Dataset Information

MRNA expression profiles of SK-N-AS neuroblastoma cells with induced ALK-wt, ALK-F1174L or ALK-R1275Q

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Katleen De Preter

PROVIDER: E-MTAB-3207 | ArrayExpress | 2015-05-04

REPOSITORIES: ArrayExpress

ACCESS DATA

Publications

Upregulation of MAPK Negative Feedback Regulators and RET in Mutant ALK Neuroblastoma: Implications for Targeted Treatment.

Lambertz Irina I Kumps Candy C Claeys Shana S Lindner Sven S Beckers Anneleen A Janssens Els E Carter Daniel R DR Cazes Alex A Cheung Belamy B BB De Mariano Marilena M De Bondt An A De Brouwer Sara S Delattre Olivier O Gibbons Jay J Janoueix-Lerosey Isabelle I Laureys Geneviève G Liang Chris C Marchall Glenn M GM Porcu Michael M Takita Junko J Trujillo David Camacho DC Van Den Wyngaert Ilse I Van Roy Nadine N Van Goethem Alan A Van Maerken Tom T Zabrocki Piotr P Cools Jan J Schulte Johannes H JH Vialard Jorge J Speleman Frank F De Preter Katleen K

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]

PMID: 25805801

Similar Datasets

Project description:The prognosis of advanced stage neuroblastoma patients remains poor and, despite intensive therapy, the 5-year survival rate remains less than 50%. We previously identified histone deacetylase (HDAC) 8 as an indicator of poor clinical outcome and a selective drug target for differentiation therapy in vitro and in vivo. Here we performed kinome-wide RNAi screening to identify genes that are synthetically lethal with HDAC8 inhibitors. These experiments identified the neuroblastoma predisposition gene ALK as a candidate gene. Accordingly, the combination of the ALK/MET inhibitor crizotinib and selective HDAC8 inhibitors (3-6μM PCI-34051 or 10μM 20a) efficiently killed neuroblastoma cell lines carrying wildtype ALK (SK-N-BE(2)-C, IMR5/75), amplified ALK (NB-1), and those carrying the activating ALK F1174L mutation (Kelly), and, in cells carrying the activating R1275Q mutation (LAN-5), combination treatment decreased viable cell count. The effective dose of crizotinib in neuroblastoma cell lines ranged from 0.05μM (ALK-amplified) to 0.8μM (wildtype ALK). The combinatorial inhibition of ALK and HDAC8 also decreased tumor growth in an in vivo zebrafish xenograft model. Bioinformatic analyses revealed that the mRNA expression level of HDAC8 was significantly correlated with that of ALK in two independent patient cohorts, the Academic Medical Center cohort (n=88) and the German Neuroblastoma Trial cohort (n=649), and co-expression of both target genes identified patients with very poor outcome. Mechanistically, HDAC8 and ALK converge at the level of receptor tyrosine kinase (RTK) signaling and their downstream survival pathways, such as ERK signaling. Combination treatment of HDAC8 inhibitor with crizotinib efficiently blocked the activation of growth receptor survival signaling and shifted the cell cycle arrest and differentiation phenotype toward effective cell death of neuroblastoma cell lines, including sensitization of resistant models, but not of normal cells. These findings reveal combined targeting of ALK and HDAC8 as a novel strategy for the treatment of neuroblastoma. Overall design: Total RNA was isolated from neuroblastoma cells (SK-N-BE(2)-C) treated for six days with solvent control (DMSO) or an HDAC8 inhibitor (PCI-34051; 4µM).