Background: High-risk neuroblastoma patients often respond to therapy before relapsing with lethal multidrug-resistant disease. We propose loss of ER-mitochondria physical tethering, selected for during therapy, contributes to drug resistance.
Methods: We studied isogenic neuroblastoma cell line pairs derived from the same patients both at diagnosis (DX) and progressive disease on/after chemotherapy (PD). Tumor-isolated mitochondria were studied for functional responses to death stimuli (BH3 profiling), biomass (citrate synthase activity), and mtDNA (content by qPCR; sequence by MitoChip v2.0). Electron microscopy (EM) was used to quantify ER-mitochondria morphology and tethering. The impact of chemical and genetic manipulation of tethering on mitochondrial response and chemosensitivity was assessed.
Results: DX/PD pairs showed no changes in mitochondrial biomass or mtDNA. However, 7/7 pairs showed markedly attenuated cytochrome c release at relapse in response to tBid and BimBH3 peptides (terminal death effectors downstream of therapeutic stress), corresponding to relative chemotherapy resistance (up to 800-fold across diverse agent classes). Marked reductions in ER-mitochondria tethering (up to 70%) were seen by EM in post-relapse tumors. Attenuated mitochondrial responses were phenocopied by limited protelolysis of ER from mitochondria in DX (sensitive) lines, as well as by enforced de-tethering using a cyclophilin-D inhibitor; extent of mitochondrial attenuation was proportional to chemoresistance. A similar phenotype was observed in ALK-mutant cells selected for ALK-inhibitor resistance, leading to mitochondrial attenuation and broad therapy resistance.
Conclusions: We identify a novel contributor to multidrug resistance attributable to attenuated mitochondrial stress-responses. Our data support this attenuation derives from reduced ER-mitochondrial tethering with consequent loss of ER-derived signals required for mitochondrial competency. This mechanism is not exclusive to any other contributor to therapeutic resistance and may also contribute to resistance to targeted agents. Altered ER-mitochondria tethering has been linked to neurodegeneration and diabetes suggesting regulation of this inter-organelle contact may have broad relevance to human disease.