Piperlongumine conquers temozolomide chemoradiotherapy resistance to achieve immune cure in refractory glioblastoma via boosting oxidative stress-inflamation-CD8+-T cell immunity

Abstract

Background: The failure of novel therapies effective in preclinical animal models largely reflects the fact that current models do not really mimic the pathological/therapeutic features of glioblastoma (GBM), in which the most effective temozolomide chemoradiotherapy (RT/TMZ) regimen can only slightly extend survival. How to improve RT/TMZ efficacy remains a major challenge in clinic.

Methods: Syngeneic G422^TN-GBM model mice were subject to RT/TMZ, surgery, piperlongumine (PL), αPD1, glutathione. Metabolomics or transcriptomics data from G422^TN-GBM and human GBM were used for gene enrichment analysis and estimation of ROS generation/scavenging balance, oxidative stress damage, inflammation and immune cell infiltration. Overall survival, bioluminescent imaging, immunohistochemistry, and immunofluorescence staining were used to examine therapeutic efficacy and mechanisms of action.

Results: Here we identified that glutathione metabolism was most significantly altered in metabolomics analysis upon RT/TMZ therapies in a truly refractory and reliable mouse triple-negative GBM (G422^TN) preclinical model. Consistently, ROS generators/scavengers were highly dysregulated in both G422^TN-tumor and human GBM. The ROS-inducer PL synergized surgery/TMZ, surgery/RT/TMZ or RT/TMZ to achieve long-term survival (LTS) in G422^TN-mice, but only one LTS-mouse from RT/TMZ/PL therapy passed the rechallenging phase (immune cure). Furthermore, the immunotherapy of RT/TMZ/PL plus anti-PD-1 antibody (αPD1) doubled LTS (50%) and immune-cured (25%) mice. Glutathione completely abolished PL-synergistic effects. Mechanistically, ROS reduction was associated with RT/TMZ-resistance. PL restored ROS level (mainly via reversing Duox2/Gpx2), activated oxidative stress/inflammation/immune responses signature genes, reduced cancer cell proliferation/invasion, increased apoptosis and CD3⁺/CD4⁺/CD8⁺ T-lymphocytes in G422^TN-tumor on the basis of RT/TMZ regimen.

Conclusion: Our findings demonstrate that PL reverses RT/TMZ-reduced ROS and synergistically resets tumor microenvironment to cure GBM. RT/TMZ/PL or RT/TMZ/PL/αPD1 exacts effective immune cure in refractory GBM, deserving a priority for clinical trials.

Keywords: Glioma; Immunotherapy; PD-1; Piperlongumine; ROS generation/elimination; Tumor microenvironment.