Overcoming Resistance Mechanisms in Hedgehog and Myc-amplified Medulloblastoma

项目摘要 髓母细胞瘤是一种发生于小脑的小儿脑肿瘤。MB治疗具有挑战性 由于不同的基因组成，对化疗的抵抗，药物在血脑中的运输效率低下， 屏障（BBB）和药物诱导的神经毒性。Hedgehog（Hh）和IGF/PI 3 K信号通路调节细胞凋亡 MB患者的生长、癌症干细胞（CSC）增殖和致瘤性。Hh抑制剂有效 最初用于治疗SHH-MB，但由于SMOothened中的突变，它们的重复使用产生了耐药性。 (SMO)但是可以通过使用SF 2523调节GLI来克服，所述GLI是SMO的下游，SF 2523是 BRD 4/PI 3 K双重抑制剂并抑制MYCN表达。在我们的初步研究中，我们合成了SMO 缓蚀剂2-氯-N1-[4-氯-3-（2-吡啶基）苯基]-N4，N4-双（2-吡啶基甲基）-1，4-苯- 二甲酰胺（MDB 5）。MDB 5和SF 2523能有效抑制ONS-76和HD-MB 03的增殖 细胞以剂量依赖性方式，当这些药物用于 组合.用这两种药物的组合处理HD-MB 03细胞显示出显著更高的细胞毒性。 与对照组相比， 个别药物。我们合成了mPEG-b-PCC-g-DC共聚物，其负载量为5.1±0.21和6.5±0.1%， MDB 5和SF 2523配制成纳米颗粒（NP）时的组合物。从纳米粒中持续释放药物， 其中MDB 5在50 h内释放100%，而SF 2523在80 h内仅释放60%。目标NP为 通过以10/90、20/80和30/70的比例混合COG-133-PEG-b-PCC-g-DC和mPEG-b-PCC-g-DC制备， 在30/70比率下细胞摄取最高。将负载SF 2523的COG-133-NP全身施用到 原位SHH-MB荷瘤NSG小鼠导致脑中的药物浓度显著更高， 在全身注射给药后6和24小时，与负载有该药物的非靶向NP相比， 游离药物在脑中的药物浓度可忽略不计。此外，COG-133-NP的全身给药 与MDB 5和SF 2523的非靶向NP相比，负载MDB 5和SF 2523的NP导致肿瘤负荷降低， 通过IVIS成像确定SF 2523，无肝毒性。我们的假设是BRD 4/PI 3 K和Hh 信号传导途径对SHH-MB中的CSC增殖发挥控制作用，因此代表了治疗的靶点。 利用可以抑制这些信号通路的小分子。我们的具体目标是：（i）评估 MDB 5和SF 2523对SHH和MYC驱动的MB和患者来源的异种移植物的再致敏作用 (PDX)ii）将MDB 5和SF 2523配制成具有COG-133缀合的NP，并测定生物分布 iii）确定负载有SF 2523和MDB 5的靶向NP在SHH中的作用 和MYC驱动的原位、患者来源的异种移植物（PDX）和转基因SmoA 1 MB小鼠模型。长- 术语意义该项目的成功完成将为治疗SHH提供平台技术- MB和其他脑肿瘤使用Hh和BRD 4/PI 3 K抑制剂的这种创新的基于NP的组合疗法。

PROJECT SUMMARY Medulloblastoma (MB) is a pediatric brain tumor arising from the cerebellum. MB treatment is challenging due to diverse genetic make-up, resistance to chemotherapy, inefficient drug transport across the blood brain barrier (BBB) and drug induced neurotoxicity. Hedgehog (Hh) and IGF/PI3K signaling pathways regulate cell growth, cancer stem cell (CSC) proliferation, and tumorigenicity in MB patients. 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 using SF2523, which is a BRD4/PI3K dual inhibitor and inhibits MYCN expression. In our preliminary studies, we synthesize SMO inhibitor 2-chloro-N1-[4-chloro-3-(2-pyridinyl) phenyl]-N4, N4-bis (2-pyridinyl methyl)-1, 4-benzene- dicarboxamide (MDB5). MDB5 and SF2523 effectively inhibited the proliferation of ONS-76 and HD-MB03 cells in a dose dependent manner, with significantly higher cell killing when these drugs were used in combination. Treatment of HD-MB03 cells with the combination of these two drugs showed significantly higher decrease in colony formation and cyclin D1 expression but higher increase in Bax expression, compared to individual drugs. We synthesized mPEG-b-PCC-g-DC copolymer, with 5.1±0.21 and 6.5±0.1% loading for MDB5 and SF2523 when formulated into nanoparticles (NPs). There was sustained drug release from NPs, wherein 100% of MDB5 was released in 50 h, but only 60% of SF2523 was released in 80 h. Targeted NPs were prepared by mixing COG-133-PEG-b-PCC-g-DC and mPEG-b-PCC-g-DC at 10/90, 20/80 and 30/70 ratios, with the highest cellular uptake at 30/70 ratio. Systemic administration of COG-133-NPs loaded SF2523 into orthotopic SHH-MB tumor bearing NSG mice resulted in significantly higher drug concentration in the brain at 6 and 24h post administration compared to non-targeted NPs loaded with this drug while systemic injection of free drug showed negligible drug concentration in the brain. Moreover, systemic administration of COG-133-NPs loaded with MDB5 and SF2523 resulted in decreased tumor burden compared to non-targeted NPs of MDB5 and SF2523 as determined by IVIS imaging, with no hepatic toxicity. Our hypothesis is that BRD4/PI3K and Hh signaling pathways exert control over CSC proliferation in SHH-MB and hence represents a target for therapeutic exploitation with small molecules which can inhibit these signaling pathways. Our specific aims are to i) evaluate the resensitization effects of MDB5 and SF2523 on SHH and MYC driven MB and patient derived xenograft (PDX) cells; ii) formulate MDB5 and SF2523 into NPs with COG-133 conjugation and determine biodistribution and systemic/organ toxicity; and iii) determine effects of targeted NPs loaded with SF2523 and MDB5 in SHH and MYC driven orthotopic, patient derived xenograft (PDX) and transgenic SmoA1 MB mouse models. Long- term significance. Successful completion of this project will provide a platform technology for treating SHH- MB and other brain tumors using this innovative NP-based combination therapy of Hh and BRD4/PI3K inhibitors.