Abstract 294: TFEB nuclear persistence predicts sensitivity to lysosomal inhibition in therapy-induced senescent thyroid cancer cells

Abstract Therapy-induced senescence (TIS), is a potential outcome of anti-cancer treatments, characterized by a stable cell cycle arrest. However, it is now widely accepted that this process acts as a double-edged sword: in facts, senescent cells are active drivers of cancer relapse, aggressiveness and metastasis, through the release of various pro-inflammatory factors, and the ability to resume proliferation. Therefore, selectively targeting TIS cells, a strategy named one-two punch approach, is crucial to avoid their harmful effects. To this purpose, identifying targetable characteristics of TIS cells is essential for the development of new senotherapeutics. One of the peculiarities of senescent cells is the expansion of the lysosomal compartment. To assess whether lysosomes may be a valid target for new senotherapies, we employed as a TIS model different cell lines of anaplastic thyroid cancer, an aggressive and untreatable type of tumor, undergoing senescence upon either CDK4/6 inhibitor Palbociclib or ionizing radiation treatment. Senescence induction and lysosomal expansion were assessed by a multi-marker approach, comprising β-gal assay, γH2AX nuclear foci quantification, p21 and HMGA2 expression levels, and lysosomal staining. Our findings indicated that the response to TIS varied among cell lines regarding lysosomal content increase. Although the autophagic flux, that relies on lysosomes, was elevated across all cell lines, its functionality was line-specific, as evidenced by differential p62 protein turnover. Targeting autophagy with the lysosomal inhibitor Chloroquine exhibited a senolytic effect only on senescent cells with an expanded lysosomal compartment and functional autophagy. Conversely, the lysosome-sparing autophagy inhibitor LY294002 did not replicate these results, suggesting that the senolytic effect is ascribable to lysosomal rather than autophagy inhibition. To further explore the differential expansion of the lysosomal compartment, we analyzed the localization of TFEB, the master regulator of lysosomal biogenesis, following TIS induction, through immunofluorescence. Six days after treatment, TFEB maintained a nuclear localization exclusively in the cell line with functional autophagy. Interestingly, this was associated with significantly lower protein levels of Exportin-1, an export receptor responsible for TFEB nuclear extrusion. Our results indicate that lysosomes represent a promising target for developing new senotherapeutics, but TFEB’s ability to enter and persist at the nuclear level appears to be crucial in determining this sensitivity. Understanding the molecular differences underlying the varying functionality of the lysosomal biogenesis machinery, often defective in cancer, may pave the way for the future introduction of this one-two punch approach in the clinical setting. Citation Format: Beatrice Mazzoleni, Mara Mazzoni, Angela Greco. TFEB nuclear persistence predicts sensitivity to lysosomal inhibition in therapy-induced senescent thyroid cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 294.