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ONCOLOGY REPORTS 41: 2027-2040, 2019 Disruption of MAPK1 expression in the ERK signalling pathway and the RUNX1‑RUNX1T1 fusion gene attenuate the differentiation and proliferation and induces the growth arrest in t(8;21) leukaemia cells SALEM ALI AL-SALEM BASHANFER2, MOHAMED SALEEM3, OLAF HEIDENREICH4, EMMANUEL JAIRAJ MOSES1 and NARAZAH MOHD YUSOFF1 1Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, Kepala Batas, Pulau Pinang 13200, Malaysia; 2Hematology Department, College of Medicine and Health Sciences, University of Hodiedah, Hodiedah 3730, Yemen; 3Advanced Genomics Lab Sdn Bhd, Kota Damansara, Petaling Jaya, Selangor Darul Ehsan 47810, Malaysia; 4Northern Institute for Cancer Research, Medical School, Newcastle upon Tyne NE2 4HH, UK Received June 28, 2017; Accepted June 8, 2018 DOI: 10.3892/or.2018.6926 Abstract. The t(8;21) translocation is one of the most frequent RUNX1‑RUNX1T1 and MAPK1 depletion induced cell cycle chromosome abnormalities associated with acute myeloid arrest at the G0/G1 phase. Accumulation of cells in the G1 phase leukaemia (AML). This abberation deregulates numerous was largely the result of downregulated expression of TBRG4, molecular pathways including the ERK signalling pathway CCNE2, FOXO4, CDK6, ING4, IL8, MAD2L1 and CCNG2 among others. Therefore, the aim of the present study in the case of RUNX1‑RUNX1T1 depletion and increased was to investigate the gene expression patterns following expression of RASSF1, FBXO6, DADD45A and P53 in the siRNA-mediated suppression of RUNX1‑RUNX1T1 and case of MAPK1 depletion. Taken together, the current results MAPK1 in Kasumi-1 and SKNO-1 cells and to determine the demonstrate that MAPK1 promotes myeloid cell proliferation differentially expressed genes in enriched biological pathways. and differentiation simultaneously by cell cycle progression BeadChip microarray and gene ontology analysis revealed while suppresing apoptosis. that RUNX1‑RUNX1T1 and MAPK1 suppression reduced the proliferation rate of the t(8;21) cells with deregulated expression Introduction of several classical positive regulator genes that are otherwise known to enhance cell proliferation. RUNX1‑RUNX1T1 Acute myeloid leukaemia (AML) with karyotypic discern- suppression exerted an anti-apoptotic effect through the ible translocation (t)(8;21)(q22;q22) (RUNX1-RUNX1T1) is overexpression of BCL2, BIRC3 and CFLAR genes, while observed in approximately 7% of adults (1), and is the most MAPK1 suppression induced apopotosis in t(8;21) cells by frequent non-lymphoblastic leukaemia in children (2). This the apoptotic mitochondrial changes stimulated by the activity balanced translocation disrupts two functionally distinct of upregulated TP53 and TNFSF10, and downregulated JUN genes, RUNX1 (previously termed AML1) and RUNX1T1 gene. RUNX1‑RUNX1T1 suppression supported myeloid (commonly known as ETO or MTG8) (3). Physiologically, differentiation by the differential expression of CEBPA, RUNX1 is a transcription factor that encodes to the alpha CEBPE, ID2, JMJD6, IKZF1, CBFB, KIT and CDK6, while subunit of the core biding factor (CBFα) which forms a heter- MAPK1 depletion inhibited the differentiation of t(8;21) cells odimer with the CBFβ subunit and regulates differentiation by elevated expression of ADA and downregulation of JUN. of haematopoietic stem cells into committed mature cells. The translocated RUNX1T1 moiety gets activated as a result of the fusion with RUNX1 (4,5). Together, the hybridised RUNX1-RUNX1T1 (RR) oncoprotein functions as a dominant negative form of RUNX1. As empirically shown, RR exerts a Correspondence to: Professor Narazah Mohd Yusoff or repressive effect on the promoters of genes normally activated Dr Emmanuel Jairaj Moses, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, Kepala Batas, Pulau by the RUNX1 transcription factor through interactions of its Pinang 13200, Malaysia RUX1T1 moiety with nuclear corepressors such as NCOR1 E-mail: [email protected] and Sin3A (6,7). Several studies have indicated that this altered E-mail: [email protected] transcription pattern impairs myeloid precursor cell differen- tiation, proliferation and apoptosis, and is therefore thought to Key words: acute myeloid leukaemia, t(8:21), MAPK1, ERK pathway be the underlying pathogenesis of myeloid leukaemia (8-13). However, this translocation itself is not necessarily sufficient for the development of AML. It appears that additional 2028 AL-SALEM BASHANFER et al: EFFECT OF RUNX1‑RUNX1T1 AND MAPK1 SILENCING ON THE SURVIVAL OF AML secondary mutations are required to impair haematopoietic medium without serum, were electroporated with siRNA differentiation to recapitulate the phenotypic features of AML. using a Bio-Rad Gene Pulser Xcell™ at 330 V for 10 m. The Based on recent studies there appears to be a fairly consistent sequences of siRNAs used are as follows: siRNA-MAPK1 association between the t(8;21) translocation and the classical (siMAPK1) sense, 5'-GUU CGA GUA GCU AUC AAG ATT-3' MAPK signalling pathway (9,14). and antisense, 5'-UCU UGA UAG CUA CUC GAA CTT-3'; One of many signalling pathways triggered by haemat- siRNA-RUNX1‑RUNX1T1 (siRR) sense, 5'-CCU CGA AAU opoietic cytokines is the ERK (Ras/Raf/MEK/ERK) pathway. CGU ACU GAG AAG-3' and antisense, 5'-UCU CAG UAC GAU The MAP kinase ERK2 (MAPK1), encoded by MAPK1, UUC GAG GUU-3'; and scrambled mismatch siRNA (siMM) plays an integral role in the ERK cascade in relaying extracel- from Qiagen (Hilden, Germany). lular biological signals from cell membrane to the nucleus. For isolated downregulation of RR and MAPK1 gene This mechanism modulates the normal haematopoietic cell expression, both cell types were electro-transfected with proliferation, differentiation and prevention of apoptosis (15). 200 nM of siRR and 100 nM of annealed siMAPK1 respec- The ERK pathway, most extensively studied of all MAPK tively; and for co-transfection a mixture of both siRR and pathways, is deregulated in a third of all human cancers, a siMAPK1 were used. These concentrations were derived from frequent observation in the pathogenesis of haematological dose-dependent experiments performed in our laboratory. malignancies such as AML (16-18). In wide varieties of AMLs, In experiments requiring extended transfection, cells were constitutive activation that centres on the MEK/ERK node in sequentially transfected on days 0, 4 and 7. Immediately after the pathway has been attributed to the tumourigenic effects electro-transfection, cells were diluted 20-fold in complete such as independence of proliferation and evasion of apop- culture medium and returned to the incubator. All cell groups tosis (16-19). Previous study revealed that AML with t(8;21) were electroporated in triplicates. encoded RR induces a microRNA-dependent mechanism that utilises the MAPK cascade to signal hyper-proliferation RNA isolation and integrity evaluation. Quantitative RT-PCR of myeloid progenitors and block their differentiation (20). total RNA was isolated using RNeasy Mini kit (Qiagen) Another group reported that Kasumi-1 cells that were refrac- according to the manufacturer's protocol. RNA integrity tory to apoptosis following RR silencing activate an array of was analysed on an Agilent 2100 Bioanalyser RNA 6000 signalling pathways involved in cell survival and proliferation, NanoLabChip (Agilent Technologies, Palo Alto, CA, USA) to prominently the ERK2 pathway (21). These findings under- produce an electrophoresis trace of 18S and 28S peaks using score the influence of RR on the MAPK pathway towards the 2100 Expert Software (Agilent Technologies). The RNA integ- pathobiology of AML. rity number (RIN) was calculated directly from the peak areas, Although the role of constitutive activation of the a value of 10 corresponded to intact RNA while 1 indicates a MEK/ERK pathway in the development of myeloid leukaemia total degradation. Our optimised methods showed a 28S:18S is well established, RR- and MAPK-driven intricate molecular ratio of 1.9-2.1:1 and an RIN of 9-10. and cellular pathways that maintain the pathobiology of AML remain elusive. In the present study we used siRNA-mediated Quantitative real‑time polymerase chain reaction (q‑RT‑PCR). gene silencing approach to elucidate the transcription profiles Quantitative real-time-polymerase chain reaction (qRT-PCR) of RR and MAPK1 suppressed AML cell lines and further was performed on RNA isolated at various time points analysed these expression signatures to explore the molecular post-transfection to quantify the mRNA expression levels of mechanisms involved in cell proliferation, cell cycle distribu- MAPK1 and RR genes in cells treated with respective siRNAs. tion, apoptosis and differentiation. In the present study, we Gene‑specific primers for MAPK1 (forward, 5'-CTG CTG report differential regulation of gene expression by RR and CTC AAC ACC ACC T-3' and reverse, 5'-GCC ACA TAT TCT MAPK1 as determined by genome-wide expression analysis GTC AGG AAC C-3'); and B2M (forward, 5'-GGC ATT CCT responsible for the phenotypic features of AML. GAA GCT GAC AG-3' and reverse, 5'-TCT GCT GGA TGA CGT GAG TAA-3') were designed using PrimerPlex software Materials and methods (Premier Biosoft, Palo Alto, CA, USA). Primers to quantify RR expression were: Forward, 5'-AAT CAC AGT GGA TGG Cell culture and siRNA transfection. Two suspension GCC C-3' and reverse, 5'-TGC GTC TTC ACA TCC ACA GG-3' leukaemic cell lines with t(8;21), Kasumi-1 and SKNO-1, as described by Heidenreich et al (5). were used in the present
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    immune responseregulatingcellsurfacereceptorsignalingpathway ventricular cardiacmuscletissuedevelopment t cellactivationinvolvedinimmuneresponse intrinsic apoptoticsignalingpathway single organismalcelladhesion cholesterol biosyntheticprocess myeloid leukocytedifferentiation determination ofadultlifespan response tointerferongamma muscle organmorphogenesis endothelial celldifferentiation brown fatcelldifferentiation mitochondrion organization myoblast differentiation response toprotozoan amino acidtransport leukocyte migration cytokine production t celldifferentiation protein secretion response tovirus angiogenesis Scrt1 Tcf25 Dpf1 Sap30 Ing2 Zfp654 Sp9 Zfp263 Mxi1 Hes6 Zfp395 Rlf Ppp1r13l Snapc1 C030039L03Rik Hif1a Arrb1 Glis3 Rcor2 Hif3a Fbxo21 Dnajc21 Rest Sirt6 Foxj1 Kdm5b Ankzf1 Sos2 Plscr1 Jdp2 Rbbp8 Etv4 Msh5 Mafg Tsc22d3 Nupr1 Ddit3 Cebpg Zfp790 Atf5 Cebpb Atf3 Trim21 Irf9 Irf2 Tbx21 Stat2 Stat1 Zbp1 Irf1 aGOslimPos Ikzf3 Oasl1 Irf7 Trim30a Dhx58 Txk Rorc Rora Nr1d2 Setdb2 Vdr Vax2 Nr1d1 Foxs1 Eno1 Thap3 Nfkbil1 Edf1 Srebf1 Lbr Tead1 Zfp608 Pcx Ift57 Ssbp4 Stat3 Mxd1 Pml Ssh2 Chd7 Maf Cic Bcl3 Prkdc Mbd5 Ppfibp2 Foxp2 Tal2 Mlf1 Bcl6b Zfp827 Ikzf2 Phtf2 Bmyc Plagl2 Nfkb2 Nfkb1 Tox Nrip1 Utf1 Klf3 Plagl1 Nfkbib Spib Nfkbie Akna Rbpj Ncoa3 Id1 Tnp2 Gata3 Gata1 Pparg Id2 Epas1 Zfp280b Commons Pathway Erg GO MSigDB KEGG Hhex WikiPathways SetGene Databases Batf Aff3 Zfp266 gene modules other (hypergeometric TF, from Figure Trim24 Zbtb5 Foxo3 Aebp2 XPodNet -protein-proteininteractionsinthepodocyteexpandedbySTRING Ppp1r10 Dffb Trp53 Enrichment
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    Supplementary Table 2

    Supplementary Table 2. Differentially Expressed Genes following Sham treatment relative to Untreated Controls Fold Change Accession Name Symbol 3 h 12 h NM_013121 CD28 antigen Cd28 12.82 BG665360 FMS-like tyrosine kinase 1 Flt1 9.63 NM_012701 Adrenergic receptor, beta 1 Adrb1 8.24 0.46 U20796 Nuclear receptor subfamily 1, group D, member 2 Nr1d2 7.22 NM_017116 Calpain 2 Capn2 6.41 BE097282 Guanine nucleotide binding protein, alpha 12 Gna12 6.21 NM_053328 Basic helix-loop-helix domain containing, class B2 Bhlhb2 5.79 NM_053831 Guanylate cyclase 2f Gucy2f 5.71 AW251703 Tumor necrosis factor receptor superfamily, member 12a Tnfrsf12a 5.57 NM_021691 Twist homolog 2 (Drosophila) Twist2 5.42 NM_133550 Fc receptor, IgE, low affinity II, alpha polypeptide Fcer2a 4.93 NM_031120 Signal sequence receptor, gamma Ssr3 4.84 NM_053544 Secreted frizzled-related protein 4 Sfrp4 4.73 NM_053910 Pleckstrin homology, Sec7 and coiled/coil domains 1 Pscd1 4.69 BE113233 Suppressor of cytokine signaling 2 Socs2 4.68 NM_053949 Potassium voltage-gated channel, subfamily H (eag- Kcnh2 4.60 related), member 2 NM_017305 Glutamate cysteine ligase, modifier subunit Gclm 4.59 NM_017309 Protein phospatase 3, regulatory subunit B, alpha Ppp3r1 4.54 isoform,type 1 NM_012765 5-hydroxytryptamine (serotonin) receptor 2C Htr2c 4.46 NM_017218 V-erb-b2 erythroblastic leukemia viral oncogene homolog Erbb3 4.42 3 (avian) AW918369 Zinc finger protein 191 Zfp191 4.38 NM_031034 Guanine nucleotide binding protein, alpha 12 Gna12 4.38 NM_017020 Interleukin 6 receptor Il6r 4.37 AJ002942
  • RUNX1 Together with a Compendium of Hematopoietic Regulators, Chromatin Modifiers and Basal Transcr

    RUNX1 Together with a Compendium of Hematopoietic Regulators, Chromatin Modifiers and Basal Transcr

    Leukemia (2014) 28, 770–778 OPEN & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu ORIGINAL ARTICLE CBFB–MYH11/RUNX1 together with a compendium of hematopoietic regulators, chromatin modifiers and basal transcription factors occupies self-renewal genes in inv(16) acute myeloid leukemia A Mandoli1, AA Singh1, PWTC Jansen2, ATJ Wierenga3,4, H Riahi1, G Franci5, K Prange1, S Saeed1, E Vellenga3, M Vermeulen2, HG Stunnenberg1 and JHA Martens1 Different mechanisms for CBFb–MYH11 function in acute myeloid leukemia with inv(16) have been proposed such as tethering of RUNX1 outside the nucleus, interference with transcription factor complex assembly and recruitment of histone deacetylases, all resulting in transcriptional repression of RUNX1 target genes. Here, through genome-wide CBFb–MYH11-binding site analysis and quantitative interaction proteomics, we found that CBFb–MYH11 localizes to RUNX1 occupied promoters, where it interacts with TAL1, FLI1 and TBP-associated factors (TAFs) in the context of the hematopoietic transcription factors ERG, GATA2 and PU.1/SPI1 and the coregulators EP300 and HDAC1. Transcriptional analysis revealed that upon fusion protein knockdown, a small subset of the CBFb–MYH11 target genes show increased expression, confirming a role in transcriptional repression. However, the majority of CBFb–MYH11 target genes, including genes implicated in hematopoietic stem cell self-renewal such as ID1, LMO1 and JAG1, are actively transcribed and repressed upon fusion protein knockdown. Together these results suggest an essential role for CBFb–MYH11 in regulating the expression of genes involved in maintaining a stem cell phenotype. Leukemia (2014) 28, 770–778; doi:10.1038/leu.2013.257 Keywords: CBFb–MYH11; RUNX1; histone acetylation; acute myeloid leukemia; inv(16) INTRODUCTION Heterozygous Cbfb-Myh11 knock-in mice are embryonic lethal, Core-binding transcription factors (CBFs) have roles in stem cell with definitive hematopoiesis blocked at the stem-cell level.