Tubocapsenolide A Overcomes Oxaliplatin Resistance in Colorectal Cancer by Targeting PLK1 to Impair CEP55–ESCRT‐Associated Vesicular Damage Clearance

ABSTRACT Oxaliplatin (L‐OHP) is a first‐line chemotherapeutic agent for advanced colorectal cancer (CRC) and postoperative adjuvant therapy. However, acquired L‐OHP resistance remains a clinical obstacle. Here, we identify Tubocapsenolide A (TA), a natural withanolide isolated from Tubocapsicum anomalum, as a promising antitumor agent that suppresses CRC growth and sensitizes L‐OHP‐resistant CRC to L‐OHP. Using a PROTAC‐based target deconvolution strategy, we identify PLK1 as a direct molecular target of TA. PLK1 is elevated in L‐OHP‐resistant CRC and is associated with reduced patient sensitivity to L‐OHP. Mechanistically, elevated PLK1 promotes CEP55 Ser436 phosphorylation and facilitates ESCRT recruitment to nuclear and mitochondrial budding sites. This process facilitates the clearance of L‐OHP‐induced damaged components through nuclear budding and TOM20 − /PDH + mitochondrial‐derived vesicles, thereby contributing to organelle homeostasis and cell survival. By inhibiting PLK1, TA interferes with this vesicle‐associated damage clearance process, leading to intracellular accumulation of damaged components. This damage accumulation is accompanied by nucleolar stress and mitochondrial dysfunction, ultimately contributing to cell death. Collectively, our findings identify a previously unappreciated role of the PLK1–CEP55–ESCRT axis in vesicle‐associated damage clearance during L‐OHP resistance and support this axis as a potential therapeutic target. TA represents a promising lead compound for overcoming L‐OHP resistance in CRC.