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Genome-scale CRISPR-Cas9 knockout screening in hepatocellular carcinoma with lenvatinib resistance
Yonggang Lu1, Haoming Shen2, Wenjie Huang3
1Department of Hepatobiliary Surgery, Affiliated Liutie Central Hospital of Guangxi Medical University, Guangxi, China.
Abstract:
Lenvatinib is the first target drug approved for advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common, and the mechanisms of lenvatinib resistance and resistant targets in HCC are poorly understood. By using CRISPR/Cas9 library screening, we screened out two key resistance genes, neurofibromin 1(NF1), and dual specificity phosphatase 9 (DUSP9), as critical drivers for lenvatinib resistance in HCC. With RNAi knockdown and CRISPR/Cas9 knockout models, we further clarified the mechanisms by which NF1 loss reactivates the PI3K/AKT and MAPK/ERK signaling pathways, while DUSP9 loss activates the MAPK/ERK signaling pathways, thereby inactivating FOXO3, followed by degradation of FOXO3, finally induced lenvatinib resistance. We also screened out trametinib, a small molecule pathway inhibitor for MEK, that can be used to reverse resistance induced by NF1 and DUSP9 loss in HCC cells. Trametinib was still able to halt HCC growth even when NF1 was knocked out in mice. Collectively, the findings indicate that NF1 and DUSP9 takes critical role in lenvatinib resistance and may be novel specific targets and predictive markers for lenvatinib resistance in HCC.
Insights
Neurofibromin 1 (NF1) and dual specificity phosphatase 9 (DUSP9) drive lenvatinib resistance in liver cancer (HCC). Targeting MEK with trametinib can overcome this resistance, offering new therapeutic strategies for advanced HCC.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Lenvatinib is a targeted therapy for advanced hepatocellular carcinoma (HCC).
- Drug resistance frequently develops, limiting lenvatinib's efficacy.
- Mechanisms underlying lenvatinib resistance in HCC remain incompletely understood.
Purpose of the Study:
- To identify key genes and pathways driving lenvatinib resistance in HCC.
- To elucidate the molecular mechanisms of lenvatinib resistance.
- To identify potential therapeutic strategies to overcome lenvatinib resistance.
Main Methods:
- CRISPR/Cas9 library screening to identify resistance genes.
- RNAi knockdown and CRISPR/Cas9 knockout models to study gene function.
- Analysis of signaling pathways (PI3K/AKT, MAPK/ERK) and protein degradation (FOXO3).
- In vivo studies using mouse models.
Main Results:
- Neurofibromin 1 (NF1) and dual specificity phosphatase 9 (DUSP9) were identified as critical drivers of lenvatinib resistance.
- NF1 loss reactivates PI3K/AKT and MAPK/ERK pathways.
- DUSP9 loss activates MAPK/ERK, leading to FOXO3 inactivation and degradation.
- Trametinib, a MEK inhibitor, reversed lenvatinib resistance in HCC cells and halted tumor growth in mice with NF1 knockout.
Conclusions:
- NF1 and DUSP9 play critical roles in lenvatinib resistance in HCC.
- These genes represent potential predictive markers for lenvatinib resistance.
- Targeting MEK with trametinib is a promising strategy to overcome lenvatinib resistance in HCC.
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