Abstract 2459: Chemotherapy resistance in pediatric neuroblastoma is associated with reduced ER-mitochondria tethering
Authors: Coku Jorida , Scadden Elizabeth O. , Liu Kangning , Vu Annette , Booth David M. , Chen Michelle , Kim Sharon , Reynolds C. Patrick , Hajnóczky György , Hogarty Michael D. 
Published: July 2016

Development of multidrug resistance poses a persistent problem in cancer treatment, yet its underlying causes remain obscure. A principal role for mitochondria has been sought as this organelle integrates diverse stress signals to impact cell fate. Endoplasmic reticulum (ER) and mitochondria (mito) interact at specialized coupling sites called mitochondria-associated ER membranes (MAMs). MAMs serve as micro-domains for the transfer of essential calcium and lipid signals to mitochondria and regulate apoptotic sensitivity. Tightening of ER-mito tethering constitutes an early response to cellular stress leading to apoptosis, and alterations in ER-mito tethering have been implicated in diabetes and neurodegeneration, suggesting that the deregulation of this process may have broad relevance in disease. Here, we define a novel mechanism for chemotherapy resistance due to selection for reduced ER-mito tethering. Most high-risk neuroblastoma patients initially respond to chemotherapy before relapsing with lethal therapy resistant disease acquired during the course of intensive multimodality treatment. We obtained isogenic neuroblastoma cell lines from the same 7 patients both at the time of diagnosis (chemosensitive) and at the time of relapse (chemoresistant). We evaluated mitochondrial biomass (citrate synthase activity), mtDNA content (qPCR), and mtDNA sequence (Affymetrix MitoChip v2.0) but identified no changes correlated with acquired resistance. Electron microscopy image analyses of ER-mito interfaces revealed that tumors at relapse contain up to 70% fewer ER-mito tether complexes than their matched at-diagnosis tumors, as confirmed by IB for organelle-specific proteins. Mitochondria isolated from all 7 post-relapse tumors show attenuated cytochrome c release in response to tBid and Bim BH3 peptide, terminal death effectors downstream of most therapeutic stress. This attenuated mitochondrial response can be phenocopied by limited protelolysis of mitochondria to reduce ER-mito tethers. Reduced mitochondrial apoptotic signaling in post-relapse tumors correlates directly with chemoresistance (up to 800-fold) across diverse agent classes. To functionally validate this relationship, Cyclosporine A (CsA), a cyclophilin D inhibitor, was used in tumors at diagnosis to reduce ER-mito tethering. This led to attenuated apoptotic responses in isolated mitochondria, and increased tumor cell IC50 to diverse chemotherapeutics, partially phenocopying the therapy resistant state. Our findings support a novel model of differential apoptotic signaling in therapy resistant cells that relies on the altered proximity and interactions of the mitochondria with ER that may be harnessed to design more effective anti-cancer drug therapies.

Citation Format: Jorida Coku, Elizabeth O. Scadden, Kangning Liu, Annette Vu, David M. Booth, Michelle Chen, Sharon Kim, C. Patrick Reynolds, György Hajnóczky, Michael D. Hogarty. Chemotherapy resistance in pediatric neuroblastoma is associated with reduced ER-mitochondria tethering. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2459.