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.

项目概要/摘要 人类 SWITCH/蔗糖不可发酵染色质重塑复合物，通常缩写为 SWI/SNF，由10-15个生化上不同的亚基组成。 SWI/SNF 复合体使用提供的能量 从 ATP 重新定位核小体并调节转录。我们发现 SWI/SNF 染色质 20% 的癌症（包括 30% 的黑色素瘤）中重塑基因发生突变。在进行基因研究的同时 明确表明 SWI/SNF 基因是肿瘤抑制因子，但尚不清楚这些基因的突变如何发挥作用 到癌症。我们小组和其他人的实验证据表明， SWI/SNF 成分受到干扰会严重影响该细胞的命运。研究这些很重要 在其适当的遗传背景中进行突变，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 肿瘤的演变是一个多步骤的过程，大多数肿瘤是在晚期才进行测序的。 完全进化了。我开发了一种测定法来确定进化过程中突变发生的顺序 黑色素瘤。目标 1 将利用该测定来确定 SWI/SNF 突变的精确背景 发生在黑色素瘤的进化过程中。这是创新的，因为我利用了一种尖端的方法来揭示 SWI/SNF 肿瘤生物学的一个未解决的特征。在目标 2 中，我将使用以下方法设计原代人类黑素细胞 CRISPR/Cas9 可模拟 SWI/SNF 突变发生之前和之后的遗传背景。这些父母和 然后将比较 SWI/SNF 突变子细胞系，以从功能和机制上探讨 SWI/SNF 突变在同基因背景下的影响。这个策略是创新的，因为 之前没有任何已知的细胞系能够明确地重现部分进化的黑色素瘤的遗传背景 以及发生 SWI/SNF 突变后。我假设 SWI/SNF 突变发生在可重复的环境中 在黑色素瘤进展过程中并促进侵袭性或突变表型。这个假设是 根据初步数据观察，SWI/SNF 突变发生在向侵入性转变时 黑色素瘤与总体突变负担的激增相一致。这个提议意义重大，因为 SWI/SNF 基因在黑色素瘤中经常发生突变，但关于它们在黑色素瘤中的作用几乎一无所知。 黑色素瘤发生。 SWI/SNF 蛋白本身并不是理想的药物靶点，因为它们是肿瘤 抑制因子，在正常细胞生物学中也很重要；然而，完成这些研究应该 揭示 SWI/SNF 黑色素瘤生物学的潜在机制，从而为以下疾病提供治疗机会 大量患者的肿瘤是由这些突变驱动的。最后是保护时间 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 如上所述的开发活动将促进我的独立研究生涯的成功。