A novel BH3 mimetic reveals a mitogen-activated protein kinase-dependent mechanism of melanoma cell death controlled by p53 and reactive oxygen species.

The RAS/BRAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is emerging as a key modulator of melanoma initiation and progression. However, a variety of clinical studies indicate that inhibiting the MAPK pathway is insufficient per se to effectively kill melanoma cells. Here, we report on a genetic and pharmacologic approach to identify survival factors responsible for the resistance of melanoma cells to MEK/ERK antagonists. In addition, we describe a new tumor cell-selective means to bypass this resistance in vitro and in vivo. By generating a panel of isogenic cell lines with specific defects in the apoptotic machinery, we found that the ability of melanoma cells to survive in the absence of functional MEK relies on an ERK-independent expression of the antiapoptotic factor Mcl-1 (and to a lesser extent, Bcl-x(L) and Bcl-2). Using computer-based modeling, we developed a novel Bcl-2 homology domain 3 (BH3) mimetic. This compound, named TW-37, is the first rationally designed small molecule with high affinity for Mcl-1, Bcl-x(L), and Bcl-2. Mechanistic analyses of the mode of action of TW-37 showed a synergistic tumor cell killing in the presence of MEK inhibitors. Importantly, TW-37 unveiled an unexpected role of the MAPK pathway in the control of reactive oxygen species (ROS). This function was critical to prevent the activation of proapoptotic functions of p53 in melanoma cells, but surprisingly, it was dispensable for normal melanocytes. Our results suggest that this MAPK-dependent ROS/p53 feedback loop is a point of vulnerability of melanoma cells that can be exploited for rational drug design.

Cerebral postischemic reperfusion-induced demethylation of the protein phosphatase 2A catalytic subunit.

Brain reperfusion after a period of global ischemia induces changes in the phosphorylation state of a great number of proteins. Neuronal responses to ischemia and reperfusion are quite different depending on the brain region, and phosphorylation changes may be implicated in this tissue-specific response. For this reason, we have used both biochemical and immunohistochemical methods to investigate the potential role of PP2A, the most abundant Ser/Thr phosphatase in the brain, in ischemic injury. PP2A activity as measured with phosphorylase a as substrate was slightly inhibited after 30 min ischemia followed by 30 min reperfusion, and this inhibition correlated with an increased S6K1 and ERK1/2 phosphorylation. Using a monoclonal antibody unable to recognize the methylated form of PP2Ac, we demonstrated that the catalytic subunit of PP2A (PP2Ac) was highly methylated in the brain. In addition, the postischemic reperfusion-induced changes in PP2Ac methylation were studied in sections from cerebral cortex, hippocampus and striatum. Regional differences in PP2Ac methylation were observed within control brains, and the postischemic reperfusion caused a generalized demethylation of PP2Ac. Those regions in the control brains containing highest levels of methylated PP2Ac were the most intensively demethylated after reperfusion and corresponded to the regions most vulnerable to ischemic damage.