OSU 2S induces caspase dependent apoptosis through ROS dependent PKC activation

The mechanism of this kinome reprogramming involved the proteolytic degradation of c Myc following MEK1 and MEK2 inhibition which resulted in increased キナーゼ 阻害剤 expression and activity of RTKs. MIB/MS analysis showed that reprogrammed kinase activation overcame MEK2 inhibition leading to therapeutic resistance. The MEK inhibitor kinome response signature allowed us to predict and test the efficacy of a novel small molecule kinase inhibitor combination. The combination synergistically inhibited TNBC cell line proliferation and caused apoptosis and tumor regression in the C3Tag GEMM of basal like/claudin low TNBC. TNBC clinical trials of single kinase inhibitors have largely failed, consistent with drug induced activation of alternative survival signaling pathways.<br><br> Figure 1A outlines our strategy to interrogate kinome dynamics with the goal of defining endpoints leading to rational design of combination therapies. RNA seq defined the transcript level expressed purchase Lenalidomide kinome and affinity capture of endogenous kinases followed by quantitative mass spectrometry measured kinome activity profiles in tumors and cells. The proteomic assessment was used to define the kinome response to targeted inhibition of kinases. RNAi tested growth and survival functions of the kinases activated in response to inhibitors, and the cumulative results were used to rationally predict kinase inhibitor combinations to test in models of TNBC. RNA seq defined the kinome transcript expression profile of a patients claudin low breast tumor and two claudin low TNBC lines, SUM159 and MDA MB 231.<br><br> Greater than 400 of the 518 human protein kinases were expressed in the claudin low human TNBC tumor and cell lines. Approximately 10% of the kinases LY2603618 IC-83 expressed in the claudin low patient tumor were unique compared to the claudin low cell lines, undoubtedly due to the tumors complex cellular composition. The majority of expressed kinases are common between tumor and claudin low cell lines, suggesting that interrogating the cellular kinome response to inhibitors will be relevant to patient tumors. Profiling kinase activity in tumors and cell lines was carried out using Multiplexed Inhibitor Beads, which consist of mixtures of Sepharose beads with covalently immobilized, linker adapted, kinase inhibitors of moderate selectivity for different kinases and relatively broad pan kinase inhibitors.<br><br> Kinase capture is reproducible and is a function of kinase expression, the affinity of kinases for the immobilized inhibitors, and the activation state of the kinase. Acute changes in activation dependent binding were demonstrated by the increased binding of MAPK pathway kinases in EGF stimulated cells and the increased retention of Tyr kinases from cells treated with the Tyr phosphatase inhibitor pervanadate. Our data showed that MIBs capture the majority of the expressed kinome estimated by RNA seq and detect altered kinome activity profiles in response to stimulus or clinical kinase inhibitors. Using MIBs and mass spectrometry, we have cumulatively sequence identified more than 320 kinases from cell lines and tumors. MIB/MS profiling of an invasive ductal carcinoma breast tumor and two claudin low cell lines identified approximately 50 60% of the expressed kinome.