Generation of in vitro models of low grade gliomas

弥漫性浸润性低级别胶质瘤是WHO分级为2级的肿瘤，包括少突胶质细胞瘤和星形细胞瘤。它们约占所有原发脑瘤的5%，占所有胶质瘤的15%。尽管低级别胶质瘤(LGG)最初具有惰性，但可能会引起相当大的发病率1，并经常转变为高级别的致命性实体2。LGG的主要治疗方法是手术，其次是放疗和化疗3。然而，哪些患者最有可能从术后辅助治疗中受益尚不清楚，尽管部署了所有当前的治疗方案，但复发几乎是普遍的。因此，迫切需要开发新的治疗方法来治疗LGGs。这些努力一直受到缺乏强大的临床前模型的阻碍，这些模型具有疾病相关的基因类型，允许询问治疗靶点和转化因子4。少突胶质细胞瘤的分子定义为异柠檬酸脱氢酶(IDH1和IDH2)的两个基因中的任何一个存在突变，以及完整的1p和19q染色体臂5的共同缺失。IDH突变被认为与G-CIMP甲基化表型有关，这通常包括O6-甲基鸟嘌呤-DNA-甲基转移酶(MGMT)基因6的启动子的甲基化。具有MGMT启动子甲基化的胶质瘤对烷化剂替莫唑胺(TMZ)7更敏感。当2级少突胶质瘤转化为高级别肿瘤时，它们被归类为3级间变性少突胶质瘤(AO)。2级星形细胞瘤也以IDH1突变为特征，但不表现出1p/19q染色体丢失5.此外，这些肿瘤通常以失活的TP53和ATRX病变为靶点，85%的IDH1突变/1p19q非共缺失星形细胞瘤具有高甲基化的MGMT启动子。8当2级星形细胞瘤转化时，它们可以成为3级间变性星形细胞瘤(AA)或4级胶质母细胞瘤(GBM)。有趣的是，在部分AOS和AAS中都发现了针对PI3K催化或调节亚基基因(分别为PIK3CA和PIK3R1)的致癌突变，或针对致癌转录因子MYC或MYCN的基因扩增，表明这些损伤可能参与了LGG8-11的恶性转化。此外，具有类似病变的其他实体肿瘤(即PI3K或MYC)已被证明对PI3K途径抑制剂12、13敏感，但脑穿透性PI3K途径抑制剂很少。拟议的PHD项目将专注于使用hTERT永生化的少突胶质细胞和星形胶质细胞产生新的基因定义的星形细胞瘤和少突胶质细胞瘤的体外模型。通过CRISPR/Cas9技术，永生化的人类少突胶质细胞将被设计成具有明确的遗传损伤，包括1p/19q共缺失和IDH1突变。为了评估激活PI3K损伤的转化能力，这些细胞将被与疾病相关的PIK3CA和PIK3R1突变体或MYC/MYCN过表达构建物稳定地转导。同样，永生化的人类星形胶质细胞将被改造成IDH1和TP53突变，以及ATRX基因失活(图1)。这些模型将被评估其增长潜力，并作为药物筛选的平台进行评估。这项拟议的研究重点在于建立建立可靠的低级别胶质瘤体外模型的可行性，该模型也可以用作治疗靶点筛选的平台。此外，这些基因定义的模型将被用来询问PI3K抑制剂的治疗潜力。学生将利用1)先进的细胞工程技术重建常见的遗传损伤，以及2)生物化学和分子图谱方法来了解这些损伤对恶性转化的影响。

Diffuse infiltrating low-grade gliomas are WHO grade 2 tumors that include oligodendrogliomas and astrocytomas. They account for approximately 5% of all primary brain tumours and 15% of all gliomas. Despite their initially indolent nature, low-grade gliomas (LGGs) may cause considerable morbidity1 and frequently transform into higher grade lethal entities 2. The mainstay of treatment for LGGs is surgery, which can be followed by radiotherapy with chemotherapy3. However, which patients are most likely to benefit from postsurgical adjuvant treatment is not clear, and recurrence is nearly universal despite deployment of all current treatment options. Consequently, there is an urgent need to develop novel therapeutics for the treatment of LGGs. These efforts have been hampered by the paucity of robust pre-clinical models with disease-relevant genotypes that allow the interrogation of therapeutic targets and transformation factors 4. Oligodendrogliomas are molecularly defined by the presence of mutations in either of the two genes for isocitrate dehydrogenase (IDH1 and IDH2) and co-deletion of whole 1p and 19q chromosomal arms 5. IDH mutations, in turn, are thought to be causally associated with the G-CIMP methylation phenotype which usually includes methylation of the promoter for the O6-methylguanine-DNA-methyltransferase (MGMT) gene 6. Importantly, gliomas with MGMT promoter methylation have been demonstrated to be more sensitive to the alkylating agent temozolomide (TMZ) 7. When grade 2 oligodendrogliomas transform to higher-grade tumors, they are categorized as grade 3 anaplastic oligodendrogliomas (AO). Grade 2 astrocytomas are also characterised by IDH1 mutations, but do not exhibit 1p/19q chromosomal losses 5. Additionally, these tumours are generally targeted by inactivating TP53 and ATRX lesions, and 85% of IDH1-mutant/1p19q non-co-deleted astrocytomas have a hypermethylated MGMT promoter.8 When grade 2 astrocytomas transform, they can become either grade 3 anaplastic astrocytomas (AA) or grade 4 glioblastomas (GBMs). Interestingly, oncogenic mutations that target the genes for the catalytic or regulatory subunits of PI3K (PIK3CA and PIK3R1, respectively) or gene amplification of the oncogenic transcription factors MYC or MYCN have been identified in a fraction of both AOs and AAs, suggesting that these lesions may be involved in malignant transformation of LGGs 8-11. Furthermore, other solid tumours with similar lesions (i.e. PI3K or MYC) have been shown to be sensitive to PI3K pathway inhibitors 12, 13, but brain-penetrant PI3K pathway inhibitors are scarce. The proposed PhD project will focus on generating novel in vitro models of genetically-defined astrocytomas and oligodendrogliomas using hTERT-immortalised oligodendrocytes and astrocytes. Through CRISPR/Cas9 technology, immortalised human oligodendrocytes will be engineered with defining genetic lesions including 1p/19q co-deletion and IDH1 mutation. To assess the transforming ability of activating PI3K lesions, these cells will be stably transduced with disease-relevant PIK3CA and PIK3R1 mutants, or with MYC/MYCN overexpression constructs. Similarly, immortalised human astrocytes will be engineered with IDH1 and TP53 mutations, as well as ATRX gene inactivation (Fig 1). These models will be assessed for their growth potential and evaluated as platforms for drug screens. The proposed research focuses on establishing the feasibility of generating faithful in vitro models of low grade gliomas that can also be used as platforms for therapeutic target identification screens. Additionally, these genetically-defined models will be used to interrogate the therapeutic potential of PI3K inhibitors. The student will make use of 1) advanced cell engineering techniques to recreate common genetic lesions, and 2) both biochemical and molecular profiling approaches to understand the impact of these lesions on malignant transformat