Abstract B058: Deconvoluting the intra-tumour heterogeneity and subclonal evolution of CDK4/6 inhibitor resistance in ER+ breast cancer
Authors: Ioanna Mavrommati, Fatemeh Ahmadi Moughari, Shefali Thakur, Yu Zhang, Lesley-Ann Martin, Syed Haider, Rachael Natrajan
Journal: Cancer Research (2024)

Abstract Subclonal heterogeneity and evolution are characteristics of breast cancer (BC), that play a key role in tumour development, progression, and resistance to current therapies. Preliminary single-cell sequencing studies suggest that subclonal heterogeneity drives therapy response in aggressive BC. Here, we sought to identify whether pre-existing transcriptomically defined cell subpopulations underpin resistance to CDK4/6 inhibitors (CDK4/6i) in ER+ BC, and to identify the distinct or shared resistance mechanisms between different CDK4/6i currently used in the clinic. ER+ BC cell lines, MCF7 and T47D, were infected with a lentiviral barcode library and were long-term exposed to DMSO or CDK4/6i (Abemaciclib or Palbociclib) until drug resistance was achieved. CDK4/6i resistant or DMSO cell lines were then subjected to DNA sequencing for the detection of barcodes coupled with single-cell transcriptomic and barcode sequencing, whole exome sequencing and G1/S cell cycle checkpoint analysis at a protein level. Single-cell lineage tracing identified that resistance to both CDK4/6i in MCF7 cells was stochastic, while in T47D cells, resistance to both CDK4/6i was conserved across all biological replicates. In all cell models, abemaciclib exerted a stronger selective pressure than palbociclib. Detection of barcodes from dead cells highlighted differential selective pressures of the drugs during resistance generation and upon resistance emergence. Single-cell transcriptomic sequencing identified shared and differential mechanisms of resistance between cell lines and specific CDK4/6i, suggestive of different subclonal, non-genetic mechanisms driving resistance. Some of these subclones were pre-defined (i.e. shared transcriptomic features with parental cells), whereas other resistant subclones were demarked by acquisition of pathway up-regulation. For instance, RB1 expression was abolished in the entire palbociclib-resistant T47D population, with copy number loss, indicating RB1 as a genetic driver of resistance. No evidence of RB1 loss was observed in MCF7 palbociclib-resistant clonal population, however a subclonal cluster was identified with loss of RB1 expression. Interestingly, abemaciclib-resistant MCF7 cells had lost RB1 protein expression, suggesting differential non-genetic mechanisms of resistance to CDK4/6i in MCF7 cells. Through single-cell lineage tracing, we have identified that resistance to CDK4/6i can be stochastic or conserved according to the cell model. Additionally, single-cell transcriptomic sequencing identified that in some cell models, cells that have the ability to become resistant are pre-existing, suggesting transcriptomic and epigenomic mechanisms of resistance, where cells exhibit drug-tolerant or drug-induced persistent properties. Citation Format: Ioanna Mavrommati, Fatemeh Ahmadi Moughari, Shefali Thakur, Yu Zhang, Lesley-Ann Martin, Syed Haider, Rachael Natrajan. Deconvoluting the intra-tumour heterogeneity and subclonal evolution of CDK4/6 inhibitor resistance in ER+ breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B058.