Reversing the Warburg Effect: YW3-56 Induces Leukemia Differentiation via AKT-Mediated Glucose Metabolic Reprogramming

<b>Background</b>: Protein arginine deiminase 4 (PAD4) has emerged as a promising therapeutic target for acute promyelocytic leukemia (APL) because of its role in epigenetic regulation and leukemogenesis. All-trans retinoic acid, a standard differentiation agent in APL therapy, has been shown to upregulate PAD4 expression during leukemic cell maturation. Interestingly, first-generation PAD4 inhibitors also promote differentiation, but simultaneously trigger compensatory PAD4 overexpression, underscoring the unresolved complexity of PAD4 modulation in leukemia therapy. <b>Methods</b>: In this study, we employed mass cytometry and transcriptomic-proteomic integrated analysis to investigate the underlying mechanisms of YW3-56, a dual-function PAD4 inhibitor against protein expression and enzymatic function, in NB4 leukemia cells. Functional validation was conducted using Western blot and metabolic assays. <b>Results</b>: Mass cytometry analysis revealed that YW3-56 reduced leukemia stemness (CD44/CD133), while enhancing myeloid differentiation (CD11b/CD14) and immunogenic activation (CD80/CD86). Multiomics analysis revealed a YW3-56-induced metabolic shift characterized by downregulation of glycolytic enzymes and upregulation of the tricarboxylic acid cycle and pentose phosphate pathway components, indicating a reversal of the Warburg effect. Mechanistically, this metabolic reprogramming was driven by reduced AKT expression and phosphorylation at Thr308, impaired GLUT1 expression and membrane localization, and decreased glucose uptake, which collectively promoted the differentiation of NB4 cells. Additionally, YW3-56 suppressed the downstream mTOR pathway, inducing caspase-3/PARP-mediated apoptosis and inhibiting cell proliferation. <b>Conclusions</b>: Our study demonstrated that YW3-56 exerts multimodal antileukemic effects in APL by simultaneously targeting PAD4-mediated epigenetic regulation, AKT-driven metabolic reprogramming and cellular differentiation, highlighting PAD4-AKT signaling as a promising target for APL combination therapy.