Abstract
Drug treatment causes resistance in cells with transiently high expression of drug resistance markers.
Major finding: Drug treatment causes resistance in cells with transiently high expression of drug resistance markers.
Concept: Drug-driven chromatin changes promote sequential expression of dedifferentiation and signaling genes.
Impact: Single-cell drug resistance dynamics may provide insight into intermittent drug dosing strategies.
The events driving drug resistance in cancer cells which do not harbor secondary resistance mutations have not been fully ascertained; however, evidence suggests that cell-to-cell differences in levels of proteins driving resistance may give rise to these rare nonmutated cancer cells. To elucidate the mechanisms underlying rare cell variability, Shaffer and colleagues evaluated single-cell cultures established from patient-derived melanoma. Resistant clones which arose after vemurafenib treatment did not harbor new mutations after drug treatment but did exhibit increased expression of genes associated with drug resistance, including AXL and EGFR, compared to pretreatment cells. High-throughput single-molecule RNA FISH identified rare pretreatment cells that exhibited elevated expression of drug resistance genes before drug treatment and showed that resistant colonies exhibited uniformly high expression of drug resistance genes after continuous vemurafenib treatment. Extended treatment of FACS-sorted EGFR-positive cells with vemurafenib resulted in an increased frequency of resistant colonies compared with that produced by extended treatment of unsorted cells. Single-cell RNA FISH demonstrated that multiple cancer types harbored rare cells that exhibit high, sporadic expression of drug resistance markers in a cancer type–specific manner, and bioinformatic analyses revealed that differentiation and housekeeping genes were coexpressed with drug resistance markers. Growth of EGFR-positive cells without vemurafenib followed by extended vemurafenib treatment reduced the frequency of resistant EGFR-positive cells, suggesting that cells exhibiting high expression levels of drug resistance markers are transiently drug resistant before drug treatment and become stably resistant after drug treatment. Gene expression and chromatin accessibility profiling of vemurafenib-treated EGFR-positive cells at different timepoints revealed that drug treatment induced cellular reprogramming, via changes in chromatin accessibility, that first resulted in decreased expression of differentiation genes and subsequently increased expression of signal transduction genes. These results identify and characterize drug tolerance dynamics in rare cells and suggest potential approaches for designing intermittent drug dosing regimens.