PO-461 Mitochondria-mediated anticancer effect of diphenyleneiodonium chloride (DPI) in aggressive neuroblastoma is regulated by MYCN

Abstract

IntroductionNeuroblastoma (NB) is the most common solid tumour in children under the age of five and it is responsible for 15% of paediatric cancer deaths. MYCN amplification represents the most frequent genetic alterations in high-risk NB and it is associated with poor prognosis.Our aim was to identify vulnerable, therapeutically targetable nodes that function as critical regulators or effectors of MYCN in neuroblastoma by using an integrative ‘-omics’ approach.Material and methodsWe used a panel of NB cell lines (SY5Y: non-amplified; NBLS: MYCN overexpression from a single gene copy; KCN, KCNR, Be2C: MYCN-amplified). Interaction proteomics was performed by immunoprecipitating MYCN protein complexes (Co-IP) followed by quantitative label-free mass spectrometry (Q-exactive and MaxQuant). Transcriptomic analysis of total RNA was performed by real-time PCR on an ABI Prism 7700 System (Applied Biosistems).Results and discussionsMYCN interaction proteomics revealed that MYCN is physically interacting with TFAM, a mitochondrial transcription factor. Therefore, we demonstrated that MYCN is present in the mitochondria of NB cells by Western blot and confocal microscopy (MYCN-Alexa488 and MitoTracker-Red-CMXRos staining), and that MYCN may repress or activate mitochondrial genes in cells rendered MYCN deficient by siRNA. Moreover, we found that certain mitochondrial genes are downregulated in patients with MYCN-amplified neuroblastoma, and they correlate with poor patient survival (Kaplan-Meier analysis of 709 patients). In addition, we performed a high throughput drug screening (~4000 compounds) and we found that diphenyleneiodonium chloride (DPI) inhibits the viability of MYCN-expressing NB cells. Interestingly, DPI significantly downregulated MYCN expression in the mitochondria of NB cells, and in turn, modulated mitochondrial gene expression. Moreover, DPI treatment resulted in a mitochondrial superoxide-mediated apoptosis in MYCN-amplified cells. In addition, soft agar colony formation assay demonstrated the tumour suppressive effects of DPI, and NB zebrafish models showed that treatment with DPI significantly reduced the neuroblastoma tumour size in vivo.ConclusionIn summary, we found a vulnerable node in the MYCN interactome and we demonstrated that DPI was able to reduce the MYCN level in the mitochondria of NB cells, to induce a ROS-mediated apoptosis in MYCN-amplified, and to reduce the size of the tumour in vivo. Therefore, DPI might serve as a potential novel drug to treat MYCN-amplified NB.