Lipid nanomedicine targeting multiple signaling pathways of medulloblastoma

项目总结 髓母细胞瘤(MB)是最常见的儿童脑肿瘤，起源于小脑。很多因素 影响小脑颗粒神经元前体(GNP)的增殖、分化和迁移。其中 其中，MDM2是肿瘤抑制基因TP53和GNP和HH信号之间的主要纽带。 促进MB肿瘤的生长和转移。此外，PI3K和BRD4信号在MB细胞中也起着关键作用 生长、肿瘤干细胞(CSC)增殖和肿瘤发生。此外，甲基溴的治疗是具有挑战性的，因为 化疗耐药、低效跨血脑屏障药物转运及药物的研究进展 诱发的神经毒性。HH抑制剂最初对治疗SHH-MB有效，但反复使用后逐渐发展。 平滑(SMO)基因突变导致的化疗耐药，但可以通过调节GLI来克服，GLI是 SMO下游。在我们的初步研究中，我们合成了一系列有效的BRD4/PI3K双重抑制剂 通过修饰母体化合物SF2523的结构。化合物8-(2，3-二氢苯并[b][1，4]二恶英- 6-基)-2-吗啉基-4H-色烯-4酮(简称MDP5)具有较强的抗癌活性。然后我们确定了X- BRD2重组BD1和BD2结构域在MDP5络合物中的晶体结构。而MDP5 在DOAY细胞中显示出比SF2523(12.6微米)更高的效力，MDP5和SF2523的IC50值是 对HD-MB03MB(MYC扩增)细胞具有类似的效力。MDP5减少了靶下游蛋白，如 P-AKT、MYCN、Cyclin D1，并增加p-MYCN(Ser54)所显示的MYCN蛋白的降解。我们 还发现了一个小分子JW-475A，它是一种有效的MDM2和XIAP双重抑制剂。MDP5和JW- 475A(一种MDM2和XIAP双重抑制剂)有效地抑制MB细胞的增殖，并呈剂量依赖性 当这些药物联合使用时，细胞杀伤率显著高于其他药物。MB细胞的治疗 联合使用这两种药物可显著降低集落形成能力。 个别药物。制备了负载量分别为4.9±0.1%和4.8±0.1%的聚乙二醇DSPE脂质纳米粒 适用于MDP5和JW-475A。狂犬病病毒表面修饰制备BBB穿透性靶向LNPs 糖蛋白(RVG)多肽。我们的假设是BRD4/PI3K和MDM2/XIAP的抑制 同时使用MDP5和JW-475A是一种有希望的体内抑制MB肿瘤的策略。此外， 我们将使用RVG-PEG-DSPE LNPs来包裹MDP5和JW-475A，这两种药物的跨膜转运能力很差 BBB。我们的具体目标是：1)合成新型的双功能BRD4/PI3K抑制剂MDP5 2)评价JW-475A联合MDP5的体外抗癌作用。 Iii)将MDP5和JW-475A制成RVG多肽修饰的LNPs，并测定其生物分布， 在SHH和MYC驱动的细胞和PDX为基础的原位和原位移植中的疗效和全身/器官毒性 转基因SmoA1 MB小鼠模型。具有长远意义。这个项目的成功完成将 为使用这种创新的基于LNP的联合疗法治疗脑瘤提供平台技术。

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.