DNA-PKcs RNASeq data: DNA-PKcs wilde-type or kinase-dead protein regulate basal and etoposide-induced gene expression changes

Maintenance of the genome is essential for cell survival, and impairment
of the DNA damage response is associated with multiple pathologies
including cancer and neurological abnormalities. DNA-PKcs is a DNA repair
protein and a core component of the classical nonhomologous end-joining
pathway, but it also has roles in modulating gene expression and thus, the
overall cellular response to DNA damage. Using cells producing either
wild-type (WT) or kinase-inactive (KR) DNA-PKcs, we assessed global
alterations in gene expression in the absence or presence of DNA damage.
We evaluated differential gene expression in untreated cells and observed
differences in genes associated with cellular adhesion, cell cycle
regulation, and inflammation-related pathways. Following exposure to
etoposide, we compared how KR versus WT cells responded transcriptionally
to DNA damage. Downregulated genes were mostly involved in protein, sugar,
and nucleic acid biosynthesis pathways in both genotypes, but enriched
biological pathways were divergent, again with KR cells manifesting a more
robust inflammatory response compared to WT cells. To determine what major
transcriptional regulators are controlling the differences in gene
expression noted, we used pathway analysis and found that many master
regulators of histone modifications, proinflammatory pathways, cell cycle
regulation, Wnt/β-catenin signaling, and cellular development and
differentiation were impacted by DNA-PKcs status. Finally, we have used
qPCR to validate selected genes among the differentially regulated
pathways to validate RNA sequence data. Overall, our results indicate that
DNA-PKcs, in a kinase-dependent fashion, decreases proinflammatory
signaling following genotoxic insult. As multiple DNA-PK kinase inhibitors
are in clinical trials as cancer therapeutics utilized in combination with
DNA-damaging agents, understanding the transcriptional response when
DNA-PKcs cannot phosphorylate downstream targets will inform the overall
patient response to combined treatment.