Hypermutation and malignant progression in an expanded cohort of TMZ treated LGG patients from different molecular subgroups

 描述（由申请人提供）：低级别胶质瘤（LGG）代表一组组织学和遗传学异质性的缓慢生长和侵袭性肿瘤，包括少突胶质细胞瘤（分子定义为1 p/19 q/IDH 1突变体）和星形细胞瘤（TP 53/ATRX/IDH 1突变体）。最流行的抗胶质瘤药物替莫唑胺（TMZ）延长了高级别胶质瘤（HGG）患者的总生存期，但可能导致LGG的遗传超突变（HM）和恶性进展。我们最近报道了TMZ治疗后10例复发性星形细胞瘤中有6例发生遗传性HM，所有超突变复发性进展为HGG，并在RB和AKT-mTOR通路中携带TMZ相关突变，所有这些都是HGG的驱动因素。我们最近扩大了TMZ治疗的LGG队列，包括来自24例星形细胞瘤和13例少突胶质细胞瘤的成对原发性和复发性肿瘤，从而可以更深入地调查两种不同LGG亚型的HM，肿瘤演变和恶性进展。我们的初步分析显示，13例TMZ治疗的低级别少突胶质细胞瘤中有7例在复发后发生超突变，大多数超突变复发进展为HGG，并在p53（常见于治疗前星形细胞瘤）和AKT-mTOR通路中携带TMZ相关突变。这些结果表明星形细胞瘤和少突胶质细胞瘤在TMZ诱导的HM和恶性进展后的遗传趋同。最初的途径分析产生染色质修饰剂作为HM在两种LGG亚型中共同的顶级基因类别，其中MLL甲基转移酶和SWI/SNF染色质重塑复合物是最常见的。这些家族中的每一个家族中的有害突变都是一系列癌症（包括HGG）的驱动因素。该提议的假设是1）TMZ诱导的超突变将最初不同的低级别星形细胞瘤和少突胶质细胞瘤推向恶性进展的会聚路径，以及2）会聚部分由染色质修饰基因中的复发突变介导。首先，在两种LGG亚型中与TMZ诱导的HM最相关的遗传途径或基因家族将从来自初始和复发性LGG肿瘤的外显子组突变调用确定。重要的是，将通过全面的途径富集分析来探索HM相关的候选肿瘤驱动途径。接下来，我将确定2个候选驱动突变（CDM）在染色质修饰基因中的功能作用（例如，MLL 3）通过在1 p/19 q/IDH 1突变型寡星形细胞瘤细胞中工程化CDM。CDM的结果将通过ChIP-seq检测CDM蛋白和CDM相关组蛋白标记（例如，H3 K4 me 1），以及随后通过RNA-seq的基因组表达变化。最后，将在体内研究CDM对恶性进展的作用。这些功能研究有助于扩大我们对会聚LGG进化的理解，以及TMZ诱导HM背景下恶性进展的遗传和表观遗传驱动因素。

 DESCRIPTION (provided by applicant): Low-grade glioma (LGG) represents a histologically and genetically heterogeneous group of slow-growing and invasive tumors, including oligodendroglioma (molecularly defined as 1p/19q/IDH1 mutant) and astrocytoma (TP53/ATRX/IDH1 mutant). The most prevalent anti-glioma drug, temozolomide (TMZ), extends overall survival for high-grade glioma (HGG) patients, but can drive genetic hypermutation (HM) and malignant progression in LGG. We recently reported genetic HM in 6 of 10 recurrent astrocytomas upon TMZ treatment, with all hypermutant recurrences progressing to HGG and harboring TMZ-related mutations in the RB and AKT-mTOR pathways, all of which are drivers of HGG. We have recently expanded our TMZ-treated LGG cohort to include paired primary and recurrent tumors from 24 astrocytomas and 13 oligodendrogliomas, allowing a deeper survey of HM, tumor evolution, and malignant progression in two distinct LGG subtypes. Our preliminary analyses show that 7 of 13 TMZ-treated, initially low-grade oligodendrogliomas hypermutated upon recurrence, with the majority of hypermutant recurrences progressing to HGG and harboring TMZ-related mutations in the p53 (common in pretreatment astrocytoma) and AKT-mTOR pathways. These results suggest a genetic convergence of astrocytoma and oligodendroglioma upon TMZ-induced HM and malignant progression. Initial pathway analysis yields chromatin modifiers as a top-ranked gene class common to HM in both LGG subtypes, with MLL methyltransferases and the SWI/SNF chromatin remodeling complex being the most frequent. Deleterious mutations in each of these families are driving events in an array of cancers, including HGG. The hypothesis of this proposal is 1) TMZ-induced hypermutation pushes initially distinct low grade astrocytomas and oligodendrogliomas down a convergent path to malignant progression and 2) the convergence is mediated, in part, by recurrent mutations in chromatin modifier genes. First, genetic pathways or gene families most associated with TMZ-induced HM in both LGG subtypes will be determined from exome mutation calls from initial and recurrent LGG tumors. Importantly, HM- associated candidate tumor driver pathways will be explored by comprehensive pathway enrichment analysis. Next, I will determine the functional role of 2 candidate driver mutations (CDMs) in a chromatin modifier gene (eg. MLL3) by engineering CDMs in 1p/19q/IDH1 mutant oligoastrocytoma cells. The consequence of the CDMs will be tested by ChIP-seq for CDM protein and CDM-related histone marks (eg. H3K4me1), as well as subsequent genomic expression changes by RNA-seq. Finally, the role of the CDMs on malignant progression will be investigated in vivo. These functional studies serve to expand our understanding of convergent LGG evolution, as well as genetic and epigenetic drivers of malignant progression in the context of TMZ-induced HM.