2647 - MDM2 Inhibition and the Efficacy of Radiotherapy and PD-L1 Antibody Combination Therapy by Reprogramming the Glioblastoma Tumor Microenvironment

C. Yuan^1,2, W. LI^2,3, Y. Wang^1,2, M. Hu², and X. Ding⁴; ¹Shandong First Medical University, Jinan, China, ²Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, ³Shandong Second Medical University, Weifang, China, ⁴Shandong Cancer Hospital and Institute, Jinan, Shandong, China

Purpose/Objective(s): Glioblastoma (GBM) is the most common malignancy in central nervous system. Different from other tumors, radiotherapy (RT) combine with immune checkpoint inhibitors (ICIs) are still not effective in improving GBM patients’ survival. One important reason is that the tumor microenvironment (TME) in GBM is highly inhibitory. Our previous RNA-sequence results displayed that mouse double minute 2 (MDM2) were significantly raised after RT in GBM cells. And some studies have reported that MDM2 overexpression (OE) had positive relationship with programmed death-ligand 1(PD-L1) high expression. However, the related mechanism was still limited. Therefore, the aims of this study were to further explore the relationship among RT, MDM2 and PD-L1 in GBM, and determine whether the combined efficacy of RT and ICIs can be improved by using MDM2 inhibitor. Materials/

Methods: In vitro, the effects of MDM2 on tumor cell proliferation, migration and invasion were verified using MDM2-knockdown (KD), MDM2-OE and null U251 cell lines. The expression of MDM2 and PD-L1 before and after RT in GBM cells and the relationship between them were evaluated by western blot (WB) and Polymerase Chain Reaction (PCR). RNA and Proteome sequencing were used to explore the pathway of MDM2 regulating PD-L1 in GBM cells. In vivo, in situ GBM models were constructed using GL261 cell line. All mouse were randomly assigned to control, RT, MDM2 inhibitor (SP-141), anti-PD-L1, RT+SP-141, RT+anti-PD-L1, SP-141+anti-PD-L1 and RT+SP-141+anti-PD-L1 groups. Tumor volume were evaluated every 3 days after treated. T-cell subsets (Th1, Th2, Th17 and Tregs) and myeloid-derived suppressor cells (MDSCs) (M1 and M2 phenotype) among different groups were analyzed by flow cytometry. SPSS 25.0 was used for statistical analysis of the data. The relevant graphs in this study were analyzed using a scientific 2-D graphing and statistics software.
Results: The results displayed that RT could increase the expression of MDM2 and PD-L1, significantly. And the MDM2 OE could improve the proliferation and invasion of GBM cells. The KEGG enrichment analysis in RNA-sequence showed that PI3K-AKT pathway was most significantly enriched (P =0.0007644, FDR=0.02914). WB and PCR results revealed that MDM2 can directly activate PI3K transcription, then phosphorylate AKT and raise PD-L1 expression. Using LY294002 (a PI3K/AKT inhibitor) could significantly decreased the PD-L1 expression. The proteome-sequence showed that CSF2 and IL-36 could be increased in MDM2-OE GBM cells and decreased in MDM2-KD cells. And in vivo experiment results proven that inhibiting MDM2 in GBM can increased M1-MDSCs polarization and Th1 cells infiltration in TME through increasing the expression of CSF2 and IL-36. And the inhibitory effect on tumor was strongest in RT+SP-141+anti-PD-L1 group compare to other groups(P <0.05).
Conclusion: The MDM2 OE can promote immune tolerance in GBM. Thus, targeting MDM2 could reprogramming the TME in GBM and improving the combine therapeutic effect of RT and PD-L1 antibody.