Defining the role of SWI/SNF chromatin remodeling complex mutations during melanoma progression

定义 SWI/SNF 染色质重塑复合体突变在黑色素瘤进展过程中的作用

• 批准号: 9367641
• 负责人: Alan Hunter Shain
• 金额: $ 19.89万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9367641
• 关键词: Affect; Applications Grants; Award; Biochemical; Biological; Biological Assay; Biology; CRISPR/Cas technology; Cell Line; Cells; Cellular biology; Chromatin Remodeling Factor; Data; Drosophila snf protein; Drug Targeting; Engineering; Evolution; Frequencies; Future; Genes; Genetic; Genetic Models; Genetic Transcription; Genetic study; Genomics; Goals; Grant; Homeostasis; Human; Intervention; K22 Award; Laboratories; Lesion; Malignant Neoplasms; Mutate; Mutation; Normal tissue morphology; Nucleosomes; Patients; Phenotype; Process; Publications; Reproducibility; Research; Research Infrastructure; Resources; Role; SWI/SNF Family Complex; Secure; Sucrose; TP53 gene; Testing; Therapeutic; Time; Training; Tumor Biology; Tumor Suppressor Proteins; base; career; career development; chromatin remodeling; daughter cell; drug candidate; gene function; genetic approach; genetic resource; innovation; melanocyte; melanoma; migration; mutant; novel; skills; success; tool; tumor

Project Summary/Abstract The human SWItch/Sucrose NonFermentable chromatin-remodeling complex, commonly abbreviated as SWI/SNF, is composed of 10-15 biochemically distinct subunits. SWI/SNF complexes use the energy provided from ATP to reposition nucleosomes and modulate transcription. We found that SWI/SNF chromatin- remodeling genes are mutated in 20% of all cancers, including 30% of melanomas. While genetic studies clearly implicate SWI/SNF genes as tumor suppressors, it is not clear how mutations in these genes contribute to cancer. Experimental evidence from our group and others indicates that the genetic context in which a SWI/SNF component is perturbed heavily influences the fate of that cell. It will be important to study these mutations in their proper genetic context in order to resolve their functional contributions to cancer. Unfortunately, the genetic context in which SWI/SNF mutations occur is poorly understood. This is because tumors evolve through a multistep process, and most tumors are sequenced at a late stage, after they have fully evolved. I have developed an assay to determine the order of mutations as they occur during the evolution of melanoma. In aim 1 will utilize this assay to determine the precise context in which SWI/SNF mutations occur during the evolution of melanoma. This is innovative because I utilize a cutting-edge approach to reveal an unresolved feature of SWI/SNF tumor biology. In aim 2, I will engineer primary human melanocytes using CRISPR/Cas9 to mimic the genetic context prior to and after a SWI/SNF mutation occurs. These parental and SWI/SNF-mutant daughter cell lines will then be compared to functionally and mechanistically interrogate the effects of SWI/SNF mutations in an otherwise isogenetic background. This strategy is innovative because there are no known cell lines that definitively recapitulate the genetic context of a partially evolved melanoma before and after a SWI/SNF mutation occurs. I hypothesize that SWI/SNF mutations occur in a reproducible context during melanoma progression and promote either an invasive or a mutator phenotype. This hypothesis is based on observations from preliminary data that SWI/SNF mutations occur at the transition to invasive melanoma and coincide with a spike in the overall mutation burden. This proposal is significant because SWI/SNF genes are frequently mutated in melanoma, yet almost nothing is known regarding their roles in melanomagenesis. SWI/SNF proteins are not themselves ideal drug targets because they are tumor suppressors and are also important in normal cellular biology; however, completion of these studies should reveal the mechanisms underlying SWI/SNF melanoma biology and thus provide therapeutic opportunities for the large number of patients whose tumors are driven by these mutations. Finally, the protected time associated with this unique award mechanism will allow me to carry out scientific and professional career development activities, as described, that will facilitate the success of my independent research career.

项目摘要/摘要 人类Switch/蔗糖非发酵染色质重塑复合体，通常缩写为 SWI/SNF由10-15个不同的生化亚基组成。SWI/SNF复合体利用提供的能量 从三磷酸腺苷到核小体的重新定位和转录调控。我们发现SWI/SNF染色质- 重塑基因在20%的癌症中发生突变，包括30%的黑色素瘤。虽然基因研究 明确地表明SWI/SNF基因是肿瘤抑制基因，目前还不清楚这些基因的突变是如何起作用的 致癌。来自我们小组和其他人的实验证据表明， SWI/SNF组分的紊乱严重影响了该细胞的命运。研究这些问题将是很重要的 在其适当的遗传背景下的突变，以解决其对癌症的功能贡献。 不幸的是，人们对SWI/SNF突变发生的遗传背景知之甚少。这是因为 肿瘤的进化经历了一个多步骤的过程，大多数肿瘤在它们发生后的晚期被测序 完全进化了。我已经开发了一种方法来确定在进化过程中发生的突变的顺序 黑色素瘤。在Aim 1中，将利用这种分析来确定SWI/SNF突变的确切背景 发生在黑色素瘤的进化过程中。这是创新的，因为我利用一种尖端的方法来揭示 SWI/SNF肿瘤生物学的一个尚未解决的特征。在目标2中，我将利用 CRISPR/Cas9来模拟SWI/SNF突变发生前后的遗传背景。这些父母和 然后对SWI/SNF突变的子代细胞系进行功能和机械上的比较 SWI/SNF突变在其他同源背景下的影响。这一战略是创新的，因为 以前没有已知的细胞系可以明确地概括部分进化的黑色素瘤的遗传背景 在SWI/SNF突变发生后。我假设SWI/SNF突变发生在可重现的背景下 在黑色素瘤进展期间，并促进侵袭性或突变型表型。这一假设是 根据初步数据的观察，SWI/SNF突变发生在向侵袭性转变的过程中 黑色素瘤与总体突变负担的峰值不谋而合。这项建议意义重大，因为 SWI/SNF基因在黑色素瘤中经常发生突变，但对它们在黑色素瘤中的作用几乎一无所知。 黑色素瘤形成。SWI/SNF蛋白本身并不是理想的药物靶点，因为它们是肿瘤 抑制物，在正常细胞生物学中也很重要；然而，这些研究的完成应该 揭示SWI/SNF黑色素瘤生物学基础的机制，从而提供治疗机会 大量患者的肿瘤是由这些突变驱动的。最后，受保护的时间 与这种独特的奖励机制相关联，将使我能够开展科学和专业的职业生涯 发展活动，如上所述，将促进我的独立研究事业的成功。