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.
PROJECT NARATIVE Neuroblastoma is childhood cancer that can spontaneously regress without therapy or relentlessly progress in spite of intensive chemotherapy. For continual cell growth cancer cells must maintain the ends of chromosomes (called telomeres) which erode if not maintained by telomere maintenance mechanisms (TMM). We will study TMM in neuroblastoma to develop novel biomarkers to aid in defining prognosis for patients and to develop novel therapeutic targets.
