Signal Transduction Events and the Regulation of Cell Growth

该项目旨在通过研究生长、生存和转移调节信号转导事件，确定抗癌药物开发的分子靶点，开发一种新的癌症治疗方法。我们的工作分为基础研究和转化研究，通过临床前开发研究核心，我们已经建立了一个转化药物开发设施。我们的工作目前集中在(1)β -catenin调节造血细胞的分子机制和β -catenin作为血液恶性肿瘤靶点的鉴定(2)开发和实施新的药效学分析，包括抗血管生成治疗的分析。(1)在研究洛伐他汀的抗癌作用时，我们发现对洛伐他汀促凋亡活性的敏感性的关键决定因素是β -连环蛋白的完整性。洛伐他汀是一种被NCI引入I期临床试验的药物，作为我们研究的直接翻译。这促使我们研究β -连环蛋白在细胞凋亡中的作用。我们使用血液恶性肿瘤作为我们的模型，发现-连环蛋白在这些细胞中起着意想不到的重要作用。我们的数据表明-连环蛋白调节白血病细胞的存活、增殖和粘附特性。这些数据是首次确定β -连环蛋白作为血液恶性肿瘤抗癌药物开发的靶标（Chung等）。血液100:982-990,2002)。为了实现我们的假设，即-catenin信号在血液恶性肿瘤中不受调节，并且每种恶性肿瘤都与一种特征性的不受调节机制有关，我们与泌尿肿瘤科的Tomohiro Kajiguchi合作，研究了-catenin在两种形式的白血病，肥大细胞白血病和FLT3 AML中的作用。我们发现-连环蛋白是酪氨酸激酶c-kit的底物，而酪氨酸激酶c-kit在肥大细胞白血病中不受调控。本研究表明c-kit在人肥大细胞白血病中上调Wnt信号，β -catenin是治疗肥大细胞增多症和肥大细胞白血病（Leuk）的新靶点。Res. 32:761-770, 2007)。通过突变或过表达激活FLT3在髓性白血病发生中起关键作用。我们证明FLT3在FLT3阳性的AML细胞系和原发性白血病细胞中调控β -catenin酪氨酸磷酸化、核定位和靶基因表达（leukemia 21:2476-2484, 2007）。我们已经和dr。NCI代谢分支的John Janik和John Morris根据代谢分支协议研究成人t细胞白血病患者β -连环蛋白信号传导的机制。急性ATL预后非常差，尽管几十年来人们已经知道ATL的病原是HTLV-1病毒，并且HTLV-1编码的Tax在HTLV-1诱导的恶性转化中起关键作用。虽然Tax在最初的转化过程中起着关键作用，但在急性ATL中往往检测不到Tax的表达。因此，在ATL最先进和最迅速发展的形式中，以税收为目标似乎不是一个可行的策略。我们发现：(1)原代急性ATL细胞表达β -catenin， (2) β -catenin在缺乏Tax癌蛋白的情况下表达，(3)β -catenin蛋白定位于Tax阴性ATL细胞的细胞核。(4)我们的合作者John Brady使用Affymetrix阵列对原发性ATL患者样本进行转录分析，发现β -catenin转录伙伴TCF4和β -catenin/TCF4靶基因survivin的高水平表达。最近，生存率已被证明是ATL中最不利的预后因素。我们已经成功地转染了原代ATL细胞，并使用该技术转染了野生型β -连环蛋白和一组阻断核β -连环蛋白信号传导以及控制siRNA和β -连环蛋白siRNA的构建物。这些实验表明，在原代ATL细胞中，survivin和强效抗凋亡基因Bfl-1受β -catenin的转录调控。对原代ATL细胞中β -catenin过度表达和激活途径的分析表明，β -catenin稳定和上调β -catenin核定位的解除调控事件的复杂模式，包括Akt磷酸化和CD45沉默。最近有研究表明，塞来昔布等非甾体抗炎药可显著下调核β -catenin水平，阻断核β -catenin信号传导。我们筛选了一组针对原代ATL细胞和htlv -1感染细胞系的非甾体抗炎药，发现塞来昔布具有最有利的效价与毒性比，抑制β -连环蛋白核信号传导并诱导细胞死亡。总之，这些数据确定核β -连环蛋白作为一种新的治疗靶点在先进的，不依赖税收ATL。作为一项开放的ATL试验和两项目前正在编写的β -catenin靶基因survivin抑制剂方案的共同研究人员，我们正在实施ATL中β -catenin信号传导的研究。(2)临床前开发研究核心一直在与校内、校外和行业研究人员合作进行一系列I期和II期临床试验。我是14项临床试验的副研究员。对于这些试验，我们与PI合作开发新的药效学终点，包括循环内皮祖细胞和成熟内皮细胞的分析。今年我们分析了120多名患者的这些参数。我们对抑制激素难治性前列腺癌细胞生长的信号转导通路的基础研究使我们确定组蛋白去乙酰化酶是该肿瘤的关键靶点。我们开发了一种新的药效学方法来评估体内HDAC抑制剂的活性。NCI已经为我们的工作申请了专利，这项工作具有独特的能力，可以分析HDAC抑制剂在手指棒一样少的血液中的活性，并且可以通过同时检查10个参数来观察联合治疗的药效学反应。我们已经在今年发表的两项临床试验中实施了这项技术（Gojo等人）。《中华医学杂志》，2007年第9期。巨蟹座，13:5411-5417,2007)。我们已经和dr。布罗德研究所的Jay Bradner和Stuart Schreiber使用我们的技术开发新的HDAC抑制剂，并与Nereus制药公司的Michael Palladino博士合作研究HDAC抑制剂与新型Nereus蛋白酶体抑制剂NPI-0052的联合使用。今年，我们已经与Syndax制药公司完成了CRADA协议，以支持实验室中的HDAC抑制剂研究，我们的HDAC抑制剂多参数流动测定被NCI选为2007年的杰出技术。我们在《Current Opinion in Oncology》杂志上发表了一篇特邀评论，分析了HDAC作为分子靶点的最新进展。我们已经开发了一种新的药理学方法，一种基于细胞的组织微阵列，用于评估体内抗癌药物的活性（drug Dev. Res. 68:226- 234,2007）。我们证明，细胞微阵列可用于以高通量方式测量药物反应，允许分析[摘要截断为7800个字符]。

This project is designed to develop a new approach to cancer treatment through the study of growth, survival, and metastasis regulatory signal transduction events that identify molecular targets for anticancer drug development. Our work is divided into basic research and translational research through the Preclinical Development Research Core, a translational drug development facility that we have established. Our work is currently focused on (1) the molecular mechanisms of hematopoietic cell regulation by beta-catenin and the identification of beta-catenin as a target in hematologic malignancies (2) development and implementation of novel pharmacodynamic assays, including assays for antiangiogenic therapy. (1) While studying the anticancer action of lovastatin, a drug that was brought to Phase I clinical trial at the NCI as a direct translation of our research, we found that a critical determinant of sensitivity to the proapoptotic activity of lovastatin was the integrity of beta-catenin protein. This led us to examine the role of beta-catenin in apoptosis. We used hematologic malignancies as our model and found that beta-catenin plays an unexpectedly vital role in these cells. Our data demonstrated that beta-catenin regulates leukemia cell survival, proliferation, and adhesive properties. These data were the first to identify beta-catenin as a target for anticancer drug development in hematologic malignancies (Chung et al. Blood 100:982-990, 2002). To pursue our hypothesis that beta-catenin signaling is deregulated in hematologic malignancies, and that each malignancy is associated with a characteristic mechanism of deregulation, in collaboration with Tomohiro Kajiguchi of the Urologic Oncology Branch we have studied beta-catenin in two forms of leukemia, mast cell leukemia and FLT3 AML. We found that beta-catenin is a substrate for the tyrosine kinase c-kit, which is deregulated in mast cell leukemia. This study demonstrated that c-kit upregulates Wnt signaling in human mast cell leukemia, and that beta-catenin is a novel target for the treatment of mastocytosis and mast cell leukemia (Leuk. Res. 32:761-770, 2007). FLT3 activation via mutation or overexpression plays a key role in myeloid leukemogenesis. We demonstrated that FLT3 regulates beta-catenin tyrosine phosphorylation, nuclear localization, and target gene expression in FLT3-positive AML cell lines and primary leukemia cells (Leukemia 21:2476-2484, 2007). We have established a collaboration with Drs. John Janik and John Morris of the Metabolism Branch, NCI to investigate the mechanism of beta-catenin signaling in adult T-cell leukemia patients on Metabolism Branch protocols. Acute ATL has a very poor prognosis, despite the fact that it has been known for decades that the etiologic agent of ATL is the HTLV-1 virus, and that HTLV-1-encoded Tax plays a key role in HTLV-1-induced malignant transformation. Although Tax plays a critical role in the initial transformation process, Tax expression is frequently undetectable in acute ATL. Thus, targeting of Tax would not appear to present a viable strategy in the most advanced and rapidly progressive form of ATL. We have discovered that (1) primary acute ATL cells express beta-catenin, (2) beta-catenin expression occurs in the absence of the Tax oncoprotein, (3) beta-catenin protein localizes to the cell nucleus in Tax-negative ATL cells, and (4) transcriptional analysis of primary ATL patient samples by our collaborator John Brady using Affymetrix arrays demonstrates high levels of expression of the beta-catenin transcriptional partner TCF4 and the beta-catenin/TCF4 target gene survivin. Recently survivin has been shown to be the most negative prognostic factor in ATL. We have succeeded in transfecting primary ATL cells, and have used this technique to transfect wild-type beta-catenin and a panel of constructs that block nuclear beta-catenin signaling as well as control siRNA and beta-catenin siRNA. These experiments demonstrated that in primary ATL cells survivin and the potent antiapoptotic gene Bfl-1 are under the transcriptional control of beta-catenin. Analysis of the pathways leading to beta-catenin overexpression and activation in primary ATL cells demonstrated a complex pattern of deregulatory events that stabilize beta-catenin and upregulate beta-catenin nuclear localization including Akt phosphorylation and CD45 silencing. Recently it has been demonstrated that NSAIDs such as celecoxib significantly down-regulate nuclear beta-catenin levels and block nuclear beta-catenin signaling. We screened a panel of NSAIDs against primary ATL cells and HTLV-1-infected cell lines and found that celecoxib had the most-favorable ratio of potency to toxicity, inhibited beta-catenin nuclear signaling and induced cell death. Together these data identify nuclear beta-catenin as a novel therapeutic target in advanced, Tax-independent ATL. We are implementing our studies of beta-catenin signaling in ATL as co-investigators on one open ATL trial and on two protocols currently being written for inhibitors of the beta-catenin target gene survivin. (2) The Preclinical Development Research Core has been working with intramural, extramural and industry investigators on a range of phase I and phase II clinical trials. I am an associate investigator on 14 clinical trials. For each of these trials we work with the PI to develop novel pharmacodynamic endpoints, including analysis of circulating endothelial progenitor cells and mature endothelial cells. This year we have analyzed over 120 patients for these parameters. Our basic research on signal transduction pathways that can inhibit the growth of hormone-refractory prostate cancer cells led us to the identification of histone deacetylase as a critical target in this neoplasm. We have developed a novel pharmacodynamic assay for assessment of HDAC inhibitor activity in vivo. The NCI has applied for a patent on our work, which is uniquely capable of analyzing HDAC inhibitor activity in as little blood as in a finger-stick, and can look at combination therapy pharmacodynamic responses by examining 10 parameters simultaneously. We have implemented this technology in 2 clinical trials published this year (Gojo et al. Blood 109:2781-2790, 2007 and Kummar et al., Clin. Cancer Res. 13:5411-5417, 2007). We have established a collaboration with Drs. Jay Bradner and Stuart Schreiber of the Broad Institute to use our technology to develop new HDAC inhibitors, and a collaboration with Dr. Michael Palladino of Nereus Pharmaceuticals to study HDAC inhibitors in combination with the novel Nereus proteasome inhibitor NPI-0052. This year we have finalized a CRADA agreement with Syndax Pharmaceuticals to support HDAC inhibitor research in the lab, and our HDAC inhibitor multiparameter flow assay was chosen by the NCI as an outstanding technology of 2007. We have analyzed recent progress in HDAC as a molecular target in an invited review in press in Current Opinion in Oncology. We have developed a new pharmacodynamic method, a cell-based tissue microarray for assessment of anticancer drug activity in vivo (Drug Dev. Res. 68:226-234, 2007). We demonstrate that the cell microarray can be used to measure drug response in a high-throughput manner, allowing analysis of an [summary truncated at 7800 characters]