Histone deacetylase inhibitors (HDACi) are novel
epigenetic-based drugs that induce a range of anti-tumour responses including
apoptosis. The HDACi, vorinostat, has
achieved remarkable clinical success in some patients but it remains unclear
why certain patients remain unresponsive.
Constitutive STAT activation or over-expression of pro-survival Bcl-2 proteins
have been identified as potential biomarkers of HDACi resistance. Therefore, the current study aims to further
elucidate vorinostat resistance mechanisms through a functional genomics screen
to identify novel genes that sensitise cells to vorinostat-induced
apoptosis.
We have conducted a genome-wide synthetic lethal siRNA screen to identify genes that when knocked down co-operate with vorinostat to induce apoptosis in otherwise resistant cells. The primary SMARTpool siRNA screen yielded 450 gene hits, of which 106 validated in a secondary screen by deconvolution into the four individual constituent siRNAs. Tertiary screening was conducted to evaluate specificity of these genes to vorinostat compared to conventional chemotherapeutics. We identified ten genes that sensitised specifically to vorinostat in two or more of the four cell lines tested. These were validated using flow cytometry-based apoptosis assays and knockdown greater than 90% was observed for all genes using qRT-PCR. One of the 10 validated HDACi sensitiser genes was GLI1, a known oncogene not previously known to regulate HDACi activity. Treatment of vorinostat-resistant cells in vitro with the GLI small molecule inhibitor, GANT61, phenocopies GLI1 knockdown and we are conducting xenograft therapy experiments with vorinostat and GANT61 and GLI1 inducible shRNA to confirm these findings in vivo. GLI1 expression is induced by HDACi treatment and we are currently utilitising NGS RNAseq technology to investigate the precise mechanism by which GLI1 loss of function restores HDACi sensitivity. Therefore, we have identified at least one potential novel drug target for development of new therapies that may be used in combination with HDACi to treat resistant tumours.