A two-step organoid screen identifies transient p53 activation as a senolytic strategy for radiation-induced salivary gland dysfunction

Radiotherapy significantly improves the survival of head and neck cancer patients. However, it frequently damages the salivary glands, causing persistent hyposalivation and xerostomia. Radiation-induced cellular senescence within the salivary gland stem/progenitor cell niche contributes to hyposalivation, suggesting that selective elimination of senescent cells could promote tissue recovery. However, the effectiveness of senolytic compounds is strongly context dependent, varying with tissue of origin and potentially impairing regenerative cells required for functional restoration. Here, we developed a two-step organoid-based screening methodology that integrates the preferential induction of apoptosis in irradiated, senescence enriched, salivary gland organoids with the preservation of stem/progenitor cell regenerative capacity. As a first step, the senolytic activity of candidate drugs was tested to identify drugs that induced apoptosis in senescent cells of irradiated organoids but were not or less toxic to unirradiated organoids. As a second step, the confirmed senolytic compounds were tested for effects on stem cell self-renewal potential. Using this approach, we identified Nutlin3a, an MDM2 inhibitor and p53 activator, as the compound with the most favorable toxicity profile in salivary glands. Last, we confirmed the anti-senescence and pro-regenerative potential of Nutlin3a in an in vivo mouse model of radiation-induced hyposalivation, where localized Nutlin3a delivery resulted in a robust increase in saliva production, as early as two weeks after administration. These findings establish an organoid-based strategy for identifying tissue-adapted senolytic interventions and propose transient local p53 activation with Nutlin3a as a potential approach to mitigate radiation-induced salivary gland dysfunction.