Flavopiridol Targets Transcription Factor/DNA Complexes

描述（由申请人提供）：黄匹利多是一种正在进行抗肿瘤临床试验的小分子CDK抑制剂，可诱导人类癌细胞系凋亡，与多种其他抗肿瘤药物协同作用，并在黄匹利多治疗的患者中诱导异种移植肿瘤消退和应答。虽然CDK抑制显然是黄嘌呤诱导的细胞停滞的一个因素，但黄嘌呤诱导的细胞毒性的机制尚不确定。因为1)NCI COMPARE分析表明DNA是黄酮吡醇的重要细胞毒性靶点，2)黄酮吡醇不会抑制拓扑异构酶，造成DNA损伤或共价修饰DNA，我们假设黄酮吡醇诱导的细胞毒性可能归因于其破坏蛋白质/DNA相互作用的能力，在评估这一假设时，我们发现黄酮吡醇1)与双链DNA结合，与其他DNA相互作用的抗肿瘤药物具有相似的亲和力常数。2)在体外三种模型系统中破坏STAT-3/DNA的相互作用，3)在体外转录水平下调STAT-3下游的抗凋亡蛋白，包括Mcl-1, 4)下调体外恶性LGLs（大颗粒淋巴细胞）和黄烷醇治疗的AML患者分离的白血病细胞中的抗凋亡蛋白Mcl-1。我们现在建议根据以下具体目的，更全面地研究黄烷醇诱导的细胞毒性可能是由于其破坏生存关键转录因子/DNA相互作用的能力所致：进一步定义黄烷醇诱导的转录因子/DNA相互作用中断的特异性。2. 进一步表征黄吡醇破坏STAT-3/DNA结合的能力，并确定其分子基础。3. 黄嘌呤诱导的stat介导的转录中断对黄嘌呤诱导的细胞毒性的贡献评估。4. 初步检查黄匹吡醇是否可能代表治疗LGL恶性肿瘤患者的潜在疗法。通过提出的研究，我们希望不仅能够获得有助于阐明黄烷醇诱导细胞毒性的机制的见解，而且还有助于理解转录因子/DNA复合物作为有希望的新型抗肿瘤靶点。

DESCRIPTION (provided by applicant): 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.