The goal of this proposal is to develop a rational basis for a novel antileukemic strategy combining pharmacologic cyclin-dependent kinase (CDK) inhibitors with clinically relevant differentiation-inducers (DIs). This approach has been prompted by the discovery that flavopiridol (FP; NSC 649890), a potent CDK ATP-binding domain inhibitor, fails to lower the maturation threshold for human leukemia cells (e.g., U937, HL-60) despite promoting cell cycle arrest; instead, it interacts synergistically with a variety of DIs, e.g., PMA, bryostatin 1, and multiple histone deacetylase inhibitors (HDIs), including sodium butyrate (SB) and SAHA, to trigger mitochondrial damage and apoptosis. Combination of FP with DIs is associated with dysregulation of multiple signaling and cell cycle regulatory pathways, including interference with the induction of the endogenous CDK inhibitor p21 CIPI, degradation of p27KIP1, accelerated dephosphorylation and proteolysis of pRb, derepression of E2F, and enhanced activation of p42/44 MAPK.
The specific aims of this proposal are (1) to test the hypothesis, using p21 CIP1 antisense, nuclear localization signal and CDK binding domain mutants, as well as inducible constructs, that dysregulation of this CDKI contributes functionally to FP/DI-induced apoptosis; (2) to determine what role, if any, dysregulation of pRb dephosphorylation, cleavage, and E2F activation play in potentiation of DI-associated apoptosis by FP; and (3) to investigate, using stable transfectants ectopically expressing Bcl-2/Bcl-xL as well as phosphorylation-deficient mutants, mechanisms by which FP and DIs circumvent the block to mitochondrial damage and apoptosis conferred by these proteins. For each of these studies, stable cell lines inducibly expressing Raf-1 or MEK1 will be employed to define the role of the PKC/Raf/MEKIMAPK cascade in FP/DI-mediated lethality. Lastly, the effect of combinations of FP with various DIs will be compared in primary human leukemic blasts and normal progenitors (e.g., CD34+, DR-, 71-) to establish whether a basis for therapeutic selectivity exists. It is hoped that information derived from this proposal will lay the foundation for the implementation of a novel therapeutic strategy combining pharmacologic CDK inhibitors with DIs in the treatment of leukemia and possibly other hematologic malignancies.
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