Manipulating chromosome structure to suppress genome instability

项目总结/摘要 是基因组不稳定性的一种形式，是癌细胞的共同特征。CIN，根据定义， 大多数实体瘤表现出染色体结构和数目畸变的特征， 基因组不稳定性染色体不稳定性（CIN），即染色体的频繁和持续的获得和丢失。 染色体 导致非整倍体细胞的产生并具有重要意义。CIN已被提议促进 肿瘤细胞通过促进遗传异质性而进化，从而实现促进生长的变化 和转移。此外，CIN产生的基因组多样性促进癌细胞的发展 对治疗药物有抵抗力并且更容易复发。因此，CIN与不良 患者预后鉴定和治疗表现出CIN的癌症的局限性源于缺乏 了解导致CIN的潜在分子机制， 以治疗相关的方式治疗CIN。 我最近证明了pRB，一种在许多人中功能受损的肿瘤抑制因子， 癌症，促进基因组稳定性。我已经证明pRB缺失导致染色质结构缺陷， 这些异常促进了DNA损伤的增加和有丝分裂保真度的恶化。然而，腐败 pRB通路的激活不是染色质结构受损的唯一机制， 染色质结构的调节物可能类似地影响基因组稳定性。一种染色质 结构受到影响是通过调节染色质和组蛋白甲基化状态。事实上，一个不断增长的身体 大量证据表明，细胞表观遗传特征的改变与非整倍性相关。在 此外，我的初步研究表明，调节甲基转移酶亚类的水平， 损害染色质结构和基因组完整性。总之，这些数据表明，普遍放松管制 染色质结构的变化与基因组不稳定性的发生有着广泛的关系。然而，这并没有被 以系统的方式分析，以及染色质结构失调的机制， 基因组不稳定性的原因尚不清楚。 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， 促进肿瘤细胞中染色体的精确分离。在此过程中，我确定了Wapl，一个负面的 作为CIN的新型调节剂的内聚调节剂。在下面的建议中，这个新颖的策略将使我能够 这是第一次测试抑制CIN是否会降低肿瘤细胞的发展能力， 因此，本发明的药物组合物在体内具有化学治疗抗性，并且因此在传统治疗后不太可能复发。此外，本发明还提供了一种方法， 使用生物信息学和细胞生物学方法相结合，我将研究结构缺陷是否 可以利用与CIN相关的细胞毒性来增加对DNA损伤剂的敏感性， 生存最后，我将确定染色质结构的调节，其失调影响基因组的稳定性 使用我开发的一种新的筛选方法。成功实现本报告所述目标， 该提案不仅将为人类癌症中CIN的发生提供新的和有价值的见解，而且还将 揭示了在肿瘤中操纵这一共同特征的治疗分支。 我的职业目标是在一家领先的研究所获得一个研究人员的职位，在那里我将定义肿瘤 抑制机制，维持正常的染色体结构和基因组的完整性。不过我 如果我的专业知识得到加强， 在细胞生物学技术与新的培训机会，在小鼠模型的癌症，生物信息学和高 通量筛选方法。正是有了这些获得的技能，我将能够识别癌症相关的 影响基因组稳定性并评估其作为新型肿瘤的作用的染色质结构调节剂 抑制剂这个项目的成功将大大提高了优秀的培训委员会，我 在我走向独立的过程中给予我指导和建议。该委员会包括我的 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·贝恩斯博士和 博士大卫佩尔曼，他们都是各自领域的专家。此外，特殊的研究 MGH和哈佛医学区拥有所有必要的物理资源 完成拟议的培训和研究所需的资源。K99/R 00奖将为我提供 这种先进的培训所需的保护时间，让我继续促进我的成长， 戴森博士和恩格尔曼博士的指导。我希望这一建议的指导阶段，其中包括 使用小鼠模型研究在实体瘤中操作CIN的治疗相关性， 启动一种新的筛选方法，将需要1-2年，并导致至少一个主要出版物。的 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因子。这些数据将用于证明未来 我计划在独立阶段的第三年开始时提交R 01赠款中提出的研究。

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.