In Silico Prediction of EZHIP Post-Translational Modification Sites and Small-Molecule High-Throughput Screening for Quantitative EZHIP Modulation
Abstract
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.
METHODS: We 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 PhosphoSitePlus 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.
RESULTS: Computational 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…
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