Synthetic chromatin engineering for STAT1 reactivation toward restoring cisplatin sensitivity in breast cancer

Chemoresistant cancers acquire transcriptomic and epigenetic alterations through changes in transcription factor activity and chromatin accessibility. Integrative in silico analyses identified STAT1 as a recurrently suppressed transcription factor in cisplatin-resistant breast cancer. Using cisplatin-resistant triple-negative breast cancer (TNBC) MDA-MB-231 cells, we further confirmed reduced STAT1 expression and activity, accompanied by defective nuclear translocation, indicating that suppression of STAT1 signaling contributes to cisplatin resistance in breast cancer. To restore STAT1 activity, we sought to promote its nuclear localization and transcriptional activation through chromatin engineering. Based on the identification of PHF6 as a direct STAT1-binding protein through in silico and in vitro analyses, we engineered Dual Reader 3 (DR3), a PHF6-derived synthetic chromatin regulator designed to enhance both STAT1 interaction and chromatin engagement through histone H2B ubiquitination. DR3 induced STAT1-associated transcriptional programs, including inflammatory and tumor-suppressive miRNAs. Mechanistically, DR3 physically promoted STAT1 nuclear translocation, activated STAT1 through a non-canonical S727-dependent mechanism independent of Tyr701 phosphorylation, and enhanced chromatin accessibility at STAT1-responsive regulatory regions through histone H2B ubiquitination. CUT&RUN analysis further revealed coordinated transcriptional and epigenetic remodeling associated with DR3-mediated STAT1 activation, including upregulation of HIPK2, which enhanced cellular sensitivity to cisplatin by strengthening the intrinsic DNA damage response. Finally, we investigated whether STAT1 reactivation could alleviate acquired cisplatin resistance. In vivo delivery of DR3 restored cisplatin sensitivity in resistant breast tumors. By coupling STAT1 nuclear translocation with chromatin remodeling, DR3 reactivated STAT1-dependent transcriptional programs and restored cisplatin sensitivity in breast cancer. Together, this study establishes transcription factor reactivation through synthetic chromatin engineering as a potential therapeutic strategy for overcoming chemoresistance in solid tumors.