Alternate telomere maintenance mechanisms in high-risk neuroblastoma as prognostic indicators and therapeutic targets

摘要 儿童癌症神经母细胞瘤（NB）的临床行为从自发消退延伸到自发消退。 未经治疗的转移性疾病到无情的肿瘤进展导致死亡，尽管多模态密集 疗法需要新的治疗方法，因为约50%的高风险NB患者死于疾病。端粒是 真核细胞染色体末端的核蛋白复合物，通过保护 染色体的末端区域免受DNA编码序列的侵蚀。癌细胞需要端粒 维持机制（TMM）和大多数激活端粒酶，一种核糖核蛋白，含有反向- 在一个实施方案中，所述方法包括使用由TERT基因编码的转录酶和由TERC基因编码的RNA模板。某些癌症 是端粒酶阴性的，并采用了知之甚少的端粒（ALT）的交替延长 染色体外端粒DNA c环是ALT的标志物。神经母细胞瘤表现为 ALT通常在α地中海贫血/X连锁精神发育迟滞（ATRX）基因中发生突变。我们建立 一组表现ALT的NB细胞系和患者来源的异种移植物（PDX）和我们的初步数据 表明ALT可以独立于ATRX突变而存在，且ALT与多种药物相关 抗性和DNA修复基因表达增加。与儿童肿瘤科合作 COG组的研究表明，高TERTmRNA表达与疾病风险相关， 低风险神经母细胞瘤的进展。我们将根据端粒维持机制对高危NB进行分类 (TMM)通过评估端粒酶表达、端粒含量和c环，并将这些与原发性高血压相关， 神经母细胞瘤对ATRX突变的风险、临床结果和通过RNA鉴定的基因表达特征 测序我们将证实我们的初步数据，表明ATM激酶在ALT NB中被激活，作为一种免疫调节剂。 ALT驱动因子，并刺激参与DNA修复的基因过表达，促进对 化疗和放疗。这些数据将使TMM能够将高风险NB分类为高TERT、ALT （TERT-低，c-circle+）和更短的端粒（EST）表型（TERT-低和c-circle阴性），并且将 能够确定TMM与临床结果的关系。我们会击倒，抑制，或过度表达 NB细胞系（ALT和非ALT）中的ATM激酶，以证明其在TMM和ALT表型中的作用， 驱动DNA修复基因的表达，以及对化疗和放疗的抗性。使用shRNA和一种 ATM激酶抑制剂我们将在ALT NB细胞系和PDX中证明ATM激酶是分子的， ALT神经母细胞瘤的治疗靶点。该项目采用了最大的ALT细胞系和PDX面板 它将为神经母细胞瘤定义新的生物标志物和治疗靶点。

ABSTRACT Clinical behavior of the childhood cancer neuroblastoma (NB) extends from spontaneous regression of untreated metastatic disease to relentless tumor progression causing death in spite of multi-modality intensive therapy. Novel therapies are needed as ~50% of high-risk NB patients die from disease. Telomeres are nucleoprotein complexes at eukaryotic chromosome ends that are essential for genomic stability by protecting the terminal regions of chromosomes from erosion of coding DNA sequences. Cancer cells require a telomere maintenance mechanism (TMM) and most activate telomerase, a ribonuclear protein containing a reverse- transcriptase encoded by the TERT gene and an RNA template encoded by the TERC gene. Some cancers are telomerase negative and employ a poorly understood alternate lengthening of telomeres (ALT) mechanism; extra-chromosomal telomeric DNA c-circles are a marker of ALT. Neuroblastomas that manifest ALT often have mutations in the alpha thalassemia/mental retardation x-linked (ATRX) gene. We established a panel of NB cell lines and patient-derived xenografts (PDXs) that manifest ALT and our preliminary data indicate that ALT can exist independent of ATRX mutations and that ALT is associated with multi-drug resistance and with increased expression of DNA repair genes. In collaboration with the Children's Oncology Group (COG) we demonstrated that high TERT mRNA expression was associated with risk of disease progression in low-risk neuroblastomas. We will classify high-risk NB by telomere maintenance mechanisms (TMM) by assessing telomerase expression, telomere content, and c-circles, and relate these in primary high- risk neuroblastomas to ATRX mutations, clinical outcome, and gene expression signatures identified by RNA sequencing. We will confirm our preliminary data indicating that ATM kinase is activated in ALT NB, serves as a driver of ALT, and stimulates overexpression of genes involved in DNA repair, promoting resistance to chemotherapy and radiation. These data will enable classification by TMM of high-risk NB into TERT-high, ALT (TERT-low, c-circle +), and an ever-shorter telomere (EST) phenotype (TERT-low and c-circle negative) and will enable determining the relationship of TMM to clinical outcome. We will knock-down, inhibit, or over-express ATM kinase in NB cell lines (ALT and non-ALT) to demonstrate its role in TMM and in the ALT phenotype, in driving expression of DNA repair genes, and in resistance to chemotherapy and radiation. Using shRNA and an ATM kinase inhibitor we will demonstrate in ALT NB cell lines and PDXs that ATM kinase is molecular therapeutic target in ALT neuroblastomas. This project employs the largest panel of ALT cell lines and PDXs established for any cancer type and will define novel biomarkers and therapeutic targets for neuroblastoma.