Mechanisms of targeting cellular self-renewal in glioblastoma

摘要 RNA是小分子或细胞功能化学探针的未充分利用的靶标。将RNA编码 对于过表达的转录抑制因子是有希望的但无效的癌症治疗靶点。 B细胞特异性Moloney小鼠白血病病毒整合位点1（BMI 1）的过表达，一个主多梳 转录抑制因子和细胞自我更新的驱动因子，是许多癌症中常见的致癌事件， 包括胶质母细胞瘤（GBM）。我们和其他人已经表明，靶向BMI 1可能具有广泛有效的抗- 肿瘤反应。我们使用细胞和斑马鱼报告筛选来鉴定几种分子， 改变BMI 1转录后过程，但也选择性降低BMI 1蛋白水平，调节BMI 1靶点 并在功能上抑制细胞自我更新。随后的结构-活性关系研究揭示了基本的 药效团和调节RNA翻译的潜力。经过化学优化，我们合成了一套 新型小分子，其中包括RU-A15和RU-A16，靶向BMI 1并杀死肿瘤干细胞样细胞 从各种癌症中分离出来。我们的目标是i）建立RU-A15的作用机制， RU-A16; ii）研究它们的体内作用;和iii）鉴定BMI 1靶点作为敏感性或耐药性的标志物 到靶向治疗为了实现这一点，我们利用正常人星形胶质细胞生成了一组独特的模型， 对照、患者来源的GBM球、类器官和原位患者来源的异种移植物（PDX）， 比传统细胞系更准确地代表GBM生物学。我们假设BMI 1探针选择性地 干扰BMI 1 RNA加工的转录后调节，导致BMI 1蛋白耗尽 水平和有效的抗GBM活性，包括消除细胞自我更新，抑制GBM启动 和/或生长和对标准治疗的敏感性。我们将利用这些独特的模型来揭示 用RU-A15和RU-A16靶向BMI 1，并在生物发光GBM PDX中体内验证这些作用， 建立了胶质瘤基因工程小鼠模型（GEMM）。在目标1中，我们将利用物理化学 使用突变和图谱策略确定BMI 1探针对野生型或突变RNA的影响的测定 并评估治疗对球体细胞周期、染色质结合和关键BMI 1靶标的影响。在目标2中， 将检查药代动力学、动力学、效力和选择性，并评估BMI 1探针的有效性 在GBM PDX中与替莫唑胺（TMZ）和辐射（IR）相比。在目标3中，我们将评估 BMI 1探针与TMZ/IR治疗在胶质瘤原位PDX和GEMM中的应用从这些进步中，我们将发展 用于RNA靶向的高度验证的BMI 1探针，并阐明BMI 1作为治疗靶点的重要性， 在GBM和可能的许多具有异常BMI 1功能的癌症中。我们的策略是使用协作翻译 验证（或反驳）BMI 1探针未来在大型患者队列中临床应用的方法。

ABSTRACT RNAs are underexploited targets for small molecules or chemical probes of cellular functions. The RNA coding for overexpressed transcriptional repressors are promising yet invalidated targets for cancer therapy. Overexpression of B-cell-specific Moloney murine leukemia virus integration site-1 (BMI1), a master polycomb transcriptional repressor and driver of cellular self-renewal, is a common oncogenic event in many cancers, including glioblastoma (GBM). We and others have shown that targeting BMI1 could have widely effective anti- tumor responses. We used cellular and zebrafish reporter screens to identify several molecules that not only alter BMI1 post-transcriptional processes but also selectively reduce BMI1 protein levels, modulate BMI1 targets and functionally inhibit cellular self-renewal. Subsequent structure-activity relationship studies revealed the basic pharmacophore and potential to modulate RNA translation. Upon chemical optimization, we synthesized a set of novel small molecules, among them are RU-A15 and RU-A16 that target BMI1 and kill tumor stem-like cells from various cancers at low nM concentrations. We aim to i) establish the mechanism of action of RU-A15 and RU-A16; ii) study their in vivo effects; and iii) identify BMI1 targets to serve as markers for sensitivity or resistance to targeted therapy. To achieve this, we generated a set of unique models utilizing normal human astrocytes as controls, patient derived GBM spheres, organoids and orthotopic patient derived xenografts (PDXs) that more accurately represent GBM biology than traditional cell lines. We hypothesize that the BMI1 probes selectively interfere with the post-transcriptional regulation of BMI1 RNA processing, leading to depletion of BMI1 protein levels and potent anti-GBM activities, including abrogation of cellular self-renewal, inhibition of GBM initiation and/or growth and sensitization to standard therapy. We will utilize these unique models to reveal the effects of targeting BMI1 with RU-A15 and RU-A16 and validate these effects in vivo in bioluminescent GBM PDXs and established genetically engineered mouse model (GEMM) of glioma. In Aim 1, we will utilize physico-chemical assays to determine the effects of the BMI1 probes on wild-type or mutant RNA using mutate-and-map strategy and assess the effects of treatment on sphere cell cycle, chromatin binding and key BMI1 targets. In Aim 2, we will examine the pharmacokinetic, dynamic, potency and selectivity and assess the efficacy of the BMI1 probe in GBM PDXs vs temozolomide (TMZ) and irradiation (IR). In Aim 3, we will assess the effects of the combined BMI1 probe with TMZ/IR therapy in orthotopic PDXs and GEMM of glioma. From these advances, we will develop highly-validated BMI1 probes for RNA targeting and clarify the significance of BMI1 as a therapeutic target both in GBM and likely many cancers with aberrant BMI1 functions. Our strategy uses a collaborative translational approach to validate (or refute) future use of the BMI1 probe in clinical applications in large cohorts of patients.