Manipulating chromosome structure to suppress genome instability

项目摘要/摘要 ，是一种基因组不稳定的形式，这是癌细胞的共同特征。CIN，根据定义， 大多数实体瘤都表现出结构和数量上的染色体异常，其特征是 基因组不稳定。染色体不稳定(CIN)，频繁而持久的整体得失 染色体 结果产生了非整倍体细胞，并具有重要的意义。CIN已被提议用于促进 通过促进遗传异质性来促进肿瘤细胞的进化，从而使促进生长的变化成为可能 和转移。此外，CIN产生的基因组多样性促进了癌细胞的发展 这些人对治疗方法有抵抗力，更容易肿瘤复发。因此，CIN与贫穷相关。 患者预后。在识别和治疗表现为CIN的癌症方面的局限性源于缺乏 对导致CIN和无法操纵的潜在分子机制的理解 以治疗相关的方式治疗CIN。 我最近证明了pRb，一种肿瘤抑制因子，其功能在许多人类身上都受到了损害 癌症，促进基因组的稳定。我已经证明了pRb的丢失会导致染色质结构的缺陷，并且 这些异常会增加DNA损伤和有丝分裂保真度的腐败。然而，腐败 并不是染色质结构受损的唯一机制，还有其他 染色质结构的调节也可能同样影响基因组的稳定性。染色质的一种方式 结构是通过调节染色质和组蛋白甲基化状态而受到影响的。事实上，一个不断增长的身体 大量证据表明，细胞表观遗传学特征的改变与非整倍体相关。在……里面 此外，我的初步研究表明，调节甲基转移酶子集的水平可以 损害染色质结构和基因组完整性。总而言之，这些数据表明，全面放松监管 染色质结构的改变与基因组不稳定性的发生有着广泛的关系。然而，这并不是 以系统的方式分析，以及染色质结构被解除管制和 导致基因组不稳定的因素仍不清楚。 值得注意的是，我已经证明，通过恢复染色体结构，我可以抑制DNA损伤和 促进肿瘤细胞染色体的准确分离。在这样做的过程中，我发现了Wapl，一个负面的 凝聚力调节因子作为CIN的新调节因子。在下面的提议中，这一新颖的战略将使我 首次测试抑制CIN是否会降低肿瘤细胞的生长能力 体内存在化疗耐药性，因此传统治疗后复发的可能性较小。此外, 结合使用生物信息学和细胞生物学的方法，我将检查结构缺陷 与CIN相关的基因可被用来增加对DNA损伤剂的敏感性并减少细胞 生死存亡。最后，我将确定染色质结构的调节器，它们的解除管制会影响基因组的稳定性 使用我开发的一种新的筛选方法。成功完成本文件中所述的目标 这一提议不仅将为人类癌症中CIN的发生提供新的和有价值的见解，而且 揭示了在肿瘤中操纵这一共同特征的治疗后果。 我的职业目标是在一家领先的研究所获得一个研究教员的职位，在那里我将为肿瘤做定义 维持正常染色体结构和基因组完整性的抑制机制。然而，我的 通过增加我的专业知识，在这个领域成功地向独立过渡将得到极大的支持 在细胞生物学技术中为小鼠癌症模型提供新的培训机会，生物信息学和高级 吞吐量筛选方法。正是凭借这些获得的技能，我将能够识别与癌症相关的 影响基因组稳定性的染色质结构调节剂及其作为新肿瘤的作用 抑制者。这个项目的成功将得到杰出的培训委员会的大力支持 在我走向独立的过程中，他们聚集在一起指导我并给我提供建议。这个委员会包括我的 共同导师尼古拉斯·戴森博士和杰弗里·恩格尔曼博士，以及丹尼尔·哈伯博士、西里尔·贝内斯博士和 大卫·佩尔曼博士，他们都是各自领域的专家。此外，例外的研究 MGH提供的环境和哈佛医疗区拥有所有必要的物理资源 完成拟议的培训和研究研究所需的经费。K99/R00奖将为我提供 这一高级培训所需的保护时间，并允许我继续在 戴森博士和恩格尔曼博士的导师。我预计这项提案的指导阶段将包括 使用小鼠模型研究操纵CIN对实体瘤的治疗相关性，以及 启动一种新的筛选方法，将需要1-2年时间，并至少出版一份主要出版物。这个 在该奖项的独立阶段之后，我将能够识别和描述其他与癌症相关的、 染色体结构和基因组稳定性的调节。这些数据加在一起将用来证明未来 我计划在独立阶段第三年开始时提交的R01拨款中提出的研究建议。

Project Summary/Abstract , is one form of genome instability that is a common feature of cancer cells. CIN, by definition, The majority of solid tumors exhibit both structural and numerical chromosome aberrations characteristic of genomic instability. Chromosome instability (CIN), the frequent and persistent gains and losses of whole chromosomes results in the generation of aneuploid cells and has important implications. CIN has been proposed to facilitate the evolution of tumor cells by promoting genetic heterogeneity, thereby enabling changes that promote growth and metastasis. Furthermore, genomic diversity generated by CIN promotes the development of cancer cells that are resistant to therapeutics and are more prone to tumor relapse. Consequently, CIN correlates with poor patient prognosis. Limitations in identifying and treating cancers that exhibit CIN stem from a lack of understanding of the underlying molecular mechanisms that contribute to CIN and the inability to manipulate CIN in a therapeutically relevant manner. I have recently demonstrated that pRB, a tumor suppressor whose function is compromised in many human cancers, promotes genome stability. I have shown that pRB loss leads to defects in chromatin structure, and that these abnormalities promote increased DNA damage and corruption of mitotic fidelity. However, corruption of the pRB pathway is not the only mechanism by which chromatin structure is compromised, and other regulators of chromatin structure are likely to similarly impact genome stability. One manner in which chromatin structure is impacted is through regulation of chromatin and histone methylation status. Indeed, a growing body of evidence has suggested that modification of epigenetic characteristics of cells correlates with aneuploidy. In addition, my preliminary studies have shown that modulation of levels of a subset of methyl-transferases can compromise chromatin structure and genome integrity. Together, these data suggest that general deregulation of chromatin structure is broadly relevant to the genesis of genomic instability. However, this has not been analyzed in a systematic manner, and the mechanisms by which chromatin structure are deregulated and contributes to genome instability remain unknown. Significantly, I have shown that by restoring chromosomal structure, I can suppress both DNA damage and promote accurate chromosome segregation in tumor cells. In doing so, I have identified Wapl, a negative regulator of cohesion as a novel regulator of CIN. In the following proposal, this novel strategy will enable me to test, for the first time, whether suppression of CIN renders tumor cells less capable of developing chemotherapeutic resistance in vivo, and thus less likely to relapse following traditional treatments. In addition, using a combination of bioinformatics and cell biological approaches, I will examine whether structural defects associated with CIN can be exploited to increase sensitivity to DNA damaging agents and decrease cell survival. Finally, I will identify regulators of chromatin structure whose deregulation impacts genome stability using a novel screening approach I have developed. The successful completion of the aims described in this proposal will not only provide new and valuable insights into the genesis of CIN in human cancers, but also reveal the therapeutic ramifications of manipulating this common feature in tumors. My career goal is to obtain a research faculty position at a leading institute where I will define the tumor suppressive mechanisms that maintain normal chromosome structure and genomic integrity. However, my successful transition to independence in this field would be significantly bolstered by augmenting my expertise in cell biological techniques with new training opportunities in mouse models of cancer, bioinformatics and high throughput screening approaches. It is with these acquired skills that I will be able to identify cancer-relevant regulators of chromatin structure that impact genome stability and assess their roles as novel tumor suppressors. The success of this project will be greatly enhanced by the outstanding training committee that I have assembled to mentor and advise me as I progress towards independence. This committee includes my co-mentors Dr. Nicholas Dyson and Dr. Jeffrey Engelman, as well as Dr. Daniel Haber, Dr. Cyril Benes, and Dr. David Pellman, all of whom are experts in their respective fields. In addition, the exceptional research environment available at MGH and the Harvard Medical Area has all of the necessary physical resources required for the completion of the proposed training and research studies. The K99/R00 award would afford me the protected time needed for this advanced training and allow me to continue to foster my growth under the mentorship of Dr. Dyson and Dr. Engelman. I expect that the mentored phase of this proposal, which includes using mouse models to investigate the therapeutic relevance of manipulating CIN in solid tumors, and the initiation of a novel screening approach, will take 1-2 years and result in at least one major publication. The following independent phase of the award will allow me to identify and characterize additional, cancer-relevant, regulators of chromosome structure and genome stability. Together these data will be used to justify future studies proposed in an R01 grant that I plan to submit at the start of the third year of the independent phase.