Abstract 6770: TFEB-dependent signals confer resistance against chemotherapeutic drug-induced nucleolar stress

Abstract In addition to their main role in inhibiting cell growth, chemotherapeutic agents are also known to trigger protective signals that equip cancer cells with resistance mechanisms. Notably, treatment with chemotherapeutics has been associated with increased lysosome-related functions, such as autophagy, which protect cells from death. Our previous research showed that gemcitabine enhances autophagy and lysosomal function, particularly in HeLa and pancreatic cancer cells. However, the precise mechanisms through which this nucleoside analog elicits a protective autophagic response are still elusive. While gemcitabine is designed to interfere with DNA replication, it has also been noted to cause nucleolar stress. In our current study, we sought to determine if gemcitabine induces nucleolar stress and, if so, to examine whether this stress mimics the effects of gemcitabine by triggering a cytoprotective response. Our findings show that: 1) Gemcitabine induces nucleolar stress, indicated by the nucleoplasmic localization of nucleolin and nucleolar morphological changes. 2) Treatment of cells with the nucleolar stress inducer CX5461 leads to an autophagy and lysosomal response, monitored by increased LC3 and LAMP1 puncta, along with elevated levels of mature cathepsin B. 3) Additionally, CX5461-treated cells show an enriched nuclear localization of TFEB, the master regulator of autophagy and lysosomal function, detected by immunofluorescence and subcellular fractionation. The nuclear enrichment of TFEB correlates with the enrichment of faster migrating forms of TFEB (indicative of post-translational modification changes), as detected by immunoblotting. 4) Like gemcitabine, CX5461 impairs cell growth. 5) Nonetheless, clonogenic assays indicate that cells with reduced TFEB function (shTFEB) are more sensitive to CX5461 than control cells. 6) In parallel, shTFEB cells exhibit higher levels of apoptotic markers in response to CX5461 treatment, suggesting that TFEB confers resistance to nucleolar stress. Overall, our data provide the first evidence that nucleolar stress triggers a TFEB-dependent protective response. Conducted in cells without functional wild-type p53 signaling, our findings suggest that TFEB could act as a sensor of nucleolar stress, relaying a protective response and helping cells engage in timely and coordinated actions to cope with stress. Therefore, our results support the involvement of TFEB in chemoresistance. These TFEB-protective signals may be particularly relevant to cancers with high TFEB expression levels, such as pancreatic cancer. Citation Format: Marjorie Rolland, Florence McDuff, Laure Bessy, Benoit Marchand, Marie-Josee Boucher. TFEB-dependent signals confer resistance against chemotherapeutic drug-induced nucleolar stress [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 6770.