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miR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma

Abstract

Drug tolerance is an acute defence response preceding a fully drug-resistant state and tumour relapse; however, there are few therapeutic agents targeting drug tolerance in the clinic. Here we show that miR-147b initiates a reversible state of tolerance to the epidermal growth factor receptor (EGFR) inhibitor osimertinib in non-small-cell lung cancer. With miRNA-seq analysis, we find that miR-147b is the most upregulated microRNA in osimertinib-tolerant and EGFR-mutated lung cancer cells. Whole-transcriptome analysis of single-cell-derived clones reveals a link between osimertinib tolerance and pseudohypoxia responses irrespective of oxygen levels. Further metabolomics and genetic studies demonstrate that osimertinib tolerance is driven by miR-147b-mediated repression of VHL and succinate dehydrogenase, which are linked to the tricarboxylic acid cycle and pseudohypoxia pathways. Finally, pretreatment with a miR-147b inhibitor delays osimertinib-associated drug tolerance in patient-derived 3D structures. This link between miR-147b and the tricarboxylic acid cycle may provide promising targets for preventing tumour relapse.

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Fig. 1: NSCLC cells adopt a tolerance strategy against EGFR TKIs.
Fig. 2: miR-147b initiates drug tolerance.
Fig. 3: A miR-147b–VHL axis mediates drug tolerance through impaired VHL activity.
Fig. 4: A miR-147b–SDHD axis mediates drug tolerance through SDH enzyme activity in the TCA cycle.
Fig. 5: Blocking miR-147b overcomes drug tolerance.

Data availability

Information and data for PDX models from the JAX PDX Resource are publicly available from the PDX Portal hosted by the Mouse Tumor Biology Database (http://www.tumor.informatics.jax.org/mtbwi/pdxSearch.do)59. Data from this study have been deposited in the Gene Expression Omnibus (GEO) under the following accessions: GSE103155 (microarray; single-cell-derived clones) and GSE103352 (miRNA-seq). The results shown in this manuscript were in part based on data generated by the TCGA Research Network32: http://cancergenome.nih.gov/. Analyses for an association between miRNA profiles and EGFR mutations as well as an association between VHL and miR-147b in a cohort of human lung adenocarcinoma cell lines were based on a public RNA-seq dataset31 available in the ArrayExpress database under accession E-MTAB-2706. The heat map for miRNA expression was generated according to the Heatmapper server (http://www2.heatmapper.ca/expression/)79. Genetic mutation status was confirmed by canSAR portal (v3.0 beta; https://cansar.icr.ac.uk/) and the Catalogue of Somatic Mutations In Cancer (COSMIC; http://cancer.sanger.ac.uk/cosmic/sample/overview?id=722040). The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank S. Kobayashi for discussion and providing EGFR-mutated lung cancer cell lines. We thank members of the Slack laboratory for comments and sharing materials, the Yale SPORE in Lung Cancer team for feedback, A. Jiao, D. Foster and S. M. Lee for reading and providing feedback on the manuscript, and H. Yang, M. Gong, D. Cook and E. Poulin for technical support. F.J.S. and K.P. acknowledge NIH-YALE SPORE in Lung Cancer grant P50CA196530. Additional support to F.J.S. was provided by grant P50CA196530-03S1 and the BIDMC–JAX collaboration project and by support from the Ludwig Center at Harvard. C.J.B. acknowledges support from NIH grant P30CA034196. J.M.A. acknowledges support from NIH grants 5P01CA120964 and 5P30CA006516. W.C.Z. acknowledges an NIH-YALE SPORE in Lung Cancer Career Development Program Award and NRSA grant 5T32HL007893-20. D.B.C. acknowledges NIH 5R37CA218707 as a funding source.

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W.C.Z. and F.J.S. directed the project; W.C.Z., J.M.W., K.-H.C., H.H., C.J.B. and F.J.S. wrote the manuscript; W.C.Z. performed miRNA-seq and microarray analyses, 3D structure derivation, cell culture experiments and in vivo experiments; J.M.W., K.-H.C., C.J.B., M.A.M. and K.P. established the PDX; H.H., M.Y. and J.M.A. performed metabolomics profiling; T.S. assisted with the small-molecule treatment experiments; W.C.Z. directed miRNA-147b target prediction and validation by genetic and metabolic approaches; and D.B.C. provided advice and project support.

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Correspondence to Frank J. Slack.

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Most authors declare no competing interests. DBC reports personal fees (consulting fees) and non-financial support (institutional research support) from Takeda/Millennium Pharmaceuticals, personal fees (consulting fees) and non-financial support (institutional research support) from Astrazeneca, personal fees (honoraria) and non-financial support (institutional research support) from Pfizer, non-financial support (institutional research support) from Merck Sharp & Dohme Corporation and non-financial support (institutional research support) from Merrimack Pharmaceuticals, all outside the submitted work.

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Whole-transcriptome analysis for single-cell-derived gefitinib-tolerant clones in PC9

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Zhang, W.C., Wells, J.M., Chow, KH. et al. miR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma. Nat Metab 1, 460–474 (2019). https://doi.org/10.1038/s42255-019-0052-9

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