The goal of this proposal is to elucidate the mechanism(s) underlying recent observations that inhibitors of the MEK/MAPK signal transduction pathway a) markedly potentiate paclitaxel- and taxotere-induced lethality toward leukemic cells in a sequence-dependent manner and b) circumvent resistance coffered by Bcl-2/Bcl-xL-mediated blockade of the cell death pathway. We will test the hypothesis that these phenomenon involve perturbations in stress-survival signaling, post-translational modification of anti-apoptotic proteins (Bcl-2/Bcl-xL), or cell cycle dysregulation, (e.g., promotion of mitotic arrest and/or disruption of the mitotic spindle checkpoint).
In Aim #1, we will relate enhancement of taxane-mediated apoptosis by subsequent exposure to selective MEK/MAPK inhibitors (PD98059, U0126, and SL327) and agents that interrupt the PKC/MAPK axis (e.g., bryostatin 1, CGP41251) to specific alterations in JNK/MAPK signaling and G2M arrest events, particular CDK1 activation.
In Aim #2, an inducible Raf-1 activation system will be used to test functionally the hypothesis that such inhibitors enhance paclitaxel by inhibiting the Raf-1 downstream targets MEK1/2 and MAPK.
In Aim #3, cells over- expressing actions of MEK/MAP kinase inhibitors involve modulation of Bcl-2/Bcl-xL phosphorylation status and/or a diminution in the threshold of taxane-induced mitochondrial dysfunction.
In Aim #4, the hypotheses that MEK/MAP kinase inhibitors act by antagonizing CDK1 activation (thereby promoting mitotic arrest) or, alternatively, induction of the cyclin-dependent kinase inhibitor p21/CIP1 in taxane-pretreated cells will be tested utilizing the CDK1 inhibitor butyrolactone I and p21/CIP1 antisense-expressing cell lines respectively. Finally, comparisons will be made between the effects of MEK/MAPK inhibitors on taxane-induced lethality toward normal (VFU-GM, HPP-CFC) versus leukemic (L-CFU) progenitors to identify a possible basis for therapeutic selectivity. This information could provide a rationale for using novel MEK/MAP kinase inhibitors with in vivo activity to enhance the anti-tumor activity of taxanes in hematological and ultimately non-hematological malignancies.
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