Abstract 3012: Single-cell transcriptomic characterization of luminal breast cancer cell lines with acquired resistance to the CDK4/6 inhibitor palbociclib
Authors: Benelli Matteo , Boccalini Giulia , Romagnoli Dario , Bonechi Martina , Biagioni Chiara , Schiff Rachel , Angelis Carmine De , Leo Angelo Di , Migliaccio Ilenia , Malorni Luca 
Published: August 2019

CDK4/6 inhibitors, such as palbociclib, target the cyclin-dependent kinases 4 and 6, essential for cell cycle regulation. CDK4/6 inhibitors are established treatment options in patients with endocrine receptor positive, HER2 negative metastatic breast cancer (BC).

We recently conducted a transcriptomic study of seven palbociclib resistant luminal BC cell lines, demonstrating a common transcriptional signature of resistance to palbociclib, including the over-expression of CCNE1 and under-expression of RB1. Genomic analysis based on ddPCR and whole exome sequencing (WES) demonstrated CCNE1 over-expression due to gene amplification in only two cell lines, and RB1 under-expression due to gene deletion in 3 out of 7 cell lines. This prompts the hypothesis that more complex molecular mechanisms of CCNE1 over-expression / RB1 under-expression may exist.

To improve the characterization of the molecular mechanisms underlying the resistance to palbociclib, we performed single-cell transcriptomic profiling (single-cell RNA-seq, scRNA-seq) of seven BC cell line resistant to palbociclib and their sensitive counterparts. Cells were isolated using the Bio-Rad ddSEQ single cell isolator and sequenced in order to obtain about 100,000 reads per cell. A total of 10,557 cells with at least 2,000 genes read per cell were selected for downstream analysis, corresponding to 5,116 palbociclib sensitive cells and 5,441 palbociclib resistant cells.

Considering the 5,000 most variable genes, dimensionality reduction analysis based on t-distributed stochastic neighbor embedding (tSNE) clearly segregated cells depending on their cell type, as expected based on previously generated bulk RNA-seq data. Using previously identified top cell-type specific differentially expressed genes between resistant and sensitive cell lines (absolute log2(fold-change) &gt; 0.5), dimensionality reduction analysis correctly classified 98.2% of cells based on their sensitivity/resistant status, demonstrating the quality of our scRNA-seq data. Interestingly, we observed a small fraction (&lt;1.5%) of misclassified sensitive cells showing resistant-like phenotypes. Co-expression analysis suggests modulation of key gene expression programs between sensitive and resistant cells, including E2F signaling. Further analyses to validate these data are ongoing and the results will be presented during the meeting.

This study represents, to our knowledge, the first attempt to generate a single-cell transcriptomic profiling of a large panel of luminal BC cell lines resistant to palbociclib. Results from this study might lead to the identification of new biomarkers of de-novo and acquired resistance to palbociclib, which would assist to better personalize treatment of patients with endocrine receptor positive BC.

Citation Format: Matteo Benelli, Giulia Boccalini, Dario Romagnoli, Martina Bonechi, Chiara Biagioni, Rachel Schiff, Carmine De Angelis, Angelo Di Leo, Ilenia Migliaccio, Luca Malorni. Single-cell transcriptomic characterization of luminal breast cancer cell lines with acquired resistance to the CDK4/6 inhibitor palbociclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3012.