Adaptive resistance of melanoma cells to RAF inhibition via reversible induction of a slowly dividing de‐differentiated state

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.

Epigenetics in Melanoma Development and Drug Resistance

“…Interestingly, a similar transition into an H3K4me3 low /H3K27me3 low /H3K9me3 high state was triggered by hypoxia and nutrient starvation and IDTCs generated by these stressors exhibited tolerance to BRAF inhibitors or cisplatin treatment, suggesting an epigenetically regulated drug-independent generic stress response that allows cells to cope with difficult environmental conditions [91]. Similar to our proposed IDTCs, a slow cycling, reversible NGFR high state that displays features of de-differentiation has also been described, which has been shown to be susceptible to inhibition of epigenetic modifiers as bromodomain inhibitors, that block recognition of acetylated histones, suppressed the slowly cycling NGFR high state [92].…”

A cancer pharmacogenomic screen powering crowd-sourced advancement of drug combination prediction

“…Alternatively, more generic signatures of dynamic (e.g. transcriptional) output may first be used to identify a mechanistic rationale 19,20,21,22 to which causative genetic or epigenetic events can then be inferred and aligned as predictive features 23,24 . A surprising result of our Challenge, however, suggested only modest improvement to prediction from inclusion of all data in SC1A compared to only genetics in SC1B.…”

“…The dynamic nature of mechanistic models may offer an advantage by enabling consideration of the heterogeneity that exists across even apparently 'clonal' cell line populations 21 . The increasing availability of published pre-derived mechanistic models for many cancer relevant pathways may soon make such an approach more viable.…”

A biomaterial screening approach reveals microenvironmental mechanisms of drug resistance

“…Mechanistically, kinase inhibitor treatment can decrease shedding of RTKs, thus enhancing bypass signaling 4 . Drug treatment can also cause changes in the cell cycle, promoting resistance in the quiescent cells adapting to the treatment 5 . These examples demonstrate that quick and widespread changes in signaling can occur in response to anti-cancer drugs.…”