Abstract
Activating BRAF mutations are thought to drive melanoma tumorigenesis and metastasis by constitutively activating MEK and ERK. Small molecule inhibitors (SMIs) of BRAF or MEK have shown promise as melanoma therapeutics. However, the development of resistance to these inhibitors in both the short- and long-term is common; warranting investigation into how these SMIs influence ERK signaling dynamics. Quantitative single cell imaging of ERK activity in living cells reveals both intra- and inter-cell heterogeneity in this activity in isogenic melanoma populations harboring a BRAFV600E mutation. This heterogeneity is largely due to a cell-cycle dependent bifurcation of ERK activity. Moreover, we show there are also cell-cycle dependent responses in ERK activity following BRAF or MEK inhibition. Prior to, but not following, CDK4/6-mediated passage through the Restriction Point (RP) ERK activity is sensitive to BRAF and MEK inhibitors. In contrast, for cells that have passed the RP, ERK activity will remain elevated in the presence of BRAF or MEK inhibition until mitosis. Our results show that ERK activity - even in the presence of activating BRAF mutations - is regulated by both positive and negative feedback loops that are engaged in cell-cycle dependent fashions. CDK4/6 inhibition sensitizes ERK activity to BRAF or MEK inhibition by preventing passage the transition from a BRAF/MEK dependent to independent state. Our results have implications for the use of MEK and BRAF inhibitors as melanoma therapeutics, and offer a rational basis for the use of these inhibitors in combination with CDK4/6 inhibition during cancer therapy.
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