AMP-activated protein kinase(AMPK)is a conserved energy sensor that plays roles in diverse biological processes via phosphorylating various substrates.Emerging studies have demon-strated the regulatory roles of AMPK in DNA repair,but the underlying mechanisms remain to be fully understood.Herein,using mass spectrometry-based proteomic technologies,we systematically investigate the regulatory network of AMPK in DNA damage response(DDR).Our system-wide phosphoproteome study uncovers a variety of newly-identified potential substrates involved in diverse biological processes,whereas our system-wide histone modification analysis reveals a link between AMPK and histone acetylation.Together with these findings,we discover that AMPK pro-motes apoptosis by phosphorylating apoptosis-stimulating of p53 protein 2(ASPP2)in an irradia-tion(IR)-dependent manner and regulates histone acetylation by phosphorylating histone deacetylase 9(HDAC9)in an IR-independent manner.Besides,we reveal that disrupting the his-tone acetylation by the bromodomain BRD4 inhibitor JQ-1 enhances the sensitivity of AMPK-deficient cells to IR.Therefore,our study has provided a resource to investigate the interplay between phosphorylation and histone acetylation underlying the regulatory network of AMPK,which could be beneficial to understand the exact role of AMPK in DDR.