Credentialing next-generation human glioma models for precision therapeutics

认证下一代人类神经胶质瘤模型的精准治疗

• 批准号: 10830654
• 负责人: Frank Furnari
• 金额: $ 13.15万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10830654
• 关键词: Affinity; Award; Bioinformatics; Biology; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Combined Modality Therapy; Coupled; Credentialing; Data; Drug Targeting; Drug resistance; Engineering; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Failure; Foundations; Genetic Engineering; Glioma; Goals; Human; Immobilization; Investigational Therapies; Isotopes; Kinetics; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Modeling; Molecular; Monitor; Parents; Peptides; Phosphorylation; Phosphotransferases; Pre-Clinical Model; Precision therapeutics; Primary Brain Neoplasms; Protein-Serine-Threonine Kinases; Proteins; Proteomics; Reaction; Sampling; Sepharose; Serine; Technology; Therapeutic Uses; Tyrosine; Tyrosine Kinase Inhibitor; Update; Work; combat; design; experience; genome editing; induced pluripotent stem cell; inhibitor; insight; interest; microchip; model design; mutant; next generation; next generation sequencing; novel; preclinical development; response; small molecule; success; time use; treatment response; tumor

ABSTRACT This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT- CA-23-045. Despite notable successes in other cancers, precision therapeutics have failed in EGFR-driven gliomas, the most common and deadly primary brain tumors. Reasons for failure include the lack of preclinical models that faithfully recapitulate the biology of EGFR-driven gliomas and adaptive responses to therapy. Through our multi-PI (MPI) parent award R01 CA258248, we are developing and molecularly credentialing novel EGFR mutant-driven human glioma models for use in preclinical development of tyrosine kinase inhibitor (TKI)-based therapies. The foundation of our modeling work comes from the Furnari Lab (UCSD), who developed a novel platform for engineering glioma models from human induced pluripotent stem cells (iPSC) using CRISPR genome editing. The Miller Lab has extensive experience in small molecule experimental therapeutics using genetically engineered (GE) glioma models, next- generation sequencing, and proteomics. Our parent award proposed to leverage our established collaboration with the Johnson Lab (UNC) to profile kinase expression en masse using multiplex inhibitor beads coupled with mass spectrometry (MIB-MS). We previously used this approach to show that dynamic kinome reprogramming contributes to targeted drug resistance in glioma models. In this Multi-PI supplement, we will extend our originally proposed work through integration of two distinct, but complementary kinome profiling approaches. The Johnson Lab has developed an updated version of MIB- MS termed SureQuant. MIB-MS requires affinity-based enrichment of active kinases using broad-spectrum inhibitors chemically coupled to Sepharose beads and is limited to relative quantification of kinase expression. SureQuant uses isotopically “heavy” peptides, designed from MIB-enriched, proteolytically digested human kinases, and parallel reaction monitoring (PRM) to absolutely quantify kinase expression in otherwise un-enriched protein samples. This advance will be coupled with a second complimentary approach: the PamStation technology from the Kinome Core (Willey Lab) at UAB. This approach monitors the kinetics of tyrosine or serine/threonine kinase activity (phosphorylation) in real time using specific peptide substrates immobilized on microchips. Thus, whereas SureQuant accurately measures kinase expression, PamStation monitors substrate-level kinase activity. We hypothesize that a combination of these two approaches, and bioinformatic integration of the resulting data, will provide novel insights into the glioma kinome and its dynamic response to targeted EGFR TKI.

抽象的 该申请是根据特殊利益通知（NOSI）提交的 CA-23-045。尽管在其他癌症中取得了显着的成功，但精确疗法在EGFR驱动中还是失败了 神经胶质瘤，最常见和致命的原发性脑肿瘤。失败的原因包括缺乏 忠实地概括了EGFR驱动的神经胶质瘤的生物学和对适应性反应的临床前模型 治疗。通过我们的多PI（MPI）父级R01 CA258248，我们正在开发和分子 证书的新型EGFR突变驱动的人神经胶质瘤模型用于临床前开发 基于酪氨酸激酶抑制剂（TKI）的疗法。我们的建模工作的基础来自 Furnari Lab（UCSD），他开发了一个新颖的平台，用于人类诱导的工程神经胶质瘤模型 使用CRISPR基因组编辑的多能干细胞（IPSC）。米勒实验室有丰富的经验 小分子实验疗法使用一般设计的（GE）神经胶质瘤模型，其次 生成测序和蛋白质组学。我们的父母奖提议利用我们已建立的 使用多重抑制剂与Johnson Lab（UNC）合作介绍激酶表达式 珠子与质谱（MIB-MS）结合。我们以前使用这种方法来表明这种动态 Kinome重新编程有助于神经胶质瘤模型的靶向耐药性。在这个多pi中 补充，我们将通过整合两个不同的，但 完全活化的分析方法。约翰逊实验室已开发了MIB的更新版本 MS称为肯定。 MIB-MS需要使用广谱的基于亲和力的活性激酶富集 化学耦合与塞彭珠珠的抑制剂，仅限于激酶的相对定量 表达。肯定使用的是用同位素“重”肽，该肽由MIB富集，蛋白水解设计 消化的人激酶和平行反应监测（PRM），以绝对量化激酶表达 在其他未添加的蛋白质样品中。此进步将与第二个免费 方法：UAB的Kinome Core（Willey Lab）的Pamstation技术。这种方法监视 酪氨酸或丝氨酸/苏氨酸激酶活性（磷酸化）的动力学实时使用特定 固定在微芯片上的肽底物。那肯定可以准确测量激酶 表达，pamstation监测底物级激酶活性。我们假设是 这两种方法以及所得数据的生物信息集成，将为您提供新的见解 神经胶质瘤及其对靶向EGFR TKI的动态反应。