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
View: Text | PDF
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

×

Figure 8

Local delivery of LNP-siPP2Ac promotes tumor immunogenicity in MB.

Options: View larger image (or click on image) Download as PowerPoint
Local delivery of LNP-siPP2Ac promotes tumor immunogenicity in MB. (A) (...
(A) (A) Dynamic light scattering analysis of PP2A LNP. Mean particle diameter was 124.9 ± 1.3 nm with a polydispersity index (PDI) of 0.136 ± 0.016. (B) Time-course analysis of fluorescently labeled LNP uptake in #2416 cells quantified by flow cytometry. (C) Immunofluorescence images of #2416 cells treated with fluorescently labeled PP2A LNP or vehicle control. Cells were stained with wheat germ agglutinin (WGA) and DAPI. (D) RT-qPCR analysis of Ppp2ca (PP2Ac) mRNA in #2416 cells treated with CTL LNP or PP2A LNP. (E) Immunoblot analysis of PP2Ac, phospho-p65 (Ser536), and total p65 in #2416 cells treated with CTL LNP or PP2A LNP for 72 hours. Total protein staining served as a loading control. Densitometric values were normalized to total protein and expressed relative to CTL LNP. (F and G) SA-β-gal activity (F) and MHC-I surface expression (G) in #2416 cells treated with CTL LNP or PP2A LNP for 72 hours. (H) RT-qPCR analysis of senescence- and SASP-associated genes in #2416 cells 72 hours after treatment with CTL LNP or PP2A LNP. (I) Cytoplasmic dsDNA in #2416 cells treated with CTL LNP or PP2A LNP, followed by irradiation (RTx) at the indicated doses. (J) Schematic of the intratumoral delivery schedule and Kaplan-Meier survival analysis. C57BL/6 or CD8-deficient (Cd8a^tm1Mak/J) mice bearing orthotopic #2416 tumors received 3 μL CTL LNP or PP2A LNP on days 7, 10, and 13 after implantation (n = 8–16/group; combined results from 2 independent experiments). (K) Representative immunofluorescence images and quantification of SA-β-gal in #2416 tumors harvested at endpoint. SA-β-gal (green) and nuclei (DAPI, blue). Scale bar: 100 μm. Data are shown as mean ± SEM. Statistical significance was determined using unpaired 2-tailed t tests, 1-way ANOVA followed by Tukey’s multiple-comparisons test, or the log-rank test, as appropriate. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.