GBM Avatar Screening Paradigm for Rational Pharmacokinomic Glioma Therapy

这个探索性的研究项目将利用一种替代战略，可以普遍适用于选择 最合适的药物或药物组合来治疗高度侵袭性的恶性脑肿瘤，称为胶质母细胞瘤 多形性（GBM）。每年有超过17，000名患者被诊断出GBM，并且基本上无法治愈。 尽管有超过75年的研究，我们的“最好的”护理标准疗法包括手术切除，放射治疗和 化疗，但不能治愈，仅提供4%的5年生存率，每年死亡人数为12，000+。 大多数细胞功能在所有细胞内都受到酶促磷酸化其他蛋白质的激酶的调节。 激酶磷酸化蛋白被激活或抑制以"驱动"大多数细胞过程（例如， 增殖、转移、存活）和调节对治疗和其它应激的细胞应答。小 分子抑制剂（SMI）是增长最快的一类阻断细胞活性的新药。 激酶，但在其作用中有些混杂。已知的人类蛋白激酶有518种， 在细胞中有活性（不受调节）或被其他激酶激活。激酶是自磷酸化的或 在一些实施方案中，激酶被其他激酶连续磷酸化以产生所谓的"信号传导途径"的"级联"。抑制 某些信号通路中激酶的正确组合将导致肿瘤细胞的死亡。我们有一个 在小鼠中从手术肿瘤组织建立的人GBM患者来源的Xenolines（PDX）组 并在小鼠脑中生长，收获并冷冻保存。一半（~27）已广泛 在基因组学、转录组学和运动组学上表征，并形成"参考组";另一半 （~24）在低传代时未表征，并用作我们的"验证组"。这些PDX将作为患者 药物选择的"化身"。我们将从六（6）只低传代参考品的冷冻组织中提取蛋白质 PDX肿瘤组，以测量四（4）种选定的（血脑屏障可渗透的）SMI抑制 在肿瘤中具有酶活性的特异性激酶。我们将使用体外试验，其中少量（1- 用1 - 20 μ M的每种SMI "掺入"来自每种PDX的蛋白质，并在PamStation-12中分析， 定量518种蛋白激酶的酶活性，并确定SMI是否"击中其靶标"或具有"脱靶"效应。 潜在有效SMI的选择将使用低传代PDX进行验证，其激酶组谱 将被确定，然后与参考组中的肿瘤匹配。这将模拟临床情况， 对患者的肿瘤进行动力学分析，并且该分析与PDX的分析相匹配，PDX将具有先前定义的特征。 SMI反应性特征。我们预计，在这项探索性研究中开发的数据将证明， 这种“化身”方法可以准确地预测患者的潜在治疗方法或其组合 其肿瘤类似于参考和验证样本组中的一种或多种PDX（约50 + PDX）。我们将使用 这些数据支持R01水平的应用，以验证我们在小鼠和小鼠原位肿瘤中的发现。 测试来自GBM患者的肿瘤组织，以预期设计和进行试点临床试验。

This exploratory research project will utilize an alternative strategy that can be generally applied to select the most appropriate drug or drug combination to treat highly aggressive malignant brain tumors called glioblastoma multiforme (GBM). GBMs are diagnosed in over 17,000 patients each year and are essentially incurable. Despite over 75 years of research, our "best" standard of care therapies involve surgical resection, radiation and chemotherapy, but are not curative, providing only a 4% 5-year survival rate with an annual death toll of 12,000+. Most cellular functions are regulated inside all cells by kinases that enzymatically phosphorylate other proteins. Kinase-phosphorylated proteins are activated or suppressed to "drive" most cellular processes (e.g., proliferation, metastasis, survival) and to modulate cellular responses to therapies and other stresses. Small molecule inhibitors (SMIs) represent the fastest growing class of new drugs that block activities of cellular kinases, but are somewhat promiscuous in their effects. There are 518 known human protein kinases and many are either active in the cell (unregulated) or are activated by other kinases. Kinases are auto-phosphorylated or phosphorylated by other kinases serially to generate “cascades” down so called "signaling pathways". Inhibition of the right combination of kinases in certain signaling pathways will lead to death of tumor cells. We have a panel of human GBM Patient-Derived Xenolines (PDX) that were established in mice from surgical tumor tissues and were grown in the brains of mice, harvested and cryopreserved. Half (~27) have been extensively characterized genomically, transcriptomically and kinomically and form the “Reference Panel”; the other half (~24) are uncharacterized, at low passage and serve as our “Validation Panel”. These PDX will serve as patient "avatars" for drug selection. We will extract proteins from frozen tissues of six (6) of the low-passage Reference Panel PDX tumors to measure the ability of four (4) selected (blood-brain-barrier permeable) SMIs, to inhibit specific kinases that are enzymatically active in tumors. We will use ex vivo testing in which a small amount (1- 15g) of protein from each PDX is `spiked' with 1-20M of each SMI and analyzed in the PamStation-12 that can quantify enzymatic activity of 518 protein kinases and determine if a SMI "hits its target" or has “off-target” effects. The selection of potentially effective SMIs will be validated using low passage PDX for which the kinome profile will be determined, then matched to tumors in the Reference Panel. This will mimic the clinical situation in which a patient's tumor is kinomically profiled, and that profile matched to that of PDX that will have a previously defined SMI-responsiveness profile. We anticipate that the data developed in this exploratory research will demonstrate that this “avatar” approach can accurately predict potential therapeutics or combinations thereof for patients whose tumors resemble one or more PDX in the Reference and Validation Panels (~50+ PDX). We will use these data to support a R01-level application to validate our findings using orthotopic tumors in mice and in testing tumor tissues from GBM patients in anticipation of designing and conducting a pilot clinical trial.