Flavopiridol Targets Transcription Factor/DNA Complexes

描述(申请人提供)：正在进行抗肿瘤临床试验的小分子CDK抑制剂，可诱导人类癌细胞株的凋亡，与其他多种抗肿瘤药物有协同作用，并已诱导异种移植瘤的消退和治疗患者的反应。尽管细胞周期蛋白依赖性激酶的抑制显然是黄烷醇诱导的细胞停滞的一个因素，但黄烷醇诱导的细胞毒性的机制(S)尚不清楚。由于1)NCI比较分析表明DNA是黄烷醇的一个重要的细胞毒性靶点，2)黄烷醇不抑制拓扑异构酶，不造成DNA损伤或共价修饰DNA，我们假设黄烷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，在评估这一假说时，我们发现1)黄烷醇与双链DNA结合，亲和力常数与其他DNA相互作用的抗肿瘤药物相似，2)在体外三个模型系统中中断STAT-3/DNA相互作用。3)在体外转录水平下调包括Mcl-1在内的STAT-3下游抗凋亡蛋白；4)在体外下调恶性大颗粒淋巴细胞和急性髓系白血病患者分离的白血病细胞中抗凋亡蛋白Mcl-1的表达。我们现在建议根据以下特定的目的来更全面地检验黄烷醇诱导的细胞毒性可能是由于其破坏关键的转录因子/DNA相互作用的能力所致的假说：1.进一步定义黄烷醇诱导的转录因子/DNA相互作用的特异性。2.进一步鉴定黄烷醇破坏STAT-3/DNA结合的能力，并确定其分子基础。3.评价黄烷醇诱导的STAT转录中断在黄烷醇诱导的细胞毒作用中的作用。4.初步探讨黄烷醇是否有可能成为治疗LGL恶性肿瘤的潜在疗法。通过所提出的研究，我们不仅希望获得有助于阐明黄烷醇诱导的细胞毒性的机制(S)的见解，而且也有助于理解转录因子/脱氧核糖核酸复合体作为新的抗肿瘤靶点的前景。

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.