Epigenetic modulator inhibition overcomes temozolomide chemoresistance and antagonizes tumor recurrence of glioblastoma
Byoung-San Moon, Mingyang Cai, Grace Lee, Tong Zhao, Xiaofeng Song, Steven L. Giannotta, Frank J. Attenello, Min Yu, Wange Lu
Byoung-San Moon, Mingyang Cai, Grace Lee, Tong Zhao, Xiaofeng Song, Steven L. Giannotta, Frank J. Attenello, Min Yu, Wange Lu
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Research Article Oncology

Epigenetic modulator inhibition overcomes temozolomide chemoresistance and antagonizes tumor recurrence of glioblastoma

Abstract

Glioblastoma multiforme (GBM) heterogeneity causes a greater number of deaths than any other brain tumor, despite the availability of alkylating chemotherapy. GBM stem-like cells (GSCs) contribute to GBM complexity and chemoresistance, but it remains challenging to identify and target GSCs or factors that control their activity. Here, we identified a specific GSC subset and show that activity of these cells is positively regulated by stabilization of methyl CpG binding domain 3 (MBD3) protein. MBD3 binds to CK1A and to BTRCP E3 ubiquitin ligase, triggering MBD3 degradation, suggesting that modulating this circuit could antagonize GBM recurrence. Accordingly, xenograft mice treated with the CK1A activator pyrvinium pamoate (Pyr-Pam) showed enhanced MBD3 degradation in cells expressing high levels of O6-methylguanine-DNA methyltransferase (MGMT) and in GSCs, overcoming temozolomide chemoresistance. Pyr-Pam blocked recruitment of MBD3 and the repressive nucleosome remodeling and deacetylase (NuRD) complex to neurogenesis-associated gene loci and increased acetyl–histone H3 activity and GSC differentiation. We conclude that CK1A/BTRCP/MBD3/NuRD signaling modulates GSC activation and malignancy, and that targeting this signaling could suppress GSC proliferation and GBM recurrence.

Authors

Byoung-San Moon, Mingyang Cai, Grace Lee, Tong Zhao, Xiaofeng Song, Steven L. Giannotta, Frank J. Attenello, Min Yu, Wange Lu

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Figure 4

CK1A-mediated phosphorylation of serine residues in MBD3 degron motif promotes MBD3 degradation.

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CK1A-mediated phosphorylation of serine residues in MBD3 degron motif pr...
(A) Group-based Prediction System software (version 2.1) predicts that CK1A phosphorylates MBD3 serines 39, 45, 85, and 106 at degron domains. (B) IP using Myc or Flag-M2 beads from respective lysates of HEK293T cells harboring Flag-tagged CK1A in the presence or absence of Myc-MBD3 or harboring HA-MBD3 plus either control vector or Flag-CK1A. Whole-cell lysates (WCLs) were immunoblotted (IB) with the indicated antibodies. (C) WCLs of T98G GSCs transfected with Myc-tagged MBD3 or control vectors plus increasing levels of Flag-tagged CK1A vector were immunoprecipitated with the indicated beads recognizing Myc. (D) Upper: USC02 GSCs were transfected with control (shCtrl) or shCK1A shRNA and treated with cycloheximide (CHX) for indicated times (h, hours) before harvest. Lower: HEK293T cells were treated with DMSO or the CK1A inhibitor D4476 (25 μM) and treated with CHX for indicated times before harvest. (E and F) Effect of CK1A activation or inhibition on MBD3 protein stability. T98G GSCs were treated with varying doses of pyrvinium pamoate (Pyr-Pam) (E) or 10 μM Pyr-Pam and 0–50 μM D4476 as indicated (F). WCLs were immunoblotted with the indicated antibodies. Data are presented as the mean ± SD and are representative of at least 3 independent experiments. **P < 0.005; ***P < 0.0005 by 2-way ANOVA with Bonferroni’s post hoc test.