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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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 7

ACh modulates DTP cell formation through activating WNT signaling.

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ACh modulates DTP cell formation through activating WNT signaling. (A) H...
(A) Heatmap of WNT signaling–related gene expression values in PC9-derived DTP versus parental PC9 cells (n = 2) with RNA-seq. Rows are z scores calculated for each gene in both cell types. (B) Log2-transformed fold change in the expression of WNT ligands and WNT target genes comparing PC9-derived DTP cells to parental PC9 cells with RNA-seq. (C) Relative mRNA levels of CD133, AXIN2, WNT3A, and WNT6 in control (n = 4) and MRD (n = 3) PC9 xenografts. (D and E) Representative image and quantification of immunohistochemical staining of WNT6 and WNT9A on the indicated control and MRD tumor sections from PC9 xenografts. n = 3 mice per group. Scale bar: 50 μm. (F) Relative mRNA levels of WNT signaling–related genes in PC9 cells treated with osimertinib or indicated concentrations of darifenacin alone or in combination for 6 days (n = 3). (G) Western blot showing β-catenin and nonphosphorylated β-catenin in nuclear extract (NE) and cytosol extract (CE) of PC9 cells treated with 2 μM osimertinib or DMSO, alone or in combination with 20 μM darifenacin or 20 μM vesamicol. (H) Relative viability of PC9-derived DTP cells cotreated with darifenacin and WNT signaling activator CHIR99021 or WNT3A (n = 5 or 6). (I) Relative viability of PC9 cells treated with 2 μM osimertinib and indicated concentrations of darifenacin and WNT signaling inhibitor LGK974 or ICG-001, alone or in combination, for 6 days (n = 5). In C, E, F, H, and I, data are shown as mean ± SEM. Significance was assessed using 2-tailed Student’s t test (C and E) or 1-way ANOVA with Tukey’s test (F, H, and I).