Elucidation Of Cancer Therapy Resistance Mechanisms Due To Altered Endoplasmic Reticulum-Mitochondria Tethering

Many neuroblastoma patients die from progression of multidrug resistant disease, the etiology of which remains poorly understood. Mitochondria (mito) integrate diverse stress and survival signals to determine whether a cell lives or dies. Importantly, apoptotic sensitivity is modulated at mitochondria by interactions with endoplasmic reticulum (ER) at specialized contact sites essential for calcium (Ca2+) and lipid transfer between the organelles. ER-mitochondria contact sites (ERMCs) are enriched for protein complexes, including MFN2 and PACS2, that bridge the organelles. Here, I define a novel mechanism for chemotherapy resistance caused by reductions in ERMC tethers and provide functional validation for this relationship. I studied neuroblastomas from diagnosis (DX, largely chemosensitive) and relapse (REL, chemoresistant) obtained from the same patients. Functional mitochondrial profiling showed that REL neuroblastoma mitochondria are markedly reduced in apoptotic responses to stress and this correlates with chemoresistance across drug classes. These differences are highly reproducible and were not caused by changes in mitochondria biomass or mtDNA content. Instead, REL cells show reduced ERMC numbers and/or increased gap-distance compared with patient-matched DX cells. The impact of reduced ERMC connectivity was confirmed using multiple orthogonal methods. MFN2 or PACS2 silencing in DX cells attenuated mitochondrial responses, phenocopied resistance and reduced ERMC tethering. As a consequence, ERMC Ca2+ transfer was decreased in REL cells. On the other hand, enhancing ERMC connectivity using synthetic linkers restored Ca2+ transfer. ERMCs serve as physiologic regulators of apoptosis, and I show in patient-matched tumor models that these contacts are markedly reduced in therapy resistant cells. Some resistant cells had reduced numbers of ERMC tethers (and reduced Ca2+ transfer), while other resistant cells had preserved numbers of tethers (and preserved Ca2+ transfer), but an abnormally increased gap distance. The outcomes of this work reveal the contributions of a “socially distanced ER-mito phenotype” to cancer therapy resistance, a novel model for the development of clinical tools to measure ERMC interactions, and the identification of therapeutic opportunities to revert resistance