Flavopiridol, a small molecule CDK inhibitor undergoing antineoplastic clinical trials, induces apoptosis in human cancer cell lines, is synergistic with a variety of other antineoplastic agents and has induced xenograft tumor regressions as well as responses in flavopiridol-treated patients. Although CDK inhibition is clearly a factor in flavopiridol-induced cytostasis, the mechanism(s) responsible for flavopiridol-induced cytotoxicity are uncertain. Because 1) NCI COMPARE analysis implicates DNA as an important cytotoxic target of flavopiridol and 2) flavopiridol does not inhibit topoisomerases, inflict DNA damage or covalently modify DNA, we hypothesized that flavopiridol-induced cytotoxicity may be attributable to its ability to disrupt protein/DNA interactions, in evaluating this hypothesis, we have found that flavopiridol 1) binds to double-stranded DNA with similar affinity constant to other DNA-interacting antineoplastic agents, 2) disrupts STAT-3/DNA interactions in vitro in three model systems, 3) downregulates antiapoptotic proteins downstream of STAT-3 including Mcl-1 in vitro at the transcriptional level and 4) downregulates the antiapoptotic protein Mcl-1 in malignant LGLs (large granular lymphocytes) ex vivo and in leukemic cells isolated from flavopiridol-treated patients with AML. We now propose to more fully examine the hypothesis that flavopiridol-induced cytotoxicity may result from its ability to disrupt survival-critical transcription factor/DNA interactions according to the following Specific Aims: 1. Further definition of the specificity of flavopiridol-induced disruption of transcription factor/DNA interactions. 2. Further characterization of the ability of flavopiridol to disrupt STAT-3/DNA binding and definition of its molecular basis. 3. Evaluation of the contributions of flavopiridol-induced disruption of STAT-mediated transcription to flavopiridol-induced cytotoxicity. 4. Preliminary examination of whether flavopiridol may represent a potential therapy in the treatment of patients afflicted with LGL malignancies. Through the proposed studies, we hope not only to gain insights that will help to clarify the mechanism(s) responsible for flavopiridol-induced cytotoxicity, but also to contribute to the understanding of transcription factor/DNA complexes as promising novel antineoplastic targets. ? ?
