【作者】 王丹；

【导师】 郗乔然；

【作者基本信息】 清华大学 ， 生物学， 2023， 博士

【摘要】 弥漫内生型脑桥胶质瘤(Diffuse intrinsic pontine gliomas,DIPG)是一种高度侵袭、不易手术,死亡率高的儿童脑肿瘤,目前没有有效的治疗药物。约80%的DIPG存在组蛋白H3K27M突变,会导致全基因组H3K27me3的低表达和转录异常,H3K27M突变也被认为是DIPG肿瘤发生的主要驱动因素。目前针对DIPG的新型化疗药物集中于对组蛋白甲基化和乙酰化平衡状态的整体调节,从而恢复由于H3K27M突变引发的级联效应。但是由于临床转化效果不佳,DIPG仍然需要新的治疗方案。除了 H3K27M突变外,在90%的H3.3K27M的DIPG中存在p53信号通路的异常,包括TP53自身突变(占比65%～69%)及PPM1D突变(占比20%～28%)。PPM1D突变能降低p53的稳定性,使p53失活,从而促进DIPG的肿瘤进展。表明重新激活p53的抑癌功能可能是DIPG的潜在治疗靶点。在本研究中,我们利用DIPG的病人样本建立了多株DIPG原代细胞系。然后,我们选择了一个表观遗传类的小分子化合物库,通过药物筛选,最终确定吉西他滨作为DIPG的潜在治疗药物。接下来,我们验证了吉西他滨能有效抑制DIPG在体外和小鼠体内的成瘤能力,并揭示了吉西他滨能引起DNA损伤,激活p53并增加p53在凋亡相关基因上的可及性,从而诱导细胞凋亡的作用机制。此外,我们还发现吉西他滨依赖于H3K27M诱导DIPG的细胞凋亡。但是,吉西他滨在引起DNA损伤的同时,还能诱导内源性逆转录病毒元件(Endogenous retrovirus,ERV)的表达,并激活cGAS-STING信号及下游RELB介导的非经典NF-κB信号通路,使肿瘤存活。我们再次经过药物筛选,发现Fimepinostat与吉西他滨表现出良好的药物协同性,而且Fimepinostat能抑制吉西他滨激活的非经典NF-κB信号通路和DNA损伤修复基因的表达,因而造成持续性的DNA损伤,与吉西他滨在体外和体内实验中均表现出良好的协同抗肿瘤作用。综上所述,本论文提出,使用吉西他滨重新激活p53的肿瘤抑制功能联合Fimepinostat抑制非经典NF-κB信号通路,可能是治疗H3.3K27M突变的DIPG患者的潜在治疗方案。更多还原

【Abstract】 Diffuse intrinsic pontine glioma(DIPG)is the most aggressive pediatric brainstem tumor with a median survival of less than 12 months;seeking effective therapy is still the focus of the field.Nearly 80%DIPG cells harbor H3K27M mutations,which caused global reduction in H3K27me3 levels,and H3K27M are considered as driver mutations for DIPG.This understanding has recently inspired substantial efforts to develop drugs for treating DIPG based on global modulation of the histone methylation/acetylation balance.Whilst promising,the findings from such studies have had limited translational impacts.Hence,screening drugs for treating DIPG is highly demanding.In addition to the characteristic H3K27M mutations,up to 90%of H3.3K27M mutant DIPG harbor alterations in the p53 pathway,including mutations in TP53(accounting for 65～69%)itself,as well as protein phosphatase,Mg2+/Mn2+-dependent 1D(PPMID,accounting for 20%～28%).PPM1D truncating mutations promoted tumor progression in DIPG by destabilizing and inactivating p53.These evidences suggest that reactivating p53 tumor suppressive function in H3.3K27M DIPG cells might be a solution for DIPG therapy.Here,we used treatment-na(?)ve patient-derived DIPG cell line provided by Tiantan hospital,and performed epigenetic compounds screening to identify that gemcitabine as a candidate.As a pyrimidine analog,gemcitabine triggers DNA damage,stabilized and activated p53 including increased chromatin accessibility for p53 at apoptosis-related loci in xenograft murine models.Notably,the H3.3K27M mutation facilitates gemcitabineinduced apoptosis in DIPG.However,we found that gemcitabine simultaneously induces a pro-survival program in DIPG through activating RELB-mediated non-canonical NF-κB signaling.Specifically,gemcitabine induced the transcription of ERV(endogenous retrovirus),which might activate cGAS-STING(cyclic GMP-AMP synthase-stimulator of interferon genes)signaling and downstream non-canonical NF-κB signaling.A drug screen with gemcitabine-treated cells revealed that Fimepinostat effectively suppresses the gemcitabine-induced RELB-mediated non-canonical NF-κB signaling in these cells.Finally,we show that a combination therapy comprising gemcitabine and Fimepinostat synergistically confers potent anti-tumor benefit in H3.3K27M DIPG.In summary,this study showed that enhanced p53 tumor suppressive function by gemcitabine and suppression of non-canonical NF-κB signaling using Fimepinostat are potent therapeutic interventions for H3.3K27M DIPG.更多还原