Atractylenolide I activates the DNA damage-apoptosis axis in colorectal cancer patient-derived organoids by dual-targeting XRCC5 and CLTC.

[BACKGROUND] Colorectal cancer (CRC) is a leading malignancy worldwide. Patient-derived organoids (PDOs) represent a cutting-edge model for cancer research. Atractylenolide I (ATT-1), a key bioactive lactone component of Baizhu (Atractylodes macrocephala), has demonstrated anti-cancer potential, but its mechanism of action in clinically relevant CRC PDO models remains unknown.

[PURPOSE] To evaluate the anti-tumor efficacy and molecular mechanism of ATT-1 using colorectal cancer PDOs.

[METHODS] We investigated ATT-1 in CRC PDOs, assessing its effects on viability, proliferation (EdU), apoptosis (TUNEL), and DNA damage (γ-H2AX). Combination therapies with oxaliplatin or NU7441 were also evaluated in the PDO models. Multi-omics analyses (RNA-seq and metabolomics) profiled transcriptional and metabolic alterations. Direct target engagement was identified through LiP-MS and CETSA, and validated via shRNA knockdown. Molecular docking and dynamics simulations characterized the binding interactions. In vivo efficacy, safety, and combination therapy were validated in mouse xenograft models.

[RESULTS] ATT-1 selectively inhibited the proliferation of CRC PDOs, inducing apoptosis and inhibiting DNA damage repair. Multi-omics profiling revealed disruption of lipid metabolism, nucleotide synthesis, and cell cycle progression. We identified CLTC and XRCC5 as direct protein targets of ATT-1, validated by LiP-MS, CETSA, and functional knockdown assays. ATT-1 synergized with oxaliplatin and NU7441, enhancing anti-tumor efficacy in both PDO and xenograft models without significant hepatorenal toxicity. Furthermore, ATT-1 exhibited favorable pharmacokinetic properties.

[CONCLUSION] This study identifies ATT-1 as a promising multi-targeted therapeutic for colorectal cancer by leveraging PDOs for direct, de novo target discovery. We uniquely identified two novel, high-affinity targets, CLTC and XRCC5, and elucidated a convergent dual-targeting mechanism wherein ATT-1 binding disrupts DNA damage repair and triggers apoptosis. This novel mechanism and its potent synergy with standard chemotherapy in physiologically relevant models provide a compelling strategy for integrating traditional Chinese medicine into modern precision oncology.