Histone deacetylase inhibitors (HDACi) are novel epigenetic-based drugs that induce a range of anti-tumour responses including apoptosis. The pan-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 synthetic lethal screen using a whole-genome RNA interference library to identify genes that when knocked down co-operate with vorinostat to induce tumour cell apoptosis in otherwise resistant cells. The primary SMARTpool siRNA screen yielded 450 gene hits, of which 105 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 which sensitised specifically to vorinostat in two or more of the four cell lines tested. These were validated using FACS-based apoptosis assays for phosphatidylserine externalisation (annexin V), DNA fragmentation (subG1 DNA content) and intracellular staining for cleaved caspase 3. Knockdown greater than 90% was observed for all genes using RT-qPCR. One of the 10 validated HDACi sensitiser genes was GLI1, a known oncogene not previously known to regulate the activity of HDACi. Treatment of vorinostat-resistant cells with the GLI small molecule inhibitor, GANT61, phenocopies GLI1 knockdown and we are currently investigating the mechanism by which GLI1 loss of function sensitises otherwise resistant tumour cells to vorinostat-induced apoptosis. 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.