FOXO3 mutation predicting gefitinib-induced hepatotoxicity in NSCLC patients through regulation of autophagy

Shaoxing Guan, Xi Chen, Youhao Chen, Guohui Wan, Qibiao Su, Heng Liang, Yunpeng Yang, Wenfeng Fang, Yan Huang, Hongyun Zhao, Wei Zhuang, Shu Liu, Fei Wang, Wei Feng, Xiaoxu Zhang, Min Huang, Xueding Wang, Li Zhang, Shaoxing Guan, Xi Chen, Youhao Chen, Guohui Wan, Qibiao Su, Heng Liang, Yunpeng Yang, Wenfeng Fang, Yan Huang, Hongyun Zhao, Wei Zhuang, Shu Liu, Fei Wang, Wei Feng, Xiaoxu Zhang, Min Huang, Xueding Wang, Li Zhang

Abstract

Hepatotoxicity is a common side effect for patients treated with gefitinib, but the related pathogenesis is unclear and lacks effective predictor and management strategies. A multi-omics approach integrating pharmacometabolomics, pharmacokinetics and pharmacogenomics was employed in non-small cell lung cancer patients to identify the effective predictor for gefitinib-induced hepatotoxicity and explore optional therapy substitution. Here, we found that patients with rs4946935 AA, located in Forkhead Box O3 (FOXO3) which is a well-known autophagic regulator, had a higher risk of hepatotoxicity than those with the GA or GG variant (OR = 18.020, 95%CI = 2.473 to 459.1784, P = 0.018) in a gefitinib-concentration dependent pattern. Furthermore, functional experiments identified that rs4946935_A impaired the expression of FOXO3 by inhibiting the promotor activity, increasing the threshold of autophagy initiation and inhibiting the autophagic activity which contributed to gefitinib-induced liver injury. In contrast, erlotinib-induced liver injury was independent on the variant and expression levels of FOXO3. This study reveals that FOXO3 mutation, leading to autophagic imbalance, plays important role in gefitinib-induced hepatotoxicity, especially for patients with high concentration of gefitinib. In conclusion, FOXO3 mutation is an effective predictor and erlotinib might be an appropriately and well-tolerated treatment option for patients carrying rs4946935 AA.

Keywords: Autophagy; FOXO3; Gefitinib; Hepatotoxicity; Pharmacogenomics; Pharmacokinetics; Pharmacometabolomic.

© 2022 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
The concentration of gefitinib/metabolites was not associated with gefitinib-induced hepatotoxicity in the general subjects (n = 180). (A) The grades of hepatotoxicity induced by gefitinib in NSCLC patients; (B–F) Neither the concentration of gefitinib nor the metabolites was associated with hepatotoxicity in the general subjects (n = 180). ns: no significant.
Figure 2
Figure 2
G>A rs4946935 was associated with gefitinib-induced hepatotoxicity. (A) Among 194 SNPs, only G>A rs4711998 in IL17, C>T rs4795896 in CCL11, G>A rs4946935 in FOXO3 and G>A rs12722604 in IL2RA were associated with gefitinib-induced hepatotoxicity; (B) G>A rs4711998, located in IL17A, was associated with gefitinib-induced hepatotoxicity; (C) C>T rs4795896, located in CCL11, was associated with gefitinib-induced hepatotoxicity; (D) G>A rs4946935, located in FOXO3, was correlated with gefitinib-induced hepatotoxicity; (E) G>A rs1272260, located in IL2RA, was correlated with gefitinib-induced hepatotoxicity; (F) G>A rs4946935, located in FOXO3, was significantly associated with gefitinib-induced hepatotoxicity by multivariate logistic regression; (G) Plasma AST/ALT level was significantly correlated with the concentration of gefitinib in FOXO3 AA carriers. ∗P < 0.05; ∗∗P < 0.01.
Figure 3
Figure 3
rs4946935_A impaired the expression of FOXO3. (A) All tag SNPs of FOXO3 in HCB; (B) G>A rs4946935 was located in intron 3 of FOXO3 on Chromosome 6; (C) FOXO3 rs4946935 was correlated to expression levels of FOXO3 in human spleen and brain caudate according to GTEx database; (D) FOXO3 rs4946935 was correlated to expression levels of FOXO3 in NSCLC patients; (E) Luciferase activity of rs4946935_ A and _G of FOXO3 reporter vectors in LO2 cells; (F–H) Overexpression of FOXO3 significantly increased the IC50 of gefitinib while knockout of FOXO3 decreased in LO2 cells. Data represent mean ± SD of three or more independent experiments; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns: no significance.
Figure 4
Figure 4
gefitinib-induced hepatotoxicity was FOXO3-dependent by inhibiting autophagy. (A) The expression of FOXO3 was correlated with autophagy-related genes in liver tissue according to GTEx dataset; (B) FOXO3 directly regulates the expression of ATG3, ATG4A, ATG5, ATG7, ATG10, ATG12, ATG14, ATG16L1, and MAP1LC3B after treatment with gefitinib in LO2 cells; (C) Overexpression of FOXO3 decreased the threshold of autophagy initiation as indicated by LC3-II/I expression level; (D) Electron micrographs of FOXO3-knock out and -overexpression LO2 cells under incubation of gefitinib; (E) Inhibition of autophagy significantly suppressed proliferation of FOXO3 overexpression hepatocytes under incubation of gefitinib; (F) Inhibition of autophagy significantly increased the cytotoxicity of gefitinib in FOXO3 overexpression hepatocytes. Data represent mean ± SD of three or more independent experiments; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns: no significance.

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