Genome‐wide CRISPR/Cas9 screening for therapeutic targets in NSCLC carrying wild‐type TP53 and receptor tyrosine kinase genes

Abstract

Background Targeted drugs have greatly improved the therapeutic outcome of non‐small cell lung cancer (NSCLC) patients compared with conventional chemotherapy, whereas about one‐third of patients are so far not suitable for targeted therapy due to lack of known driver oncogenes such as a mutated receptor tyrosine kinase (RTK) genes. In this study, we aimed to identify therapeutic targets for this subgroup of NSCLC patients. Methods We performed genome‐wide CRISPR/Cas9 screens in two NSCLC cell lines carrying wild‐type TP53 and receptor tyrosine kinase (wtTP53‐RTK) genes using a GeCKO v2.0 lentiviral library (containing 123411 sgRNAs and targeting 19050 genes). MAGeCKFlute was used to analyse and identify candidate genes. Genetic perturbation and pharmacological inhibition were used to validate the result in vitro and in vivo. Results The Genome‐wide CRISPR/Cas9 screening identified MDM2 as a potential therapeutic target for wtTP53‐RTK NSCLC. Genetic and pharmacological inhibition of MDM2 reduced cell proliferation and impaired tumour growth in the xenograft model, thus confirming the finding of the CRISPR/Cas9 screening. Moreover, treatment by a selective MDM2 inhibitor RG7388 triggered both cell cycle arrest and apoptosis in several NSCLC cell lines. Additionally, RG7388 and pemetrexed synergistically blocked the cell proliferation and growth of wtTP53‐RTK tumours but had limited effects for other genotypes. Conclusions We identified MDM2 as an essential gene and a potential therapeutic target in wtTP53‐RTK NSCLC via a genome‐wide CRISPR/Cas9 screening. For this subgroup, treatment by RG7388 alone or by its combination with pemetrexed resulted in significant tumour inhibition.

CRISPR/Cas9 screening reveals that ASB2 inhibits p53-dependent ferroptosis to mediates Lenvatinib resistance in hepatocellular carcinoma

“…Chune Yu et al reported that a genome-wide CRISPR-Cas9 knockdown screen identi ed GRB7 as a driver of MEK inhibitor resistance in KRAS mutant colon cancer [19].Qianqian Wang et al identi ed MDM2 as an essential gene and potential therapeutic target of WT-p53-RTK Non-small cell lung adenocarcinoma(NSCLC) by genome-wide CRISPR/Cas9 screening. RG7388 alone or in combination with pemetrexed resulted in signi cant tumor suppression [20]. Xue Sui et al identi ed a novel cisplatin-resistant esophageal oncogene, ERCC8, based on genome-scale CRISPR/Cas9 screening [21].…”

Genome‐scale CRISPR–Cas9 screen identifies PAICS as a therapeutic target for EGFR wild‐type non‐small cell lung cancer

“…Genome‐wide CRISPR knockout screening was performed as described 58,59 . As the GeCKO v2.0 library contains 123411 sgRNAs targeting 19,050 genes, about 1.6 × 10 8 NCI‐H460, A549, and BEAS‐2B cells need to be infected to achieve 300‐fold coverage at a multiplicity of infection of 0.3–0.5.…”

“…Genome-wide CRISPR knockout screening was performed as described. 58,59 As the GeCKO v2.0 library contains 123411 sgRNAs targeting 19,050 genes, about 1.6 × 10 8 NCI-H460, A549, and BEAS-2B cells need to be infected to achieve 300-fold coverage at a multiplicity of infection of 0.3-0.5. Cells were seeded in 12-well plates for 24 h and then infected with lentivirus by centrifuging at 450 g for 2 h at 37 • C in the presence of 8 µg/mL polybrene to increase transduction efficiency.…”

Applications of CRISPR screening to lung cancer treatment

“…Two genome-scale CRISPR/Cas9 screenings were conducted in the wild-type TP53 and RTK-containing cell lines with BEAS-2B cells serving as a control. The results showed that MDM2 was a potential therapeutic target ( Wang et al, 2022 ). A genome-scale screen was used to identify tumor suppressor genes through knockout by CRISPR/Cas9 system.…”