MDM4 coordinates YWHAG-mediated phosphorylation-ubiquitination crosstalk to stabilize PFKFB2 and drive glycolysis and proliferation in oral squamous cell carcinoma

Mouse double minute 4 (MDM4) is a well-established regulator of the tumor suppressor p53, but its independent role in the context of TP53 mutations in oral squamous cell carcinoma (OSCC) has not been elucidated. This study aims to investigate the metabolic regulatory function of MDM4 in TP53-mutant OSCC and explore its potential as a therapeutic target. Bioinformatic analysis, qRT-PCR, western blotting, metabolomics, and Seahorse assays were used to assess MDM4-mediated metabolic regulation. Mechanistic studies included co-immunoprecipitation, site-directed mutagenesis, and ubiquitination assays to examine regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2). In vitro and in vivo experiments were performed to evaluate the antitumor effects of combined inhibition of MDM4 and PFKFB2. We demonstrate that MDM4 enhances tumor cell proliferation and glycolytic activity in TP53-mutant OSCC. Mechanistically, MDM4 stabilizes the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2) by promoting its interaction with the adaptor protein YWHAG and inhibiting K48-linked ubiquitination. This interaction protects phosphorylated PFKFB2 (Ser466/Ser483) from ubiquitin-mediated degradation and promotes the binding of YWHAG to PFKFB2, thereby preventing its K490 ubiquitination. Silencing PFKFB2 significantly reduces the glycolysis-promoting effects of MDM4, and combined inhibition of both MDM4 and PFKFB2 leads to a synergistic suppression of tumor growth both in vitro and in vivo. These findings uncover a novel p53-independent role of MDM4 in metabolic regulation in TP53-mutant OSCC and identify the MDM4-YWHAG-PFKFB2 axis as a potential therapeutic vulnerability. Targeting this pathway may provide a promising therapeutic strategy for TP53-mutant OSCC and support MDM4 as a candidate therapeutic target. Schematic illustration of the mechanism by which MDM4 promotes glycolysis and proliferation in TP53-mutant OSCC. In TP53-mutant OSCC, MDM4 enhances glycolysis by stabilizing PFKFB2. Mechanistically, MDM4 reinforces the interaction between YWHAG and PFKFB2 phosphorylated at S466/S483, thereby protecting PFKFB2 from ubiquitination at K490 and subsequent degradation. As a consequence, stabilized PFKFB2 increases the level of F-2,6-BP, which activates PFK1 and promotes glycolytic flux. Enhanced glycolysis further increases ATP production, ultimately supporting tumor cell growth and proliferation in TP53-mutant OSCC. Abbreviations: ATP, adenosine triphosphate; F-1,6-BP, fructose-1,6-bisphosphate; F-2,6-BP, fructose-2,6-bisphosphate; F6P, fructose-6-phosphate; G6P, glucose-6-phosphate; K490, lysine 490; MDM4, mouse double minute 4; OSCC, oral squamous cell carcinoma; PFK1, phosphofructokinase-1; PFKFB2, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2; S466, serine 466; S483, serine 483; TP53, tumor protein p53; YWHAG, 14-3-3γ. MDM4 promotes glycolytic reprogramming and tumor progression in TP53-mutant OSCC. MDM4 stabilizes PFKFB2 through YWHAG-dependent inhibition of K48-linked ubiquitination. Co-targeting MDM4 and PFKFB2 exerts synergistic antitumor effects in OSCC.