Generation of in vitro models of low grade gliomas

低级别胶质瘤体外模型的生成

• 批准号: 2886769
• 金额: --
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2886769
• 关键词: Generation; vitro; models; low; grade

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

弥漫性浸润低级神经胶质瘤是其中2级肿瘤，其中包括少突胶质瘤和星形胶质细胞瘤。它们约占所有原发性脑肿瘤的5％，占所有神经胶质瘤的15％。尽管最初具有懒惰的性质，但低级神经胶质瘤（LGGS）可能会引起相当大的发病率1，并经常转化为更高级的致命实体2。LGGS治疗的主要是手术，随后可以进行化学疗法进行放疗3。但是，哪些患者最有可能受益于术后辅助治疗，尽管部署了所有当前治疗选择，但复发几乎是普遍的。因此，迫切需要开发新型的LGG治疗方法。这些努力受到了与疾病相关的基因型的差异，允许对治疗靶标的询问和转化因子的询问4.寡聚胶质瘤的分子是由两种基因中的两种基因的突变来定义的，这些基因在异位酸酯脱氢酶（IDH1和IDH1和IDH1）中的两种基因中都存在分子定义，从而使寡胶质胶质瘤（IDH1和IDH1和CODH2）均具有分子定义，从而阻碍了这些努力。 5。反过来，IDH突变被认为与G-CIMP甲基化表型有因果关系，通常包括O6-甲基鸟嘌呤-DNA-DNA-DNA-甲基转移酶（MGMT）Gene 6的启动子的甲基化6。少突胶质瘤转化为高级肿瘤，它们被归类为3级变性性寡头胶质瘤（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变换，它们可以成为3级构型星形胶质细胞瘤（AA）或4级胶质母细胞瘤（GBMS）。有趣的是，针对PI3K（分别PIK3CA和PIK3R1）的催化或调节亚基的基因的致癌突变，或在AAS和AAS的一部分中鉴定出了致癌转录因子MYC或MYCN的基因扩增，这表明这些病态可能涉及恶意转化。此外，其他具有相似病变的实体瘤（即PI3K或MYC）已被证明对PI3K途径抑制剂12、13敏感，但脑培训PI3K途径抑制剂很少。拟议的博士学位项目将专注于使用HTERT-IMMORTALALISE的少突胶质细胞和星形胶质细胞生成遗传定义的星形胶质细胞瘤和少突胶质瘤的新型体外模型。通过CRISPR/CAS9技术，永生的人类少突胶质细胞将通过定义的遗传病变进行设计，包括1P/19Q共脱落和IDH1突变。为了评估激活PI3K病变的转化能力，这些细胞将通过疾病相关的PIK3CA和PIK3R1突变体或MYC/MYCN过表达构建体稳定转导。同样，永生的人类星形细胞将通过IDH1和TP53突变以及ATRX基因失活进行设计（图1）。这些模型将根据其增长潜力进行评估，并评估为药物筛查的平台。拟议的研究重点是建立生成低级神经胶质瘤体外模型的可行性，这些模型也可以用作治疗目标识别筛查的平台。此外，这些遗传定义的模型将用于询问PI3K抑制剂的治疗潜力。学生将利用1）先进的细胞工程技术来重现常见的遗传病变，以及2）生化和分子分析方法，以了解这些病变对恶性转化的影响