Top-down proteomic characterization of MEK/ERK in MAPK-driven cancers

MAPK 驱动的癌症中 MEK/ERK 自上而下的蛋白质组学表征

• 批准号: 10248327
• 负责人: Bryon Shane Drown
• 金额: $ 6.37万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10248327
• 关键词: Antineoplastic Agents; Area; BRAF gene; Behavior; Biology; Cancer Histology; Catalogs; Cell Culture Techniques; Cell Line; Cells; Chemicals; Collaborations; Colorectal; Colorectal Cancer; Computers and Advanced Instrumentation; Data; Data Analyses; Digestion; Disease; Dose-Limiting; Drug resistance; Elements; Environment; Feedback; Future; Gene Expression Regulation; Generations; Genetic Transcription; Genotype; Growth; Growth and Development function; Immunoprecipitation; In Vitro; Ions; KRAS2 gene; Knowledge; Laboratories; Lead; Link; MAP2K1 gene; MAPK3 gene; MEKs; Malignant Neoplasms; Malignant neoplasm of lung; Mass Spectrum Analysis; Melanoma Cell; Metastatic Melanoma; Metastatic to; Methods; Mitogen-Activated Protein Kinases; Modeling; Modification; Mutation; Normal Cell; Oncogenic; Organic Chemistry; Outcome; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Phosphorylation Site; Post-Translational Protein Processing; Program Development; Proteins; Proteomics; RNA Splicing; Ras Signaling Pathway; Recurrence; Regulation; Research Proposals; Research Training; Resistance; Sampling; Schools; Scientist; Signal Pathway; Signal Transduction; Stimulus; Structure; Surveys; Techniques; Toxic effect; Training; Training Programs; Translating; Translations; Universities; Ursidae Family; Variant; Work; addiction; analytical method; cancer cell; cancer type; career; design; drug development; improved; inhibitor/antagonist; insight; interest; knowledge base; melanoma; metastatic colorectal; next generation; novel therapeutics; patient derived xenograft model; patient response; resistance mechanism; response; skills; targeted treatment; training opportunity; tumor

Abstract The MAPK signaling pathway is a central element of normal development and growth and is a common driver in many cancer types. Although typically described solely by their canonical phosphorylation sites, the downstream components of the MAPK pathway, including MEK1/2 and ERK1/2, also integrate signals from other inputs. This behavior is especially important in elucidating the mechanisms of intrinsic and acquired resistance to drug therapies that target the MAPK pathway. Despite the clear importance of signal integration, almost nothing is known about the combinations of post-translational modifications (PTMs) present on MEK1/2 and ERK1/2 in cancer, particularly in response to drug treatment. These PTM combinations are not effectively detected by standard bottom-up proteomic approaches due to proteolytic digestion and resulting protein inference problem. Here, we propose utilizing an alternative approach comprising immunoprecipitation and subsequent top-down mass spectrometry (IP-TDMS), in which the protein of interest is enriched, ionized, and fragmented to provide precise PTM characterization of intact modified protein forms, or proteoforms. This IP-TDMS approach will be initially executed in cell culture models of resistant metastatic melanoma and colorectal carcinoma. In order to determine if these proteoforms are unique to cancer type and drug responsiveness, IP-TDMS will next be translated to patient derived xenografts. Findings from this study are anticipated to provide considerable insight into how tumors can overcome addiction to MAPK signaling and potential new drug development leads. The proposed work provides an excellent training opportunity in mass spectrometry and proteomics, and the Kelleher laboratory is the ideal environment for this training. The Kelleher group has worked at the forefront of top-down mass spectrometry for the last decade and has extensive knowledge and capability in this area. Having received training in chemical biology and organic chemistry in graduate school, I have constructed a detailed training plan in close collaboration with Prof. Kelleher to rapidly build expertise in this new field. The Kelleher group has a highly structured training program for with the advanced instrumentation in the lab that will establish mass spectrometry as a core expertise. This training program will afford me with the capacity to acquire and analyze targeted proteomics data, a critical skill set for establishing my independent career. Northwestern University features state-of-the-art facilities and many collaborative opportunities with world renowned scientists. We are highly optimistic of the prospect of this combined research proposal and training plan given the intra- laboratory and institutional support for this work.

摘要 MAPK信号通路是正常发育和生长的中心要素，并且是细胞增殖和分化的共同驱动力。 许多癌症类型。虽然通常仅通过其典型磷酸化位点来描述，但下游磷酸化位点的 MAPK通路的组分，包括MEK 1/2和ERK 1/2，也整合来自其他输入的信号。这 行为在阐明内在和获得性耐药机制方面尤为重要 针对MAPK通路的治疗。尽管信号整合的重要性显而易见，但几乎没有什么是 已知存在于MEK 1/2和ERK 1/2上的翻译后修饰（PTM）的组合， 癌症，尤其是药物治疗。这些PTM组合不能有效检测， 标准的自下而上的蛋白质组学方法由于蛋白水解消化和导致的蛋白质推断问题。 在这里，我们建议利用另一种方法，包括免疫沉淀和随后的自上而下 质谱（IP-TDMS），其中感兴趣的蛋白质被富集、电离和片段化以提供 完整修饰的蛋白质形式或蛋白质形式的精确PTM表征。这种IP-TDMS方法将 最初在耐药转移性黑色素瘤和结肠直肠癌的细胞培养模型中进行。为了 确定这些蛋白质形式是否是癌症类型和药物反应所特有的，IP-TDMS下一步将 转化为患者来源的异种移植物。这项研究的结果预计将提供相当多的见解 肿瘤如何克服对MAPK信号的依赖以及潜在的新药开发线索。 拟议的工作提供了一个很好的培训机会，在质谱和蛋白质组学， 凯莱赫实验室是这种培训的理想环境。凯莱赫集团一直致力于 自上而下的质谱分析在过去的十年中，并在这一领域拥有广泛的知识和能力。具有 在研究生院接受了化学生物学和有机化学的培训，我构建了一个详细的 培训计划与教授凯莱赫迅速建立在这个新领域的专业知识。凯莱赫酒店 小组有一个高度结构化的培训计划，在实验室中使用先进的仪器， 质谱分析作为核心专业知识。这个培训项目将使我有能力获得和 分析目标蛋白质组学数据，这是建立我独立职业生涯的关键技能。西北 大学拥有最先进的设施和许多与世界知名科学家合作的机会。 我们对这一结合研究方案和培训计划的前景非常乐观， 为这项工作提供实验室和机构支持。

项目成果

Mapping the Proteoform Landscape of Five Human Tissues.

• DOI: 10.1021/acs.jproteome.2c00034
• 发表时间: 2022-05-06
• 期刊: JOURNAL OF PROTEOME RESEARCH
• 影响因子: 4.4
• 作者: Drown, Bryon S.;Jooss, Kevin;Melani, Rafael D.;Lloyd-Jones, Cameron;Camarillo, Jeannie M.;Kelleher, Neil L.
• 通讯作者: Kelleher, Neil L.