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)的临床行为源于 未经治疗的转移性疾病导致肿瘤持续进展导致死亡，尽管是多种方式的密集 心理治疗。由于约50%的高危NB患者死于疾病，因此需要新的治疗方法。端粒是 真核细胞染色体末端的核蛋白复合体，通过保护 由于编码DNA序列的侵蚀而导致的染色体末端区域。癌细胞需要端粒 维持机制(TMM)和大多数激活端粒酶，一种含有逆转录酶的核糖核蛋白。 TERT基因编码的转录酶和TERC基因编码的RNA模板。一些癌症 端粒酶阴性并采用鲜为人知的交替延长端粒(ALT) 机制：染色体外端粒DNA c环是ALT的标志。表现为神经母细胞瘤 ALT通常在α地中海贫血/智力低下x连锁(ATRX)基因上有突变。我们建立了 一组显示ALT和我们的初步数据的NB细胞系和患者来源的异种移植(PDX) 表明ALT可以独立于ATRX突变而存在，且ALT与多种药物有关 抗性和DNA修复基因表达增加。与儿童肿瘤学合作 组(COG)：我们证实TERT mRNA高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... 神经母细胞瘤与ATRX突变、临床结果和通过RNA鉴定的基因表达特征的风险 测序。我们将确认我们的初步数据，表明ATM激酶在ALT NB中被激活，作为一种 ALT的驱动力，并刺激DNA修复相关基因的过度表达，促进对 化疗和放射治疗。这些数据将使TMM能够将高危NB分类为TERT-HIGH、ALT (TERT-低，c-环+)和越来越短的端粒(EST)表型(TERT-低，c-环阴性)和Will 能够确定TMM与临床结果的关系。我们将击倒、抑制或过度表达 在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，并对化疗和放射耐药。使用shRNA和AN ATM激酶抑制剂我们将在ALT NB细胞系和PDX中演示ATM激酶是分子 ALT神经母细胞瘤的治疗靶点。这个项目使用了最大的ALT细胞系和PDX面板 为任何癌症类型建立，并将为神经母细胞瘤定义新的生物标记物和治疗靶点。

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.