Generation of in vitro models of low grade gliomas

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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