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.