(M1230-10-59) Identifying Novel Synthetic Lethal Drug Targets to Overcome Chemotherapy Resistance in Triple Negative Breast Cancer by Genome-Wide CRISPR/Cas9 Screen Paralleled with Transcriptome RNAseq

Purpose: Triple-negative breast cancer (TNBC) is one kind of breast cancer. Even though TNBC accounts for 15-20% of total breast cancer patients, it is still one of the most lethal breast cancers. Because of the lack of therapeutic targets, chemotherapy is still the first-line treatment for TNBC. However, more than half of TNBC patients develop drug resistance after chemotherapy. Thus, to better understand the chemo-drug resistance mechanism and identify novel drug targets to overcome drug resistance in breast cancer. In our study, firstly, we use bioinformatics tools and statistical analysis to identify the most representative TNBC cell lines for TNBC patients based on the TNBC genomic profiles. We then performed a genome-wide CRISPR/Cas9 screen and bulk RNAseq to explore the potential synthetic lethal targets to Cisplatin/ Doxorubicin on the TNBC cell line model.

Methods: By screening the GEO database, we selected genomic profiles of 51 TNBC patients and 21 TNBC cell models for similarity analysis. In addition, we have also performed Hierarchical clustering analysis, single-sample GSEA analysis, and Spearman's rank correlation to identify the most representative chemo resistance cell line models. As a result, we discovered the MDA-MB-231 cell line to be the most representative of TNBC chemo-resistant patients at gene expression level and in terms of pathway enrichment. To understand the chemo-drug resistance mechanism and to identify the novel drug targets to overcome Cisplatin/ Doxorubicin treatment, we performed genome-wide CRISPR-Cas9 knockout screens in MDA-MB-231. After 21 days of treatment with Cisplatin/ Doxorubicin, the sgRNA library was prepared and sequenced at around 80 million reads per sample to achieve the hundreds of coverages over the TKOV3 library. In the meantime, we performed after-treatment with the same drug conditions on the normal MDA-MB-231 cell lines. Then, bulk RNAseq was performed to capture the transcriptomic changes and validate the CRISPR-Cas9 screen result.

Results: We generated a list of candidate genes using the Megeck algorithm, and these genes are a potential match for the synthetic lethal partnership with Cisplatin/ Doxorubicin. Our adverse selection confirmed hundreds of essential genes. Some have already been identified, such as BCL2L1, ATM, and CDC25B, NBN. These hundred genes show enrichment in the chemo resistance pathways, such as DNA repair, G2/M DNA damage checkpoint, AMPK signaling pathway, and mTOR signaling pathway. Our study also found novel gene-targeted genes such as ATR, MCM9, and NEPPS. siRNA knockdown study shows that lower expression of these genes improves the potency of CIS/DOX on MDA-MB-231 cell lines. In addition, enrichment pathways of differential expression genes in transcriptomic RNAseq profile show many differences compared with the enrichment analysis results in Crispr screening. This finding suggests a complex regulatory system in cell response to DNA damage drugs.

Conclusion: Combining bioinformatics similarity analysis and Genome-wide CRISPR-Cas9 screen with transcriptome RNAseq, we identified the essential genes that have potential synthetic lethal effects with Cisplatin/ Doxorubicin. Our study provides new opportunities to overcome chemo resistance in TNBC patients.