AMPK interacts with PRC1.1 complex on chromatin to regulate NADK expression in response to metabolic stress in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) remains a leading cause of pediatric cancer-related mortality. ALL cells are particularly vulnerable to metabolic stress following activation of AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis. We and others have demonstrated that AMPK interacts with chromatin-associated proteins to modulate gene expression in response to energy stress. A comprehensive AMPK interactome analysis using TurboID proximity labeling proteomics identified the non-canonical Polycomb Repressive Complex 1.1 (PRC1.1) as an AMPK-associated complex. Co-immunoprecipitation confirmed interactions between AMPK and PRC1.1 components (PCGF1, RING1, KDM2B, BCOR, BCORL1, USP7, SKP1, and RYBP) in KASUMI-2 (Bp-ALL) and KE-37 (T-ALL) cells, which were enhanced following treatment with allosteric AMPK activators (PF-06409577, 991). To evaluate the functional consequence of AMPK activation on PRC1.1 activity, we measured H2AK119ub levels under metabolic stress and observed a marked increase. In contrast, AMPKalpha1/alpha2 double knockout (DKO) HEK293T cells treated with AMPK activators exhibited no change, confirming AMPK dependency. ChIP-seq analysis in KASUMI-2 and KE-37 cells identified Nicotinamide adenine dinucleotide kinase ( NADK ) as an AMPK-associated chromatin target. ChIP-qPCR demonstrated accumulation of AMPK, BCOR, and KDM2B at the NADK promoter under metabolic stress, correlating with increased H2AK119ub and reduced RNA polymerase II occupancy, indicative of transcriptional repression. RT-qPCR and immunoblot analyses confirmed NADK downregulation in ALL cells exposed to metabolic stress, while pharmacological inhibition of AMPK (BAY-3827) or RING1 (PRT4165) upregulated NADK expression. Similarly, NADK repression was abrogated in HEK293T AMPKalpha1/alpha2-deficient cells, and pharmacological inhibition of USP7 (CDDO-Me and Eupalinolode B) increased NADK expression in ALL cells. NADK catalyzes phosphorylation of NAD⁺ to NADP⁺, supporting NADPH generation essential for anabolic metabolism and redox homeostasis. NADK inhibition depletes NADPH, induces oxidative stress, and suppresses proliferation. We propose that AMPK-mediated transcriptional repression of NADK represents an adaptive mechanism to enforce metabolic homeostasis under energy stress. Consistently, combined treatment with the AMPK activator ASP4132 and the NADK inhibitor thionicotinamide induced synergistic cytotoxicity in ALL cells, suggesting a potential therapeutic strategy targeting AMPK-PRC1.1-NADK signaling.