Credentialing next-generation human glioma models for precision therapeutics

摘要 本申请是为回应特别利益通知(NOSI)而提出的，该通知被确认为非- CA-23-045。尽管在其他癌症方面取得了显著的成功，但精准疗法在EGFR驱动的癌症中失败了 胶质瘤是最常见和最致命的原发脑瘤。失败的原因包括缺乏 临床前模型真实地概括了EGFR驱动的胶质瘤的生物学和适应性反应 心理治疗。通过我们的多PI(MPI)家长奖R01 CA258248，我们正在开发和分子 新的EGFR突变驱动的人脑胶质瘤模型用于临床前研究 以酪氨酸激酶抑制剂(TKI)为基础的治疗。我们建模工作的基础来自于 Furnari Lab(UCSD)，他开发了一个新的平台，用于从人诱导的脑胶质瘤模型中工程 使用CRISPR基因组编辑的多能干细胞(IPSC)。米勒实验室在以下方面拥有丰富的经验 使用基因工程(GE)胶质瘤模型的小分子实验疗法，下一步- 世代测序和蛋白质组学。我们的父级奖项建议利用我们已建立的 与约翰逊实验室(UNC)合作，使用多重抑制剂整体分析激酶的表达 珠联用质谱仪(MIB-MS)。我们以前使用这种方法来展示动态的 Kinome重新编程有助于胶质瘤模型中的靶向耐药性。在此多PI中 作为补充，我们将通过整合两个不同的、但 互补的基因组图谱分析方法。约翰逊实验室开发了MIB的更新版- 她被命名为SureQuant。MIB-MS要求使用广谱基于亲和力的活性激酶的富集化 化学偶联到琼脂糖珠上的抑制剂，仅限于相对定量的激酶 表情。SureQuant使用的是从富含MIB的蛋白质水解物中设计的同位素“重”肽 消化的人激酶和平行反应监测(PRM)以绝对量化激酶的表达 在其他未浓缩的蛋白质样本中。这笔预付款将与第二份免费赠品一起提供 方法：UAB的Kinome Core(Willey Lab)的PamStation技术。此方法监视 酪氨酸或丝氨酸/苏氨酸激酶活性(磷酸化)的实时动力学 固定在微芯片上的多肽底物。因此，尽管SureQuant准确地测量了激酶 表达，PamStation监测底物水平的激酶活性。我们假设是一组 这两种方法，以及由此产生的数据的生物信息学集成，将为 胶质瘤细胞系及其对靶向EGFR TKI的动态反应。

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.