IN-VITRO CYTOTOXICITY AND CELL CYCLE ANALYSIS OF TWO NOVEL BIS-1, 2, 4-TRIAZOLE DERIVATIVES: 1,4-BIS[5-(5-MERCAPTO-1,3,4-OXADIAZOL-2-yl-METHYL)-THIO-4-(p-TOLYL)-1, 2,4-TRIAZOL-3-yl]-BUTANE (MNP-14) AND 1,4-BIS[5-(CARBETHOXY-METHYL)-THIO-4-(p-ETHOXY PHENYL)-1,2,4-TRIAZOL-3-yl]-BUTANE (MNP-16)

In the present study, we have tested the cytotoxic and DNA damage activity of two novel bis-1,2,4 triazole derivatives, namely 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio4-(p-tolyl)-1,2 ,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl) -1,2,4-triazol-3-yl]-butane (MNP-16). The effect of these molecules on cellular apoptosis was also determined. The in-vitro cytotoxicity was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as well as Trypan blue dye exclusion methods against human acute lymphoblastic leukemia (MOLT4) and lung cancer cells (A549). Our results showed that MNP-16 induced significant cytotoxicity (IC50 of 3-5 mu M) compared with MNP-14. The cytotoxicity induced by MNP-16 was time and concentration dependent. The cell cycle analysis by flow cytometry (fluorescence-activated cell sorting [FACS]) revealed that though there was a significant increase in the apoptotic population (sub-G1 phase) with an increased concentration of MNP-14 and 16, there was no cell cycle arrest. Further, the comet assay results indicated considerable DNA

Toll-Like Receptors 7, 8, and 9 Expression and Function in Primary Human Cervical Cancer Langerhans Cells Evidence of Anergy

Objective: Human papillomavirus oncoproteins E6 and E7 down modulate Toll-like receptor (TLR) 9 expression in infected keratinocytes. We explored the status of expression and function of TLR7, TLR8, and TLR9 in primary human Langerhans cells (LCs) isolated from cervical tumors. Methodology: Single-cell suspensions were made from fresh tissues of squamous cell carcinoma (International Federation of Gynecology and Obstetrics stage IB2); myeloid dendritic cells were purified using CD1c magnetic activated cell separation kits. Langerhans cells were further flow sorted into CD1a(+)CD207(+) cells. Acute monocytic leukemia cell line THP-1-derived LCs (moLCs) formed the controls. mRNA from flow-sorted LCs was reverse transcribed to cDNA and TLR7, TLR8, and TLR9 amplified. Monocyte-derived Langerhans cells and cervical tumor LCs were stimulated with TLR7, TLR8, and TLR9 ligands. Culture supernatants were assayed for interleukin (IL) 1 beta, IL-6, IL-10, IL-12p70, interferon (IFN) alpha, interferon gamma, and tumor necrosis factor (TNF) alpha by Luminex multiplex bead array. Human papillomavirus was genotyped. Results: We have for the first time demonstrated that the acute monocytic leukemia cell line THP-1 can be differentiated into LCs in vitro. Although these moLCs. expressed all the 3 TLRs, tumor LCs expressed TLR7 and TLR8, but uniformly lacked TLR9. Also, moLCs secreted IL-6, IL-1 beta, and tumor necrosis factor alpha to TLR8 ligand and interferon alpha in response to TLR9 ligand; in contrast, tumor LCs did not express any cytokine to any of the 3 TLR ligands. Human papillomavirus type 16 was one of the common human papillomavirus types in all cases. Conclusions: Cervical tumor LCs lacked TLR9 expression and were functionally anergic to all the 3: TLR7, TLR8, and TLR9 ligands, which may play a crucial role in immune tolerance. The exact location of block(s) in TLR7 and TLR8 signaling needs to be investigated, which would have important immunotherapeutic implications.

Análise morfológica e citogenética de medula óssea em pacientes com síndrome mielodisplásica primária

A síndrome mielodisplásica primária (SMD) compreende um grupo de doenças hematopoéticas clonal de célula tronco pluripotente cacacterizada por vários graus de pancitopenia e alterações morfológica das células hematopoeticas e risco aumentado de transformação para leucemia mielóide aguda. A citogenética e a morfologia da medula óssea desempenham um papel fundamental para o diagnóstico e o prognóstico desses pacientes. Alterações cromossômicas são encontradas em aproximadamente 30-50% dos casos. Devido à importância da análise desses fatores para escolha terapêutica, torna-se necessário definir as alterações morfológicas e citogenéticas que possam contribuir para o prognóstico. Esse trabalho visa correlacionar as características morfológicas e citogenéticas da medula óssea em pacientes com SMD primária com as classificações OMS e FAB e com o IPSS. Foram estudados 32 pacientes com SMD primária diagnosticados entre 2000 e 2009 no HUPE-UERJ. As características clínicas foram analisadas através do levantamento de prontuários. A análise citogenética foi feita pela técnica de bandeamento GTG em células da medula ossea. A análise morfológica da biópsia de medula óssea e do mielograma foram realizadas através da revisão de lâminas. Vinte e três pacientes foram classificados em estágios iniciais da doença (22 AR, 1 ARSA) e 9 em estágio avançado AREB de acordo com a FAB. Alterações cromossômicas foram detectadas em 16 pacientes (50%). As mais frequentes foram: del(11)(q23) e del(17p). Dos pacientes com doença avançada, seis (66%) apresentaram aumento significativo da relação M:E (p=0,003) e sete (77%) possuíam alterações arquiteturais acentuadas (p<0,001) em comparação ao grupo de doença inicial. Pacientes classificados como intermediário 2 e alto risco pelo IPSS tiveram importante perda arquitetural (p<0,001), número significativamente maior de micromegacariócitos (p=0,017) e seis (85%) sofreram transformação leucêmica (p=0,006). ALIP foi significantemente aumentada nos pacientes de pior prognóstico (p=0,0 1) e naqueles com doença avançada (p=0,001). Nossos resultados apresentaram implicações potenciais para o diagnóstico e o prognóstico da SMD primária. As alterações morfológicas foram associadas com as classificações FAB, OMS e com os grupos de risco segundo o IPSS.

中药蜘蛛香的化学成分研究及以羧甲基魔芋葡苷聚糖-壳聚糖为细胞膜的天冬酰胺酶人工细胞研究

本论文由三章组成。第一章介绍了中药蜘蛛香的化学成分的研究成果，第二章为羧甲基魔芋葡苷聚糖-壳聚糖为细胞膜的天冬酰胺酶人工细胞的研究，第三章综述了人工细胞在生物医学领域的应用。 第一章报道了中药蜘蛛香（Valeriana wallichii）根部乙醇提取物的化学成分，采用正、反相硅胶层析等分离方法和MS、NMR等多种波谱手段，从中共分离鉴定出17个化合物，分别为缬草素(valtrate，1)，valechlorine（2），homobadrinal（3），baldrinal（4），乙酰缬草素(acevaltrate 5)，valeriotetrate C（6），valeriotetrate B（7），对羟基苯乙酮(4'-hydroxy-acetophenone 8)，7-hydroxy valtrate（9），8-methylvalepotriate（10），1,5-dihydroxy-3,8-epoxyvalechlorine A（11），二氢缬草素(didrovaltrate 12)，胡萝卜苷（13），橙皮苷 (hesperidin 14)，prinsepiol-4-O-β-D-glucopyranoside（15），longiflorone（16），乙基糖苷（17）。其中化合物6、7、10、和11为新化合物，化合物9、15、16为首次从该植物中得到。新化合物11为含有氯原子的刚性骨架环烯醚萜，并且确定了其绝对构型。 第二章报道了以羧甲基魔芋葡苷聚糖（CKGM）和壳聚糖（CS）为膜的固定化L-天冬酰胺酶人工细胞研究成果。利用羧甲基魔芋葡苷聚糖和壳聚糖两种生物相容性很好的天然多糖之间的静电吸引力，在非常温和的条件下制备成具有半透过性膜的人工细胞，将治疗儿童急性成淋巴细胞性白血病(ALL)的药物L-天冬酰胺酶包裹在内。通过考察温度和pH对人工细胞的影响,结果表明以CKGM- CS为膜的L-天冬酰胺酶人工细胞对温度和pH的稳定性和耐受性均高于自由酶，说明CKGM-CS对酶具有保护作用，而且小分子底物和产物可以自由进出膜内外，而包裹在膜内的生物大分子则不能泄露出来。 第三章综述了微囊化人工细胞的研究进展。 This dissertation consists of three parts. In the first part, the chemical constituents from the root of Valeriana wallichii were reported. In the second part, preparation and characteristics of L-Asparaginase Artificial cell were reported. The third part is a review on progress of microcapsule artificial cell. The first chapter is about the isolation and identification of the chemical constituents from the root of V. wallichii. Seventeen compounds were isolated from the ethanol extract of roots of V. wallichii through repeated column chromatography on normal and reversed phase silica gel. By the spectroscopic and chemical evidence, their structures were elucidated as valtrate (1), valechlorine (2), homobadrinal (3), baldrinal (4), acevaltrate (5), valeriotetrate C (6), valeriotetrate B (7), 4'-hydroxy-acetophenone (8), 7-hydroxy valtrate (9), 8-methylvalepotriate (10), 1,5-dihydroxy-3,8-epoxyvalechlorine A (11), didrovaltrate (12), daucosterol (13), hesperidin (14), prinsepiol-4-O-β-D-glucopyranoside (15), longiflorone (16), and ethyl glucoside (17). Among them, 6, 7, 10, and 11 are new compounds. 15, 16 and 9 were isolated from this plant for the first time. The absolute configuration of compound 11, an unusual iridoid bearing a C-10 chlor-group and an oxo-bridge connecting C-3 and C-8 resulting in a rigid skeleton, was confirmed. The second chapter is about the semi-permeable microcapsule of carboxymethyl konjac glucomannan-chitosan for L-asparaginase immobilization. Carboxymethyl konjac glucomannan-chitosan (CKGM-CS) microcapsules, which have good biocompatibility, prepared under very mild conditions via polyelectrostatic complexation, were used for immobilize L-asparaginase-a kind of drug for acute lymphoblastic leukemia (ALL). The activity and stability under different temperature and pH of the enzyme loaded-microcapsules were studied. The results indicated the immobilized enzyme has better stability and activity contrasting to the native enzyme. The study illustrates that the L-asparaginase could be protected in CKGM-CS microcapsules, the substrate and product could pass through the system freely.

Study on bioactivity of extracts from marine sponges in Chinese Sea

The bioactivity screening of fractions from two inter-tidal sponges collected from the north of China Yellow Sea and one sponge collected from the South Chinese Sea was reported in this study. In sponge Hymeniacidon perleve there were 9 fractions out of 15 from CHCl3 extract with anti Staphylococcus aureus activity, 9 fractions out of 19 from BuOH extract with anti Escherichia coli activity, and three fractions from CHCl3 extract which had moderate to strong activity in inhibiting Bacillus subtilis, Candida albicans, and Aspergilus niger. The fractions of Reniochalina sp. showed bioactivity against bacteria and fungi. The fractions of Acanthella acuta Schmidt showed bioactivity against S. aureus and fungi. One compound from H. perleve obtained by the bioactively directing isolation was tested for bioactivity against the human hepatoma cell line Qgy7701 (IC50 10.1 mug/ml), Burkitt's lymphoma cell line Raji (IC50 9.76 mug/ml) and chronic myelogenous leukemia K562 (IC50 1.90 mug/ml). (C) 2003 Elsevier B.V. All rights reserved.

Metabolic profiling studies on the toxicological effects of realgar in rats by H-1 NMR spectroscopy

The toxicological effects of realgar after intragastrical administration (1 g/kg body weight) were investigated over a 21 day period in male Wistar rats using metabonomic analysis of H-1 NMR spectra of urine, serum and liver tissue aqueous extracts. Liver and kidney histopathology examination and serum clinical chemistry analyses were also performed. H-1 NMR spectra and pattern recognition analyses from realgar treated animals showed increased excretion of urinary Kreb's cycle intermediates, increased levels of ketone bodies in urine and serum, and decreased levels of hepatic glucose and glycogen, as well as hypoglycemia and hyperlipoidemia, suggesting the Perturbation of energy metabolism. Elevated levels of choline containing metabolites and betaine in serum and liver tissue aqueous extracts and increased serum creatine indicated altered transmethylation. Decreased urinary levels of trimethylamine-N-oxide, phenylacetylglycine and hippurate suggested the effects on the gut microflora environment by realgar.

HDAC inhibitors cause site-specific chromatin remodeling at PU.1-bound enhancers in K562 cells

BACKGROUND: Small molecule inhibitors of histone deacetylases (HDACi) hold promise as anticancer agents for particular malignancies. However, clinical use is often confounded by toxicity, perhaps due to indiscriminate hyperacetylation of cellular proteins. Therefore, elucidating the mechanisms by which HDACi trigger differentiation, cell cycle arrest, or apoptosis of cancer cells could inform development of more targeted therapies. We used the myelogenous leukemia line K562 as a model of HDACi-induced differentiation to investigate chromatin accessibility (DNase-seq) and expression (RNA-seq) changes associated with this process. RESULTS: We identified several thousand specific regulatory elements [~10 % of total DNase I-hypersensitive (DHS) sites] that become significantly more or less accessible with sodium butyrate or suberanilohydroxamic acid treatment. Most of the differential DHS sites display hallmarks of enhancers, including being enriched for non-promoter regions, associating with nearby gene expression changes, and increasing luciferase reporter expression in K562 cells. Differential DHS sites were enriched for key hematopoietic lineage transcription factor motifs, including SPI1 (PU.1), a known pioneer factor. We found PU.1 increases binding at opened DHS sites with HDACi treatment by ChIP-seq, but PU.1 knockdown by shRNA fails to block the chromatin accessibility and expression changes. A machine-learning approach indicates H3K27me3 initially marks PU.1-bound sites that open with HDACi treatment, suggesting these sites are epigenetically poised. CONCLUSIONS: We find HDACi treatment of K562 cells results in site-specific chromatin remodeling at epigenetically poised regulatory elements. PU.1 shows evidence of a pioneer role in this process by marking poised enhancers but is not required for transcriptional activation.

Elucidation of the interactions between the transcription factor E2A and the transcriptional co-activator CBP/p300

E2A is a transcription factor that plays a particularly critical role in lymphopoiesis. The chromosomal translocation 1;19, disrupts the E2A gene and results in the expression of the fusion oncoprotein E2A-PBX1, which is implicated in acute lymphoblastic leukemia. Both E2A and E2A-PBX1 contain two activation domains, AD1 and AD2, which comprise conserved ΦxxΦΦ motifs where Φ denotes a hydrophobic amino acid. These domains function to recruit transcriptional co-activators and repressors, including the histone acetyl transferase CREB binding protein (CBP) and its paralog p300. The PCET motif within E2A AD1 interacts with the KIX domain of CBP/p300, the disruption of which abrogates the transcriptional activation by E2A and the transformative properties of E2A-PBX1. The generation of a peptide-based inhibitor targeting the PCET:KIX interaction would serve useful in further assessing the role of E2A and E2A-PBX1 in lymphopoiesis and leukemogenesis. An interaction between E2A AD2 and the KIX domain has also been recently identified, and the TAZ domains of CBP/p300 have been shown to interact with several transcription factors that contain ΦxxΦΦ motifs. Thus the design of an inhibitor of the E2A:CBP/p300 interaction requires the full complement of interactions between E2A and the various domains of CBP/p300 to be elucidated. Here, we have used nuclear magnetic resonance (NMR) spectroscopy to determine that AD2 interacts with KIX at the same site as PCET, which indicates that the E2A:KIX interaction can be disrupted by targeting a single binding site. Using an iterative synthetic peptide microarray approach, a peptide with the sequence DKELQDLLDFSLQY was derived from PCET to interact with KIX with higher affinity than the wild type sequence. This peptide now serves as a lead molecule for further development as an inhibitor of the E2A:CBP/p300 interaction. Fluorescence anisotropy, peptide microarray technology, and isothermal titration calorimetry were employed to characterize interactions between both TAZ domains of CBP/p300 and the PCET motif and AD2 of E2A. Alanine substitution of residues within PCET demonstrated that the ΦxxΦΦ motif is a key mediator of these interactions, analogous to the PCET:KIX interaction. These findings now inform future work to establish possible physiological roles for the E2A:TAZ1 and E2A:TAZ2 interactions.

CD33 reponses are blocked by SOCS3 through accelerated proteasomal-mediated turnover

CD33 is a member of the sialic acid–binding immunoglobulin-like lectin (Siglec) family of inhibitory receptors and a therapeutic target for acute myeloid leukemia (AML). CD33 contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM), which can recruit SHP-1 and SHP-2. How CD33 expression is regulated is unclear. Suppressor of cytokine signaling 3 (SOCS3) is expressed in response to cytokines, LPS, and other PAMPs, and competes with SHP-1/2 binding to ITIMs of cytokine receptors, thereby inhibiting signaling. In this study, using peptide pull-down experiments, we found that SOCS3 can specifically bind to the phosphorylated ITIM of CD33. Additionally, following cross-linking SOCS3 can recruit the ECS E3 ligase resulting in accelerated proteasomal degradation of both CD33 and SOCS3. Our data suggest that the tyrosine motifs in CD33 are not important for internalization, while they are required for degradation. Moreover, SOCS3 inhibited the CD33-induced block on cytokine-induced proliferation. This is the first receptor shown to be degraded by SOCS3 and where SOCS3 and its target protein are degraded concomitantly. Our findings clearly suggest that during an inflammatory response, the inhibitory receptor CD33 is lost by this mechanism. Moreover, this has important clinical implications as tumors expressing SOCS3 may be refractory to -CD33 therapy.

Hematopoietic malignancies associated with viral and alcoholic hepatitis

Hepatitis C virus (HCV) and hepatitis B virus (HBV) have been associated with hematopoietic malignancies, but data for many subtypes are limited. From the U.S. Surveillance, Epidemiology, and End Results-Medicare database, we selected 61,464 cases (=67 years) with hematopoietic malignancies and 122,531 population-based controls, frequency-matched by gender, age, and year (1993-2002). Logistic regression was used to compare the prevalence of HCV, HBV, and alcoholic hepatitis in cases and controls, adjusted for matching factors, race, duration of Medicare coverage, and number of physician claims. HCV, HBV, and alcoholic hepatitis were reported in 195 (0.3%), 111 (0.2%), and 404 (0.7%) cases and 264 (0.2%), 242 (0.2%), and 798 (0.7%) controls, respectively. HCV was associated with increased risk of diffuse large B-cell lymphoma [odds ratio (OR) 1.52, 95% confidence interval (95% CI) 1.05-2.18], Burkitt lymphoma (OR 5.21, 95% CI 1.62-16.8), follicular lymphoma (OR 1.88, 95% CI 1.17-3.02), marginal zone lymphoma (OR 2.20, 95% CI 1.22-3.95), and acute myeloid leukemia (OR 1.54, 95% CI 1.00-2.37). In contrast, HBV was unrelated to any hematopoietic malignancies. Alcoholic hepatitis was associated with decreased risk of non-Hodgkin lymphoma overall, but increased risk of Burkitt lymphoma. In summary, HCV, but not other causes of hepatitis, was associated with the elevated risk of non-Hodgkin lymphoma and acute myeloid leukemia. HCV may induce lymphoproliferative malignancies through chronic immune stimulation. Copyright © 2008 American Association for Cancer Research.


--------------------------------------------------------------------------------

Reaxys Database Information|

--------------------------------------------------------------------------------

Hoxa6 potentiates short-term hemopoietic cell proliferation and extended self-renewal.

Hemopoietic progenitor cells express clustered homeobox (Hox) genes in a pattern characteristic of their lineage and stage of differentiation. In general, HOX expression tends to be higher in more primitive and lower in lineage-committed cells. These trends have led to the hypothesis that self-renewal of hemopoietic stem/progenitor cells is HOX-dependent and that dysregulated HOX expression underlies maintenance of the leukemia-initiating cell. Gene expression profile studies support this hypothesis and specifically highlight the importance of the HOXA cluster in hemopoiesis and leukemogenesis. Within this cluster HOXA6 and HOXA9 are highly expressed in patients with acute myeloid leukemia and form part of the "Hox code" identified in murine models of this disease. We have examined endogenous expression of Hoxa6 and Hoxa9 in purified primary progenitors as well as four growth factor-dependent cell lines FDCP-Mix, EML, 32Dcl3, and Ba/F3, representative of early multipotential and later committed precursor cells respectively. Hoxa6 was consistently higher expressed than Hoxa9, preferentially expressed in primitive cells and was both growth-factor and cell-cycle regulated. Enforced overexpression of HOXA6 or HOXA9 in FDCP-Mix resulted in increased proliferation and colony formation but had negligible effect on differentiation. In both FDCP-Mix and the more committed Ba/F3 precursor cells overexpression of HOXA6 potentiated factor-independent proliferation. These findings demonstrate that Hoxa6 is directly involved in fundamental processes of hemopoietic progenitor cell development.

Identification of candidate small-molecule therapeutics to cancer by gene-signature perturbation in connectivity mapping.

Connectivity mapping is a recently developed technique for discovering the underlying connections between different biological states based on gene-expression similarities. The sscMap method has been shown to provide enhanced sensitivity in mapping meaningful connections leading to testable biological hypotheses and in identifying drug candidates with particular pharmacological and/or toxicological properties. Challenges remain, however, as to how to prioritise the large number of discovered connections in an unbiased manner such that the success rate of any following-up investigation can be maximised. We introduce a new concept, gene-signature perturbation, which aims to test whether an identified connection is stable enough against systematic minor changes (perturbation) to the gene-signature. We applied the perturbation method to three independent datasets obtained from the GEO database: acute myeloid leukemia (AML), cervical cancer, and breast cancer treated with letrozole. We demonstrate that the perturbation approach helps to identify meaningful biological connections which suggest the most relevant candidate drugs. In the case of AML, we found that the prevalent compounds were retinoic acids and PPAR activators. For cervical cancer, our results suggested that potential drugs are likely to involve the EGFR pathway; and with the breast cancer dataset, we identified candidates that are involved in prostaglandin inhibition. Thus the gene-signature perturbation approach added real values to the whole connectivity mapping process, allowing for increased specificity in the identification of possible therapeutic candidates.

Microarray-based classifiers and prognosis models identify subgroups with distinct clinical outcomes and high risk of AML transformation of myelodysplastic syndrome

The diagnosis of myelodysplastic syndrome (MDS) currently relies primarily on the morphologic assessment of the patient's bone marrow and peripheral blood cells. Moreover, prognostic scoring systems rely on observer-dependent assessments of blast percentage and dysplasia. Gene expression profiling could enhance current diagnostic and prognostic systems by providing a set of standardized, objective gene signatures. Within the Microarray Innovations in LEukemia study, a diagnostic classification model was investigated to distinguish the distinct subclasses of pediatric and adult leukemia, as well as MDS. Overall, the accuracy of the diagnostic classification model for subtyping leukemia was approximately 93%, but this was not reflected for the MDS samples giving only approximately 50% accuracy. Discordant samples of MDS were classified either into acute myeloid leukemia (AML) or

Two routes to leukemic transformation after a JAK2 mutation-positive myeloproliferative neoplasm

Acute myeloid leukemia (AML) may follow a JAK2-positive myeloproliferative neoplasm (MPN), although the mechanisms of disease evolution, often involving loss of mutant JAK2, remain obscure. We studied 16 patients with JAK2-mutant (7 of 16) or JAK2 wild-type (9 of 16) AML after a JAK2-mutant MPN. Primary myelofibrosis or myelofibrotic transformation preceded all 7 JAK2-mutant but only 1 of 9 JAK2 wild-type AMLs (P = .001), implying that JAK2-mutant AML is preceded by mutation(s) that give rise to a "myelofibrosis" phenotype. Loss of the JAK2 mutation by mitotic recombination, gene conversion, or deletion was excluded in all wild-type AMLs. A search for additional mutations identified alterations of RUNX1, WT1, TP53, CBL, NRAS, and TET2, without significant differences between JAK2-mutant and wild-type leukemias. In 4 patients, mutations in TP53, CBL, or TET2 were present in JAK2 wild-type leukemic blasts but absent from the JAK2-mutant MPN. By contrast in a chronic-phase patient, clones harboring mutations in JAK2 or MPL represented the progeny of a shared TET2-mutant ancestral clone. These results indicate that different pathogenetic mechanisms underlie transformation to JAK2 wild-type and JAK2-mutant AML, show that TET2 mutations may be present in a clone distinct from that harboring a JAK2 mutation, and emphasize the clonal heterogeneity of the MPNs.

Comprehensive genomic screens identify a role for PLZF-RAR alpha as a positive regulator of cell proliferation via direct regulation of c-MYC

The t(11; 17)(q23;q21) translocation is associated with a retinoic acid (RA)-insensitive form of acute promyelocytic leukemia (APL), involving the production of reciprocal fusion proteins, promyelocytic leukemia zinc finger-retinoic acid receptor alpha (PLZF-RAR alpha) and RAR alpha-PLZF. Using a combination of chromatin immuno-precipitation promotor arrays (ChIP-chip) and gene expression profiling, we identify novel, direct target genes of PLZF-RAR alpha that tend to be repressed in APL compared with other myeloid leukemias, supporting the role of PLZF-RAR alpha as an aberrant repressor in APL. In primary murine hematopoietic progenitors, PLZF-RAR alpha promotes cell growth, and represses Dusp6 and Cdkn2d, while inducing c-Myc expression, consistent with its role in leukemogenesis. PLZF-RAR alpha binds to a region of the c-MYC promoter overlapping a functional PLZF site and antagonizes PLZF-mediated repression, suggesting that PLZF-RAR alpha may act as a dominant-negative version of PLZF by affecting the regulation of shared targets. RA induced the differentiation of PLZF-RAR alpha-transformed murine hematopoietic cells and reduced the frequency of clonogenic progenitors, concomitant with c-Myc down-regulation. Surviving RA-treated cells retained the ability to be replated and this was associated with sustained c-Myc expression and repression of Dusp6, suggesting a role for these genes in maintaining a self-renewal pathway triggered by PLZF-RAR alpha. (Blood. 2009; 114: 5499-5511)