胶质母细胞瘤（GBM）是最具侵袭性的原发性脑肿瘤，具有高复发率和不良预后。手术和化疗放疗联合替莫唑胺（TMZ）是标准治疗，但在大多数情况下，肿瘤会对进一步治疗产生耐药性，患者最终因病情恶化而死亡。因此，迫切需要开发耐受性良好、有效靶向化疗耐药性胶质瘤的药物。NEO214是通过共价结合磷酸酯酶4（PDE4）抑制剂罗氏丙，与柠檬醇相关的天然单萜衍生物合成的。我们之前的前期研究表明，NEO214具有抗癌活性，可以穿过血脑屏障，并且耐受性显著。本研究中，我们研究了其作用机制，并发现抑制自噬/自噬是其抗癌效应在胶质母细胞瘤细胞中的关键因素。我们展示了NEO214可以阻止自噬溶酶体融合，从而阻塞自噬通路并触发胶质瘤细胞死亡。此过程涉及机械靶点雷帕霉素激酶（MTOR）活性的激活，导致转录因子EB（TFEB）的细胞质积累，TFEB是自噬-溶酶体通路中关键基因的重要调控因子，从而降低自噬-溶酶体相关基因的表达。当与氯喹和TMZ联合使用时，NEO214的抗癌影响进一步增强，并对TMZ耐药性细胞产生作用。总之，我们的研究结果表明NEO214是一种新型自噬抑制剂，可以用于克服胶质母细胞瘤的化疗耐药性。 缩写词：ATG：自噬相关；BAFA1：巴非霉素A1；BBB：血脑屏障；CQ：氯喹；GBM：胶质母细胞瘤；LAMP1：溶酶体相关膜蛋白1；MAP1LC3/LC3：微管相关蛋白1轻链3；MGMT：O-6-甲基鸟嘌呤-DNA甲基转移酶；MTOR：机械靶点雷帕霉素激酶；MTORC：MTOR复合物；POH：柠檬醇；SQSTM1/p62：封闭体1；TFEB：转录因子EB；TMZ：替莫唑胺。

Glioblastoma (GBM) is the most aggressive primary brain tumor, exhibiting a high rate of recurrence and poor prognosis. Surgery and chemoradiation with temozolomide (TMZ) represent the standard of care, but, in most cases, the tumor develops resistance to further treatment and the patients succumb to disease. Therefore, there is a great need for the development of well-tolerated, effective drugs that specifically target chemoresistant gliomas. NEO214 was generated by covalently conjugating rolipram, a PDE4 (phosphodiesterase 4) inhibitor, to perillyl alcohol, a naturally occurring monoterpene related to limonene. Our previous studies in preclinical models showed that NEO214 harbors anticancer activity, is able to cross the blood-brain barrier (BBB), and is remarkably well tolerated. In the present study, we investigated its mechanism of action and discovered inhibition of macroautophagy/autophagy as a key component of its anticancer effect in glioblastoma cells. We show that NEO214 prevents autophagy-lysosome fusion, thereby blocking autophagic flux and triggering glioma cell death. This process involves activation of MTOR (mechanistic target of rapamycin kinase) activity, which leads to cytoplasmic accumulation of TFEB (transcription factor EB), a critical regulator of genes involved in the autophagy-lysosomal pathway, and consequently reduced expression of autophagy-lysosome genes. When combined with chloroquine and TMZ, the anticancer impact of NEO214 is further potentiated and unfolds against TMZ-resistant cells as well. Taken together, our findings characterize NEO214 as a novel autophagy inhibitor that could become useful for overcoming chemoresistance in glioblastoma.Abbreviations: ATG: autophagy related; BAFA1: bafilomycin A1; BBB: blood brain barrier; CQ: chloroquine; GBM: glioblastoma; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MGMT: O-6-methylguanine-DNA methyltransferase; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; POH: perillyl alcohol; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TMZ: temozolomide.