Protein phosphatase 2A regulates senescence and immunogenicity in medulloblastoma models
Winson S. Ho, Isha Mondal, Jingjing Liu, Raymond Sun, Jiawei Huo, Chao Gao, Oishika Das, Daren Tieu, Jingqi Sun, Hanchen Lin, Peng Zhang, Jiyang Yu, Rongze Olivia Lu
Winson S. Ho, Isha Mondal, Jingjing Liu, Raymond Sun, Jiawei Huo, Chao Gao, Oishika Das, Daren Tieu, Jingqi Sun, Hanchen Lin, Peng Zhang, Jiyang Yu, Rongze Olivia Lu
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Research Article Immunology Oncology

Protein phosphatase 2A regulates senescence and immunogenicity in medulloblastoma models

Abstract

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Current therapies are associated with substantial morbidity, and prognosis remains poor in high-risk subgroups, particularly those with TP53 mutations or relapsed disease. Cellular senescence is a tumor-suppressive program implicated in MB, but its role in antitumor immunity remains incompletely understood. We found that protein phosphatase 2A (PP2A) regulated immunogenic senescence in MB. Genetic ablation of the PP2A catalytic subunit PP2Ac or depletion of the regulatory subunit PP2A-B56α induced senescence in MB models. PP2Ac-deficient senescent cells exhibited increased MHC class I expression and enhanced immunogenicity. In syngeneic orthotopic models, PP2Ac loss prolonged survival in an immune- and CD8+ T cell–dependent manner. Analysis of patient datasets showed that senescence-associated gene signatures correlated with improved survival. Single-cell transcriptomic analysis further revealed that senescent MB cells were heterogeneous and that reduced PP2A activity was associated with an immunogenic senescence state. Because the PP2A inhibitor LB-100 has limited potency and off-target effects, we developed a lipid nanoparticle (LNP) platform to deliver siRNA targeting PPP2CA. LNP–small-interfering PP2Ac efficiently silenced PP2Ac in vitro and, when delivered locally in vivo, prolonged survival in a CD8+ T cell–dependent manner. Together, these findings identify PP2A as a regulator of immunogenic senescence in MB and support PP2Ac targeting as a therapeutic strategy.

Authors

Winson S. Ho, Isha Mondal, Jingjing Liu, Raymond Sun, Jiawei Huo, Chao Gao, Oishika Das, Daren Tieu, Jingqi Sun, Hanchen Lin, Peng Zhang, Jiyang Yu, Rongze Olivia Lu

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Figure 2

Loss of PP2Ac induces cellular senescence in MB cells.

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Loss of PP2Ac induces cellular senescence in MB cells. (A) Flow cytometr...
(A) Flow cytometric quantification of SA-β-gal activity in WT and PP2Ac-KO D425 and #2416 cells. (B) WT D425 and #2416 cells were treated with LB-100 for 48 hours at the indicated concentrations, followed by analysis of SA-β-gal activity by flow cytometry. (C) GSEA plots showing enrichment of the Fridman senescence signature in PP2Ac-KO versus WT cells in both cell lines. (D) RT-qPCR analysis of senescence-associated genes in WT and PP2Ac-KO D425 and #2416 cells. (E) Representative images and quantification of SA-β-gal staining in WT and PP2Ac-KO D425 cells assessed by light microscopy. (F) Immunoblot analysis of phosphorylated NF-κB (p-p65, Ser536), total NF-κB (p65), and senescence-associated proteins in WT and PP2Ac-KO D425 cells. Densitometric quantification was performed by normalizing to total protein, with WT levels set to 1. (G) Quantification of IL-8 protein levels in culture supernatants from WT and PP2Ac-KO D425 cells after 48 hours, measured by bead-based immunoassay. (H) WT D425 cells were cultured for 48 hours in CM derived from WT or PP2Ac-KO cells. SA-β-gal was subsequently measured by flow cytometry. Representative histograms are shown. (I) Immunoblot validation of doxycycline-inducible CRISPR-Cas9–mediated PP2Ac KO in D425 cells expressing control sgRNA (sgCTL) or PP2Ac-targeting sgRNA (sgPP2Ac). Cells were treated with doxycycline (1 μg/mL) for 3 days. (J) D425 cells expressing inducible Cas9 and sgPP2Ac were treated with doxycycline for 7 days, followed by the NF-κB inhibitor BAY 11-7082 or vehicle for 3 days. SA-β-gal activity was assessed on day 10 by flow cytometry. Data are shown as mean ± SEM. Statistical significance was determined using unpaired 2-tailed t tests, 1-way ANOVA followed by Dunnett’s or Tukey’s multiple-comparisons tests, as appropriate for each panel. Immunoblots shown are representative of 2 independent experiments. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.