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)组蛋白去乙酰化酶作为抗癌药物开发的靶点；(2)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；(3)开发新的药理学分析，包括抗血管生成治疗的分析。(1)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进行了基础研究，发现组蛋白去乙酰化酶是该肿瘤的关键靶点。在本财政年度，我们已经完成了一种新的药效学试验的开发，用于评估体内HDAC抑制剂的活性。NCI已经为我们的工作申请了专利，该工作具有独特的分析HDAC抑制剂活性的能力，可以通过同时检查7个参数来观察联合治疗的药效学反应。我们已经在三个临床试验中使用了这项技术，其中两个已经发表。我们已经和dr。布罗德研究所的杰·布莱德纳和斯图尔特·施赖伯利用我们的技术开发新的HDAC抑制剂。(2)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。这些数据确定了β -连环蛋白作为血液恶性肿瘤抗癌药物开发的新靶点。我们之前发表过β -连环蛋白促进成人t细胞白血病（ATL）细胞系HUT102的增殖。我们与NCI代谢科的John Janik和John Morris建立了合作关系，对他们的ATL患者进行研究，探讨β -catenin信号在ATL中的作用机制。成人t细胞白血病/淋巴瘤的急性临床亚型预后非常差，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)原发性急性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蛋白在Tax阴性ATL细胞中定位于细胞核，(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的表达水平很高。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晚期，不依赖税收ATLL。为了进一步验证我们的假设，即β -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殊的不受调节机制相关，我们与泌尿肿瘤科的Tomohiro Kajiguchi合作，研究了β -catenin在另外两种形式的白血病（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。这些实验表明c-kit在人肥大细胞白血病中上调Wnt信号，并且β -catenin是治疗肥大细胞增多症和肥大细胞白血病的新靶点。这项研究发表在《白血病研究》杂志上。FLT3突变或过表达是AML中最常见的突变。我们发现FLT3在FLT3阳性的AML细胞中调节β -连环蛋白酪氨酸磷酸化、核定位和靶基因表达。这项研究发表在2007年9月的《白血病》杂志上。(3)临床前开发研究核心一直在与内部研究人员合作进行一系列I期和II期临床试验。我是6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。我们的多参数流动药效学分析正在申请国际专利，并已被校内科学家选择在2007年9月25日的NCI技术展示会上展示。流动试验是HDAC抑制剂MS-275 I期试验的组成部分，该试验于2007年4月发表在《血液》杂志上，也是MS-275在实体瘤患者中的第二项临床试验，目前正在《临床癌症研究》杂志上发表。我们开发了一种新的药效学方法，一种基于细胞的组织微阵列来评估体内抗癌药物的活性。描述这种技术的手稿在《药物开发研究》杂志上发表。我们证明了该技术在体外治疗的PBMC以及接受SNDX-275一期临床试验的PBMC和骨髓抽吸物中对组蛋白去乙酰化酶抑制剂SNDX-275治疗的蛋白质超乙酰化反应分析中的实用性。我们证明了细胞微阵列可以用于高通量方式测量药物反应，允许在一个或两个玻片上分析整个试验。细胞微阵列技术将组织微阵列平台的优势引入到单个细胞的药效学评估中，例如从骨髓抽吸、细针抽吸或恶性积液中分离的细胞，以及肿瘤试验中最常研究的替代物PBMC的分析

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) histone deacetylase as a target for anticancer drug development, and (2) the molecular mechanisms of hematopoietic cell regulation by beta-catenin and the identification of beta-catenin as a target in hematologic malignancies (3) development of novel pharmacodynamic assays, including assays for antiangiogenic therapy. (1) 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. During this fiscal year we have finished development of a novel pharmacodynamic assay for assessment of HDAC inhibitor activity in vivo. The NCI has applied for a patent for 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 7 parameters simultaneously. We have used this technology in 3 clinical trials, two of which have been written for publication. 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. (2) 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 identified beta-catenin as a novel target for anticancer drug development in hematologic malignancies. We had published previously that beta-catenin promotes proliferation of the adult T-cell leukemia (ATL) cell line HUT102. We have established a collaboration with John Janik and John Morris of the Metabolism Branch, NCI to study their ATL patients and investigate the mechanism of beta-catenin signaling in ATL. The acute clinical subtype of adult T-cell leukemia/lymphoma have 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 the acute form of 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 ATLL. To pursue further 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 additional 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. These experiments 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. This work is in press in Leukemia Research. FLT3 mutation or overexpression is the most common mutation in AML. We showed that FLT3 regulates beta-catenin tyrosine phosphorylation, nuclear localization, and target gene expression in FLT3-positive AML cells. This work was published in September 2007 in Leukemia. (3) The Preclinical Development Research Core has been working with intramural investigators on a range of phase I and phase II clinical trials. I am an associate investigator on 6 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 mulitparameter flow pharmacodynamic assay is in international phase patent filing and has been selected by intramural scientists for presentation at the NCI Technology Showcase September 25th, 2007. The flow assay was an integral part of the phase I trial of the HDAC inhibitor MS-275 published in Blood in April 2007, and in a second clinical trial of MS-275 in solid tumor patients, currently in press in Clinical Cancer Research. We have developed a new pharmacodynamic method, a cell-based tissue microarray for assessment of anticancer drug activity in vivo. The manuscript describing this technique is in press in Drug Development Research. We demonstrate the utility of this technique for analysis of protein hyperacetylation in response to treatment with the histone deacetylase inhibitor SNDX-275 in PBMC treated in vitro and in PBMC and bone marrow aspirates from patients on SNDX-275 phase I clinical trials. We demonstrate that the cell microarray can be used to measure drug response in a high-throughput manner, allowing analysis of an entire trial on one or two glass slides. The cell microarray technique brings the advantages of the tissue microarray platform to the pharmacodynamic assessment of single cells, such as those isolated from bone marrow aspirates, fine needle aspirates or malignant effusions, and to analysis of PBMC, the most commonly studied surrogate in oncology trials