USP33-mediated deubiquitination stabilizes YTHDF2 and promotes glioblastoma malignancy and temozolomide resistance

Ubiquitin-specific protease 33 (USP33) has been implicated in tumor progression, but its downstream substrates and pharmacological regulation in glioblastoma (GBM) remain poorly defined. Analysis of the TCGA-GBM/LGG cohort revealed a strong positive correlation between USP33 and the N6-methyladenosine reader YTHDF2, and high expression of either gene was associated with unfavorable overall survival. In U87 and U251 cells, USP33 overexpression increased YTHDF2 protein abundance, whereas CRISPR/Cas9-mediated USP33 depletion reduced YTHDF2 protein levels without significantly altering its mRNA expression. Co-immunoprecipitation assays demonstrated an association between USP33 and YTHDF2. Moreover, USP33 loss accelerated YTHDF2 degradation, increased its polyubiquitination, and reduced its abundance through a proteasome-dependent mechanism, supporting YTHDF2 as a previously unrecognized deubiquitination substrate of USP33. Functional rescue experiments showed that YTHDF2 re-expression partially restored proliferation, clonogenic growth, invasion, cell survival, and temozolomide resistance in USP33-deficient GBM cells. To identify pharmacological inhibitors of USP33, we established an Ub-AMC-based enzymatic screening platform and screened a bioactive library containing 4221 compounds. Deoxyshikonin emerged as a prioritized hit and inhibited USP33 deubiquitinase activity with an IC₅₀ of 15.2 μM. In GBM cells, Deoxyshikonin restored YTHDF2 ubiquitination, reduced YTHDF2 protein abundance, and suppressed USP33-driven proliferative phenotypes. Collectively, these findings identify YTHDF2 as a functional substrate of USP33 and suggest that pharmacological inhibition of USP33-mediated YTHDF2 stabilization may represent a potential strategy for limiting GBM malignancy and temozolomide resistance.