Lipid nanomedicine targeting multiple signaling pathways of medulloblastoma

项目摘要 髓母细胞瘤（MB）是最常见的儿童脑肿瘤起源于小脑。许多因素 影响小脑颗粒神经元前体（GNP）的增殖、分化和迁移。之间 因此，MDM 2是GNP中肿瘤抑制因子TP 53和hedgehog（Hh）信号传导之间的主要联系， 促进MB肿瘤生长和转移。此外，PI 3 K和BRD 4信号通路也在MB细胞中发挥关键作用。 生长、癌症干细胞（CSC）增殖和肿瘤发生。此外，MB处理具有挑战性，因为 化疗耐药性的发展、药物穿过血脑屏障（BBB）的低效转运和药物依赖性的降低。 诱发神经毒性。Hh抑制剂最初对治疗SHH-MB有效，但其重复使用发展 由于smoothened（SMO）突变导致的化疗耐药性，但可以通过调节GLI来克服， 在SMO的下游。在我们的初步研究中，我们合成了一系列有效的BRD 4/PI 3 K双重抑制剂， 通过对母体化合物SF 2523的结构进行修饰，化合物8-（2，3-二氢苯并[B][1，4]二恶英- 6-基）-2-吗啉代-4H-苯并吡喃-4酮（简称MDP 5）。然后，我们确定了X- 来自BRD 2的重组BD 1和BD 2结构域与MDP 5复合的X射线晶体结构。虽然MDP 5 与SF 2523（12.6 µM）相比，MDP 5和SF 2523在DOAY细胞中显示出更高的效力， 对HD-MB 03 MB（MYC扩增）细胞的效力相似。MDP 5降低了靶向下游蛋白，如 p-AKT、MYCN、Cyclin D1，并增加MYCN蛋白的降解，由p-MYCN（ser 54）指示。我们 还发现了一种小分子JW-475 A，它是一种有效的双重MDM 2和XIAP抑制剂。MDP 5和JW- 475 A（MDM 2和XIAP双重抑制剂）以剂量依赖性方式有效地抑制MB细胞的增殖。 当这些药物联合使用时，细胞杀伤率显著提高。MB细胞的处理 这两种药物的组合显著降低了殖民地形成能力， 个别药物。我们制备了载量为4.9±0.1%和4.8±0.1%的基于PEG-DSPE的脂质纳米粒（LNPs MDP 5和JW-475 A。用狂犬病病毒修饰表面，制备了穿透血脑屏障的靶向LNPs 糖蛋白（RVG）肽-肽。我们的假设是BRD 4/PI 3 K和MDM 2/XIAP的抑制 同时使用MDP 5和JW-475 A代表了体内抑制MB肿瘤的有希望的策略。此外，本发明还 我们将使用RVG-PEG-DSPE LNP包封MDP 5和JW-475 A，它们的药物转运能力较差， 的BBB。我们的具体目标是i）合成作为双功能BRD 4/PI 3 K抑制剂的新型MDP 5衍生物 ii）在体外评估JW-475 A与MDP 5组合的抗癌功效; iii）将MDP 5和JW-475 A配制成用RVG肽修饰的LNP并测定它们的生物分布， 在SHH和MYC驱动的细胞和基于PDX的原位和非原位细胞中的治疗功效和全身/器官毒性。 转基因SmoA 1 MB小鼠模型。长期意义。该项目的成功完成将 提供了一个平台技术，用于使用这种创新的基于LNP的组合疗法治疗脑肿瘤。

PROJECT SUMMARY Medulloblastoma (MB) is the most common childhood brain tumor arising from the cerebellum. Many factors influence the proliferation, differentiation, and migration of cerebellar granular neuronal precursor (GNP). Among them, MDM2 is a major nexus between tumor suppressor TP53 and hedgehog (Hh) signaling in GNPs and promotes MB tumor growth and metastasis. In addition, PI3K and BRD4 signaling also play key roles in MB cell growth, cancer stem cell (CSC) proliferation, and tumorigenesis. Further, MB treatment is challenging due to the development of chemoresistance, inefficient drug transport across the blood brain barrier (BBB) and drug induced neurotoxicity. Hh inhibitors are effective initially to treat SHH-MB, but their repeated use develops chemoresistance due to mutations in smoothened (SMO) but can be overcome by modulating GLI, which is downstream of SMO. In our preliminary studies, we synthesized a series of potent BRD4/PI3K dual inhibitors by modifying structure of parent compound SF2523. One of the compounds 8-(2,3-dihydrobenzo[b][1,4]dioxin- 6-yl)-2-morpholino-4H-chromen-4one (abbreviated as MDP5) was found highly potent. We then determined X- ray crystal structures of the recombinant BD1 and BD2 domains from BRD2 in complex with MDP5. While MDP5 showed higher potency in DOAY cells compared to SF2523 (12.6 µM), IC50 values for MDP5 and SF2523 were similar potency on HD-MB03 MB (MYC amplified) cells. MDP5 decreased the target downstream proteins like p-AKT, MYCN, Cyclin D1, and increased the degradation of MYCN protein indicated by p-MYCN (ser 54). We also discovered a small molecule JW-475A which is a potent dual MDM2 and XIAP inhibitor. MDP5 and JW- 475A (a dual MDM2 and XIAP inhibitor) effectively inhibited the proliferation of MB cells in a dose dependent manner, with significantly higher cell killing when these drugs were used in combination. Treatment of MB cells with the combination of these two drugs significantly decreased the colony formation capacity compared to individual drugs. We prepared PEG-DSPE based lipid nanoparticles (LNPs) with 4.9±0.1% and 4.8±0.1% loading for MDP5 and JW-475A. BBB penetrating targeted LNPs were prepared by surface decorating with rabies virus glycoprotein (RVG) peptide-peptide. Our hypothesis is that inhibition of BRD4/PI3K and MDM2/XIAP simultaneously using MDP5 and JW-475A represents a promising strategy to inhibit MB tumor in vivo. Further, we will use RVG-PEG-DSPE LNPs to encapsulate MDP5 and JW-475A, which have poor drug transport across the BBB. Our specific aims are to i) Synthesize novel MDP5 derivatives as dual function BRD4/PI3K inhibitors and characterize in vitro activity; ii) Evaluate anti-cancer efficacy of JW-475A in combination with MDP5 in vitro.; iii) Formulate MDP5 and JW-475A into LNPs decorated with RVG peptide and determine their biodistribution, therapeutic efficacy, and systemic/organ toxicity in in SHH and MYC driven cells and PDX-based orthotopic and transgenic SmoA1 MB mouse models. Long-term significance. Successful completion of this project will provide a platform technology for treating brain tumors using this innovative LNP-based combination therapy.