CRISPR/Cas9 genome-wide screening identifies KEAP1 as a sorafenib, lenvatinib, and regorafenib sensitivity gene in hepatocellular carcinoma

Abstract

Sorafenib is the first-line drug used for patients with advanced hepatocellular carcinoma (HCC). However, acquired sorafenib resistance in cancer patients limits its efficacy. Here, we performed the first genome-wide CRISPR/Cas9-based screening on sorafenib-treated HCC cells to identify essential genes for non-mutational mechanisms related to acquired sorafenib resistance and/or sensitivity in HCC cells. KEAP1 was identified as the top candidate gene by Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK). KEAP1 disrupted HCC cells were less sensitive than wild-type cells in short- and long-term sorafenib treatments. Compared to wild-type cells, KEAP1-disrupted cells showed lower basal and sorafenib-induced reactive oxygen species (ROS) levels and were more resistant to oxidative stress-induced cell death. The absence of KEAP1 led to increased activity of Nrf2, a key transcription factor controlling antioxidant responses, as further evidenced by increased expression of Nrf2-controlled genes including NQO1, GPX2 and TXNRD1, which were positively associated with chemoresistance. In addition, KEAP1 disruption counteracted the reduction of cell viability and the elevation of ROS caused by lenvatinib, a drug that recently showed clinical efficacy as a first-line treatment for unresectable HCC. Finally, Keap1 disruption also increased the resistance of cells to regorafenib, a recently approved drug to treat HCC as a second line therapy. Taken together, our data indicate that deregulation of the KEAP1/Nrf2 pathway following KEAP1 inactivation contributes to sorafenib, lenvatinib, and regorafenib resistance in human HCC cells through up-regulation of Nrf2 downstream genes and decreased ROS levels.

Oxidative Stress Management in Chronic Liver Diseases and Hepatocellular Carcinoma

“…However, while these agents have shown promise for controlling advanced HCC, drug resistance is an increasing problem. A genome-wide CRISPR/Cas9-based screening of a sorafenib-treated HCC cell line identified KEAP1 as the top candidate drug resistance-related gene [149]. Given that KEAP1 disruption resulted in increased Nrf2 activity and Nrf2-driven genes and decreased ROS production, oxidative stress reduction is one of the mechanisms underlying resistance to sorafenib.…”

Roles of Nrf2 in Liver Diseases: Molecular, Pharmacological, and Epigenetic Aspects

“…HCC cells with disrupted KEAP1 were less sensitive than wild-type cells to short- and long-term treatment with sorafenib. KEAP1 inactivation led to sorafenib, lenvatinib, and regorafenib resistance in HCC cells through induction of Nrf2 target genes and reduction of ROS levels [ 88 ]. Other compounds such as the flavonoids apigenin and luteolin, as well as the potent Nrf2 inhibitor chrysin, among others, have been shown to reduce the expression of Nrf2 and its target genes, which suggests that these compounds may be considered as potential anti-cancer agents [ 86 ].…”

ASH2L drives proliferation and sensitivity to bleomycin and other genotoxins in Hodgkin’s lymphoma and testicular cancer cells

“…The cells were selected with 5 µg/ml blasticidin for 7 days. The GeCKO v2 library was purchased from Addgene (2plasmid system catalog number 1000000049), and amplified in bacteria as previously described (60) and following the recommendations of the library creators (61). The library was packaged into viral particles (see lentivirus production section below).…”