Abstract
Treatment of BRAF‐mutant melanomas with MAP kinase pathway inhibitors is paradigmatic of the promise of precision cancer therapy but also highlights problems with drug resistance that limit patient benefit. We use live‐cell imaging, single‐cell analysis, and molecular profiling to show that exposure of tumor cells to RAF/MEK inhibitors elicits a heterogeneous response in which some cells die, some arrest, and the remainder adapt to drug. Drug‐adapted cells up‐regulate markers of the neural crest (e.g., NGFR), a melanocyte precursor, and grow slowly. This phenotype is transiently stable, reverting to the drug‐naïve state within 9 days of drug withdrawal. Transcriptional profiling of cell lines and human tumors implicates a c‐Jun/ECM/FAK/Src cascade in de‐differentiation in about one‐third of cell lines studied; drug‐induced changes in c‐Jun and NGFR levels are also observed in xenograft and human tumors. Drugs targeting the c‐Jun/ECM/FAK/Src cascade as well as BET bromodomain inhibitors increase the maximum effect (E max) of RAF/MEK kinase inhibitors by promoting cell killing. Thus, analysis of reversible drug resistance at a single‐cell level identifies signaling pathways and inhibitory drugs missed by assays that focus on cell populations.
Highlights
Treatment of BRAF-mutant melanomas with MAP kinase pathway inhibitors is paradigmatic of the promise of precision cancer therapy and highlights problems with drug resistance that limit patient benefit
To study the dynamics of BRAFV600E inhibition in melanoma cells, we performed live-cell imaging on two vemurafenib-sensitive cell lines at concentrations near the IC50 for cell killing (COLO858 and MMACSF; IC50 ~0.1–0.5 lM; we subsequently expanded the analysis to additional lines, as described below)
It has previously been show that cells expressing NGFR represent an intermediate in the process by which melanocytes differentiate from neural crest cells (Mica et al, 2013) and NGFR is used clinically as a histopathological marker to distinguish desmoplastic melanomas, tumors that are negative for conventional melanocytic markers, from other skin neoplasms (Lazova et al, 2010)
Summary
Treatment of BRAF-mutant melanomas with MAP kinase pathway inhibitors is paradigmatic of the promise of precision cancer therapy and highlights problems with drug resistance that limit patient benefit. We use live-cell imaging, single-cell analysis, and molecular profiling to show that exposure of tumor cells to RAF/ MEK inhibitors elicits a heterogeneous response in which some cells die, some arrest, and the remainder adapt to drug. Drugadapted cells up-regulate markers of the neural crest (e.g., NGFR), a melanocyte precursor, and grow slowly. This phenotype is transiently stable, reverting to the drug-naïve state within 9 days of drug withdrawal. Transcriptional profiling of cell lines and human tumors implicates a c-Jun/ECM/FAK/Src cascade in de-differentiation in about one-third of cell lines studied; drug-induced changes in c-Jun and NGFR levels are observed in xenograft and human tumors. Drugs targeting the c-Jun/ECM/FAK/Src cascade as well as BET bromodomain inhibitors increase the maximum effect (Emax) of RAF/MEK kinase inhibitors by promoting cell killing. Analysis of reversible drug resistance at a single-cell level identifies signaling pathways and inhibitory drugs missed by assays that focus on cell populations
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