Multiomics analysis of adaptation to repeated DNA damage in prostate cancer cells.

DNA damage is frequently utilized as the basis for cancer therapies; however, resistance to DNA damage remains one of the biggest challenges for successful treatment outcomes. Critically, the molecular drivers behind resistance are poorly understood. To address this question, we created an isogenic model of prostate cancer exhibiting more aggressive characteristics to better understand the molecular signatures associated with resistance and metastasis. 22Rv1 cells were repeatedly exposed to DNA damage daily for 6 weeks, similar to patient treatment regimes. Using Illumina Methylation EPIC arrays and RNA-seq, we compared DNA methylation and transcriptional profiles between the parental 22Rv1 cell line and the lineage exposed to prolonged DNA damage. Here we show that repeated DNA damage drives the molecular evolution of cancer cells to a more aggressive phenotype and identify molecular candidates behind this process. Total DNA methylation was increased while RNA-seq demonstrated these cells had dysregulated expression of genes involved in metabolism and the unfolded protein response (UPR) with Asparagine synthetase (ASNS) identified as central to this process. Despite the limited overlap between RNA-seq and DNA methylation, oxoglutarate dehydrogenase-like (OGDHL) was identified as altered in both data sets. Utilising a second approach we profiled the proteome in 22Rv1 cells following a single dose of radiotherapy. This analysis also highlighted the UPR in response to DNA damage. Together, these analyses identified dysregulation of metabolism and the UPR and identified ASNS and OGDHL as candidates for resistance to DNA damage. This work provides critical insight into molecular changes which underpin treatment resistance and metastasis.

Determinants of a transcriptionally competent environment at the GM-CSF promoter.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is produced by T cells, but not B cells, in response to immune signals. GM-CSF gene activation in response to T-cell stimulation requires remodelling of chromatin associated with the gene promoter, and these changes do not occur in B cells. While the CpG methylation status of the murine GM-CSF promoter shows no correlation with the ability of the gene to respond to activation, we find that the basal chromatin environment of the gene promoter influences its ability to respond to immune signals. In unstimulated T cells but not B cells, the GM-CSF promoter is selectively marked by enrichment of histone acetylation, and association of the chromatin-remodelling protein BRG1. BRG1 is removed from the promoter upon activation concomitant with histone depletion and BRG1 is required for efficient chromatin remodelling and transcription. Increasing histone acetylation at the promoter in T cells is paralleled by increased BRG1 recruitment, resulting in more rapid chromatin remodelling, and an associated increase in GM-CSF mRNA levels. Furthermore, increasing histone acetylation in B cells removes the block in chromatin remodelling and transcriptional activation of the GM-CSF gene. These data are consistent with a model in which histone hyperacetylation and BRG1 enrichment at the GM-CSF promoter, generate a chromatin environment competent to respond to immune signals resulting in gene activation.