Regorafenib inhibits EphA2 phosphorylation and leads to liver damage via the ERK/MDM2/p53 axis

Nature Communications · 11 authors, 8 centres

AI SUMMARY

FIDELITY 36%

POPULATIONC57BL/6J mice (male and female) and human hepatocyte cell lines (HL-7702, primary human hepatocytes)

INTERVENTIONRegorafenib (200 or 400 mg/kg/day for 3-6 weeks in mice; 1-8 μM for 24-48 h in cells); EphA2 knockdown (siRNA or AAV8-shEphA2); EphA2-S897A/S897D overexpression; schisandrin C (10-50 μM in cells; 10 mg/kg/day in mice)

COMPARISONVehicle (0.5% CMC-Na or DMSO); negative control siRNA; vector control; sorafenib; other kinase inhibitors

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TL;DR

Regorafenib causes hepatotoxicity by inhibiting EphA2 Ser897 phosphorylation, which reduces ERK/MDM2 signaling and stabilizes p53, leading to mitochondrial-dependent apoptosis in hepatocytes. This mechanism is distinct from sorafenib and involves a non-therapeutic target of regorafenib. Recovering EphA2 Ser897 phosphorylation with schisandrin C alleviates liver injury without compromising regorafenib's anticancer efficacy.

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**Background:** Regorafenib, a multikinase inhibitor approved for metastatic colorectal cancer, GIST, and hepatocellular carcinoma, carries a black box warning for severe hepatotoxicity. The mechanism underlying this liver injury is poorly understood. This study investigates the role of EphA2, a non-therapeutic target of regorafenib, in mediating hepatotoxicity through p53 stabilization.

**Methods:** C57BL/6J mice were treated with regorafenib (200 or 400 mg/kg/day) for 3-6 weeks. Human primary hepatocytes and HL-7702 cells were exposed to regorafenib (1-8 μM). Apoptosis was assessed by Annexin V-PI flow cytometry, TUNEL staining, and c-PARP western blot. Mitochondrial function was evaluated by JC-1 staining (MMP), MitoTracker (mitochondrial mass), and ATP levels. p53 stability was examined via cycloheximide chase and ubiquitination assays. EphA2 was knocked down using siRNA or AAV8-shEphA2 in mice. EphA2 phosphorylation mutants (S897A, S897D, Y588A, K646M) were overexpressed. ERK/MDM2 signaling was analyzed by western blot and immunofluorescence. Schisandrin C (10-50 μM in vitro; 10 mg/kg/day in vivo) was tested for rescue.

**Key Results:** Regorafenib (400 mg/kg/day for 6 weeks) elevated serum ALT and AST (p<0.05) and caused nuclear shrinkage, hepatocyte apoptosis, and mitochondrial dysfunction (decreased MMP, ATP, mitochondrial mass). p53 protein increased 2-3 fold in liver tissues and hepatocytes without changes in mRNA, due to reduced ubiquitination and prolonged half-life (from ~15 min to >60 min). EphA2 knockdown (siRNA or AAV8-shEphA2) rescued regorafenib-induced ALT/AST elevation (from ~120 U/L to ~40 U/L for ALT), apoptosis (apoptosis rate reduced from ~30% to ~10%), and p53 accumulation. Regorafenib inhibited EphA2 Ser897 phosphorylation (pS-EphA2) by ~70% at 8 μM for 24 h, while Tyr588 phosphorylation was also reduced but not involved in apoptosis. EphA2-S897A (phosphorylation-dead) mutant increased p53 and induced apoptosis, whereas EphA2-S897D (phosphomimetic) reduced p53. Mechanistically, pS-EphA2 inhibition decreased ERK Tyr204 phosphorylation (pY-ERK) by ~50%, leading to reduced MDM2 Ser166 phosphorylation and cytoplasmic retention of MDM2, preventing p53 nuclear export and degradation. Schisandrin C (30-50 μM) restored pS-EphA2, pY-ERK, and pS-MDM2, reduced p53, and attenuated regorafenib-induced ALT/AST elevation (from ~120 U/L to ~60 U/L for ALT), apoptosis, and mitochondrial dysfunction in mice, without affecting regorafenib's anticancer activity in HCC and colon cancer cell lines.

**Clinical Implications:** This study identifies EphA2 Ser897 phosphorylation as a critical regulator of p53 stability and a key mediator of regorafenib-induced hepatotoxicity. The findings suggest that screening for EphA2 Ser897 inhibition could predict hepatotoxicity of kinase inhibitors. Schisandrin C, a natural product that restores pS-EphA2, represents a potential therapeutic strategy to prevent or treat regorafenib-induced liver injury without compromising anticancer efficacy. Clinical pilot data with Wu wei zi granules (containing Schisandra chinensis) in 15 patients with grade 1-2 ALT/AST elevation showed controlled liver enzymes without regorafenib dose adjustment, warranting further investigation.

PICO

PPOPULATION

C57BL/6J mice (male and female) and human hepatocyte cell lines (HL-7702, primary human hepatocytes)

IINTERVENTION

Regorafenib (200 or 400 mg/kg/day for 3-6 weeks in mice; 1-8 μM for 24-48 h in cells); EphA2 knockdown (siRNA or AAV8-shEphA2); EphA2-S897A/S897D overexpression; schisandrin C (10-50 μM in cells; 10 mg/kg/day in mice)

OOUTCOME

Serum ALT/AST levels; hepatocyte apoptosis (Annexin V-PI, TUNEL, c-PARP); mitochondrial dysfunction (MMP, ATP, mitochondrial mass); p53 protein level and ubiquitination; EphA2, ERK, MDM2 phosphorylation; liver histology (H&E)

STUDY TYPE

other

SPECIALTY

ophthalmology

SUMMARISED BY

AI pipeline

FIDELITY CHECK

36% · F

ORIGINAL PAPER

PMC10182995

Regorafenib inhibits EphA2 phosphorylation and leads to liver damage via the ERK/MDM2/p53 axis

Nature Communications

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ABSTRACT

Abstract The hepatotoxicity of regorafenib is one of the most noteworthy concerns for patients, however the mechanism is poorly understood. Hence, there is a lack of effective intervention strategies. Here, by comparing the target with sorafenib, we show that regorafenib-induced liver injury is mainly due to its nontherapeutic target Eph receptor A2 (EphA2). EphA2 deficiency attenuated liver damage and cell apoptosis under regorafenib treatment in male mice. Mechanistically, regorafenib inhibits EphA2 Ser897 phosphorylation and reduces ubiquitination of p53 by altering the intracellular localization of mouse double minute 2 (MDM2) by affecting the extracellular signal-regulated kinase (ERK)/MDM2 axis. Meanwhile, we found that schisandrin C, which can upregulate the phosphorylation of EphA2 at Ser897 also has protective effect against the toxicity in vivo. Collectively, our findings identify the inhibition of EphA2 Ser897 phosphorylation as a key cause of regorafenib-induced hepatotoxicity, and chemical activation of EphA2 Ser897 represents a potential therapeutic strategy to prevent regorafenib-induced hepatotoxicity. Subject terms: Toxicology, Drug safety, Hepatocytes, Apoptosis

CITATION EXPORT

Hao Yan, Wentong Wu, Yuhuai Hu et al.. Regorafenib inhibits EphA2 phosphorylation and leads to liver damage via the ERK/MDM2/p53 axis. Nature Communications. (2023). https://doi.org/10.1038/s41467-023-38430-8

AUTHORS

11 · 8 CENTRES

Hao Yan, Wentong Wu, Yuhuai Hu, Jinjin Li, Jiangxin Xu, Xueqin Chen, Zhifei Xu, Xiaochun Yang, Bo Yang, Qiaojun He, Peihua Luo

1Center for Drug Safety Evaluation and Research of Zhejiang University, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058 China
2Innovation Institute for Artificial Intelligence in Medicine of Zhejiang University, Hangzhou, 310018 China
3Laboratory of Fruit Quality Biology/Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology/The State Agriculture Ministry Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Zhejiang University, Hangzhou, 310058 China
4Department of Oncology, Affiliated Hangzhou Cancer Hospital, Zhejiang University School of Medicine, Key Laboratory of Clinical Cancer Pharmacology and Toxicology Research of Zhejiang Province, Hangzhou, 310002 China
5Cancer Center, Zhejiang University, Hangzhou, 310058 China
6Institute of Pharmacology & Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058 China
7Department of Pharmacology and Toxicology, Hangzhou Institute of Innovative Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310018 China
8Key Laboratory of Clinical Cancer Pharmacology and Toxicology Research of Zhejiang Province, Affiliated Hangzhou Cancer Hospital, Zhejiang University School of Medicine, Hangzhou, 310002 China