Aberrant activation of HGF/MET signaling as a therapeutic target in 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，但复杂核型AML等高风险疾病对目前的治疗仍有很大的难治性，并且大多数是致命的。通过使用候选基因方法确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，我利用复杂核型AML细胞中的短发夹rna （shRNAs）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）的消耗，受体酪氨酸激酶MET的配体，特异性地抑制AML的生长，而不是其他血液肿瘤细胞。MET是一种强效癌基因，其异常激活与癌症发生密切相关，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尚不清楚，HGF/MET信号传导不被认为在AML中发挥作用。为了在患者标本中验证这一观察结果，我对诊断性骨髓活检进行了免疫组织化学检查。我观察到HGF在大约15%的AML患者中异常表达并与MET激活相关，包括大多数复杂核型疾病患者。对来自此类患者的细胞系的分析表明，HGF的表达与其受体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，可显著降低表达HGF而不表达HGF的AML细胞系的增殖和诱导死亡。这表明该通路存在功能依赖或“癌基因依赖”，并提示治疗性抑制HGF/MET信号传导可用于改善AML的治疗。然而，目前尚缺乏对该途径促进AML细胞生长和存活的分子机制的详细了解。通过基因工程AML细胞系来消耗和表达特定的信号分子，并分离对HGF/MET抑制有抗性的细胞系，我将确定介导HGF/MET“癌基因成瘾”的信号成分，以及克服对HGF/MET信号治疗抑制的抗性的策略。这些研究将结合使用功能性纳米免疫测定、磷酸化蛋白质组学和基因组学方法对小鼠AML模型体内HGF/MET抑制的抗白血病效果进行研究，不仅确定临床靶向这一途径的最佳策略，而且确定如何将其与其他AML信号传导靶向抑制剂最佳地整合。这些技术进步将绕过与AML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对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进行详细分析，从而提供对各种生物和疾病现象非常重要的关键机制见解。在体外和体内的研究结果将进一步利用原始患者标本进行研究，为未来AML靶向治疗的临床前研究和临床试验奠定基础。具体目标1：剖析AML细胞生长和存活中HGF/MET依赖性的分子信号通路，并确定HGF/MET抑制耐药的机制（1-3年）。特异性目标2：评估体内小鼠AML模型中HGF/MET抑制的抗白血病效果，无论是单独抑制还是协同抑制其他致白血病酪氨酸激酶，包括FLT3和KIT（3-5年）。申请人Alex Kentsis博士是丹娜-法伯癌症研究所（DFCI）的儿科血液学/肿瘤学研究员，他概述了他在生物物理学和临床血液学/肿瘤学方面的5年职业规划。在癌症细胞生物学和白血病转化研究领域公认的领导者Thomas Look博士的指导下，Kentsis博士寻求利用强大的功能基因组学和蛋白质组学方法，结合体外系统和小鼠体内模型来研究异常HGF/MET信号在AML中的作用。Kentsis博士将接受由该领域国际公认专家组成的咨询委员会的指导。最后，该计划理想地在DFCI儿科肿瘤科实施，因为它在丰富和协作的环境中培训医生科学家的杰出记录。在K08奖的支持下，Kentsis博士的项目将引领临床有效的HGF/MET靶向治疗AML的发展。

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. PUBLIC HEALTH RELEVANCE: 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. To identify new therapeutic targets, I carried out a genome-wide functional screen, and discovered that hepatocyte growth factor (HGF) and its receptor tyrosine kinase MET are aberrantly activated in AML cells, particularly among patients with the highest-risk disease. This research proposal will investigate the molecular mechanisms responsible for HGF/MET-dependent AML cell growth and survival, means for its therapeutic targeting in patients, and optimal strategies to overcome potential resistance to therapy, leading to the development of improved, rationally combined targeted therapy for the treatment of AML. The written critiques and criteria scores of individual reviewers are provided in essentially unedited form in the "Critique" section below. Please note that these critiques and criteria scores were prepared prior to the meeting and may not have been revised subsequent to any discussions at the review meeting. The "Resume and Summary of Discussion" section above summarizes the final opinions of the committee.