than acute promyelocytic leukemia (APL), respond to ATRA. We hypothesize that specific genes must be activated or inhibited in AML for drugs like ATRA to induce differentiation, and that gene activation or inhibition may be the result of specific epigenetic modification. We also hypothesize that AML with different genetic alterations may respond differently to specific epigenetic inhibitors. Our initial studies have focused on two MLL-driven AML cell lines, MV4;11 and THP-1, showing translocations t(4;11) and t(9;11), respectively, and one non-MLL related AML cell line, U937. MV4;11, THP-1, and U937 were treated with specific epigenetic modifiers, including tranylcypromine (TCP), an inhibitor of histone demethylase KMD1A/LSD1, N-acetyl-dinaline (CI-994), a general histone deacetylase inhibitor, and 3-deazaneplanocin A (DZNep), a S-adenosylhomocystein hydrolase inhibitor that depletes EZH2 and thus its associated H3K27me3 activity. Evidence for differentiation was noted in a variety of assays, including reduced cell proliferation as measured directly and by MTT assay, upregulation of myeloid-specific cell surface markers such as CD11b as measured by fluorescence-activated cell sorting (FACS), myeloid-related nuclear morphological changes noted with cytospin analysis of cells, and decreased AML-associated gene expression with qPCR. All three drugs seemed to sensitize U937 and THP-1 cells to differentiate when treated with ATRA. MV4;11 cells were sensitized to differentiate by CI-994 and DZNep with or without ATRA, although there was no indication of CD11b upregulation. However, MV4;11 was not sensitized by TCP to differentiate with or without ATRA. These experiments suggest epigenetic inhibitors may increase sensitivity to ATRA differentiation therapy, but since the two MLL cell lines responded differently the response may still be dependent on the specific MLL partner gene driving the AML. Citation Format: Kalsi Heimdal, Bikalpa Ghimire, Edjay Ralph Hernandez, Heidi J. Gill Super. Epigenetic manipulation can sensitize AML cells to differentiate with ATRA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3064.

Abstract SY35-03: Novel anti-tumor activity of targeted LSD1 inhibition

Abstract Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. These complexes include Histone Deacetylases (HDAC1/2) and Co-Repressor for Element-1-Silencing Transcription factor (CoREST). LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression and has been shown to play a role in development. LSD1 can interact with pluripotency factors in human embryonic stem cells and is essential for decommissioning enhancers in stem cell differentiation. Beyond embryonic settings, LSD1 is also critical for hematopoietic differentiation. LSD1 dysregulation plays a key role in cancer and LSD1 is overexpressed in multiple tumor types, including acute myeloid leukemia (AML). Recent literature suggests inhibition of LSD1 reactivates the all-trans retinoic acid differentiation pathway in AML. Together, these observations suggest LSD1 is an important regulator of the epigenome that modulates gene expression through post-translational modification of histones and its presence in transcriptional complexes. The current study describes the anti-tumor effects of a novel LSD1 inhibitor (GSK2879552). GSK2879552 is a potent, selective, mechanism-based irreversible inhibitor of LSD1 currently in clinical development. Screening of over 150 cancer cell lines revealed that while many tumor types appear resistant to LSD1 inhibition, specific tumor types were uniquely sensitive. For example, AML cells have a distinct requirement for LSD1, and LSD1 inhibition results in a prodifferentiation effect in this setting. Preclinical characterization of the anti-tumor activities of GSK2879552 both in vitro and in vivo in AML as well as the solid tumor setting will be discussed. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Citation Format: Ryan G. Kruger. Novel anti-tumor activity of targeted LSD1 inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY35-03. doi:10.1158/1538-7445.AM2014-SY35-03

Distinguishing between cancer cell differentiation and resistance induced by all-trans retinoic acid using transcriptional profiles and functional pathway analysis.

All-trans retinoic acid (ATRA) induces differentiation in various cell types and has been investigated extensively for its effective use in cancer prevention and treatment. Relapsed or refractory disease that is resistant to ATRA is a clinically significant problem. To identify the molecular mechanism that bridges ATRA differentiation and resistance in cancer, we selected the multidrug-resistant leukemia cell line HL-60[R] by exposing it to ATRA, followed by sequential increases of one-half log concentration. A cytotoxicity analysis revealed that HL-60[R] cells were highly resistant to ATRA, doxorubicin, and etoposide. A comparative genome hybridization analysis of HL-60[R] cells identified gains of 4q34, 9q12, and 19q13 and a loss of Yq12 compared with in the parental HL-60 cell line. Transcriptional profiles and functional pathway analyses further demonstrated that 7 genes (FEN1, RFC5, EXO1, XRCC5, PARP1, POLR2F, and GTF2H3) that were relatively up-regulated in HL-60[R] cells and repressed in cells with ATRA-induced differentiation were related to mismatch repair in eukaryotes, DNA double-strand break repair, and nucleotide excision repair pathways. Our results suggest that transcriptional time series profiles and a functional pathway analysis of drug resistance and ATRA-induced cell differentiation will be useful for identifying promyelocytic leukemia patients who are eligible for new therapeutic strategies.

High-Resolution Array CGH Profiling Identifies Na/K Transporting ATPase Interacting 2 (NKAIN2) as a Predisposing Candidate Gene in Neuroblastoma

Neuroblastoma (NB), the most common solid cancer in early childhood, usually occurs sporadically but also its familial occurance is known in 1-2% of NB patients. Germline mutations in the ALK and PHOX2B genes have been found in a subset of familial NBs. However, because some individuals harbouring mutations in these genes do not develop this tumor, additional genetic alterations appear to be required for NB pathogenesis. Herein, we studied an Italian family with three NB patients, two siblings and a first cousin, carrying an ALK germline-activating mutation R1192P, that was inherited from their unaffected mothers and with no mutations in the PHOX2B gene. A comparison between somatic and germline DNA copy number changes in the two affected siblings by a high resolution array-based Comparative Genomic Hybridization (CGH) analysis revealed a germline gain at NKAIN2 (Na/K transporting ATPase interacting 2) locus in one of the sibling, that was inherited from the parent who does not carry the ALK mutation. Surprisingly, NKAIN2 was expressed at high levels also in the affected sibling that lacks the genomic gain at this locus, clearly suggesting the existance of other regulatory mechanisms. High levels of NKAIN2 were detected in the MYCN-amplified NB cell lines and in the most aggressive NB lesions as well as in the peripheral blood of a large cohort of NB patients. Consistent with a role of NKAIN2 in NB development, NKAIN2 was down-regulated during all-trans retinoic acid differentiation in two NB cell lines. Taken together, these data indicate a potential role of NKAIN2 gene in NB growth and differentiation.

Differentiation treatment by all-trans retinoic acid reduces phenotype of glioma stem cells

To explore the effects of all-trans retinoic acid (ATRA) on glioma stem cell phenotype. The glioma stem cell (GSC) from surgically resected human glioma specimens were isolated and enriched by neurosphere assay and then its differentiation was induced with all-trans retinoic acid (ATRA, 1 µmol/L) for 1 week. Markers were determined by flow cytometry, Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Side population cells were analyzed by flow cytometry. Growth characteristics were detected by neurosphere formation assay and cell cycle analysis. GSC and the differentiated cells (1×10(5)) were implanted stereotactically and intracranially into the Balb/c nude mice to compare the survival time. All data were analyzed with the SPSS software version 17.0. ATRA potently induced the differentiation of GSC and reduced glioma stem cell phenotype. And there were an elevated expression of glial fibrillary acidic protein (GFAP) and a reduced expression of such stem cell makers as CD133 and Nestin. The side population rate decreased. ATRA inhibited the neurosphere formation of GSC and induced the arrest of cell growth. ATRA could prolong the survival time. GSC may be differentiated efficiently by ATRA. The phenotype of GSC decreases obviously after the differentiation of ATRA and the survival time is prolonged. Thus ATRA may be applied for targeted therapies of glioma stem cell.

Src Family Kinase Inhibitor PP2 Has Different Effects On ATRA or Arsenic Trioxide-Induced Differentiation of Acute Promyelocytic Leukemia Cell Line

AbstractAbstract 4800Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) with distinctive biologic and clinical features that is now highly curable. Leukemic promyelocytes have the unique ability to undergo differentiation after exposure to retinoic acid and both differentiation and apoptosis after exposure to arsenic trioxide (As2O3, ATO). Emerging evidence has implicated Src family kinases (SFKs) as regulators of proliferation and survival of myeloid lineage cells. Recent studies showed that inhibition of SFKs resulted in enhancement of retinoic acid-induced myeloid differentiation. In this study, we demonstrated that the SFK inhibitor PP2 enhanced the differentiation of NB4 cells when combined with ATO as well when combined with as all-trans-retinoic acid (ATRA) and the difference in the retinoic acid-induced gene expression between the cells treated with PP2 in combination with ATRA and the cells treated with PP2 in combination with ATO.SFK inhibitor PP2 significantly enhanced ATRA- or ATO-induced differentiation of NB4 cells. The synergistic effect was significantly stronger when PP2 was combined with ATRA than when PP2 was combined with ATO. Flow cytometric analysis demonstrated a significant increase in CD11b-positive granulocytes up to 60.73% and 31.58%, respectively. These results were confirmed by morphologic analysis using Wright stain and NBT staining. These effects were not related to apoptosis. Annexin-V-fluorescein staining revealed that PP2 combined with ATRA or PP2 combined with ATO did not induce apoptosis in NB4 cells. Retinoic acid-induced gene expression was different in both groups. ICAM-1 expression was significantly increased in the cells treated with PP2 in combination with ATRA whereas cathepsin D expression was significantly increased in the cells treated with PP2 in combination with ATO. These findings suggested that the synergistic effect of SFK inhibitor PP2 in combination with ATRA was significantly stronger than that of PP2 in combination with ATO on NB4 myeloid leukemia cell differentiation and this difference was related to the disparate activation of retinoic acid-induced genes. Disclosures:No relevant conflicts of interest to declare.

Cardiac stunning as a manifestation of ATRA differentiation syndrome in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is a specific type of acute myelogenous leukemia characterized by the reciprocal translocations between the long arms of chromosomes 15 and 17 [t(15;17)] leading to the fusion of the retinoic acid receptor and the promyelocytic leukemia (PML) genes, called PML/RARalpha. The PML/RARalpha gene product acts as a transcription repressor, resulting in the blocking of the differentiation of APL blasts at the stage of promyelocytes. Pharmacological doses of all-trans retinoic acid (ATRA) reverse this blockage and induce disease remission. However, although ATRA is well tolerated in the majority of the APL patients treated with ATRA and idarubicin regimen (AIDA), approximately one quarter of them develop a complication denominated ATRA differentiation syndrome (DS), formerly known as retinoic acid syndrome [1–3]. The onset of DS ranges from 2 to 21 days (median of 10 days) after initiating ATRA therapy. The full development of DS is characterized by unexplained fever, weight gain, edema, interstitial pulmonary infiltrates with dyspnea, pleural and pericardial effusions, episodic hypotension, and acute renal failure. The most frequent manifestations are respiratory distress and fever, affecting more than 80% of DS patients. Acute myocardial ischemia is not commonly observed in DS. As far as we know, there is only one case report describing this complication [4]. Through early diagnosis, DS responds well to the administration of dexamethasone, with decrease in mortality from 30% to less than 10% in the patients suffering from it. The pathogenesis of this complication is not completely clarified, but increased expression of adhesion molecules on blasts and on endothelial cells and the release of inflammatory cytokines may be the main explanation [5].

Massive Ascites Associated with All-trans Retinoic Acid Treatment in Therapy-Related Acute Promyelocytic Leukemia

A 77-year-old man who developed pancytopenia was administered granulocyte colony-stimulating factor (G-CSF) by another doctor, and referred to us for the evaluation of pancytopenia. He had hepatocellular carcinoma and was treated with transcatheter arterial chemoembolization (TACE) containg epirubicin (total dose: 300 mg over the last two years). Bone marrow aspiration smear demonstrated hypercellular marrow with promyelocytes. Cytogenetic analysis demonstrated del(7), t(15;17)(q22;q12), and a fluorescence in-situ hybridization (FISH) study demonstrated chimeric fusion genes of PML-RAR-alpha. He was diagnosed with therapy-related acute promyelocytic leukemia (APL), and treated with all trans-retinoic acid (ATRA). He showed the progressive accumulation of ascites with liver damage, without pulmonary symptoms of ATRA differentiation syndrome. After 60 days of ATRA treatment, complete hematological and cytogenetic responses were achieved. However, the patient died of septic circulatory failure.

The cavity-to-cavity migration of leukaemic cells through 3D honey-combed hydrogels with adjustable internal dimension and stiffness

Whilst rigid, planar surfaces are often used to study cell migration, a physiological scenario requires three-dimensional (3D) scaffolds with tissue-like stiffness. This paper presents a method for fabricating periodic hydrogel scaffolds with a 3D honeycomb-like structure from colloidal crystal templates. The scaffolds, made of hydrogel-walled cavities interconnected by pores, have separately tuneable internal dimensions and adjustable gel stiffness down to that of soft tissues. In conjunction with confocal microscopy, these scaffolds were used to study the importance of cell compliance on invasive potential. Acute promyelocytic leukaemia (APL) cells were differentiated with all- trans retinoic acid (ATRA) and treated with paclitaxel. Their migration ability into the scaffolds' size-restricted pores, enabled by cell softening during ATRA differentiation, was significantly reduced by paclitaxel treatment, which interferes with cell shape recovery. These findings demonstrate the usability of the scaffolds for investigating factors that affect cell migration, and potentially other cell functions, in a realistic 3D tissue model.

Auer rods and differentiation in acute promyelocytic leukemia

Dalal et al (2007) recently reported that the residual detection of Auer rods one month after treatment of an Acute Promyelocytic Leukemia (APL) patient was a sign of maturing granulocytes under differentiation therapy but not of refractory leukemia. Auer rods deriving from the crystallisation of myeloperoxidase (MPO) granules are the hallmark of Acute Myeloid Leukemia (AML), but can also be observed in the Myelodysplastic Syndromes in which they have been associated with a more aggressive disease (Seymour & Estey, 1995). Among AML, two characteristics are specific to APL (AML-M3 subtype in the French-American-British classification): 1) the presence in more than 90% of cases of the oncogenic PML-RARα fusion protein and 2) an exquisite sensitivity to the differentiation induced by All-trans retinoic acid (ATRA) (Cassinat & Chomienne, 2001). We report herein an unexpected correlation between the presence of Auer rods in APL blast cells at diagnosis and ATRA differentiation efficacy. We have been routinely assessing primary APL cells at diagnosis to evaluate their sensitivity to differentiation induction by ATRA. As previously reported (Cassinat et al, 2001), although the percentage (%) of differentiated cells (estimated by the percentage of nitroblue tetrazolium (NBT) positive cells) after 6 d of culture was very high in almost all cases, the levels were more heterogeneous after 3 d of culture (ranging between 0% and 100%) indicating different levels of differentiation sensitivity. 128 consecutive patient samples showed an inverse correlation between the percentage of cells harbouring Auer rods and the percentage of differentiated cells at day 3 (P < 0·015, Pearson test). When patients were studied as three groups according to the proportion of cells with Auer rods (Fig 1A), the median percentages of differentiated cells after 3 d of culture were significantly lower in patients with a higher level of Auer rods (P = 0·014, Kruskal-Wallis non parametric test). (A) Results of ATRA-induced differentiation (measured by NBT test after 3 d of culture) according to the percentage of Auer rod-positive cells at diagnosis. Box 1: 0 to 1% Auer rod-positive cells (n = 48 patients). Box 2: 2 to 15% Auer rod-positive cells (n = 55 patients). Box 3: at least 15% Auer rod-positive cells (n = 25 patients). (B) Distribution of the percentage of Auer rod-positive cells in patients that relapsed (n = 8) or not (n = 30), in the group of patients with a PML-RARα transcript of the Bcr1 subtype. In 62 of these patients, clinical outcome was obtained from the APL93 and APL 2000 trials registries (median follow-up, 9 and 4 years, respectively) (Fenaux et al, 1999; Ades et al, 2006). It appeared that patients with higher levels of Auer rod-positive cells tended to have a higher risk of relapse (Table I). Accordingly, the median percentage of Auer rod-positive cells was higher in the group of patients (n = 12) that relapsed compared to patients that did not relapse (n = 50) (8·5% vs. 4·5%). This difference only trended toward significance when the whole population was considered (P = 0·14; Kruskal-Wallis non parametric test), but was in accordance with our previous observation that in vitro differentiation could be correlated to patient prognosis (Cassinat et al, 2001). Interestingly, when a more homogeneous population, such as patients harbouring a Bcr1 transcript (the most frequent transcript) were analysed (n = 38) (Fig 1B), the percentage of Auer rod-positive cells in the group of patients that relapsed was significantly higher compared to the patients that did not relapse (13·5% vs. 5%, P = 0·045, Kruskal-Wallis non parametric test). These data suggest a link between the presence of Auer rods in APL cells at diagnosis and the sensitivity of these cells to the differentiation effect of ATRA and hence with the clinical outcome. Given that Auer rods are supposed to be a result of MPO crystallisation and their presence is inconstant in AML patients, one hypothesis could be that presence of Auer rods is dependent on a particular MPO genotype. Indeed, several polymorphisms have been reported in the MPO promoter or coding regions and correlation may exist with several types of cancer, such as lung cancer (Gresner et al, 2007) or leukemia (Zhang et al, 2007). We have recently shown that MPO expression during ATRA treatment differs between patients with good or poor differentiation response (Quere et al, 2007). Thus, the present study could be the basis for testing the hypothesis that the presence of Auer rods could reflect a specific genotype correlated to ATRA sensitivity.

Induction of Both CCAAT/Enhancer Binding Protein β and ε Are Required for All Trans Retinoic Acid-Induced Granulocytic Differentiation of Myeloid Leukemia Cells.

All trans retinoic acid (ATRA) induces remission in patients with acute promyelocytic leukemia (APL) by induction of granulocytic differentiation. Since CCAAT/enhancer binding protein (C/EBP) α, β and ε play important roles in normal granulocytic differentiation we compared their expression and regulation in ATRA differentiation inducible NB4 and HL-60 cells to their ATRA differentiation resistant subclones R4 and HL-60/Res cells. All four cell lines robustly express C/EBPα but have low or absent C/EBPβ and ε expression. ATRA treatment increases the levels of C/EBPβ and ε protein in NB4 and HL-60 cells but not in the R4 and HL-60/Res cells which is correlated with the degree of differentiation induction. Knockdown of C/EBPβ or ε using shRNA decreases ATRA differentiation induction of HL-60 cells. HL-60 cells with BCR-ABL stable transfection lose expression of C/EBPα, and ATRA-induced differentiation and expression of C/EBPβ and ε are no longer seen. K562 cells which express BCR-ABL do not have detectable or inducible C/EBPα, β and ε after ATRA treatment and are resistant to ATRA differentiation induction. Ectopic expression of C/EBPα-estrogen receptor (ER) or C/EBPβ-ER, but not C/EBPε-ER, induces granulocytic differentiation in K562 cells after addition of estradiol or tamoxifen which activates these fusion factors. C/EBPα-ER or C/EBPβ-ER infected K562 cells is followed by induction of C/EBPε expression and knockdown of C/EBPε in C/EBPα-ER or C/EBPβ-ER infected K562 cells decreases induction of differentiation. The induction of C/EBPα, β, and ε expression by ATRA is investigated in ten additional myeloid leukemia cell lines and it is found that the expression of C/EBPβ and ε, but not C/EBPα, are induced in THP-1 and ML-1 cells which are responsive to ATRA differentiation induction. These results indicate that induction of C/EBPβ is required and C/EBPε plays a collaborative role with C/EBPβ in ATRA-induced differentiation of myeloid leukemia cells.

Combination of all-trans retinoic acid and taxol regressed glioblastoma T98G xenografts in nude mice

Glioblastoma is the most prevalent and highly malignant brain tumor that continues to defy current treatment strategies. This investigation used all-trans retinoic acid (ATRA) and taxol (TXL) as a combination therapy for controlling the growth of human glioblastoma T98G xenografted in athymic nude mice. Histopathological examination revealed that ATRA induced differentiation and combination of ATRA and TXL caused more apoptosis than either treatment alone. Combination therapy decreased expression of telomerase, nuclear factor kappa B (NFkappacapital VE, Cyrillic), and inhibitor-of-apoptosis proteins (IAPs) indicating suppression of survival factors while upregulated Smac/Diablo. Combination therapy also changed expression of Bax and Bcl-2 proteins leading to increased Bax:Bcl-2 ratio, mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF), and activation of caspase-9. Increased activities of calpain and caspase-3 degraded 270 kD alpha-spectrin at the specific sites to generate 145 kD spectrin breakdown product (SBDP) and 120 kD SBDP, respectively. Further, increased activity of caspase-3 cleaved inhibitor-of-caspase-activated DNase (ICAD). In situ double immunofluorescent labelings showed overexpression of calpain, caspase-12, caspase-3, and AIF during apoptosis, suggesting involvement of both caspase-dependent and caspase-independent pathways for apoptosis. Our investigation revealed that treatment of glioblastoma T98G xenografts with the combination of ATRA and TXL induced differentiation and multiple molecular mechanisms for apoptosis.

Regulation of progranulin expression in myeloid cells

Progranulin (pgrn; granulin-epithelin precursor, PC-cell-derived growth factor, or acrogranin) is a multifunctional secreted glycoprotein implicated in tumorigenesis, development, inflammation, and repair. It is highly expressed in macrophage and monocyte-derived dendritic cells. Here we investigate its regulation in myeloid cells. All-trans retinoic acid (ATRA) increased pgrn mRNA levels in myelomonocytic cells (CD34(+) progenitors; monoblastic U-937; monocytic THP-1; progranulocytic HL-60; macrophage RAW 264.7) but not in nonmyeloid cells tested. Interleukin-4 impaired basal expression of pgrn in U-937. Differentiation agents DMSO, and, in U-937 only, phorbol ester [phorbol 12-myristate,13-acetate (PMA)] elevated pgrn mRNA expression late in differentiation, suggestive of roles for pgrn in more mature terminally differentiated granulocyte/monocytes rather than during growth or differentiation. The response of pgrn mRNA to ATRA differs in U-937 and HL-60 lineages. In U-937, ATRA and chemical differentiation agents greatly increased pgrn mRNA stability, whereas, in HL-60, ATRA accelerated pgrn mRNA turnover. The initial upregulation of pgrn mRNA after stimulation with ATRA was independent of de novo protein synthesis in U-937 but not HL-60. Chemical blockade of nuclear factor-kappaB (NF-kappaB) activation impaired ATRA-stimulated pgrn expression in HL-60 but not U-937, whereas in U-937 it blocked PMA-induced pgrn mRNA expression, suggestive of cell-specific roles for NF-kappaB in determining pgrn mRNA levels. We propose that: 1) ATRA regulates pgrn mRNA levels in myelomonocytic cells; 2) ATRA acts in a cell-specific manner involving the differential control of mRNA stability and differential requirement for NF-kappaB signaling; and 3) elevated pgrn mRNA expression is characteristic of more mature cells and does not stimulate differentiation.

Curcumin Enhances Differentiation of All-Trans Retinoic Acid (ATRA)-Sensitive and ATRA-Resistant Acute Promyelocytic (APL) Cells.

The introduction of ATRA-based differentiation therapy has significantly enhanced outcomes in patients with APL. However, retinoic acid syndrome and ATRA-resistance remain significant concerns. It would therefore be useful to develop drugs that reduce the therapeutic doses of ATRA needed, and would be effective in ATRA-resistant cases. We have shown previously that curcumin, the yellow compound isolated from spice turmeric, suppresses the initiation and promotion stages of cancer development. In the present study we evaluated whether curcumin affects differentiation of NB4 APL cells. The NB4 cells were derived from a patient with APL, and differentiate in response to ATRA, while NB4-R1 cells are resistant to ATRA. Treatment of NB4 cells with 5 μM curcumin enhanced ATRA-mediated differentiation. Differentiation was assessed by evaluating CD11b expression, nitroblue tetrazolium (NBT) reduction and by morphologic examination. This curumin-mediated enhanced differentiation was apparent at 1 μM as well as 0.1 μM of ATRA. Curcumin alone did not cause differentiation of the NB4 cells, although higher concentrations of curcumin caused apoptosis. We then examined the effect of curcumin on the ATRA-resistant NB-R1 cells. Addition of ATRA and curcumin together induced differentiation of the NB4-R1 cells, whereas either agent alone did not cause differentiation. The differentiation was characterized by increased CD11b expression, NBT reduction and the typical morphologic changes. In addition, differentiation of the NB4-R1 cells was accompanied by restoration of the PML-oncogenic domains (PODs). These results indicate that curcumin may be another unconventional therapeutic agent in APL, following the successful use of ATRA and arsenic trioxide.

WEB-2086 and WEB-2170 trigger apoptosis in both ATRA-sensitive and -resistant promyelocytic leukemia cells and greatly enhance ATRA differentiation potential

PAF-receptor antagonists WEB-2086 and WEB-2170 (WEBs) have been previously shown to induce differentiation in murine and human leukemia cells. The present study describes the apoptotic-differentiative effect of WEBs in all-trans-retinoic acid (ATRA)-sensitive (NB4) and -resistant (NB4-007-6 and NB4-MR4) acute promyelocytic leukemia (APL) cell lines as well as blasts from patients with t(15;17) APL. NB4 cells exposed to 0.5-1 mM WEBs underwent striking growth arrest and massive apoptosis without appreciable differentiation; IC50 values after 3-day treatment of NB4 were 0.4 and 0.25 mM for WEB-2086 and WEB-2170, respectively. WEBs induced apoptosis also in the two ATRA-resistant NB4-007-6 and NB4-MR4 cell lines and in blasts from patients with t(15;17) APL. Moreover, subapoptotic WEBs acted synergistically with low-dose (0.025-0.05 microM) ATRA; this allowed to increase ATRA differentiation potential up to 40-fold and to improve both number and intensity of NBT-positive NB4 cells at definitely higher levels than with 1 muM ATRA alone. The powerful antiproliferative-apoptotic activities of WEBs in vitro on ATRA-sensitive, ATRA-resistant APL cells and blasts from patients with APL as well as drug capabilities to enhance ATRA differentiation potential suggested that these agents also due to their recognized tolerability in vivo might improve, alone or in combination, clinical treatment of APL.

Retinoic acid and dexamethasone regulate the expression of PEDF in retinal and endothelial cells

Both all- trans-retinoic acid (ATRA) and pigment epithelial-derived factor (PEDF) regulate cell proliferation and differentiation. Treatment of human Y-79 retinoblastoma and A-RPE 19 pigment epithelial cells with ATRA increased the levels of PEDF protein and RNA. Endothelial cells from bovine retina and human umbilical cord expressed PEDF and the levels were also increased by ATRA. Mouse Müller glial cells and rat C6 glioma cells showed at least a 2·5 fold increase in PEDF RNA levels after ATRA treatment, as measured by quantitative PCR. The PEDF promoter contains a retinoic acid receptor element (RARE). Plasmids containing a PEDF promoter regulating a luciferase gene were transfected into D407 and C6 cells and the luciferase activity measured after incubation in the presence or absence of ATRA. In both cell types ATRA increased the level of luciferase activity suggesting the RARE is functional. Dexamethasone was also effective at increasing PEDF RNA levels in both mouse Muller glial cells and C6 rat glioma cells. To test the effects of PEDF on retinoic acid function, expression of retinoic acid receptors in Y-79 and A-RPE 19 cells was measured by PCR. In Y79 cells, PEDF treatment increased the expression levels of RARα and RXRγ receptors and in the A-RPE 19 cells it resulted in a decrease in expression of the RARβ and RXRβ receptors. This study clearly indicates an interaction between PEDF and ATRA. The cell differentiation activities of PEDF may operate through mechanisms orchestrated by retinoids, and the converse may also be true. The differentiation, anti-mitotic, and apoptotic actions of PEDF and ATRA may utilize parallel pathways that converge at key junctional transduction molecules to coordinate cellular quiescence and maintain tissue mass in the presence of signals that stimulate abnormal cell proliferation. It will be an interesting therapeutic strategy to co-administer PEDF and retinoic acid in developing protocols for neovascular diseases in the eye and in cancer.

The cleavage product deltaPML-RARalpha contributes to all-trans retinoic acid-mediated differentiation in acute promyelocytic leukemia cells.

PML-RARalpha protein, the leukemogenic product of t(15,17) in acute promyelocytic leukemia, is cleaved into a truncated form termed deltaPML-RARalpha during all-trans retinoic acid (ATRA)-induced differentiation of NB4 cells. DeltaPML-RARalpha is not formed in ATRA differentiation resistant NB4 subclones. As(2)O(3) inhibits deltaPML-RARalpha formation and differentiation-induction when given in combination with ATRA. Treatment with hexamethylene bisacetamide (HMBA) combined with ATRA enhances ATRA-induced differentiation in ATRA-insensitive NB4-CI and arsenic-resistant NB4/As cells, and is associated with stabilization of PML-RARalpha protein and increased deltaPML-RARalpha formation. Unlike forced expression of PML-RARalpha, forced deltaPML-RARalpha expression based on an estimated deletion of the N-terminal PML portion does not repress RARE-tk-luc reporter activity mediated by endogenous retinoic acid receptors. The cleavage of PML-RARalpha is blocked by RARalpha antagonist Ro-41-5253 and cycloheximide and therefore requires a RARalpha transactivation-dependent pathway. Proteasome inhibitor MG-132 and caspase inhibitor Z-VAD-FMK do not block ATRA-induced PML-RARalpha cleavage and differentiation. These data suggest that (a) ATRA treatment induces PML-RARalpha cleavage by induction of unknown enzymes independent of proteasome- and caspase-mediated pathways; (b) deltaPML-RARalpha might function differently from both PML-RARalpha and RARalpha; (c) failure to cleave PML-RARalpha and form deltaPML-RARalpha after ATRA treatment may contribute to ATRA resistance in APL cells.

Cytogenetics, FISH and RT-PCR Analysis of Acute Promyelocytic Leukemia: Structure of the Fusion Point in a Case Lacking Classic t(15;17) Translocation

The chimeric gene product PML-RAR α, the result of a reciprocal t(15;17) translocation, plays an important role in the pathogenesis of acute promyelocytic leukemia (APL). In the present study on clinical effects of cytogenetics and molecular events in APL, we performed chromosome analysis, fluorescence in situ hybridization (FISH) using gene-specific probe, and reverse transcription-polymerase chain reaction (RT-PCR) analysis in 10 patients with APL. Patients were treated with all- trans retinoic acid (ATRA) and/or chemotherapy, and all achieved complete remission. Cytogenetic analysis revealed the classic translocation t(15;17) in nine of 10 patients, and a remaining patient had an apparently normal karyotype. Interphase FISH was performed in nine patients, and revealed the presence of PML-RAR α fusion gene in all patients. RT-PCR analysis in 10 patients showed that eight expressed long (L)-form type, one expressed a short (S)-form type, and the other expressed a variable (V)-form type. Metaphase FISH of the patient with normal karyotype revealed a juxtaposed PML-RAR α fusion signal on one chromosome 17 homologue, an RAR α signal on the other chromosome 17 homologue, and one PML signal on each chromosome 15 homologue. Moreover, V-form of the PML-RAR α transcript was detected, and a portion of RAR α intron 2 was found inserted in the breakpoint region. ATRA differentiation induction therapy was effective in treating this patient, a result infrequently reported in cytogenetic and molecular investigations of APL.