Single-cell transcriptomics reveals biomarker heterogeneity linked to CDK4/6 Inhibitor resistance in breast cancer cell lines.

Cyclin dependent kinases 4 and 6 inhibitors have brought great improvements in the treatment of luminal breast cancer, but resistance is a major clinical hurdle. Multiple biomarkers of resistance have been proposed, but none is currently utilized in clinical practice. By performing single-cell RNA sequencing of seven palbociclib-naïve luminal breast cancer cell lines and palbociclib-resistant derivatives, we show that established biomarkers and pathways related to CDK4/6i resistance present marked intra- and inter- cell-line heterogeneity. Transcriptional features of resistance could be already observed in naïve cells correlating with levels of sensitivity (IC50) to palbociclib. Resistant derivatives showed transcriptional clusters that significantly varied for proliferative, estrogen response signatures or MYC targets. This marked heterogeneity was validated in the FELINE trial where, compared to the sensitive ones, ribociclib-resistant tumors developed higher clonal diversity at genetic level and showed greater trascriptional variability for genes associated with resistance. A potential signature of resistance inferred from the cell-line models, positively enriched for MYC targets and negatively enriched for estrogen response markers, was probed on the FELINE trial, separating sensitive from resistant tumors and revealing higher heterogeneity in resistant versus sensitive cells. These data suggest that heterogeneity for CDK4/6 inhibitors resistant markers might facilitate the development of resistance and challenge the validation of clinical biomarkers.