利用CRISPR-Cas9筛选技术探索BMP信号通路中导致弥漫内生型脑干胶质瘤协同致死效应的关键基因研究

Diffuse intrinsic pontine glioma(DIPG)is a malignant pediatric brain tumor with devastating prognosis. The pathogenesis is totally different from hemispheric Glioblastoma multiform(GBM), and there is still no effective therapeutics. In DIPG cell lines with ACVR1 mutation, activated BMP signaling promote the tumorigenesis. However, the data from our preliminary experiment showed that, in the DIPG cell lines with wildtype ACVR1, the BMP pathway was not active, while after the administration of BMP4 to mimic the effect of ACVR1 mutation, the activated BMP signaling neither facilitate the growth of the tumor, nor promote the astrocytic lineage differentiation, but lead to the apoptosis of the cells. A synthetic lethal interaction occurs between two genes when the perturbation of either gene alone is viable but the perturbation of both genes simultaneously results in the loss of viability. The hypothesis was that the synthetic lethality related apoptosis of DIPG cells was caused by the transcriptional manipulation of BMP signaling downstream target genes . The purpose of this research is to discover the key target genes in BMP transcriptional network by CRISPR-Cas9 screen, and verify the gene functions unveiled by the screen in vitro and in vivo, so as to detect the new therapeutic targets for DIPG.

弥漫内生型脑干胶质瘤（DIPG）是一类预后极差的儿童中枢神经系统恶性肿瘤，其发病机制不同于大脑半球胶质母细胞瘤，目前尚无有效的治疗手段。在携带ACVR1特异性突变的DIPG细胞中BMP信号通路持续激活，起到了肿瘤促进作用；我们在预实验中发现ACVR1野生型的DIPG细胞中未发现BMP通路的激活，应用BMP4处理细胞，模拟ACVR1突变激活该通路后既没有促进肿瘤生长，又没有造成肿瘤干细胞向星形细胞分化，而是导致了肿瘤细胞凋亡。细胞中某两个基因如对其单独进行干预细胞维持存活状态，而两个基因同时受到干预则造成细胞凋亡的现象被称为协同致死效应。本研究提出了BMP信号通路通过对下游关键靶基因的转录调控导致协同致死效应的假说，拟应用CRISPR-Cas9筛选技术在BMP信号通路下游寻找导致协同致死效应的关键靶基因，并在体内及体外验证其作用机制，以期为DIPG的靶向治疗寻找到新的治疗靶点。

弥散内生型桥脑胶质瘤 (DIPG)是一类预后极差的儿童中枢神经系统恶性胂瘤，其发病机制不同于大脑半球胶质母细胞瘤，目前尚无有效的治疗手段。在携带ACVR1特异性突变的DIPG细胞中BMP信号通路持续激活，起到了肿瘤促进作用；而多数患者为ACVR1野生型，H3.3K27M突变型DIPG。本项目通过转录组分析、表观遗传分析和基础实验验证，首次系统性地研究了BMP信号通路在ACVR1野生型，H3.3K27M突变型DIPG中的抑癌作用机制，探索了针对该机制治疗DIPG的新型药物Palbociclib、Idasanutlin和Dacinostat的可行性；通过单细胞测序鉴定并实验验证了在BMP信号通路下游所调控基因中导致DIPG协同致死效应的关键基因HMGB2，针对该类基因的靶向治疗及药物联用有望成为DIPG治疗的新手段。为未来推动上述药物进入临床试验，为这类严重威胁儿童健康的恶性肿瘤寻找到有效的治疗方法奠定基础，推动DIPG治疗的发展。

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