Abstract 580: Increased DNA repair capacities and p53/MDM2 pathway aberrations hallmark neuroblastoma cell lines with the alternative lengthening of telomeres (ALT) phenotype.

Abstract

Abstract Introduction: Neuroblastoma (NB) is a malignant pediatric tumor of the sympathetic nervous system. Approximately 10-20% of neuroblastoma tumors maintain telomere length by activating a telomerase-independent alternative lengthening of telomeres (ALT) mechanism, and that these tumors are associated with high-stage disease, and a poor prognosis. Methods: mRNA expression of TERT (the catalytic component of telomerase) and TERC (RNA template), determination of MYCN copy number, and telomerase activity (TA) was measured by RT-PCR. Telomere content (TC) and C-Circle (CC) content was assayed by quantitative PCR, p53 function was assessed by quantifying induction of p21 protein by flow cytometry after irradiation. Expression profiling was carried out with custom-designed Taq-Man Low Density Arrays (TLDAs) (ABI) that quantified expression of 61 DNA-repair genes in a panel of 12 NB cell lines, 4 ALT and 8 telomerase-positive. Of the telomerase-positive lines, 2 were drug-sensitive, 6 multi-drug resistant. Cell line identities were confirmed using short tandem repeat (STR) genotyping. Response to cytotoxic drugs was assessed using the DIMSCAN fluorescence imaging system. ATRX protein expression was measured with immunoblots. Results: From a panel of 40 human NB cell lines we identified 4 ALT NB lines, LA-N-6, SK-N-FI, CHLA-90, and COG-N-291, that had elevated TC > 2-fold that of TA+ NB cell lines (p<.005), two of which demonstrated telomere repeat C-circles, and all lacked MYCN genomic amplification. All 4 ALT+ NB cell lines maintain telomere length with significantly low levels of telomerase activity (p<.005) and mRNA expression of TERT relative to telomerase-positive NB cell lines; 3 of the 4 ALT lines lacked p53 function and LA-N-6, the sole p53-functional ALT line carries a homozygous deletion of p14(ARF). ALT lines had a significant increase (p<.05) in the expression of 28 of the 61 DNA-repair genes analyzed by TLDA relative to telomerase-positive lines (including those that manifest multidrug resistance); 10 of these genes are involved in the nucleotide excision repair pathway. All 4 ALT cell lines showed a high degree of multi-drug resistance, with the mean concentration cytotoxic to 90% of the ALT cells (IC90) being higher than clinically achievable plasma levels for melphalan (2-fold), etoposide (4-fold), topotecan (5-fold), and carboplatin (4-fold). CHLA-90 had undetectable ATRX protein levels, whereas the other three ALT+ NB cell lines expressed ATRX. Conclusions: ALT-based telomere maintenance in NB cell lines was associated with loss of p53 function, increased expression of DNA-repair genes, and resistance to DNA-damaging chemotherapy. Loss of ATRX expression is not essential for ALT to develop in NB. Novel, p53-independent therapies should be considered to treat neuroblastomas harboring the ALT phenotype. Citation Format: Ahsan Farooqi, Ashly Hindle, Balakrishna Koneru, Jerry Shay, Patrick Reynolds. Increased DNA repair capacities and p53/MDM2 pathway aberrations hallmark neuroblastoma cell lines with the alternative lengthening of telomeres (ALT) phenotype. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 580. doi:10.1158/1538-7445.AM2013-580