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Fiona Patricia Bailey, Helen Kalirai, Haleh Shahidipour, Kim Clarke, Sarah E. Coupland, Patrick A Eyers; The response of uveal melanoma (UM) cells to Bromodomain and Extra Terminal (BET) inhibitors. Invest. Ophthalmol. Vis. Sci. 2017;58(8):566.
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© ARVO (1962-2015); The Authors (2016-present)
UM is an aggressive ocular malignancy in which ~50% of patients develop fatal metastases. Our aim is to generate ‘kinase expression fingerprints’ of GNAQ/11 mutated (mt) and wild type (WT) UM cell lines, and probe kinome responses to the BET-inhibitor JQ1.
Using digital nanoString profiling and kinome network models, baseline mRNA expression profiles were obtained for the human kinome in GNAQ/GNA11 mt UM cell lines 92.1 (GNAQ G209L) Mel270, OMM2.5 (both GNAQ Q209P) and OMM1 (GNA11 Q209L); the GNAQ/11 WT conjunctival melanoma cell lines CRMM1 (BRAF V600E) and CRMM2 (BRAF WT), and 5 non-melanoma cell lines (GNAQ/11 and BRAF WT). Cell line responses to TKIs, MEK or BET-domain inhibitors were analysed by MTT assay. Cell-cycle perturbations were examined by flow cytometry.
In ocular melanoma cells, expression of several kinase mRNAs (ROR2, EGFR, CAMK1D and FGFRL1) was markedly decreased compared to non-melanoma cells, irrespective of GNAQ/11 status. Conversely, CDK2 mRNA expression was higher. All UM/CRMM cell lines showed sub-μM EC50 responses to the BETi JQ1, in contrast to TKIsThe kinome response to JQ1 was dependent upon GNAQ/11 status. In GNAQ/11 mt UM cell lines, only 0.4 % of kinome mRNAs were upregulated more than 2-fold; in contrast, JQ1 induced upregulation of 3.5 % and 14.8 % of the kinome in CRMM1 and CRMM2 cells (GNAQ/11 WT), respectively.JQ1 significantly reduced BRD4 mRNA expression in UM cells but not in CRMM cells, or the non-melanoma cell line HCT116. Conversely, BRD2 mRNA expression significantly increased in OMM1 (GNA11 Q209L) and CRMM1/2 cells.CDK4 mRNA levels decreased >3-fold in the GNAQ/11 mt UM cells exposed to JQ1. We found that JQ1 induced a G1 cell-cycle arrest in 92.1 and OMM1 cells, and reduced FOXM1 protein levels and FOXM1-regulated kinase mRNAs. In UM patient samples, FOXM1 mRNA expression positively correlated with the expression of the FOXM1-regulated kinases that JQ1 reduced in UM cell lines.
Our data suggest that GNAQ/11 mutation status of ocular melanomas might dictate how cells respond to BET inhibitors, and provides mechanistic insight into kinome-wide responses to this class of compound. We demonstrate that JQ1 reduces the levels of both BRD4 and CDK4 mRNA in GNAQ/11 mt cells and causes a G1 cell-cycle arrest. Furthermore, JQ1 reduces FOXM1 protein levels and impairs expression of the FOXM1 transcriptional network of kinases.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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