Abstract A26: MYCN modifies p53 transcriptional responses to oppose apoptosis and activate cell cycle progression
Authors: Agarwal Saurabh , Rajapakshe Kimal , Coarfa Cristian , Shohet Jason 
Published: May 2015

Introduction: The MYCN transcription factor plays a central role in neuroblastoma pathogenesis. This aggressive pediatric malignancy is p53 wild type and repression of p53 functions is required for tumorigenesis. MYC family transcription factors activate and repress a larger number of genes involved in cell proliferation, metastasis and tumorigenesis through direct and indirect mechanisms. We hypothesized that MYCN promotes NB tumorigenesis through repression of p53 transcriptional function.

Design: To define MYCN mediated alterations in p53 activity, we performed RNA sequencing on NB cells treated with the MDM2 inhibitor, Nutlin 3a, in the presence or absence of MYCN expression (conditional Tet-on system). MYCN3 cells were either uninduced or induced with doxycycline for 16 h followed by treatment for 8 hours with either Nutlin 3a or control. Total RNA was isolated and used for RNA-seq on the HiSeqTM platform (Illumina).

Results: Between 6.2 × 107 to 7.6 × 107 sequence tags were obtained for each condition which uniquely mapped onto the human genome reference sequence (HG19) using the PASH algorithm. We normalized the data sets using a genomic distribution (FDR &lt; 0.001). Next, determined fold changes in gene expression in response to p53 activation with high or low MYCN levels using stringent cutoffs of FKM ≥ 1 and log fold change ≥ 1.3. We found total 103 genes whose transcription was increased in response to Nutlin treatment and 161 genes whose transcription was repressed (p value of &lt; 0.05). Comparison of p53 target gene transcript levels under these four conditions (low MYCN / low p53, low MYCN / high p53, high MYCN / low p53 and high MCYN / high p53) revealed that MYCN levels altered the transcriptional response to p53 activation with Nutlin. MYCN inhibited expression of 25 genes activated by p53 and promoted expression of 30 genes repressed by p53 (log fold change cutoff of ≤ 0.5 with a p value of &lt; 0.01). Bioinformatic analysis using DAVID and GSEA further demonstrated that p53 response genes, altered by MYCN, are primarily involved in apoptotic pathways, cell cycle, and mitotic regulation. Next, we used MYCN ChIP-seq and p53 ChIP-seq datasets to evaluate gene promoters for presence of MYCN binding and p53 binding. Remarkably, we found that the promoters of p53 activated genes which were repressed by the presence of MYCN consistently bound both transcription factors, while the p53 repressed genes bound only MYCN and didn't display p53 binding. We have confirmed these ChIP-seq results with ChIP-qPCR for a subset of genes with central roles in tumor suppression and tumorigenicity (e.g. CDKN1A, CHEK1, AEN, MDM2, Notch1). In addition, a subset of these MYCN modulated p53 transcriptional targets is over expressed in tumors from patients with high-risk disease.

Conclusions: Over all these data provide the first unbiased, genome-wide evaluation of how MYCN can modify p53 transcriptional function. Our data suggest a mechanism of direct interactions between MYCN and p53 on the promoters of p53 transcriptional targets, acting to inhibit p53 pro-apoptotic pathways. These data identify key components of the p53 transcriptional response altered by MYCN (i.e. apoptosis, mitotic checkpoints and cell cycle) and we propose that reactivation of elements provides a novel therapeutic strategy.

Citation Format: Saurabh Agarwal, Kimal Rajapakshe, Cristian Coarfa, Jason Shohet. MYCN modifies p53 transcriptional responses to oppose apoptosis and activate cell cycle progression. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A26.