Investigating mechanisms of kinase-mediated cell migration in aggressive glioblastoma

项目摘要--莫勒 多形性胶质母细胞瘤(GBM)是成人最致命的脑肿瘤，其特点是进展迅速和 由于其高度增殖性和侵袭性，预后不良。GBM是动态异构性的，具有 一组复杂的输入，最终决定一组与细胞存活、增殖、 和侵入性迁徙。作为一个整体，本项目的目标旨在提供对以下机制的见解 胶质母细胞瘤细胞迁移。该方法的中心是评估未被开发的激酶的贡献。 调控网络对癌细胞从增殖状态到迁移状态的表型转换，潜在的 进攻性疾病进展。作为真核信号转导中含量最丰富的翻译后修饰 蛋白磷酸化在调控网络和细胞维持中起着核心作用 动态平衡，然而它们在调节细胞迁移中的作用却鲜为人知。它最近被展示出来了 GBM细胞可以使用正常的生理过程进行细胞迁移，例如利用离子通道，如 NKCC1，促进运动。Ste20家族蛋白酪氨酸激酶SPS1相关的富含脯氨酸/丙氨酸的激酶(SPAK) 细胞体积变化到阳离子-氯离子共转运体(NKCC和KCC)以维持细胞内环境的稳定 并控制细胞的迁移。尽管之前的报道强调了Spak激酶作为一种 治疗GBM的潜在治疗靶点是无法产生高产量的生理上的 到目前为止，磷酸化的激酶阻碍了这一进展。利用正交化翻译的最新进展 莱因哈特实验室的系统和质谱学支持实施强大的管道 以生理磷酸化活性为靶点的铅抑制剂化合物的鉴定和验证 Spak激酶。候选SPAK抑制剂对GBM细胞迁移和生理的影响将是 使用基于微流控的一维细胞迁移分析进行评估。同时，直接的相互作用和影响 具有GBM蛋白质组的候选抑制剂化合物将使用定量质谱学进行定义 技巧。总体而言，从这条管道中确定的小分子抑制剂化合物的表征将 对激酶网络在调节癌细胞迁移中的作用提供了重要的机制洞察， 将潜在的应用扩展到更广泛的癌症生物学领域。

Project Summary – Mohler Glioblastoma multiforme (GBM) is the deadliest brain tumor in adults, characterized by rapid progression and poor prognosis due to its highly proliferative and invasive nature. GBM is dynamically heterogeneous with a complex set of inputs ultimately determining a set of outputs (phenotypes) related to cell survival, proliferation, and invasive migration. As a whole, the goals of this project are structured to provide insight into mechanisms of glioblastoma cell migration. The approach is centered on assessing the contributions of underexplored kinase regulatory networks to the phenotypic switch of cancer cells from proliferative to migratory states, underlying aggressive disease progression. As the most abundant post-translational modifications in eukaryotic signaling pathways, protein phosphorylation occupies a central role in regulatory networks and the maintenance of cellular homeostasis, yet their roles in the mediation of cell migration are poorly understood. It has recently been shown that GBM cells can use normal physiological processes for cell migration, such as exploiting ion channels like NKCC1, to promote motility. The Ste20-family kinase SPS1-related proline/alanine-rich kinase (SPAK) relays changes in cell volume to cation-chloride cotransporters (NKCCs and KCCs) to maintain cellular homeostasis and control cell migration. Although previous reports have highlighted the importance of SPAK kinase as a potential therapeutic target for the treatment of GBM, the inability to produce high yields of physiologically phosphorylated kinase, until now, impeded this progress. Leveraging recent advances in orthogonal translation systems and mass spectrometry from the Rinehart lab enables the implementation of a robust pipeline for the identification and validation of lead inhibitor compounds which target physiologically phosphorylated, active SPAK kinase. The impact of candidate SPAK inhibitors on the migration and physiology of GBM cells will be assessed using a microfluidics based 1D cell migration assays. In parallel, the direct interactions and impacts of candidate inhibitor compounds with the GBM proteome will defined using quantitative mass spectrometry techniques. Overall, characterization of small molecule inhibitor compounds identified from this pipeline will provide important mechanistic insight into the role of kinase networks in the regulation of cancer cell migration, expanding potential applications to the broader scope of cancer biology.