(M1530-06-35) COG133 Peptide-Conjugated Lipid Nanoparticles Sensitize Medulloblastoma to Radiation Therapy in Mice

Purpose: Medulloblastoma (MB) is a malignant brain tumor that often arises in the cerebellum and has a propensity for spreading to the spinal cord or other parts of the central nervous system. Craniospinal irradiation (CSI) has long been a cornerstone in the treatment of MB, especially for patients with high-risk or metastatic disease. However, CSI often leads to long-term neurocognitive deficits, including learning disabilities, and growth abnormalities, especially in children. In this study, we aimed to decrease the dose of irradiation and the proliferation of MB by using Volasertib (VSB), a Polo-like kinase 1 (PLK1) specific inhibitor and reduce the side affects associated with the treatment with maintaing efficacy of treatment.

Methods: Using UPLC-MS/MS, the amount of VSB in the tissues was determined. Briefly, BI2523 was added as an internal standard (IS) to tissue samples that had been homogenized in LC/MS grade water. For all three groups, 100 mg of brain was weighed, and spleen was weighed to be 30 mg, rest of the organs including liver, kidney, and heart was weighed to be 50 mg. Following the addition of 1.0 mL of acetonitrile, samples were vortexed and high-speed centrifugation was performed. Following the dry evaporation of the supernatant, 150 μL of acetonitrile: water (60:40, v/v) was used to reconstitute the residue. SCIEX OS® software on a 5500 QTRAP, AB, Sciex Inc. Mass spectrometer was used to acquire UPLC-MS/MS data. For UPLC method development, YMC Flow ODS-AQ 5µm 120Å 2x150 mm column was used at a flow rate of 0.25 ml/min with a gradient method with water: ACN changing from 5% to 95% in 4 mins. For VSB (m/z 479.2) and internal standard (m/z 402.8), the mass spectrometer was run in the positive mode multiple reaction monitoring (MRM). All the experiments done in animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Nebraska Medical Center and meet all the federal guidelines concerning animal experimentation to ensure proper care and use of laboratory animals for research. DAOY cells were transduced with lentivirus to express green fluorescent protein (GFP) and luciferase by following the operating protocol. Cells with stable GFP expression were sorted to obtain luciferase-expressing population ( >95 %). To establish orthotopic MB model, luciferase-expressing cells were stereotaxically injected into the cerebellum. Briefly, NSG mice (6-8 weeks old) were anesthetized, mounted on a stereotaxic frame, and exposed to the skull. A small hole was drilled in the skull, located 1 mm posterior of the lambdoid suture of the scalp over the cerebellum and 1 mm lateral of the sagittal suture. 10 µL of cell suspension (1 × 10⁷ cells/mL in PBS) were slowly injected at the rate of 20 µL/sec into the cerebellum using 25µl Hamilton syringe. The tumor was grown for 4 weeks with monitor by IVIS imaging.

Results: VSB was highly potent in-vitro with an IC₅₀ of 27.43 nM and 13 nM in HDMB03 and DAOY cells, respectively. However, in the orthotopic MB mouse model, VSB as a free drug did not improve overall survival or decrease tumor burden. Hence, we encapsulated VSB in COG133-conjugated lipid nanoparticles (LNPs) to circumvent the blood-brain barrier. We observed that COG-133 conjugated LNPs loaded with VSB increased the biodistribution of VSB by three folds than the non-targeted LNPs in the brain. Furthermore, he COG133 conjugated LNPs along with irradiation decreased tumor burden 9 fold as compared to radiation alone.

Conclusion: Our study highlights the dual therapeutic benefits of VSB as a PLK1 inhibitor and radiosensitizer, particularly when delivered via targeted LNPs. This approach offers a promising strategy to enhance the efficacy of radiotherapy while minimizing its systemic toxicity in patients with aggressive MB. The ability of VSB to sensitize MB cells to lower radiation doses holds potential for mitigating radiation-induced neurocognitive damage in pediatric patients. The translational potential of this therapy, coupled with its ability to address critical challenges in MB treatment, underscores its relevance for advancing pediatric brain tumor care.