Background Posterior fossa group A (PFA) ependymoma is a lethal pediatric brain tumor driven predominantly by epigenetic dysregulation.Enhancer of Zeste Homologs Inhibitory Protein (EZHIP) is a defining oncogenic factor in PFA ependymoma that inhibits PRC2 activity, inducing a global loss of H3K27me3 and sustaining aberrant developmental transcriptional programs.Although the metabolic modulator, metformin, reduces EZHIP protein levels, the mechanisms governing EZHIP regulation remain undefined. MethodsWe generated a stable HEK293T reporter cell expressing HA-and RFP-tagged EZHIP together with a GFP viability control, enabling quantitative and viability-normalized assessment of EZHIP abundance.In silico post-translational modification prediction was performed using Phos-phoSitePlus and NetPhos 3.1 to identify candidate regulatory residues and upstream kinases.A focused panel of pathway targeting compounds was evaluated using fluorescence-based high-throughput screening, followed by secondary validation including cell counting, LC50 (half-maximal lethal concentration) analysis, and Western blotting. ResultsComputational analyses identified multiple high-confidence serine phosphorylation sites on EZHIP and implicated AMPK, MAPK, PKC, AKT, and CK2 signaling pathways.High-throughput screening revealed that activation of the AMPK axis robustly suppressed EZHIP protein levels.Secondary validation demonstrated that biguanides activating AMPK reduced EZHIP abundance independently of cytotoxicity and restored global H3K27me3 levels.In contrast, PKC activation increased EZHIP protein abundance. ConclusionOur study identifies EZHIP as a dynamically regulated oncoprotein controlled by posttranslational signaling pathways.AMPK and PKC exert opposing effects on EZHIP stability, defining actionable regulatory mechanisms for therapeutic targeting in EZHIP-driven cancers.
Moon et al. (Thu,) studied this question.