Temporally Structured Coordination between DNA Damage Response and MAPK/PI3KSignaling Pathways under Genotoxic Stress

The DNA damage response (DDR) safeguards genomic integrity through lesion sensing,
checkpoint activation, and repair. While ATM/ATR-centered DDR signaling is well
defined, how growth-associated pathways such as MAPK/ERK and PI3K/AKT co-
engage with DDR programs across distinct genotoxic stresses remains incompletely
resolved. U2OS and HCT116 cells were challenged with ionizing radiation (4 Gy),
replication stress (hydroxyurea, 2 mM), or oxidative stress (H₂ O₂ , 200 μM, 30 min).
DDR and growth-pathway phosphorylation kinetics were quantified by immune-blot
densitometry (phosphor /total; fold-change vs. 0 h vehicle baseline). Replication fork
dynamics were assessed by DNA fiber assays under HU with ATR or PI3K inhibition.
DNA damage burden was measured by alkaline comet tail moment and 53BP1 foci at 6
h and 24 h. Long-term proliferative capacity was quantified by clonogenic survival (SF4)
after IR. Pharmacologic perturbations included trametinib (MEK), BKM120 (PI3K),
VE-821 (ATR), NU7441 (DNA-PK), and olaparib (PARP). Analyses used biological
replicate–level inference. Stressors produced expected checkpoint signatures (IR: ATM–
CHK2; HU: ATR–CHK1) with temporally overlapping ERK and AKT phosphorylation
changes. ATR inhibition markedly increased fork stalling and reduced fork progression
under HU. PI3K inhibition was associated with higher early DNA-damage readouts
(comet tail moment and 53BP1 foci at 6 h) and produced larger reductions in clonogenic
survival after IR than MEK inhibition in both cell lines. Canonical DDR activation and
growth-signaling phosphorylation changes co-occur after genotoxic, replication, and
oxidative stress and are associated with distinct functional outcomes: ATR predominates
in fork stability, whereas PI3K activity is linked to early damage burden and post-IR survival.
These findings motivate mechanistic follow-up to define causal nodes for combination
targeting in genomically unstable cells.
Keywords: DNA damage response; ATM–CHK2; ATR–CHK1; PI3K–AKT signaling;
MAPK/ERK pathway; replication stress