Cell Division Errors as a Mechanism Driving Massive Genomic Rearrangements

项目总结/摘要（描述） DNA测序技术的进步已经揭示了DNA序列中显著的结构复杂性。 人类癌症基因组，包括一类新的大规模和局部染色体内重排 创造的chromothripsis。自2011年发现以来，现在已经发现了chromothripsis的特征， 广泛的实体瘤和血液肿瘤这些变化被认为发生在一个单一的 灾难性事件;然而，其潜在的机械起源还没有得到很好的理解。有丝分裂细胞错误 分裂可以通过将错误分离的染色体捕获到异常的染色体中而引起染色体断裂。 称为微核。Ly博士先前发现，微核中的染色体受到 在有丝分裂中大量碎裂，产生的DNA片段通过典型的末端连接重新组装 在随后的间期修复。NIH独立之路奖的提案旨在 了解有丝分裂过程中染色体分离错误如何启动下游基因组级联反应， 不稳定性事件直接塑造或促成癌症基因组。在第一个目标中， 具有从头染色体断裂的功能性染色体将通过发育一个强大的 可逆的着丝粒特异性失活方法结合染色体特异性选择策略， 人类组织培养细胞。全基因组测序将用于识别 染色体误分离成微核后，从独特的克隆衍生物中分离出的染色体碎裂。在 第二个目标，染色体破碎和重组的精确机制和时空动力学 将通过多学科细胞生物学方法，包括基因破坏和活细胞， 细胞成像第三个目标将集中在如何染色体与粗重排，缺乏功能性的， 阿尔法状着丝粒能够通过表观遗传形成稳定的新着丝粒而无限繁殖 着丝粒位于非α型位点。通过利用他在设计复杂组织培养模型方面的专业知识 结合他在癌症生物学方面的背景，Ly博士的这些集体努力将建立 触发基因组不稳定性的有丝分裂错误的机制和后果-对 了解患者肿瘤中常见的复杂基因组特征的生物起源。期间 在K99阶段的指导下，Ly博士将在路德维希研究所接受额外的必要培训， 癌症研究在唐·克利夫兰博士的指导下-一个广泛认可的和既定的领导者， 细胞分裂和非整倍体领域。加州大学路德维希癌症研究所 在圣地亚哥，和周围的拉霍亚科学界作为一个特殊的气氛， 研究培训、协作科学和职业发展。一个优秀的团队合作者（博士。 大卫页和安德鲁萧）和顾问（博士理查德Kolodner，保罗Mischel，和卡伦Oegema） 已经组装，以提供额外的科学培训和职业生涯的支持博士Ly之前，期间， 在向独立的终身教职转变之后。R 00奖励阶段将设置 Ly博士长期职业目标的基础是建立一个严格的研究计划， 解决细胞生物学、癌症遗传学和基因组稳定性领域令人兴奋的未解问题 通过使用尖端的跨学科方法。

PROJECT SUMMARY/ABSTRACT (DESCRIPTION) Advances in DNA sequencing technologies have uncovered remarkable structural complexities within the human cancer genome, including a new category of massive and localized intra-chromosomal rearrangements coined chromothripsis. Since its discovery in 2011, the signatures of chromothripsis have now been detected in a broad spectrum of solid and hematological tumors. These alterations are thought to occur during a single catastrophic event; however, their underlying mechanistic origins are not well understood. Errors in mitotic cell division can provoke chromothripsis through entrapment of missegregated chromosomes into aberrant structures called micronuclei. Dr. Ly previously identified that chromosomes in micronuclei are subjected to extensive shattering in mitosis, and the resulting DNA fragments are reassembled by canonical end-joining repair in the subsequent interphase. This proposal for an NIH Pathway to Independence Award seeks to understand how chromosome segregation errors during mitosis initiate a cascade of downstream genomic instability events to directly shape or contribute to the cancer genome. In the first aim, examples of fully functional chromosomes with de novo chromothripsis will be generated through development of a powerful and reversible centromere-specific inactivation approach coupled to a chromosome-specific selection strategy in human tissue culture cells. Genome-wide sequencing will be used to identify the hallmark features of chromothripsis from unique clonal derivatives following chromosome missegregation into micronuclei. In the second aim, the precise mechanisms and spatiotemporal dynamics of chromosome shattering and reassembly events will be explored through multidisciplinary cell biological approaches, including gene disruption and live- cell imaging. The third aim will focus on how chromosomes with gross rearrangements that lack a functional alphoid centromere are able to propagate indefinitely through the epigenetic formation of a stable, new centromere at non-alphoid loci. By leveraging his expertise in engineering sophisticated tissue culture models combined with his background in cancer biology, these collective efforts by Dr. Ly will establish the mechanisms and consequences of mitotic errors in triggering genomic instability – insights that are critical for understanding the biogenesis of complex genomic features commonly manifested in patient tumors. During the mentored K99 phase of the Award, Dr. Ly will receive additional and needed training at the Ludwig Institute for Cancer Research under the guidance of Dr. Don Cleveland – a widely recognized and established leader in the fields of cell division and aneuploidy. The Ludwig Institute for Cancer Research, the University of California at San Diego, and the surrounding La Jolla scientific community serves as an exceptional atmosphere for research training, collaborative science, and career development. An excellent team of collaborators (Drs. David Page and Andrew Shiau) and consultants (Drs. Richard Kolodner, Paul Mischel, and Karen Oegema) has been assembled to provide Dr. Ly with additional scientific training and career support before, during, and after transitioning toward an independent tenure-track faculty position. The R00 Award phase will set the foundation for Dr. Ly's long-term career goals of establishing a rigorous research program that seeks to address exciting, unanswered questions in the fields of cell biology, cancer genetics, and genome stability through use of cutting-edge, interdisciplinary methodologies.