Expression profiling data of CLB-GA neuroblastoma cells upon TAE-684, Crizotinib, X-396, LDK378, Trametinib, BEZ-235 and Vandetanib
ABSTRACT: Treatment of the CLB-GA cell line with complementary ALK, MEK, PI3K/mTOR or RET inhibitors (and DMSO (VWR) as control) was performed in duplicate for each drug using the GI50 concentrations (see further): 0.5ë_M Crizotinib (Pfizer/Sigma-Aldrich); 0.06ë_M X-396 (VWR); 0.2ë_M LDK378 (Hoelzel Biotech); 0.05uM Trametinib (SelleckChem); 0.5ë_M BEZ-235 (SelleckChem); 9.5ë_M Vandetanib (SelleckChem). Cells were collected at 6h following treatment and further profiled. The selected inhibitor concentrations were used based on determined GI50 values. For generation of gene expression time series following ALK inhibition, CLB-GA cells were treated with 0.3uM TAE-684 (and DMSO as control treatment) and RNA was harvested at 0 ÛÒ 10۪ ÛÒ 30۪ ÛÒ 60۪ ÛÒ 120۪ ÛÒ 240۪ ÛÒ 360۪ time points. Samples were labeled and hybridised to the Sureprint G3 human GE or G3 Mouse GE 8x60K microarrays (Agilent Technologies), according to the manufacturer۪s guidelines and starting from 200 ng of RNA.
Clinical cancer research : an official journal of the American Association for Cancer Research 20150324 14
Activating ALK mutations are present in almost 10% of primary neuroblastomas and mark patients for treatment with small-molecule ALK inhibitors in clinical trials. However, recent studies have shown that multiple mechanisms drive resistance to these molecular therapies. We anticipated that detailed mapping of the oncogenic ALK-driven signaling in neuroblastoma can aid to identify potential fragile nodes as additional targets for combination therapies.To achieve this goal, transcriptome profiling ...[more]
Project description:Human wild-type ALK (SK-N-AS, NGP, IMR-32), ALKR1275Q (CLB-GA, LAN-5, UKF-NB-3), ALKF1174L (SK-N-SH, Kelly, SMS-KCNR) and ALK amplified (NB-1) neuroblastoma cell lines were treated in triplicate with 0.3ë_M NPV-TAE-684 (Novartis/SelleckChem) or DMSO (VWR) for 6 hours, followed by RNA isolation and gene expression profiling. For shRNA mediated knockdown, pGIPZ-ALK shRNAmir and pGIPZ-non-silencing control shRNAmir vectors were used (Open Biosystems). The ALK shRNA was directed against a part of exon 26 in the tyrosine kinase domain of ALK (target sequence: TGGAAGGAATATTCACTTCTAA). Lentiviral particles were produced according to manufacturer۪s protocol (Open Biosystems). On day 2 post-transduction of neuroblastoma cells, the transduction efficiency was determined by flow cytometry and microscopic analysis of GFP-positive cells (>90% so no further selection was performed). Cells were subsequently harvested for expression profiling. Labeling of the RNA samples and hybridisation to the Affymetrix HG-U133plus2 arrays was performed using the manufacturer۪s protocol starting from 1 åµg of RNA.
Project description:Anaplastic Lymphoma Kinase (ALK) most frequently mutated in neuroblastoma (NB) and is atractive molecular target for therapy. However, efficacy of the ALK inhibitor against ALK-amplified NB is unclear. To elucidate genetic alterations induced by treatment of the ALK inhibitor, we compared expression profile between ALK inhibitor-treated and DMSO-treated NB39nu cells using Agilent SurePrint G3 Human GE 8x60K V2 Microarray Kit Overall design: The ALK inhibitor or DMSO-treated NB39nu cells (parental and resistant), which harbor ALK amplification, were for RNA extraction and hybridization on Agilent microarrays.
Project description:Anaplastic Lymphoma Kinase (ALK) is a tyrosine kinase receptor which is a clinical target of major interest in cancer, including neuroblastoma. To better understand ALK signaling, three different neuroblastoma cell lines (CLB-BAR, CLB-GE and SK-N-AS) were cultured for 1hr and 24hrs in control conditions or after treatment with the ALK inhibitors crizotinib or lorlatinib. RNA-Seq experiments were performed to determine the expression changes resulting from ALK inhibition. Together with parallel phosphoproteomic experiments, these data unveil several important conserved oncogenic pathways in neuroblastoma.
Project description:Establishment of inducible SK-N-AS ALKwt, ALKF1174L and ALKR1275Q cell lines Human neuroblastoma SK-N-AS cells were electroporated with pcDNA6/TR (Invitrogen) using the Neonå¨ Transfection System (Life Techologies). Single cell clones were generated using blasticidin (7.5åµg/ml) and limited dilution. Using a TetR antibody (Clonetech), the clone with the highest TetR expression was selected (named SK-N-AS-TR) and used for the transfection with pT-REx-DEST30-ALK, ALKF1174L or ALKR1275Q. After transfection of SK-N-AS-TR with the ALK variants, single cell clones were raised using geneticin (500 åµg/ml) and limited dilution, while blasticidin treatment was continued as described above. Total RNA of (treated) cell lines, transgenic mice tumors and ganglia was isolated using the miRNeasy kit (Qiagen) according to the manufacturer۪s instructions, including on-column DNase treatment. Samples were labeled and hybridised to the Sureprint G3 human GE or G3 Mouse GE 8x60K microarrays (Agilent Technologies), according to the manufacturer۪s guidelines and starting from 200 ng of RNA.
Project description:8 neuroblastoma (NB) cell lines (CLB-GA, IMR-32, SH-SY5Y, N206, CHP-902R, LAN-2, SK-N-AS, SJNB-1) were profiled on the Affymetrix HGU-133plus2,0 platform before and after treatment with DAC (2'-deoxy-5-azacytidine) to investigate the influence on expression after inhibiting DNA-methylation 8 NB cell lines were included (CLB-GA, IMR-32, SH-SY5Y, N206, CHP-902R, LAN-2, SK-N-AS, SJNB-1), before and after treatment with 3 micromolars of DAC for 3 days
Project description:8 neuroblastoma (NB) cell lines (CLB-GA, IMR-32, SH-SY5Y, N206, CHP-902R, LAN-2, SK-N-AS, SJNB-1) their methylome is determined by sequencing after MBD2-capture using MethylCollector (ActiveMotif) 8 NB cell lines were included (CLB-GA, IMR-32, SH-SY5Y, N206, CHP-902R, LAN-2, SK-N-AS, SJNB-1) in this study. After shearing (fragments of about 200 bp), DNA was captured using MBD2-capture (MethylCollector - ActiveMotif) followed by library preparation and multiplexing. Captured sequence tags were sequenced paired-end (2 x 45 bp) on Illumina GAIIx.
Project description:MLLENL transformed primary cells were treated for 6h with 250nM flavopiridol, PC585 or DMSO as control. RNA was isolated and analyzed on Agilent SurePrint G3 Mouse GE 8x60K (Agilent Microarray Design ID 028005) arrays.
Project description:Human HepG2/C3A cells were exposed to indoor dust reference material SRM2585; DMBA (dimethylbenzanthracene); HBCD (hexabromocyclododecane); two different mixtures of flame retardants (all dissolved in 0.1% DMSO) or 0.1% DMSO alone for 72h. RNA was prepared and labeled with Cy3 then hybridized to Agilent SurePrint G3 Human GE v2 8x60k Microarrays, Agilent design ID 039494.
Project description:Human cell cultures A549 and HepG2/C3 were exposed to 0-100 micromolar Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) in 0.5% DMSO or to 0.5% DMSO alone for 24 h. RNA was prepared and labeled with Cy3. Agilent SurePrint G3 Human GE v2 8x60k Microarrays, Agilent design ID 039494, were used to profile transcriptional responses to TDCIPP.