(W1230-10-64) COG133-Targeted Polymeric Nanoparticles Encapsulating PLK-1 Inhibitor Blocks DNA-Damage Repair Pathways to Sensitize Medulloblastoma Cells for Radiotherapy

Purpose: Medulloblastoma (MB) is one of the most common malignant pediatric brain tumor with a 5-year overall survival rate of 70%. Treatment of MB is challenging due to the existence of cancer stem cells (CSCs), chemoresistance, and inefficient drug transport to the brain. Radiotherapy continues to be the most significantly effective treatment for primary brain tumors because of its local action and fewer adverse effects. However, radio resistance develops due to the DNA damage repair mechanism. Hence, we have selected Volasertib (VSB) which is a PLK-1-specific inhibitor that blocks the checkpoint recovery after DNA damage and arrests the cells in the G2/M phase which sensitizes the cancer cells for radiotherapy. However, VSB was discontinued from clinical trials due to its dose-limiting toxicity. To address this, we have prepared ApoE (COG133)-targeted polymeric nanoparticles which bind with LDLR receptors on the brain and help to circumvent the blood-brain barrier. Therefore, the overall purpose of this proposal is to overcome the existing chemo-radiotherapy resistance in the treatment of MB for long-term tumor control or eradication.

Methods: Initially, we checked the potential of VSB in DAOY and HDMB03 cells which represent SHH and MYC-driven MB respectively through cell viability assay. After that, we checked VSB potential to inhibit metastasis in HDMB03 cells through the Boyden Chamber assay. To check whether VSB can sensitize cells for radiotherapy, we have performed a colony formation assay which is the most acceptable method to do so. Later, we checked whether VSB could arrest the cells in the G2/M phase which is considered to be the most radiosensitive as compared to G1/S-phase through Fxcycle/RNAse staining in Flow cytometry. Also, CD133 is the surface marker highly reported for CSCs. Therefore, we checked whether VSB could inhibit CD133+ cells. Through western blot, we have analyzed the mechanistic action of VSB in MB cells. We have synthesized poly(ethylene glycol)-block-poly(2-methyl-2-benzoxy carbonyl propylene carbonate) (mPEG-b-PBC) polymeric NPs which have polycarbonate backbone chain and hence biodegradable and no adverse effects.

Results: We observed an IC₅₀ of 27.43nM and 20.92 nM in HDMB03 and DAOY cells respectively. Also, we observed that VSB lessens the cell migration at 35nM and eliminates it completely at 150nM in HDMB03 cells. Moreover, in the colony formation assay, VSB demonstrated radio sensitizing properties at 10nM with a 2Gy dose of irradiation in radioresistant DAOY cells. Furthermore, our synthesized (mPEG-b-PBC) polymeric NPs demonstrated a size of 78.32 ± 5.09 nm with a PDI of 0.112 ± 0.01. VSB shows high encapsulation efficiency of 86.25 ± 9.97% w/w with a drug loading of 8.58 ± 0.992% w/w in polymeric NPs. Also, no significant size change was observed with ApoE-targeted NPs and a 10-fold greater cellular uptake was observed as compared to nontargeted polymeric NPs in DAOY cells.

Conclusion: We observed that VSB displays high potency in the treatment of MB. It inhibits the DNA damage repair mechanism which causes resistance after radiotherapy. Most importantly, it kills CD133+ cancer stem cells which is the primary reason for relapse in cancer treatment.

References: 1. Kumar V, Wang Q, Sethi B, Lin F, Kumar V, Coulter DW, et al. Polymeric nanomedicine for overcoming resistance mechanisms in hedgehog and Myc-amplified medulloblastoma. Biomaterials. 2021;278:121138.
2. Kumar V, Kumar V, McGuire T, Coulter DW, Sharp JG, Mahato RI. Challenges and recent advances in medulloblastoma therapy. Trends in pharmacological sciences. 2017;38(12):1061-84.
3. Wakida T, Ikura M, Kuriya K, Ito S, Shiroiwa Y, Habu T, et al. The CDK-PLK1 axis targets the DNA damage checkpoint sensor protein RAD9 to promote cell proliferation and tolerance to genotoxic stress. eLife. 2017;6:e29953.
4. Wang, Q., Kumar, V., Lin, F., Sethi, B., Coulter, D. W., McGuire, T. R., & Mahato, R. I. (2020). ApoE mimetic peptide targeted nanoparticles carrying a BRD4 inhibitor for treating Medulloblastoma in mice. Journal of Controlled Release, 323, 463-474. https://doi.org/10.1016/j.jconrel.2020.04.053

Acknowledgements:The authors declare no conflict of interest. This research was supported by the State of Nebraska through the Pediatric Cancer Research Group, part of the Child Health Research Institute.

Figure 1. A) Hurdles in treatment of medulloblastoma (MB) overcome by targeted therapy of PLK1 inhibitor volasertib. B) Treatment plan orthotopic MB mouse model.

Figure 2. A) Roles of PLK1 inhibitor volasertib in DNA damage. Volasertib effectively inhibits cell proliferation, and inhibits cell migration and colony formation after incubation with HD-MB03 and DAOY cells. B) proliferation, C) migration and D) Quantification of migrated cells

Figure 3. Volasertib and radiation therapy synergistically inhibits colony formation of DAOY cells. A) Colony formation and B) quantification of colonies. C) Volasertib sensitize radiation treated DAOY MB cells by arresting them in G2/M phase D) zeta size of volasertib loaded PEG-PBC nanoparticles E) Cellular uptake of COG-133 targeted nanoparticles in DAOY cells