Comment[ArrayExpressAccession]	E-TABM-1222
Investigation Title	Characterising the cellular activity of new Fibroblast Growth Factor Receptor tyrosine kinase inhibitors and identification of a gatekeeper mutation in FGFR3 as a mechanism of acquired resistance.
Comment[Submitted Name]	Characterising the cellular activity of new Fibroblast Growth Factor Receptor tyrosine kinase inhibitors and identification of a gatekeeper mutation in FGFR3 as a mechanism of acquired resistance.
Comment[AEExperimentDisplayName]	Characterising the cellular activity of new Fibroblast Growth Factor Receptor tyrosine kinase inhibitors and identification of a gatekeeper mutation in FGFR3 as a mechanism of acquired resistance.
Comment[AEExperimentType]	transcription profiling by array
Experimental Design	genetic_modification_design
Experimental Factor Name	Phenotype
Experimental Factor Type	phenotype
Person Last Name	Andrews
Person First Name	Simon
Person Email	simon.andrews@babraham.ac.uk
Person Affiliation	The Babraham Institute
Person Roles	submitter
Quality Control Type	biological_replicate
Public Release Date	2012-10-02
Comment[ArrayExpressSubmissionDate]	2011-12-02
Publication Author List	Chell V, Balmanno K, Little AS, Wilson M, Andrews S, Blockley L, Hampson M, Gavine PR, Cook SJ.
Publication Title	Tumour cell responses to new fibroblast growth factor receptor tyrosine kinase inhibitors and identification of a gatekeeper mutation in FGFR3 as a mechanism of acquired resistance.
Experiment Description	Fibroblast growth factor receptors (FGFRs) can act as driving oncoproteins in certain cancers due to mutation, over-expression or activating gene fusions and are therefore attractive drug targets.  Here we have characterized tumour cell responses to three new inhibitors of FGFR1-3, AZ12576089, AZ12908010 and the clinical candidate AZD4547, making comparisons with the well-characterized FGFR inhibitor PD173074.  Using a panel of 16 human tumour cell lines we show that the anti-proliferative activity of AZ12908010 and AZD4547 is strongly linked to the presence of de-regulated FGFR signalling.  In contrast, AZ12576089 was also able to inhibit proliferation of cells lacking de-regulated FGFR, suggesting off-target effects.  Acquired resistance to targeted tyrosine kinase inhibitors (TKIs) is a growing problem in the clinic.  To assess how FGFR-dependent tumour cells may adapt to long-term exposure to FGFR inhibitors we generated a derivative of the KMS-11 myeloma cell line (FGFRY373C) with acquired resistance to AZ12908010 (KMS-11R cells).  Basal P-FGFR3, P-FRS2  and P-ERK1/2 and D-type cyclins were all inhibited by AZ12908010 in parental KMS-11 cells whereas these markers were constitutively elevated and refractory to drug in KMS-11R cells.  Sequencing of FGFR3 in KMS-11R cells revealed the presence of a heterozygous mutation at the gatekeeper residue, encoding FGFR3V555M.  Consistent with this KMS-11R cells were cross-resistant to AZD4547 and PD173074 but remained fully sensitive to AZ12576089, confirming that the anti-proliferative effects of AZ12576089 are not related to FGFR inhibition. These results define the selectivity and efficacy of two new FGFR inhibitors and identify a secondary gatekeeper mutation as a mechanism of acquired resistance to FGFR inhibitors that should be anticipated as clinical evaluation proceeds.
Protocol Name	P-TABM-5381	P-MTAB-23623	P-TABM-1392	P-TABM-5045	P-TABM-5071
Protocol Type	extraction	labeling	hybridization	scanning	image_acquisition
Protocol Description	Cells were lysed and RNA was extracted using TRIzol method, according to manufacturer's instruction	RNA was biotinylated and purified using the MessageAmp II aRNA Amplification kit (Ambion, Inc., Austin, TX).	Hybridization of labeled cRNA to the BeadChip, and washing and scanning were performed according to the Illumina BeadStation 500x manual. Essentially the amplified, biotin-labeled human cRNA samples were resuspended in a solution of Hyb E1 buffer (Illumina) and 25% (v/v) formamide at a final concentration of 25 ng/uL. 1.5 ug of each cRNA were hybridized.  Hybridization was allowed to proceed at 55 degrees C, for 18 hours after which, the bead array matrix was washed for 10 minutes with 1X High temperature buffer (Illumina), followed by a subsequent 10 minute wash in Wash E1BC buffer.  The arrays were then washed with 100% ethanol for 10 min to strip off any remaining adhesive on the chip. A 2 minute E1BC wash was performed to remove residual ethanol.  The arrays were blocked for 5 minutes with 1% (w/v) casein-PBS, (Pierce).  The array signal was developed via 10 minute incubation with Streptavidin-Cy3 at a final concentration of 1ug/mL solution of (GE Healthcare) in 1% casein-PBS blocking solution. The Mouse 6 Sentrix Expression BeadChip was washed a final time in Wash E1BC buffer for five minutes and subsequently dried via centrifugation for 4 minutes at a setting of 275 rcf.	The arrays were scanned on the Illumina BeadArray Reader, a confocal-type imaging system with 532 (cye3) nm laser illumination.	The array images were registered using an algorithm described previously.  Essentially the bead signals were computed with weighted averages of pixel intensities, and local background is subtracted. Sequence-type signal was calculated by averaging corresponding bead signals with outliers removed (using median absolute deviation). Preliminary data analysis and QC was carried out using the BeadStudio software (Illumina).
SDRF File	E-TABM-1222.sdrf.txt
PubMed ID	22869148
Publication DOI	10.1038/onc.2012.319