Targeting acetylcholine signaling modulates persistent drug tolerance in EGFR-mutant lung cancer and impedes tumor relapse
Meng Nie, … , Caicun Zhou, Zeping Hu
Meng Nie, … , Caicun Zhou, Zeping Hu
Published September 1, 2022
Citation Information: J Clin Invest. 2022;132(20):e160152. https://doi.org/10.1172/JCI160152.
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Research Article Metabolism Oncology

Targeting acetylcholine signaling modulates persistent drug tolerance in EGFR-mutant lung cancer and impedes tumor relapse

Abstract

Although first-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy is effective for treating EGFR-mutant non–small cell lung cancer (NSCLC), it is now understood that drug-tolerant persister (DTP) cells escaping from initial treatment eventually drives drug resistance. Here, through integration of metabolomics and transcriptomics, we found that the neurotransmitter acetylcholine (ACh) was specifically accumulated in DTP cells, and demonstrated that treatment with EGFR-TKI heightened the expression of the rate-limiting enzyme choline acetyltransferase (ChAT) in ACh biosynthesis via YAP mediation. Genetic and pharmacological manipulation of ACh biosynthesis or ACh signaling could predictably regulate the extent of DTP formation in vitro and in vivo. Strikingly, pharmacologically targeting ACh/M3R signaling with an FDA-approved drug, darifenacin, retarded tumor relapse in vivo. Mechanistically, upregulated ACh metabolism mediated drug tolerance in part through activating WNT signaling via ACh muscarinic receptor 3 (M3R). Importantly, we showed that aberrant ACh metabolism in patients with NSCLC played a potential role in predicting EGFR-TKI response rate and progression-free survival. Our study therefore defines a therapeutic strategy — targeting the ACh/M3R/WNT axis — for manipulating EGFR TKI drug tolerance in the treatment of NSCLC.

Authors

Meng Nie, Na Chen, Huanhuan Pang, Tao Jiang, Wei Jiang, Panwen Tian, LiAng Yao, Yangzi Chen, Ralph J. DeBerardinis, Weimin Li, Qitao Yu, Caicun Zhou, Zeping Hu

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Figure 5

Activated ACh metabolism and signaling promotes tolerance to EGFR inhibition in vitro and in vivo.

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Activated ACh metabolism and signaling promotes tolerance to EGFR inhibi...
(A) Colony formation assay of cells treated with indicated drugs (n = 3). (B) Relative viability of cells treated with osimertinib and 10 μM ACh (n = 5). (C) Linear regression analysis of correlation between short ChAT isoform levels and osimertinib sensitivity log10(IC50). (D) Colony formation assay of PC9 single-cell clones exposed to osimertinib (n = 3). (E) Colony formation assay of Flag-ChAT short isoform overexpression (ChAT-OE, 41 kDa) and negative control (NC) cells treated with osimertinib (n = 3). (F) PC9-xenograft mice injected with ChAT-OE or NC cells were treated with 5 mg/kg osimertinib for 9 days or with vehicle. n = 7 or 8 per group. (G) Relative viability of PC9 WT and ChAT-knockout cells treated with osimertinib and 10 μM ACh (n = 6). (H) Colony formation assay of PC9 WT and ChAT-knockout cells treated with osimertinib and ACh (n = 3). (I) PC9-xenograft mice injected with WT and ChAT-knockout cells were treated with 5 mg/kg osimertinib for 9 days or with vehicle. ACh was injected subcutaneously once daily (n = 7). (J) PC9-xenograft mice injected with WT and ChAT-knockout cells were treated with 1 mg/kg osimertinib or with vehicle. n = 5 or 6 per vehicle group, n = 11 or 14 per osimertinib treatment group. (K) Percentage survival curve generated from PC9-xenograft mice. In A, B, and D–J, data are shown as mean ± SEM. Significance was assessed using 1-way ANOVA with Dunnett’s test (A), 2-way ANOVA with Tukey’s test (B and G), 1-way ANOVA with Tukey’s test (D, E and H), 2-way ANOVA adjusted by Bonferroni’s correction (F, I and J), or 2-sided log-rank test (K).