CRISPR screens uncover protective effect of PSTK to targeted therapy induced ferroptosis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most frequent malignancies with extremely poor prognosis. Clinical efficacies of existing targeted therapy strategies were not satisfied due to targeted therapy resistance. In this study, we performed a series of CRISPR/Cas9 screens to identify synthetic lethality genes to improve the clinical response of HCC. CRISPR loss-of-function genetic screens were performed to identify synthetic lethality genes of targeted therapies (Abemaciclib, Palbociclib, Sorafenib, Lenvatinib, Regorafenib and Erastin) in HCC cell lines. RNA-seq analysis were performed to clarify potential mechanism. We identified that inhibition of phosphoseryl-tRNA kinase (PSTK) sensitized HCC cells to targeted therapies. PSTK contributes to resistance of targeted therapy induced ferroptosis. Overall design: CRISPR screen: Toronto human knockout pooled library (TKOv3) was a gift from Jason Moffat (Addgene #125517). TKOv3 contains 70948 sgRNAs targeted to 18053 protein-coding genes. Library amplification and virus preparation were performed according to the protocol of Moffat Lab. Firstly, we established stable Cas9-expressing HCC cell lines by lentiviral transduction of Cas9 coding sequence together with mCherry coding sequence. Cas9 expressing cells were sorted by flow cytometry. Hep3B-Cas9 cells or SNU-398-Cas9 cells were transduced with TKOv3 library at low MOI (0.2-0.3) for 24 hours and cultured with fresh DMEM with 3µg/mL puromycin for 72 hours. Then cells were divided into eight groups (around 1000-fold coverage of the sgRNA library at each group). One group of transfected cells was harvested as baseline of screening (T0) and other groups were treated with inhibitors (IC20) or DMSO for 14 days respectively. After screening, cells were harvested and extracted for genomic DNA. Genomic DNA were further amplified and labeled with barcodes using NEBNext® High-Fidelity 2X PCR Master. Illumina deep sequencing were carried out by Annoroad (Beijing, China). The abundance of sgRNAs was counted by Bowtie2 and the synergistic/suppressor chemical genetic interactions were analyzed by DrugZ algorithm. RNA-seq: For RNA sequencing, RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Differential expression analysis of two groups (Hep3B_PSTK_KO vs. Hep3B_control, three biological replicates) was performed using the DESeq2 R package (1.20.0).