Evolutionary dynamics of melanoma metastasis

描述(申请人提供)：转移性疾病仍然是晚期恶性肿瘤的定义特征，并且是绝大多数癌症死亡的原因。转移可以被概念化为一幅进化的图景，由达尔文进化的关键要素组成：可遗传的(EPI)基因类型、地理扩散和新的微环境选择压力。我推测，改变这些进化格局将为治疗癌症提供一种惊人的新方法，在这种方法中，癌细胞可以与宿主长期共存。要做到这一点，需要深入了解肿瘤产生新基因类型的方式，以及微环境中的自然选择如何放大这些突变。我的工作利用了斑马鱼，这是一种最近才被发现的小型脊椎动物，是一种重要的癌症模型。斑马鱼为研究转移提供了几种独特的能力：高通量转基因，无偏见的遗传筛选，以及光学透明的Casper成鱼的单细胞成像。在这项提案中，我计划解决转移性黑色素瘤的三个主要问题：1)我们能否识别允许转移进展的早期基因变化，无论它们是在原发肿瘤中还是在扩散后发生；2)转移是否需要适应性突变，这是否可以修改？以及3)我们能否识别不利于转移进展的宿主微环境？为此，我将建立一个黑色素瘤的斑马鱼模型，在该模型中，BRAFV600E等位基因在黑素细胞前体细胞中表达，在p53功能丧失的背景下(BRAFV600E；P53-/-模型)。通过将“脑弓”命运图谱系统与BRAFV600E；P53-/-FISH相结合，我将使用外显子组测序来根据谱系、空间和时间来识别与转移相关的基因组病变。这些候选的变化可以使用我在透明的Casper菌株中开发的转移分析来功能化。我假设转移过程中的选择应激源导致一种适应状态 突变，即DNA复制的错误率暂时增加，以找到应对这种压力的进化解决方案。为了测试这一点，我将生成带有突变报告的斑马鱼，然后使用这个系统来探索容易出错的Y家族DNA聚合酶介导的适应性突变是否促进了转移进展。最后，由于宿主微环境提供了最终的选择压力，我将进行一次无偏见的遗传筛选，以确定转移进展的新的基质调节因子。总之，这些研究提供了一个全面的框架，既考虑了肿瘤细胞的内在动力学，也考虑了转移性疾病的微环境动力学。我的实验室的长期目标是利用这些信息来确定可以将播散性疾病转变为稳定状态、无法进一步进展的治疗方法，从而大幅提高已确定转移患者的长期存活率。

DESCRIPTION (provided by applicant): Metastatic disease remains the defining feature of advanced malignancy, and is responsible for the vast majority of cancer deaths. Metastasis can be conceptualized as an evolutionary landscape, composed of key elements of Darwinian evolution: heritable (epi)genotypes, geographic dispersal, and novel microenvironmental selection pressures. I hypothesize that altering these evolutionary landscapes would provide a strikingly new method for treating cancer, in which the cancer cells can co-exist with the host over long periods of time. To achieve this requires a deep mechanistic understanding of the ways in which tumors generate novel genotypes, and how natural selection in the microenvironment amplifies these mutations. My work utilizes the zebrafish, a small vertebrate organism that has only recently come to light as an important cancer model. The zebrafish offers several unique capacities for studying metastasis: high-throughput transgenesis, unbiased genetic screens, and single cell imaging in the optically transparent casper adult fish. For the period of this proposal, I plan to address three primary questions in metastatic melanoma: 1) can we identify the incipient genetic changes that allow for metastatic progression, whether they arise in the primary tumor or after dissemination, 2) is adaptive mutation required for metastasis, and can this be modified?, and 3) can we identify host microenvironments that disfavor metastatic progression? To do this, I will build upon a zebrafish model of melanoma in which the BRAFV600E allele is expressed in melanocyte progenitors, in the context of p53 loss of function (the BRAFV600E;p53-/- model). By combining the "brainbow" fate mapping system with the BRAFV600E;p53-/- fish, I will use exome sequencing to identify genomic lesions associated with metastasis based on lineage, space and time. These candidate changes can be functionalized using a metastasis assay I have developed in the transparent casper strain. I hypothesize that selection stressors during metastasis lead to a state of adaptive mutation, in which the error rate of DNA replication is temporarily increased to find an evolutionary solution to that stress. To test this, I will generate zebrafish with mutation reportes, and then use this system to probe whether adaptive mutation mediated by error-prone Y family DNA polymerases promotes metastatic progression. Finally, since the host microenvironment provides the ultimate selection pressure, I will perform an unbiased genetic screen to identify novel stromal regulators of metastatic progression. Together, these studies provide a comprehensive framework that considers both tumor cell-intrinsic and microenvironmental dynamics of metastatic disease. The long-term goal of my laboratory is to utilize this information to identify therapies which can convert disseminated disease into a stable state, unable to further progress, leading to large improvements in long-term survival in patients with established metastases.