Sequential Senolytic Clearance Followed by Targeted OSK Reprogramming Maximizes Healthspan by Exploiting Senescence‑Driven Reprogramming Potentiation

Hypothesis

Intermittent senolytic clearance with dasatinib+quercetin (D+Q) followed by transient, senescent‑cell‑targeted OSK expression produces synergistic healthspan extension by first removing SASP‑mediated paracrine inhibition and then exploiting the senescence‑enhanced reprogramming competence of residual cells.

Mechanistic Rationale

Senescent cells secrete IL‑6 and other SASP factors that activate STAT3 in neighboring cells, creating a pro‑inflammatory milieu that impedes chromatin remodeling (see IL‑6‑STAT3 signaling in aging). Removing these cells with D+Q lowers IL‑6, reducing STAT3‑mediated heterochromatin stabilization and making the remaining senescent subset more receptive to OSK‑driven epigenetic opening. Furthermore, p16^INK4a‑positive cells intrinsically facilitate OSK binding via open chromatin at Cdkn2a loci (p16‑mediated reprogramming boost). Thus, a senolytic “priming” step clears the bulk of SASP while preserving a p16^INK4a+ reservoir that is epigenetically primed for OSK, yielding greater reduction of epigenetic age and SASP than either approach alone.

Testable Predictions

1. Mice receiving D+Q followed by OSK (D+Q→OSK) will show greater median lifespan increase and lower frailty index than OSK→D+Q, D+Q only, OSK only, or untreated controls.
2. Epigenetic clock reversal (e.g., Horvath mouse clock) will be significantly larger in the D+Q→OSK group, correlating with reduced p16^INK4a+IL-6+ cell frequency in tissues.
3. SASP cytokines (IL-6, IL-1β, TNF-α) will be suppressed more durably after D+Q→OSK than after OSK→D+Q, indicating that senolytic pretreatment prevents OSK‑induced SASP rebound.
4. No increase in tumorigenic incidence will be observed in D+Q→OSK mice compared with OSK‑only groups, confirming that senolytic clearance mitigates any oncogenic risk from transient OSK expression.

Experimental Design

• Use aged (20‑month) C57BL/6 mice, n=15 per arm.
• Arms: (1) Vehicle control, (2) Intermittent D+Q (5 days on/28 days off ×3 cycles), (3) Inducible OSK via AAV9‑Cdkn2a‑rtTA; doxycycline 2 weeks on/2 weeks off ×3 cycles, (4) D+Q then OSK (same dosing, OSK started after final D+Q cycle), (5) OSK then D+Q (OSK first, then D+Q).
• Monitor lifespan, frailty (grip strength, gait speed), monthly blood SASP panel, quarterly epigenetic DNA methylation from liver biopsies, and histopathology for tumors.

Potential Outcomes and Interpretation

If D+Q→OSK outperforms all other regimens in lifespan and frailty while showing maximal epigenetic age reversal and lowest SASP, the hypothesis is supported, indicating that senolytic priming unlocks the reprogramming potential of senescent cells. If OSK→D+Q is superior or no difference is observed, the hypothesis is falsified, suggesting that senescent‑cell‑derived signals are not required for OSK efficacy or that senolytics interfere with OSK activity. Either outcome will refine the sequencing strategy for combined senolytic‑reprogramming therapies in aging.

Key References [1] Dasatinib+quercetin trials: https://pmc.ncbi.nlm.nih.gov/articles/PMC12120425/ [2] OSK lifespan extension: https://pmc.ncbi.nlm.nih.gov/articles/PMC12910722/ [3] IL‑6/STAT3 in aging: https://doi.org/10.1038/s41593-019-0372-9 [4] p16‑mediated reprogramming boost: https://www.liebertpub.com/doi/10.1089/cell.2023.0072 [5] Senocyte IL‑6 enhances reprogramming: https://www.aging-us.com/article/204896/text [6] Chemical OSK mimics: https://www.science.org/doi/10.1126/scitranslmed.adg1777 [7] Targeted OSK to senescent cells: https://doi.org/10.1111/acel.12711