Intratumor heterogeneity and therapeutic resistance

项目总结/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。存在不同的 对任何给定疗法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，了解肿瘤内异质性的机制是 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 研究瘤内异质性对HER 2+乳腺癌中HER 2靶向治疗的影响 (Aim 1）。在我的初步研究中，我使用了STAR-FISH，这是我开发的一种新方法，可以同时 在完整组织切片中检测单细胞水平的点突变和基因扩增，以评估 匹配治疗初治和治疗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 患者结局。因此，在目前的建议中，我将测试从这些研究中得出的假设： 肿瘤内异质性可能对靶向治疗有显著影响。我将研究这种影响 接受新型抗HER 2方案的HER 2+乳腺癌样本（目的1.A）。我还将剖析 PIK 3CA点突变在HER 2+乳腺癌细胞表观遗传和表型可塑性中的作用（目的 1.B）。为了揭示维持这些肿瘤内异质性的机制，我将描述 遗传上不同的亚群之间的相互作用（目标2）。为了实现 这个建议，我将使用新颖独特的技术，在我以前的研究验证。 在该奖项的独立阶段，我将把我的研究工作集中在高度侵略性的大脑上。 肿瘤的这些肿瘤显示出高度的瘤内异质性， 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 动机是研究胶质瘤中的遗传瘤内异质性（目的3）。我会集中讨论 低级别胶质瘤进展为胶质母细胞瘤过程中的遗传多样性特征和I 将分析靶向治疗诱导的肿瘤内异质性变化及其对患者的影响 （目标3.A）。我还将使用一种新的方法来进行表观遗传分析， 胶质母细胞瘤细胞亚群，以研究遗传和表观遗传之间的相互作用 异质性（目标3.B）。总之，拟议的研究将揭示新的治疗意义 这将有助于设计更有效的癌症治疗方法。 在我的科学生涯中，我表现出了高生产力，奉献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。我有一个长期的兴趣， 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，可以评估遗传 在单细胞水平上原位检测肿瘤内异质性，以及用于自动化图像分析的新工具。的 该方法在HER 2+乳腺癌中的应用产生了题为“原位单细胞”的手稿 分析鉴定了HER 2+乳腺癌中PIK 3CA突变和HER 2扩增的异质性”， 最近发表在《自然遗传学》上。在奖励的指导阶段，我将扩大 这些研究旨在探讨克隆相互作用的机制及其在新靶向药物作用下的变化， 治疗我还将获得额外的技术技能，包括ChIP-seq和RNA-seq性能， 计算，这对我的独立研究计划中提出的实验至关重要。而且我 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。 丹娜-法伯癌症研究所提供了一个培育环境，成功完成我的 科学和职业目标。作为世界领先的癌症研究机构之一， 哈佛癌症中心，DFCI促进了临床医生和科学家在地方，国家， 和国际水平。多个研讨会和讲习班使学员能够与专家互动，讨论 研究思路和职业发展。乳腺癌和脑癌的孢子项目将为我提供 在研究和治疗设计的科学领导者的支持下。优秀的实验室设备和核心 DFCI的设施将有助于完成拟议的实验。除了机构 支持，我将提供科学和职业发展的意见，由导师小组内 科学和指导委员会。在这个委员会中支持我的科学家们都来自 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， 推进拟议项目。 我的背景，强大的指导委员会和DFCI优秀的制度环境是关键， 圆满完成了提出的目标。建议的研究结果将扩大我们的 了解肿瘤内异质性在治疗耐药性中的作用，从而改善新的 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.