Abstract P4-03-07: Combined genome-scale CRISPR-Cas9 knockout screening with transcriptome sequencing to identify paclitaxel related drivers in triple negative breast cancer

Background. Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancers, for which the only standard therapeutics is chemotherapy containing Taxol. However, quite a number of TNBC patients cannot get the expected drug response after paclitaxel treatment and the resistant mechanism has not been clear yet. Other than the traditional “genotype-to-phenotype” means, the high-throughput functional screening, such as CRISPR-CAS9 library, selects genes with the phenotype of interest. Here, we combine the novel screening model with the drug-resistant genotype to explore the decisive role in paclitaxel effect. Methods. Breast cancer cell line MDA-MB-231(231 WT ) was treated by paclitaxel from 1ug/ml to 5ug/ml to establish a paclitaxel-resistant cell type (231 PTX ) for transcriptome sequencing. Genome-scale CRISPR-Cas9 sgRNA library was made into lentivirus to affect MDA-MB-231 cells expressed Cas9 protein (231 cas9 ). Then 231 cas9-sgRNA was treated by low dose of paclitaxel for 14 days and was read by next generation sequencing. RNA sequencing data was processed to TPM values and sgRNA data to gene ranking and p value. The threshold of “231 PTX TPM/231 WT TPM” was above 2 or below 1/2 and the gene p value was smaller than 0.05. Biological technology applied in this study includes western blot (WB), immunofluorescence (IF), real time PCR and cell proliferation assay. In vivo, 20 balb/c mouse were injected MDA-MB-231 in situ for tumor formation and were treated with paclitaxel/normal saline for six times. Results. Crosstalk between these two sequencing data had result of 124 genes related to paclitaxel resistance (fold change> 2 and p value H9 KO ) contributed to nearly 2-fold decrease IC50 value (1.7nM versus 3.7nM, p value H9 KO cells. After treatment with paclitaxel, the mark of polymerized tubulin, acetylation tubulin and the mark of cell cycle G2/M, cyclin B1 were notably increased when HDAC9 knockout in both MD-MB1-231 and BT-100 cell lines. In vivo assays found that HDAC9 knockout induced the declined tumorigenesis and more sensitive breast tumors to paclitaxel. Conclusions . Combined Genome-scale CRISPR-Cas9 knockout screening with transcriptome sequencing is efficient to investigate potent drug targets. In vitro assays suggest that HDAC9 is conductive to paclitaxel resistance in TNBC cells. In vivo results imply inhibition HDAC9 may beneficial to paclitaxel therapeutic response. Citation Format: Lian B, Xin H, Zhimin S. Combined genome-scale CRISPR-Cas9 knockout screening with transcriptome sequencing to identify paclitaxel related drivers in triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-07.