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

Systematic, network-based characterization of therapeutic target inhibitors.

A large fraction of the proteins that are being identified as key tumor dependencies represent poor pharmacological targets or lack clinically-relevant small-molecule inhibitors. Availability of fully generalizable approaches for the systematic and efficient prioritization of tumor-context specific protein activity inhibitors would thus have significant translational value. Unfortunately, inhibitor effects on protein activity cannot be directly measured in systematic and proteome-wide fashion by conventional biochemical assays. We introduce OncoLead, a novel network based approach for the systematic prioritization of candidate inhibitors for arbitrary targets of therapeutic interest. In vitro and in vivo validation confirmed that OncoLead analysis can recapitulate known inhibitors as well as prioritize novel, context-specific inhibitors of difficult targets, such as MYC and STAT3. We used OncoLead to generate the first unbiased drug/regulator interaction map, representing compounds modulating the activity of cancer-relevant transcription factors, with potential in precision medicine

Comparison of CMoA, GES and GI₅₀ profile similarities.

<p>(A) Venn diagram of the top 206 similar compound pairs (top 2.5%) using DTPA, DTGE and GI₅₀ sensitivity profiles. (B) top: Enrichment of the top 206 pairs based on DTPA similarity in the vector of 8256 (129*128/2) compound pairs ranked by DTGE similarity, and vice versa; middle: Enrichment of the top 206 pairs based on DTPA similarity in the vector of 8256 (129*128/2) compound pairs ranked by GI₅₀ correlation, and vice versa; bottom: Enrichment plot of the top 206 pairs using DTGE similarity in the vector of 8256 (129*128/2) compound pairs ranked by GI₅₀ correlation, and vice versa.</p

OncoLead: Network-based protein activity inference.

<p>(A) Drug perturbation induced genome-wide transcriptional changes are interpreted, based on multiple networks including ARACNE network, CHEA network, STRING network, and Gene knock-down (KD) network, with the VIPER algorithm, to infer changes in the activities of the regulatory proteins. The resulting four different protein activity matrixes were integrated into a single final protein activity matrix. In this way, VIPER analysis transforms drug-perturbation gene expression signatures into unbiased genome-wide regulator protein activity representations of CMoA. The left part is a simple illustration of how VIPER algorithm works based on the ARACNE network. First, ARACNE reverse engineers context-specific regulatory networks by leveraging a large collection of gene expression profiles (N > 100) from the same cellular context. Then, regulator’s activity is inferred by computing the enrichment of the genes in its regulon (from ARACNE) in every drug treatment signature sorted from the most over-expressed (colored in orange) to the most under-expressed (colored in green) genes. When there is positive or negative enrichment, the regulator is up-regulated or down-regulated (colored in red/blue). Regulator’s activity is represented by the normalized enrichment score. (B) IRS score decreases when progressively degrading the networks for MCF7 drug signatures. (C) Relative representation of how accurate each interactome is as a model for the transcriptional regulation in each of the three cell lines MCF7, PC3, and HL60 in CMAP database. Shown is the IRS in relative units for TRs inferred by OncoLead on each interactome (x-axis) / GES combination (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005599#sec013" target="_blank">Methods</a> for details). Percent IRS scores were obtained by dividing each specific IRS score by the largest score obtained across the three interactomes used in the analysis. (D) Distribution of the significant TRs inferred by OncoLead when adding increasing ratios of random noises to the Irinotecan signature in MCF7 cell line.</p

Reactive oxygen species, oxidative stress, and cell death correlate with level of CoQ10 deficiency

Coenzyme Q10 (CoQ10) is essential for electron transport in the mitochondrial respiratory chain and antioxidant defense. The relative importance of respiratory chain defects, ROS production, and apoptosis in the pathogenesis of CoQ10 deficiency is unknown. We determined previously that severe CoQ10 deficiency in cultured skin fibroblasts harboring COQ2 and PDSS2 mutations produces divergent alterations of bioenergetics and oxidative stress. Here, to better understand the pathogenesis of CoQ10 deficiency, we have characterized the effects of varying severities of CoQ10 deficiency on ROS production and mitochondrial bioenergetics in cells harboring genetic defects of CoQ10 biosynthesis. Levels of CoQ10 seem to correlate with ROS production; 10–15% and >60% residual CoQ10 are not associated with significant ROS production, whereas 30–50% residual CoQ10 is accompanied by increased ROS production and cell death. Our results confirm that varying degrees of CoQ10 deficiency cause variable defects of ATP synthesis and oxidative stress. These findings may lead to more rational therapeutic strategies for CoQ10 deficiency.—Quinzii, C. M., López, L. C., Gilkerson, R. W., Dorado, B., Coku, J., Naini, A. B., Lagier-Tourenne, C., Schuelke, M., Salviati, L., Carrozzo, R., Santorelli, F., Rahman, S., Tazir, M., Koenig, M., DiMauro, S., Hirano, M. Reactive oxygen species, oxidative stress, and cell death correlate with level of CoQ10 deficiency

Molecular basis of infantile reversible cytochrome c oxidase deficiency myopathy.

Childhood-onset mitochondrial encephalomyopathies are usually severe, relentlessly progressive conditions that have a fatal outcome. However, a puzzling infantile disorder, long known as 'benign cytochrome c oxidase deficiency myopathy' is an exception because it shows spontaneous recovery if infants survive the first months of life. Current investigations cannot distinguish those with a good prognosis from those with terminal disease, making it very difficult to decide when to continue intensive supportive care. Here we define the principal molecular basis of the disorder by identifying a maternally inherited, homoplasmic m.14674T>C mt-tRNA(Glu) mutation in 17 patients from 12 families. Our results provide functional evidence for the pathogenicity of the mutation and show that tissue-specific mechanisms downstream of tRNA(Glu) may explain the spontaneous recovery. This study provides the rationale for a simple genetic test to identify infants with mitochondrial myopathy and good prognosis