Overexpression of histone deacetylases (HDACs) has been reported in many cancers. As such, histone deacetylase inhibitors (HDIs) have emerged as potent antitumor agents that prevent HDACs from removing acetyl groups from both histone and non-histone proteins, thereby maintaining transcriptionally active DNA. Romidepsin (Rd) is an HDI that targets primarily class I HDACs and has shown efficacy in T-cell lymphomas. However, many solid tumors have been found to be intrinsically resistant to Rd. To investigate potential mechanisms of resistance, we performed a stepwise selection of MCF7 breast cancer cells in romidepsin in the presence of 2.5 μg/ml verapamil to prevent the emergence of P-glycoprotein, an ATP-binding cassette transporter, as a mechanism of resistance. Resistant cells were stable in 300 ng/ml romidepsin. Romidepsin-resistant (RdVp) cells were more than 100-fold resistant to Rd relative to untreated control cells. In addition, measurement of cell viability with CellTiterGlow showed that RdVp cells exhibited cross-resistance to other HDIs such as panobinostat, belinostat, and vorinostat. We performed RNA-Seq to identify genes responsible for the observed resistance to Rd. Downstream data analysis was performed using Partek Genomics Suite, followed by network analysis using Ingenuity Pathway Analysis (IPA) software. Only expression changes with p< 0.01 were considered significant for pathway analysis. Out of 47788 total transcripts identified, only 1910 transcripts were significantly altered in RdVp cells relative to untreated controls. Transcription of several genes involved in the cholesterol and lipid biosynthesis pathway was increased in RdVp cells, indicating increased cholesterol synthesis to support increased cell growth. Several members of the sonic hedgehog pathway were elevated in cells resistant to Rd. The GLI1 gene that encodes a zinc finger protein was upregulated in RdVp cells by 14-fold. Treatment with a GLI1 inhibitor resulted in decreased survival of cells resistant to Rd. The sequencing data were analyzed to identify single nucleotide variations and short insertions/deletions, and only those present in coding sequences of RdVp cells were further confirmed with Sanger sequencing. We identified novel mutations in MCCC2, HIPK3, ABR, SIRT6, TRAK2, and ZNF100 in RdVp cells. Our results suggest potential targets for combination therapy to sensitize cancer cells resistant to romidepsin and enhance its antitumor efficacy in the clinic.

Citation Format: Fatima G. Ali-Rahmani, Robert Robey, Justin Lack, Bethelihem Tebase, Susan Bates, Michael Gottesman. Genomic characterization of breast cancer cells resistant to the histone deacetylase inhibitor romidepsin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-241.