Intratumor heterogeneity and therapeutic resistance

项目摘要/摘要 肿瘤内的细胞多样性是癌症治疗失败的主要原因之一。存在截然不同的 对任何一种治疗具有不同敏感性的细胞亚群增加了耐药性的机会 和肿瘤复发。因此，对肿瘤内异质性的机制的理解是 对于设计更有效的治疗方案至关重要。在建议书的指导阶段，我计划 肿瘤内异质性对HER2+乳腺癌靶向治疗的影响 (目标1)。在我的初步研究中，我使用了星鱼，这是我开发的一种新方法，允许同时 检测完整组织切片中单细胞水平的点突变和基因扩增，以评估 配对治疗前后细胞亚群的遗传异质性和空间分布 化疗样本。我们发现，化疗后细胞多样性的变化可以预测 病人结局。因此，在目前的提案中，我将检验从这些研究中出现的假设： 肿瘤内的异质性可能对靶向治疗有显著影响。我将调查这一影响 对HER2+乳腺癌进行新的抗HER2方案的研究(目标1.A)。我还将剖析 PIK3CA点突变在HER2+乳腺癌细胞表观遗传和表型可塑性中的作用 1.b)。为了揭示这些肿瘤中维持肿瘤内异质性的机制，我将描述 遗传不同亚群之间的相互作用(目标2)。要实现中描述的目标 在这项提议中，我将使用新颖而独特的技术，并在我之前的研究中得到验证。 在奖项的独立阶段，我将集中精力研究高度攻击性的大脑。 肿瘤。这些肿瘤表现出严重的瘤内异质性和目前的治疗方法。 干预措施未能显著延长这种疾病患者的生命。因此，我强烈地 目的：研究脑胶质瘤的遗传异质性(目标3)。我将重点介绍 低级别胶质瘤向胶质母细胞瘤发展过程中的遗传多样性特征 将分析靶向治疗引起的肿瘤内异质性的变化及其对患者的影响 成果(目标3.A)。我还将使用一种新的方法来进行表观遗传学分析 胶质母细胞瘤细胞亚群研究遗传和表观遗传之间的相互作用 异质性(目标3.B)。总而言之，这项拟议的研究将为治疗意义提供新的线索。 并将有助于设计更有效的癌症治疗方法。 在我的整个科学生涯中，我表现出了高生产率、献身精神和强大的技术技能， 这对成功执行拟议的研究至关重要。我对这个项目有长期兴趣 肿瘤内异质性的研究，因为我选择了癌症干细胞作为我的博士论文工作的主题 后来加入了Dana-Farber癌症研究所Polyak博士的实验室，专门研究乳腺癌 异质性研究。作为一名博士后，我开发了一种新的方法来评估基因 原位单细胞水平的肿瘤内异质性，以及用于自动图像分析的新工具。这个 将这种方法应用于HER2+乳腺癌，得到了一篇题为《原位单细胞》的手稿 分析确定了HER2+乳腺癌中PIK3CA突变和HER2扩增的异质性。 这项研究最近发表在《自然遗传学》上。在奖项的指导阶段，我将扩展 那些研究克隆相互作用机制及其在新靶点上的变化的研究 治疗。我还将获得其他技术技能，包括芯片序列和RNA序列性能以及 计算，这对我的独立研究计划中提出的实验至关重要。此外，我 将通过参加DFCI内部和外部的研讨会来提高我的管理和领导技能。 达纳-法伯癌症研究所为我的成功实现提供了一个培育环境 与科学和职业相关的目标。作为世界领先的癌症研究机构之一和 哈佛癌症中心，DFCI促进临床医生和科学家在地方、国家、 和国际水平。多个研讨会和研讨会允许学员与专家互动，讨论 研究思路和职业发展。乳腺癌和脑癌的孢子计划将为我提供 在研究和治疗设计方面得到科学领先者的支持。优良的实验室设备和核心 DFCI的设施将有助于完成拟议的实验。除了制度上的 支持，我将得到导师小组提供的科学和职业发展方面的建议 科学指导委员会。在这个委员会中支持我努力的科学家都来自 不同的领域，但他们都有共同的利益，他们不同的专业知识将是无价的 推进拟议的项目。 我的背景、强大的指导委员会和DFCI良好的制度环境是 圆满完成拟议目标。拟议研究的结果将扩大我们的 了解肿瘤内异质性在治疗耐药中的作用，导致新的 治疗设计和更好的患者结果。

PROJECT SUMMARY/ABSTRACT Cellular diversity within a tumor is one of the main causes of cancer treatment failure. Presence of distinct subpopulations of cells with different sensitivity to any given therapy increases the chance of resistance and tumor recurrence. Therefore, understanding of mechanism governing the intratumor heterogeneity is vital for designing more effective regimens. Within the mentored phase of the proposal I am planning to investigate the influence of intratumor heterogeneity on HER2-targeted therapies in HER2+ breast cancer (Aim 1). In my preliminary study I used STAR-FISH, a novel method I developed allowing for simultaneous detection of point mutation and gene amplification at single cell level in intact tissue slides, to assess genetic heterogeneity and spatial distribution of subpopulations of cells in matched therapy naïve and post- chemotherapy samples. We have found that changes in cellular diversity upon chemotherapy can predict patient outcome. Thus, in the current proposal I will test the hypothesis that emerged from those studies: intratumor heterogeneity could have a pronounced effect on targeted treatment. I will investigate this effect on samples of HER2+ breast cancers undergoing novel anti-HER2 regimen (Aim 1.A). I will also dissect the role of a point mutation in PIK3CA in epigenetic and phenotypic plasticity of HER2+ breast cancer cells (Aim 1.B). To uncover the mechanism that sustains intratumor heterogeneity in those tumors I will characterize the interactions between genetically distinct subpopulations (Aim 2). To accomplish the goals described in this proposal, I will use novel and unique techniques, validated in my previous studies. In the independent phase of the award I will concentrate my research efforts on highly aggressive brain tumors. These tumors display profound degree of intratumor heterogeneity and the current therapeutic interventions fail to significantly prolong lives of patients with this disease. Therefore, I am strongly motivated to study the genetic intratumor heterogeneity in gliomas (Aim 3). I will focus on the characterization of genetic diversity in the process of progression of low-grade glioma to glioblastoma and I will analyze targeted treatment-induced changes in intratumor heterogeneity and their influence on patient outcomes (Aim 3.A). I will also use a novel method to perform epigenetic profiling of genetically distinct subpopulations of glioblastoma cells to investigate the interplay between genetic and epigenetic heterogeneity (Aim 3.B). In summary, the proposed research will shed new light on therapeutic implications of intratumor heterogeneity and will help designing more effective cancer treatment. Throughout my scientific career I have demonstrated high productivity, devotion and strong technical skills, which are crucial for the successful execution of the proposed research. I have a long-term interest in the study of intratumor heterogeneity, as I have chosen the topic of cancer stem cells for my PhD thesis work and later joined the laboratory of Dr. Polyak at Dana-Farber Cancer Institute, specializing in breast cancer heterogeneity studies. As a postdoc, I have developed a new method allowing for assessment of genetic intratumor heterogeneity on single cell level in situ, as well as a new tool for automated image analysis. The application of this method on HER2+ breast cancer resulted in a manuscript entitled “In situ single cell analysis identifies heterogeneity for PIK3CA mutation and HER2 amplification in HER2+ breast cancer”, which was recently published in Nature Genetics. During the mentored phase of the award I will expand those studies to investigate the mechanisms of clonal interactions and their changes upon novel targeted treatment. I will also acquire additional technical skills, including ChIP-seq and RNA-seq performance and computation, which are crucial to the experiments proposed in my independent research plan. Moreover, I will improve my management and leadership skills by participating in workshops within and outside DFCI. Dana-Farber Cancer Institute provides a nurturing environment for successful accomplishment of my scientific and career related goals. As one of the world's leading cancer research institutes and a part of the Harvard Cancer Center, DFCI facilitates collaborations between clinicians and scientists on local, national, and international level. Multiple seminars and workshops allow trainees to interact with experts to discuss research ideas and career development. The SPORE programs in breast and brain cancer will provide me with support of scientific leaders in research and treatment design. Excellent laboratory equipment and core facilities at DFCI will facilitate the completion of the proposed experiments. In addition to the institutional support, I will be provided with scientific and career development advice by the panel of mentors within Scientific and Mentoring Committee. The scientists supporting my efforts in this committee are all from distinct fields, yet they all share common interests and their diverse expertise will be invaluable to the advancement of the proposed project. My background, strong mentoring committee and excellent institutional environment of DFCI are key to the successful completion of the proposed aims. The results of the proposed study will broaden our understanding of the role of intratumor heterogeneity in therapy resistance, leading to improvements in new treatment design and better patient outcomes.