Lipid nanomedicine targeting multiple signaling pathways of medulloblastoma

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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.