Aberrant activation of HGF/MET signaling as a therapeutic target in AML

HGF/MET 信号传导异常激活作为 AML 的治疗靶点

• 批准号: 8307367
• 负责人: Alex Kentsis
• 金额: $ 14.26万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8307367
• 关键词: Accounting; Acute Myelocytic Leukemia; Acute Promyelocytic Leukemia; Adult; Advisory Committees; Alternative Therapies; Antibodies; Award; Biochemistry; Biological; Biological Assay; Biophysics; Bone marrow biopsy; Candidate Disease Gene; Cell Line; Cell Survival; Cells; Cellular biology; Cessation of life; Chemicals; Child; Chromosome abnormality; Clinical; Clinical Trials; Combination Drug Therapy; Complex; Critiques; Dana-Farber Cancer Institute; Dasatinib; Dependence; Development; Diagnosis; Diagnostic; Disease; Elements; Engineering; Environment; FLT3 gene; Foundations; Future; Gene Expression; Gene Expression Profiling; Genetic; Genomic Instability; Genomics; Growth; Hematologic Neoplasms; Hematology; Hepatocyte Growth Factor; Immunohistochemistry; In Vitro; Individual; Investigation; Karyotype; Lead; Libraries; Ligands; Lymphoma; MET Oncogene; Mediating; Mentors; Mentorship; Methods; Modeling; Molecular; Multiple Myeloma; Mus; Mutate; Mutation; Oncogenes; Oncogenic; Pathway interactions; Patients; Pediatric Hematology/Oncology; Pediatric Oncology; Phosphotransferases; Physicians; Play; Protein Tyrosine Kinase; Proteomics; RNA Interference; Receptor Protein-Tyrosine Kinases; Refractory; Relapse; Research Proposals; Resistance; Resistance development; Risk; Role; Scientist; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; Specimen; Surveys; System; Testing; Therapeutic; Therapeutic Intervention; Training; Transduction Gene; Treatment Efficacy; Tyrosine Kinase Inhibitor; Writing; autocrine; cancer cell; carcinogenesis; career; cell growth; clinically relevant; combinatorial; functional genomics; genome-wide; high risk; improved; in vivo; in vivo Model; inhibitor/antagonist; innovation; insight; kinase inhibitor; leukemia; meetings; mouse model; nanoimmunoassay; new therapeutic target; oncogene addiction; oncology; preclinical study; receptor; small hairpin RNA; therapeutic target; therapy resistant; tool

DESCRIPTION (provided by applicant): Despite improvement in treatment of acute myeloid leukemia (AML), high-risk disease such as complex karyotype AML remains largely refractory to current therapy, and is mostly fatal. Identification of effective therapeutic targets by using candidate gene approaches has been limited by the number and variety of genetic defects associated with AML. To identify new therapeutic targets, I carried out a genome-wide functional screen by using a retroviral library of short hairpin RNAs (shRNAs) in complex karyotype AML cells. I discovered that shRNA mediated depletion of hepatocyte growth factor (HGF), ligand of the receptor tyrosine kinase MET, specifically inhibits growth of AML but not other hematologic cancer cells. MET is a potent oncogene, whose aberrant activation is widely implicated in carcinogenesis, causing enhanced growth, survival, and genomic instability of cancer cells. However, mechanisms of carcinogenic MET signaling are currently not well understood, and HGF/MET signaling is not thought to play a role in AML. To validate this observation in patient specimens, I carried out immunohistochemistry of diagnostic bone marrow biopsies. I observed that HGF is aberrantly expressed and associated with activation of MET in about 15% of patients with AML, including most patients with complex karyotype disease. Analysis of cell lines derived from such patients showed that HGF expression was associated with autocrine activation of its receptor MET. Depletion of HGF or MET using shRNA or inhibition of MET using tyrosine kinase inhibitors and neutralizing anti-HGF antibody profoundly reduced proliferation and induced death of AML cells lines that express HGF but not those that lack HGF expression. This indicates the functional dependence or "oncogene addiction" to this pathway, and suggests that therapeutic inhibition of HGF/MET signaling may be used to improve the treatment of AML. However, detailed understanding of the molecular mechanisms by which this pathway promotes AML cell growth and survival is currently lacking. By genetically engineering AML cell lines to deplete and express specific signaling molecules, and isolating cell lines that are resistant to HGF/MET inhibition, I will identify signaling components that mediate HGF/MET "oncogene addiction," and strategies to overcome resistance to therapeutic inhibition of HGF/MET signaling. These studies will be combined with the investigation of antileukemic efficacy of HGF/MET inhibition in murine models of AML in vivo using functional nanoimmunoassay, phosphoproteomic and genomic methods to identify not only the optimal strategy to target this pathway clinically, but also how to optimally integrate it with other targeted inhibitors of AML signaling. These technical advances will circumvent limitations associated with empiric discovery of novel therapeutic targets in AML, and will identify the principal signaling pathways required for AML cell growth and survival. The cell line systems and mouse models will also allow for detailed analysis of gene expression and proteomic changes that accompany signaling by receptor tyrosine kinases, thus providing key mechanistic insights that will be important to a wide variety of biological and disease phenomena. Results of the in vitro and in vivo studies will be further investigated using primary patient specimens, and will lay the foundation for future preclinical studies and clinical trials of targeted therapies of AML. The specific aims are: Specific Aim 1: Dissect the molecular signaling pathways responsible for the HGF/MET dependence of AML cell growth and survival, and identify mechanisms that account for resistance to HGF/MET inhibition (years 1-3). Specific Aim 2: Assess the antileukemic efficacy of HGF/MET inhibition in murine AML models in vivo, both by itself and in concert with the inhibition of other leukemogenic tyrosine kinases, including FLT3 and KIT (years 3-5). The applicant, Dr. Alex Kentsis, a pediatric hematology/oncology fellow at the Dana-Farber Cancer Institute (DFCI) has outlined a 5-year career plan that will build upon his background in biophysics and clinical hematology/oncology. Under the mentorship of Dr. Thomas Look, a recognized leader in cancer cell biology and translational investigations of leukemia, Dr. Kentsis seeks to utilize powerful functional genomic and proteomic approaches using a combination of in vitro systems and murine models in vivo to study the role of aberrant HGF/MET signaling in AML. Dr. Kentsis will be mentored by an Advisory Committee of internationally recognized experts in the field. Finally, the plan is ideally carried out in the Department of Pediatric Oncology at DFCI, given its distinguished record for training physician-scientists in a rich and collaborative environment. With the support provided by the K08 award, Dr. Kentsis' project will lead to the development of clinically effective HGF/MET targeted therapy for AML.

描述（由申请人提供）：尽管急性髓性白血病（AML）的治疗有所改善，但高风险疾病（如复杂核型AML）在很大程度上仍然是当前治疗难以治愈的，并且大多数是致命的。通过使用候选基因方法鉴定有效的治疗靶点受到与AML相关的遗传缺陷的数量和种类的限制。为了确定新的治疗靶点，我在复杂核型的AML细胞中使用短发夹RNA（shRNA）的逆转录病毒文库进行了全基因组功能筛选。我发现shRNA介导的肝细胞生长因子（HGF）（受体酪氨酸激酶MET的配体）的耗竭特异性抑制AML的生长，但不抑制其他血液癌细胞的生长。MET是一种有效的致癌基因，其异常激活广泛参与致癌作用，导致癌细胞的生长、存活和基因组不稳定性增强。然而，致癌MET信号传导的机制目前还不清楚，并且认为HGF/MET信号传导在AML中不起作用。为了验证在患者标本中的观察结果，我对诊断性骨髓活检进行了免疫组化。我观察到HGF异常表达，并与约15%的AML患者（包括大多数复杂核型疾病患者）的MET活化相关。对来自这些患者的细胞系的分析表明，HGF表达与其受体MET的自分泌激活有关。使用shRNA消耗HGF或MET或使用酪氨酸激酶抑制剂和中和抗HGF抗体抑制MET显著降低表达HGF的AML细胞系的增殖并诱导其死亡，但不影响缺乏HGF表达的AML细胞系。这表明对该途径的功能依赖性或“癌基因成瘾”，并表明HGF/MET信号传导的治疗性抑制可用于改善AML的治疗。然而，目前缺乏对该途径促进AML细胞生长和存活的分子机制的详细了解。通过基因工程改造AML细胞系以耗尽和表达特定信号分子，并分离对HGF/MET抑制具有抗性的细胞系，我将鉴定介导HGF/MET“癌基因成瘾”的信号成分，以及克服对HGF/MET信号传导治疗抑制的抗性的策略。这些研究将与使用功能性纳米免疫测定、磷酸蛋白组学和基因组学方法在AML小鼠模型中研究HGF/MET抑制的体内抗白血病疗效相结合，以确定不仅是临床靶向该途径的最佳策略，而且如何最佳地将其与AML信号传导的其他靶向抑制剂整合。这些技术进步将规避与AML中新治疗靶点的经验性发现相关的限制，并将确定AML细胞生长和存活所需的主要信号通路。细胞系系统和小鼠模型还将允许详细分析伴随受体酪氨酸激酶信号传导的基因表达和蛋白质组学变化，从而提供对各种生物和疾病现象至关重要的关键机制见解。体外和体内研究的结果将使用原代患者标本进行进一步研究，并将为AML靶向治疗的未来临床前研究和临床试验奠定基础。具体目标是：具体目标1：剖析负责AML细胞生长和存活的HGF/MET依赖性的分子信号传导途径，并确定导致对HGF/MET抑制耐药的机制（1-3年）。具体目标二：评估HGF/MET抑制作用本身以及与其他致白血病酪氨酸激酶（包括FLT 3和KIT）抑制作用联合作用在体内小鼠AML模型中的抗白血病功效（第3-5年）。申请人，Dana-Farber癌症研究所（DFCI）的儿科血液学/肿瘤学研究员Alex Dallsis博士概述了一个5年职业计划，该计划将建立在他的生物物理学和临床血液学/肿瘤学背景基础上。在癌症细胞生物学和白血病转化研究领域公认的领导者托马斯·Look博士的指导下，Dr. Albersis寻求利用强大的功能性基因组和蛋白质组学方法，使用体外系统和体内小鼠模型的组合来研究异常HGF/MET信号传导在AML中的作用。一个由该领域国际公认专家组成的咨询委员会将指导Dr.最后，该计划理想地在DFCI的儿科肿瘤学系进行，因为它在丰富和协作的环境中培训医生科学家方面有着出色的记录。在K 08奖的支持下，Dr. Alfresis的项目将导致临床有效的HGF/MET靶向治疗AML的开发。