Abstract LB131: Genome-wide CRISPR-Cas9 screen parallel with transcriptome RNAseq to identify synthetic lethal drug targets to cisplatin/doxorubicin in triple-negative breast cancer
Authors: Shao Shuai , Tang Shan , Wu Xue , Zhao Yue , Yang Huo , Cheng Lijun , Li Lang 
Published: June 2022

Triple-negative breast cancer (TNBC) accounts for 15-20% of cases yet disproportional accounts for 35% of breast cancer deaths. Chemotherapy remains the first-line treatment for TNBC. Despite the great research efforts, above half of TNBC patients have developed drug resistance after chemotherapies. Thus, the study of chemo-resistance and identification of novel targets are critical to improving TNBC treatment. In our study, we compared genomic profiles between TNBC cell lines and patients' samples and selected the most representative one, MDA-MB-231,for TNBC chemotherapy-poor responders. Then, Genome-wide CRISPR-Cas9 screen and RNAseq were performed in MDA-MB-231 to identify potential synthetic lethal targets to Cisplatin/Doxorubicin.

To identify cell lines that correspond to poor responder patients, we selected genomic profiles of 17 good responders, 36 poor responders, and 21 TNBC cell lines from GEO database. We used Hierarchical clustering analysis, Spearman's rank correlation, and GSEA analysis to determine the representative cell lines in gene expression level and pathway level. With the results from our analysis, MDA-MB-231 is most representative among 21 TNBC cell lines.

To identify essential genes and pathways for cell survival under Cisplatin/Doxorubicin treatment, we performed genome-wide CRISPR-Cas9 knockout screens in MDA-MB-231 and introduced CRISPR library TKOV3 into cells by lentivirus. After puromycin selection, the surviving cells were treated with Cisplatin/Doxorubicin for 21 days and sgRNAs were sequenced at ~80 million reads per sample to achieve a 600x coverage over the TKOV3 library. To accurate the CRISPR-Cas9 screen results and capture the transcriptomic changes during the Cisplatin/Doxorubicin treatment, we performed RNAseq from MDA-MD-231 after treating in same drug conditions matched with the CRISPR screen. By the MAGeCK algorithm, we generated the list of candidate genes that can form a synthetic lethal partnership with Cisplatin/Doxorubicin. Our negative selection screen confirmed that loss of essential genes from DNA damage repair and regulation of DNA replication pathways, such as BCL2L1, ATM, CDC25B, NBN, sensitizes cells to Cisplatin/Doxorubicin, which has been reported in DNA damage drug synthetic lethal studies. Meanwhile, our analysis also revealed hundreds of unrecognized genes involved in the G2/M DNA damage checkpoint, AMPK signaling pathway, mTOR signaling pathway, and Hsp90-mediated pathways. In addition, the pathways related to transcriptomic response to Cisplatin/Doxorubicin from RNAseq data show many differences with essential pathways shown in the CRISPR screen, which proposed a complex regulation system in cell response to DNA damage drug.

In general, Genome-wide CRISPR-Cas9 screen along with transcriptome RNAseq is efficient to identify essential genes that have potential synthetic lethal interaction with Cisplatin/Doxorubicin. This provides new opportunities for combination therapies in TNBC chemo-resistant patients.

Citation Format: Shuai Shao, Shan Tang, Xue Wu, Yue Zhao, Huo Yang, Lijun Cheng, Lang Li. Genome-wide CRISPR-Cas9 screen parallel with transcriptome RNAseq to identify synthetic lethal drug targets to cisplatin/doxorubicin in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB131.