Overcoming Resistance Mechanisms in Hedgehog and Myc-amplified Medulloblastoma

项目总结

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.