Abstract 6333: Genomic, transcriptomic, and epigenetic profiling of triple-negative breast cancer cells after Navitoclax treatment
Authors: Marczyk Michal , Gunasekharan Vignesh , Zhao Jun , Qu Rihao , Li Xiaotong , Patwardhan Gauri A. , Wali Vikram B. , Gupta Abhishek K. , Pillai Manoj M. , Kluger Yuval , Hatzis Christos , Pusztai Lajos 
Published: September 2020

Abstract
Cancer cells possess various anti-apoptotic defense mechanisms that can protect them from drug induced cell death. We investigated the effects of treatment with the bcl-2/bcl-XL inhibitor Navitoclax on transcriptome, methylome, and genome of MDA-MB-231 cells. Two biological replicates were treated simultaneously and profiled using 10x Genomics single-cell RNA sequencing (scRNAseq; gene expression), bulk RNA sequencing (bRNAseq; gene expression), ATAC sequencing (ATAC; chromatin accessibility), bisulphite targeted sequencing (DNAm; DNA methylation) and shallow whole-genome sequencing (WGS; copy number variants) at baseline before treatment, after 3 days of 10µM navitoclax treatment, and after 10 days of cell recovery from treatment. Variance between biological replicates on transcriptome level was minimal before the treatment (R&gt;0.99), but we noticed divergence in gene expression after recovery from the treatment (both on bulk and single-cell level), mostly driven by cell cycle genes. On other platforms, biological replicates were similar in all 3 phases. After treatment, we observed more genes with decreased expression (n=151) in comparison to baseline, but after 10 days of recovery there were more up-regulated genes (n=655) in comparison to samples after treatment. Chromatin accessibility for most genomic regions increased after treatment in comparison to baseline, suggesting an acute response to the stress caused by drug treatment, and then returned to baseline level after recovery period. DNA methylation patterns showed more regions with decreased methylation after treatment (n=529) in comparison to baseline which remained detectable even after the recovery period. In WGS data, we found 752 genes with deletions and amplifications only in baseline samples representing genomic background of drug sensitive cells, mostly enriched in epithelial mesenchymal transition pathway. On single-cell level, we identified a subset of cells that were resistant to treatment and discovered 2,324 genes significantly up-regulated in these cells, that could be potential markers of resistance. In gene set analysis, these markers were enriched in MYC and E2F target gene sets, and were involved in angiogenesis and JAK-STAT pathway. We measured the expression of 16 top up-regulated markers of resistance in 4 different TNBC cell lines using qPCR and found that 5 were significantly enriched after treatment in 3 cell lines, 5 in 2 cell lines and 5 in a single cell line. Summarizing, we thoroughly described molecular effects of Navitoclax treatment and showed that most cells return to the basal transcriptional state after the drug recovery period, but bulk genome and methylome are permanently changed. Finally, we provided a list of new markers of resistance that may be useful in the studies of combinational therapies with other drugs. This work was funded by Breast Cancer Research Foundation.
Citation Format: Michal Marczyk, Vignesh Gunasekharan, Jun Zhao, Rihao Qu, Xiaotong Li, Gauri A. Patwardhan, Vikram B. Wali, Abhishek K. Gupta, Manoj M. Pillai, Yuval Kluger, Christos Hatzis, Lajos Pusztai. Genomic, transcriptomic, and epigenetic profiling of triple-negative breast cancer cells after Navitoclax treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6333.