Abstract A06: N-MYC regulates mitochondrial dynamics in neuroblastoma via direct interaction with Drp1 and LRRC59

Background: Neuroblastoma (NB) is the most common extracranial tumor of childhood (1), accounting for approximately 7% of all cancer in children (2). The morbidity and mortality are affected by a number of factors – perhaps most significantly by amplification of the oncogene, N-MYC. The N-MYC gene product (N-Myc) regulates the transcription of a vast array of genes, and when overexpressed alters the regulation of essential cellular processes such as: the cell cycle, ribosome biogenesis and apoptosis (3). Although pathways such as these are believed to be contributory to tumorigenesis, the precise manner in which amplification of N-Myc contributes to the generation and maintenance of NB has not been fully elucidated. Objective: Many of the so-called Hallmarks of Cancers are phenotypes that are influenced by the mitochondria (4). As other members of the Myc family have been shown to significantly impact mitochondrial structure and function, we have hypothesized that that overexpression of N-Myc would alter mitochondrial dynamics in NB and speculate that these changes may contribute to tumorigenesis. Design/Methods: To perform these studies we utilized a cell line with endogenous levels of N-Myc (SK-N-SH), in which we have ectopically overexpressed N-Myc and examined the cells for changes in the mitochondrial structure with electron and confocal microscopy. Decay of fluorescence of a photoactivatable mitochondrially targeted GFP was used to measure rates of mitochondrial fusion. Western blots were used to assess differences in protein expression in response to N-Myc overexpression. Finally, the BIOID assay (5,6) was used to screen for proteins that interacted with the mitochondrial fusion protein, Drp1 and protein-protein interactions were confirmed with immunoprecipitation. Results: We found a two-fold increase in mitochondrial length in NB cells that overexpress N-Myc, which corresponded to a more tubular and elongated appearance of the organelle. We found this to be a result of an estimated two-fold increase in mitochondrial fusion in response to N-Myc overexpression. While the expression of mitochondrial fusion and fission proteins were altered in response to N-Myc overexpression, most of these changes were modest. In contrast, there was a greater than six-fold inhibition of mitochondrially-targeted Drp1 in N-Myc overexpressing NB. This led us to screen for changes in the Drp1 interactome in response to N-Myc overexpression, which resulted in the identification of LRRC59, an endoplasmic reticulum (ER) membrane protein which we found to directly interact with both Drp1 and N-Myc. In fact, we demonstrated that LRRC59 played a major role in the targeting of Drp1 to the mitochondrial surface and subsequent initiation of mitochondrial fission. Overexpression of N-Myc appeared to inhibit the Drp1-LRRC59 interaction, thereby decreasing mitochondrial localization of Drp1 and subsequently reducing fission. Conclusions: N-Myc amplification in NB increased mitochondrial length as a result of an increase in mitochondrial fusion. While there may be some contribution from transcriptional regulation of the genes involved in mitochondrial dynamics, it appeared that the most significant result of N-Myc overexpression is mislocalization of the fission protein, Drp1. This inability of Drp1 to properly translocate to the mitochondrial surface appeared to be a result of a non-transcriptional effect of N-Myc -- physically inhibiting the interaction of Drp1 with a newly identified protein partner, LRRC59 -- at mitochondrial-ER junctions. This novel mechanism of N-Myc is believed to be the primary mechanism underlying the appearance of elongated mitochondria in N-Myc overexpressing NB and we speculate that these changes are significant in the pathology of these tumors. REFERENCES 1. Siegel, R., Naishadham, D. & Jemal, A. CA: a cancer journal for clinicians 63, 11–30 (2013). 2. Park, J. R., Eggert, A. & Caron, H. Hematol. Oncol. Clin. North Am. 24, 65–86 (2010). 3. Bell, E. et al. Cancer Lett 293, 144–157 (2010). 4. Galluzzi, L. et al. Molecular Aspects of Medicine 31, 1–20 (2010) 5. Roux, K. J., Kim, D. I., Raida, M. & Burke, B. J Cell Biol 196, 801–810 (2012). 6. Roux, K. J., Kim, D. I. & Burke, B. Curr Protoc Protein Sci 74, 19.23.1–19.23.14 (2013) Citation Format: G. Casinelli, S. Banerjee, M. Sharma, S. Sims-Lucas, D. Stolz, J. A. Graves. N-MYC regulates mitochondrial dynamics in neuroblastoma via direct interaction with Drp1 and LRRC59. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A06.