Abstract 3755: Genome-wide transcriptome analysis reveals the distinct gene expression profiles and specific biological effects of fluorescent polyamides, HxIP and azaHxPI

Abstract

Abstract DNA-binding polyamides target predetermined sequences to inhibit specific transcription factor-DNA interactions and modulate gene expression. Inherently non-genotoxic, these non-covalent binding small molecules exert their biological activity without inflicting DNA damage and provide the basis for potentially less toxic DNA-targeting anticancer therapeutics. However, the genome-wide specificity of polyamide binding remains an outstanding issue in delivering the desired biological response without inducing widespread off-target effects. In this study, we used RNA-seq transcriptome analysis to assess the global effects of fluorescent polyamides HxIP and azaHxPI, having previously shown that they bind with high affinity and selectivity to their respective target DNA sequences 5’-WWCGWW-3’ and 5’-WCGCGW-3’ (W = A/T)1,2. Changes to the MDA-MB-231 breast adenocarcinoma transcriptome were measured following 24 h treatment with 2 and 5 μM HxIP or azaHxPI. At 5 μM, HxIP affected 349 genes by two-fold (p<0.05), corresponding to 1.72% of the Ensembl coding gene assembly, of which 171 genes were downregulated and 178 were upregulated. AzaHxPI caused the differential expression of 496 genes (2.44%), with 176 of these genes downregulated and 320 genes upregulated. Both HxIP and azaHxPI affected only a small number of the total gene set without significantly perturbing the rest of the transcriptome, demonstrating the specific nature of polyamide induced biological effects. Only 26 genes were affected by both polyamides (3.1% overlap), suggesting that HxIP and azaHxPI stimulate unique transcriptome profiles as a result of targeting different DNA sequences. Following a 96 h treatment HxIP had no effect on cell growth (GI50 > 100 μM), whereas azaHxPI causes growth inhibition (GI50 = 20.3 μM) and induces apoptosis. Immunoblotting analysis of γ;H2AX showed no evidence of DNA damage, further corroborated by the absence of chk2 phosphorylation. In addition, no ATR-chk1 activation was detected suggesting that azaHxPI does not induce replication stress, despite inhibiting DNA synthesis as shown using the BrdU incorporation assay. Levels of phospho-AKT (Ser472) decreased in response to polyamide treatment, whilst p21 is significantly upregulated and both may be implicated in the mechanism of azaHxPI induced-apoptosis. Taken together these results emphasise the potential application of polyamides as apoptosis-inducing DNA-targeting agents benefiting from enhanced sequence selectivity which in turn confers specific biological activity.