Targeting PML for therapy in leukemia-initiating cells

6-项目总结 这份提案描述了一项为期五年的培训计划，旨在发展癌症干细胞的学术生涯 细胞生物学。我有四年的癌症生物学家经验，两年的干细胞生物学家经验，以及 在一所大学的医院当了五年的血液学家。我现在计划扩大我的知识，技能和 干细胞与癌症研究相结合的致癌细胞研究领域。 我未来五年的目标是成为癌症干细胞生物学领域的一名独立科学家， 尤其是在启动白血病的细胞中。我计划分两个阶段实现我的目标。第一阶段(第一阶段 2-3年)的特点是紧张的板凳工作，以构建我的研究项目的基础，并 在对我来说是新的领域获得经验，例如 临床相关的小鼠模型和临床试验在美国的贡献。此外，我将开发/建立 利基特定的PML-在独立阶段删除了我自己研究的小鼠模型。在第一部分中， 计划，我将得到Pier Paolo Pandolfi博士的指导，他是一位领先的癌症生物学家，在 他在体内为白血病和实体肿瘤建模，并培训了许多独立调查人员。这个 D·G·特宁博士、D·E·阿维根博士、T·苏达博士和J·特鲁亚-费尔德斯坦博士的合作将丰富该计划。 奖项的第一部分将在贝丝以色列女执事医疗中心完成，这是一个科学环境 卓越与扎实的临床资源，以推动这一职业发展奖的成功。 第二阶段将完全致力于确立我作为独立科学家的职业生涯。其中一个主要的 这一阶段的主题将是PML在特定生态位微环境中的详细功能分析 条件基因敲除小鼠。需要五年的支持才能产生足够的数据，特别是在 考虑我计划建立的老鼠模型，让我能够追求独立的职业生涯 赛道。 研究将集中在白血病启动细胞(LICs)中调节静止的机制。 具体地说，我的目标是聚集性造血干细胞的LIC特异性治疗的发展 (HSC)疾病：慢性粒细胞白血病(CML)。 白血病的维持已被证明依赖于体内的一小部分细胞 具有自我更新特性的白血病群体称为“白血病启动细胞”(LICs)。 LIC与常规干细胞具有相同的机械特性，包括更静态的性质，这被认为是 调解他们对以化疗为基础的标准治疗的耐药性。无法有效锁定LIC可能会导致 在疾病复发时。慢性粒细胞白血病(CML)是一种被广泛研究的干细胞疾病 目前的靶向治疗并不总是根除LIC池，从而导致药物复发 停产。 明确了PML在维持CML启动细胞中的重要作用，并提出了一种新的治疗方法 通过药物抑制PML来靶向静止的LIC的方法。我已经证明了 早幼粒细胞白血病(PML)抑癌基因的表达在两者中的表达都令人惊讶地高 造血干细胞(HSCs)和CML原始细胞中PML表达的缺失预示着更有利的 慢性粒细胞白血病的转归。我随后证明了PML在维持静止和 HSCs/LICs的自我更新特性，尽管涉及的确切分子机制还知之甚少。 众所周知，PML还可以通过用砷处理细胞而在药物上受到抑制，这是一种特定的 会降低蛋白质的稳定性。利用这一点，我使用了砷介导的PML消融在一个 CML小鼠模型成功靶向LIC。 这项工作有可能对慢性粒细胞白血病的治疗和根除产生重大影响。重要的是，我的 研究结果支持这样一种观点，即砷消融PML可能是一种有效的工具，可以使CML启动细胞更多地 对抗肿瘤治疗敏感。然而，PML靶向在临床相关小鼠中的有效性 模型和在临床上需要评估。因此，为了了解下游的关键路径 PML是LIC维护所需的，为了将PML消融LIC目标转换到临床，我建议 具体目标如下：(1)进一步明确PML依赖的细胞周期调控机制；(2) 分析PML缺失在LIC与其生态位相互作用中的作用；(3)检验PML缺失对LIC与其生态位相互作用的影响 砷在临床前模型中的介导性治疗，最终支持联合砷+的临床试验 达沙替尼治疗慢性粒细胞白血病的体外评价。 这份提案中概述的培训计划将开启我的独立研究生涯。 重要的是，实现这些目标可以极大地改善慢性粒细胞白血病患者的治疗，可能允许 LIC根除后停止治疗。此外，了解PML函数可以提供其他 LIC和肿瘤干细胞消融的治疗靶点。

6- PROJECT SUMMARY This proposal describes a five-year training program for the development of an academic career in Cancer Stem Cell Biology. I have four-years of experience as a cancer biologist, two-years as a stem cell biologist, and five-years as a hematologist in a university hospital. I am now planning to expand my knowledge, skills and research field to cancer-initiating cells by combining stem cell and cancer research. My goal over the next five years is to become an independent scientist in the field of cancer stem cell biology, especially in leukemia-initiating cells. I am planning to reach my goal by a two-phase plan. The first phase (first 2-3 years) will be characterized by intense bench work to construct the basis of my research projects and to acquire experience in fields that are new to me, such as the characterization of hematological malignancies in clinically relevant mouse models and contribution of clinical trials in the U.S.A. Further, I will develop/establish niche-specific Pml-deleted mouse models for my own research during independent phase. In the first part of the program, I will be mentored by Pier Paolo Pandolfi, PhD, a leading cancer biologist who has great experience in modeling leukemia and solid tumors in vivo and who has trained numerous independent investigators. The program will be enriched by the collaboration of Drs. D.G. Tenen, D.E. Avigan, T. Suda and J. Teruya-Feldstein. The first part of the award will be completed at Beth Israel Deaconess Medical Center, a scientific environment of excellence with the solid clinical resources necessary to promote the success of this Career Development Award. The second phase will be entirely devoted to establishing my career as an independent scientist. One of the main topics in this phase would be detailed functional analysis of Pml in the microenvironment in niche-specific conditional knockout mice. Five years of support are necessary to produce sufficient data, especially in consideration of the mouse models that I am planning to establish, to allow me to pursue an independent career track. Research will focus on the study of the mechanisms regulating quiescence in leukemia-initiating cells (LICs). Specifically, I am aiming at the development of LIC-specific therapy in paradigmatic hematopoietic stem cell (HSC) disease: chronic myeloid leukemia (CML). Maintenance of leukemia has been demonstrated to be dependent upon a small sub-population of cells within the bulk leukemic population that have self-renewal properties and are termed "leukemia-initiating cells" (LICs). LICs share mechanistic properties with regular stem cells including a more quiescent nature, which is thought to mediate their resistance to standard chemotherapy-based treatment. Failure to effectively target LICs can result in disease relapse. Chronic Myelogenous Leukemia (CML) is an extensively studied stem cell disorder in which the LIC pool is not always eradicated by current targeted therapy, leading to disease relapse upon drug discontinuation. I have defined the essential role of PML in the maintenance of CML-initiating-cell, and present a new therapeutic approach for targeting quiescent LICs by pharmacological inhibition of PML. I have demonstrated that expression of the Promyelocytic Leukemia (PML) tumor suppressor is surprisingly high in both regular hematopoietic stem cells (HSCs) and in CML blasts and that loss of PML expression predicts a more favorable outcome in CML. I subsequently demonstrated that PML plays a key role in maintaining the quiescence and self-renewal properties of HSCs/LICs although the exact molecular mechanisms involved are poorly understood. PML is also known to be pharmacologically inhibited by treatment of cells with arsenic, which specifically decreases the stability of the protein. Taking advantage of this, I have used arsenic-mediated ablation of Pml in a mouse model of CML to successfully target LICs. This work has the potential to have a significant impact on treatment and eradication of CML. Importantly, my findings support the notion PML-ablation by arsenic might be an effective tool to render CML-initiating cells more sensitive to anti-tumor therapy. However, the effectiveness of PML targeting both in clinically relevant mouse model and in the clinic needs to be assessed. Therefore, in order to understand the key pathways downstream PML required for LIC maintenance and to translate PML-ablative LIC targeting to the clinic, I propose the following Specific Aims: (1) to further define the mechanisms of PML-dependent cell cycle regulation in LICs; (2) to analyze the effect of PML loss in the interaction between LICs and their niche; (3) to test the efficacy of arsenic-mediated therapy in preclinical models and finally to support a clinical trial of combination arsenic + Dasatinib treatment for CML through in vitro evaluation. The Training Program outlined in this proposal will launch my independent research career. Importantly, accomplishing these aims could greatly improve treatment of patients with CML, possibly allowing discontinuation of therapy after LIC eradication. Further, understanding PML function could provide other therapeutic targets for LIC and cancer stem cell ablation.