The context‑dependent effects of dasatinib plus quercetin (D+Q) suggest that the molecular state of senescent cells, not just their presence, dictates whether the combination triggers apoptosis or promotes survival. In proliferative senescent cells, dasatinib suppresses Src‑driven PI3K/Akt and YAP-1 signaling, while quercetin lowers BCL‑2 anti‑apoptotic activity, pushing cells toward death. In post‑mitotic tissues such as intervertebral disc cells, D+Q instead modulates the JUN‑NF-κB axis to attenuate SASP without killing the cell. We hypothesize that these divergent outcomes reflect a temporal switch in senescent cell signaling networks: early‑stage senescent cells retain high YAP-1 transcriptional activity and are primed for dasatinib‑induced apoptosis, whereas late‑stage senescent cells downregulate YAP-1 and become dependent on JUN‑NF-κB‑mediated survival pathways, making them resistant to apoptosis but still responsive to SASP suppression.

To test this, we will induce senescence in human fibroblasts using a defined DNA damage pulse (etoposide, 2 h) and harvest cells at 0 h, 24 h, 48 h, 72 h, and 96 h post‑induction. At each time point we will quantify YAP-1 nuclear localization (immunofluorescence), JUN‑NF-κB transcriptional activity (luciferase reporter), and baseline expression of pro‑survival (BCL‑2, BCL‑XL) and pro‑apoptotic (BAX, PUMA) genes. Parallel plates will receive intermittent D+Q (dasatinib 5 µM + quercetin 50 µM, 2 h pulse every 48 h) for two cycles. Outcomes measured 24 h after the final dose include: (1) apoptosis (caspase‑3/7 activity, Annexin V), (2) SASP secretion (IL‑6, IL‑8, MMP‑3 ELISA), and (3) cell viability (ATP‑based assay).

We predict that cells harvested at 0‑24 h will show high YAP-1 nuclear signal, low JUN‑NF-κB activity, and undergo significant apoptosis after D+Q (≥40 % increase vs control). Cells harvested at 72‑96 h will display diminished YAP-1, elevated JUN‑NF-κB signaling, and resist apoptosis (<10 % increase) while exhibiting reduced SASP secretion (≥30 % decline). A quantitative switch point where YAP-1 activity falls below a threshold and JUN‑NF-κB rises above another threshold will be identified using ROC analysis.

If the hypothesis holds, it will provide a mechanistic basis for timing senolytic interventions: early treatment maximizes senescent cell clearance, while later treatment may be better suited for SASP modulation without risking tissue loss in post‑mitotic compartments. Conversely, if apoptosis rates remain constant across senescence age or if JUN‑NF-κB inhibition does not alter SASP outcomes, the hypothesis will be falsified, indicating that other factors (e.g., stromal interactions or immune milieu) dominate the context‑dependent response. This work bridges the gap between empirical dosing schedules and a testable, mechanism‑driven framework for optimizing D+Q therapy across tissue types and disease stages.