Small molecule inhibition of activated MAP kinase signaling in human cancers: Biological and therapeutic implications

The MAP kinase signaling module (RAS-RAF-MEK-ERK) is a central regulator of cell proliferation, frequently deregulated in human cancers by activating mutations. Proliferation of tumors with mutant BRAF and some with mutant RAS is ERK-dependent and is suppressed by MEK inhibitors. In contrast, tumor cells with receptor tyrosine kinase activation proliferate in a MEK-independent manner. These findings have led to the development of RAF and MEK inhibitors as anticancer agents. Like MEK inhibitors, selective ATP-competitive RAF inhibitors, such as PLX4032, inhibit the proliferation of BRAFV600E tumor cells, but not that of HER kinase-dependent tumors. However, tumors with RAS mutation that are sensitive to MEK inhibition are insensitive to RAF inhibitors. Whereas MEK inhibitors inhibit ERK phosphorylation in all cell types, RAF inhibitors only inhibit ERK signaling in tumor cells expressing BRAF V600E, and paradoxically activate ERK signaling in cells with wild-type RAF. Mutant BRAF-selective responses to RAF inhibitors are also seen at the level of ERK-dependent gene expression. MEK and RAF inhibitors downregulate the transcription of a common set of genes in BRAFV600E tumors cells, while RAF inhibitors transiently induce expression of these genes in tumor cells with wild-type RAF. The mechanism of paradoxical RAF activation has recently been elucidated. In wild-type RAF cells, activation of Ras causes hetero- and homodimerization of RAF molecules, resulting in their activation, and the binding of RAF inhibitors to one protomer in these dimers allosterically trans-activates the other and stimulates downstream effectors. Tumors that express BRAFV600E have low basal levels of active RAS and therefore, low amounts of RAF dimers. Furthermore, because BRAF V600E signals as RAS-independent monomer, RAF inhibitor binding causes direct kinase inhibition and thus suppression of the pathway. These data explain why RAF inhibitors like PLX4032 selectively inhibit the growth of mutant BRAF tumors and suggest that they will not cause toxicity due to the inhibition of ERK signaling in normal cells. This also implies RAF inhibitors enjoy a broader therapeutic index compared to MEK inhibitors.;Recent clinical trials have borne out this prediction, showing that PLX4032 causes tumor regression in most patients with metastatic melanoma with mutant BRAF. Unfortunately these responses are rarely complete, with a median progression of seven months. Meanwhile, the development of low-grade squamous cell carcinoma in the skin of a third of patients may be a side effect related to the drug-mediated induction of ERK signaling in normal tissue. Several drug resistance mechanisms have been elucidated already, and include activating Ras and MEK mutations and upregulated RTK expression. We find that prolonged treatment of BRAF mutant cells with PLX4032 is associated with a rebound of ERK phosphorylation from its nadir. This rebound is accompanied by increases in the transcriptional output of the pathway and could therefore limit the therapeutic effects of the drug. In this new steady state, ERK phosphorylation and output are sensitive to MEK inhibitors but not re-addition of RAF inhibitors. The rebound of ERK signaling is associated with upstream activation in the RAS pathway. We therefore determined the effects of combined RAF and MEK kinase inhibition in a panel of melanoma cells with mutant BRAF. Combined inhibition delayed pathway rebound and resulted in more prolonged suppression of ERK output, enhanced cell death, and tumor growth inhibition compared to treatment with the RAF inhibitor alone. These results suggest that combined RAF and MEK kinase inhibition may be a useful strategy to improve the extent and duration of responses to RAF inhibitors and minimize toxicity caused by ERK activation in normal tissue.