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被充分利用的目标。RNA编码 因为过度表达的转录抑制物是癌症治疗的有希望但无效的靶点。 B细胞特异性Moloney小鼠白血病病毒整合位点-1(BMI1)的高效表达 转录抑制子和细胞自我更新的驱动力，是许多癌症中常见的致癌事件， 包括胶质母细胞瘤(GBM)。我们和其他人已经证明，靶向BMI1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屏幕识别了几个分子，不仅 改变BMI1转录后过程，但也选择性地降低BMI1蛋白水平，调节BMI1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，我们合成了一组 在新型小分子中，其中包括靶向BMI1并杀死肿瘤干细胞的RU-A15和RU-A16 在低NM浓度下从各种癌症中分离出来。我们的目标是：1)建立RU-A15的作用机制和 RU-A16；ii)研究它们在体内的作用；以及iii)确定BMI1靶点作为敏感性或耐药性的标记 到靶向治疗。为了实现这一点，我们利用正常的人类星形胶质细胞生成了一组独特的模型 对照、患者衍生的GBM球体、有机类化合物和患者原位移植(PDX) 比传统的细胞系更能准确地代表GBM生物学。我们假设BMI1选择性地探测 干扰BMI1 RNA加工的转录后调控，导致BMI1蛋白耗尽 水平和强大的抗基底膜活性，包括取消细胞自我更新，抑制基底膜启动 和/或生长和对标准疗法的敏感化。我们将利用这些独特的模型来揭示 用RU-A15和RU-A16靶向BMI1，并在体内验证这些效应 建立脑胶质瘤基因工程小鼠模型。在目标1中，我们将利用物理化学 利用突变和MAP策略确定BMI1探针对野生型或突变型RNA的影响 并评估治疗对球状细胞周期、染色质结合和关键BMI1靶点的影响。在目标2中，我们 将检查药代动力学、动力学、效力和选择性，并评估BMI1探针的疗效 GBM PDXs与替莫唑胺(TMZ)和放射治疗(IR)的比较。在目标3中，我们将评估合并后的影响 BMI1探针联合TMZ/IR治疗脑胶质瘤的原位PDX和GEMM从这些进步中，我们将发展 用于RNA靶向的高度有效的BMI1探针和阐明BMI1作为治疗靶点的意义 在GBM和可能许多具有BMI1功能异常的癌症中。我们的战略使用协作式翻译 验证(或驳斥)未来BMI1探针在大规模患者队列中的临床应用的方法。

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.