USP9X deubiquitinates ALDH1A3 and maintains mesenchymal identity in glioblastoma stem cells

The mesenchymal (MES) subtype of glioblastoma (GBM) stem cells (GSCs) represents a subpopulation of cancer cells that are notorious for their highly aggressive nature and resistance to conventional therapy. Aldehyde dehydrogenase 1A3 (ALDH1A3) has been recently suggested as a key determinant for the maintenance of MES features of GSCs. However, the mechanisms underpinning aberrant ALDH1A3 expression remain elusive. Here, we identified ubiquitin-specific protease 9X (USP9X) as a bona fide deubiquitinase of ALDH1A3 in MES GSCs. USP9X interacted with, depolyubiquitylated, and stabilized ALDH1A3. Moreover, we showed that FACS-sorted USP9X(hi) cells were enriched for MES GSCs with high ALDH1A3 activity and potent tumorigenic capacity. Depletion of USP9X markedly downregulated ALDH1A3, resulting in a loss of self-renewal and tumorigenic capacity of MES GSCs, which could be largely rescued by ectopic expression of ALDH1A3. Furthermore, we demonstrated that the USP9X inhibitor WP1130 induced ALDH1A3 degradation and showed marked therapeutic efficacy in MES GSC-derived orthotopic xenograft models. Additionally, USP9X strongly correlated with ALDH1A3 expression in primary human GBM samples and had a prognostic value for patients with the MES subgroup. Collectively, our findings unveil USP9X as a key deubiquitinase for ALDH1A3 protein stabilization and a potential target for GSC-directed therapy.

胶质母细胞瘤（GBM）干细胞（GSCs）的间充质（MES）亚型是癌细胞的一个亚群，因其高度侵袭性和对常规治疗的抗性而臭名昭著。最近，醛脱氢酶1A3（ALDH1A3）被认为是维持GSCs间充质特性的关键决定因素。然而，支撑ALDH1A3异常表达的机制仍然不清楚。在此，我们确定泛素特异性蛋白酶9X（USP9X）是间充质GSCs中ALDH1A3的一种真正的去泛素化酶。USP9X与ALDH1A3相互作用，使其去多聚泛素化并稳定ALDH1A3。此外，我们发现通过流式细胞术分选的USP9X（高表达）细胞富含具有高ALDH1A3活性和强大致瘤能力的间充质GSCs。USP9X的缺失显著下调ALDH1A3，导致间充质GSCs的自我更新和致瘤能力丧失，而这种情况可通过ALDH1A3的异位表达在很大程度上得到挽救。此外，我们证明USP9X抑制剂WP1130诱导ALDH1A3降解，并在间充质GSC来源的原位异种移植模型中显示出显著的治疗效果。另外，在原发性人类GBM样本中，USP9X与ALDH1A3的表达密切相关，并且对间充质亚型患者具有预后价值。总之，我们的研究结果揭示USP9X是ALDH1A3蛋白稳定的关键去泛素化酶，也是针对GSCs治疗的一个潜在靶点。