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 人开关/蔗糖不可发酵的染色质复合物，通常被缩写为 SWI/SNF由10-15种生物化学不同的亚基组成。 SWI/SNF复合物使用提供的能量 从ATP到重新定位核小体，并调节转录。我们发现SWI/SNF染色质 - 重塑基因在所有癌症中有20％（包括黑色素瘤的30％）突变。而遗传研究 清楚地将SWI/SNF基因视为肿瘤抑制因子，尚不清楚这些基因中的突变如何贡献 癌症。来自我们小组和其他人的实验证据表明，遗传环境 SWI/SNF成分受到干扰，严重影响了该细胞的命运。研究这些很重要 在其适当的遗传环境中的突变，以解决其对癌症的功能贡献。 不幸的是，对SWI/SNF突变发生的遗传背景知之甚少。这是因为 肿瘤通过多步骤进化，大多数肿瘤在后期进行了测序，之后 完全进化。我已经开发了一个测定法以确定突变的顺序在进化过程中发生 黑色素瘤。在AIM 1中，将利用该测定法来确定SWI/SNF突变的确切环境 发生在黑色素瘤进化期间。这是创新的，因为我利用一种尖端的方法来揭示 SWI/SNF肿瘤生物学的未解决特征。在AIM 2中，我将使用原代人类黑素细胞使用 CRISPR/CAS9在发生SWI/SNF突变之前和之后模仿遗传环境。这些父母和 然后将SWI/SNF突变的子细胞系与功能和机械询问 SWI/SNF突变在原本遗传背景下的影响。这种策略是创新的，因为那里 不是已知的细胞系，可以明确地概括了在 在发生SWI/SNF突变之后。我假设SWI/SNF突变发生在可再现的环境中 在黑色素瘤进展过程中，并促进了侵入性或突变体表型。这个假设是 根据初步数据的观察，SWI/SNF突变发生在过渡到侵入性时 黑色素瘤和与整体突变负担相兴奋。该提议很重要，因为 SWI/SNF基因在黑色素瘤中经常突变，但几乎一无所知 黑素作仿。 SWI/SNF蛋白本身并不是理想的药物靶标，因为它们是肿瘤 抑制剂，在正常细胞生物学中也很重要；但是，完成这些研究应该 揭示SWI/SNF黑色素瘤生物学的基础机制，从而为治疗机会提供 肿瘤由这些突变驱动的大量患者。最后，受保护的时间 与这种独特的奖励机制相关联，我可以从事科学和职业生涯 如上所述，开发活动将有助于我独立研究生涯的成功。