| An important feature of most cases of non-small cell lung cancer (NSCLC) is deregulation of the classical MAPK pathway (Ras/Raf/MEK/ERK cascade). The upstream epidermal growth factor receptor (EGFR) is frequently mutated and/or overexpressed and activating mutations of the K-Ras gene have been found in up to 30% of lung adenocarcinomas. BRAF, a major downstream effector of Ras, has been found to be mutated in a smaller but significant subset of lung adenocarcinomas. In a mouse model, lung-specific expression of the activating V600E BRAF mutant induces the development of lung adenocarcinoma with phenotypic similarities to human bronchioloalveolar carcinoma (BAC).
Tumors containing more than one of these mutations are extremely rare. This observation initially suggested that the aforementioned mutations provide virtually the same tumorgenic stimulus. However, recent work from our lab and others suggests that the MAPK network is considerably more complex. There is marked variability in tumor dependence upon the pathway as a function of the specific activating mutation (EGFR, K-Ras or BRAF).
Tumor addiction to a particular molecular abnormality provides opportunity for targeted therapeutic intervention with selective pharmacologic inhibitors. This concept has been validated in cell culture models, in mice and in patients with other cancers, such as CML (chronic myelogenous leukemia) and GIST (gastrointestinal stromal tumors). NSCLC has not only proven highly lethal, it has also been extremely refractory to broadly applied chemotherapy. Targeted therapy of the MAPK pathway in NSCLC is highly attractive because it may offer effective therapy with less toxicity. Recently, EGFR inhibitors conferred improved survival to patients with advanced NSCLC when combined with chemotherapy. Our lab recently demonstrated that tumors harboring BRAF mutations depend on MEK/ERK signaling and are sensitive to inhibition of the downstream target, MEK. In collaboration with Dr. G. Bollag (Plexikkon Inc) we are developing and evaluating the highly potent and selective Raf kinase inhibitor PLX4032. PLX4032 is currently in a Phase I clinical trial involving patients with metastatic melanoma in Memorial Sloan-Kettering Cancer Center and at three additional Cancer Centers in the U.S. Our preliminary data suggest that, even though Raf and MEK are considered to lie within a linear signaling pathway, the effects of Raf inhibition differ substantially from those of MEK inhibition. The most profound difference is that, unlike MEK inhibition which effectively downregulates MAPK in all cell lines, Raf inhibitors selectively inhibit MAPK signaling only in cells carrying mutant BRAF. We hypothesize that such differences in the consequences of MEK inhibition and Raf inhibition are due to feedback regulation of the MAPK pathway at the level of Raf. Moreover, we propose that Raf inhibition can be an effective therapeutic strategy for the specific subset of NSCLC patients whose tumors depend on Raf activity.
Our proposal is outlined in the following specific aims:
Aim 1. To compare the effect of the Raf kinase inhibitor PLX4032 to MEK inhibition in NSCLC in lung cancer cells and in lung tumors.
Aim 2. To determine the effect of Raf inhibition on Ras/Raf/MEK complex formation by comparing biochemical Raf inhibition with Raf inhibition in cells. To determine the effect of Raf inhibition on the heterodimerization between BRAF and c-Raf and the formation of the signaling complex with Ras and MEK.
Our project has two parallel aims that bridge basic and translational research: We will validate the use of Raf kinase inhibitor PLX4032 for targeted therapeutic intervention in NSCLC. In addition, we will use this specific inhibitor as a tool to interrogate the regulation of the MAPK pathway. Our findings will help elucidate mechanisms of regulation of Raf activity, which shall ultimately have important clinical implications.
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