Developmental phosphoproteomics identifies the kinase CK2 as a driver of Hedgehog signaling and a therapeutic target in medulloblastoma

Abstract

A major limitation of targeted cancer therapy is the rapid emergence of drug resistance, which often arises through mutations at or downstream of the drug target or through intrinsic resistance of subpopulations of tumor cells. Medulloblastoma (MB), the most common pediatric brain tumor, is no exception, and MBs that are driven by sonic hedgehog (SHH) signaling are particularly aggressive and drug-resistant. To find new drug targets and therapeutics for MB that may be less susceptible to common resistance mechanisms, we used a developmental phosphoproteomics approach in murine granule neuron precursors (GNPs), the developmental cell of origin of MB. The protein kinase CK2 emerged as a driver of hundreds of phosphorylation events during the proliferative, MB-like stage of GNP growth, including the phosphorylation of three of the eight proteins commonly amplified in MB. CK2 was critical to the stabilization and activity of the transcription factor GLI2, a late downstream effector in SHH signaling. CK2 inhibitors decreased the viability of primary SHH-type MB patient cells in culture and blocked the growth of murine MB tumors that were resistant to currently available Hh inhibitors, thereby extending the survival of tumor-bearing mice. Because of structural interactions, one CK2 inhibitor (CX-4945) inhibited both wild-type and mutant CK2, indicating that this drug may avoid at least one common mode of acquired resistance. These findings suggest that CK2 inhibitors may be effective for treating patients with MB and show how phosphoproteomics may be used to gain insight into developmental biology and pathology.

Fig. 1.. Quantitative mapping of the phosphoproteome during GNP development.

(A) Schematic of early postnatal proliferation and differentiation of GNPs. Pink, Atoh1-positive proliferative GNPs; red, postmitotic GNPs. oEGL/iEGL, outer/inner external granule layer; IGL, internal granule layer. (B) Experimental scheme for the phosphoproteomic assays. (C) Heat map representing relative phosphopeptide abundance and undirected clustering among three biological replicates and sample types (P1, P7, and P14 GNPs and Ptch^+/− MB). Color axis = R². *Cluster branch of P7 GNPs versus MB sample. (D) Distribution of relative phosphopeptide abundance at developmental transitions. Dashed lines = 1.5 SDs. Mass spectrometry (MS) was performed in n = 3 biological replicates per experimental time point (14 to 40 mice per replicate at each time point).

Fig. 2.. Phosphoproteomic data indicate increased phosphorylation at CK2 motifs at P7.

(A and B) The 16 motifs enriched in the fractions of altered phosphopeptide abundance (>1.5 SDs) in the phosphoproteomic analysis of GNPs purified from mice harvested on P1, P7, and P14 (A). The predicted kinase of each motif is noted down the right. *CK2 target motifs. **Known regulators of GNP development. Color axis: Fraction of maximum enrichment. Red box: CK2 motif characterized by motif logo (B). (C) Enriched phosphopeptides, and the corresponding protein, contributing to the SD.E motif-enriched phosphopeptides described in (A). Proteins include known (*) and currently uncharacterized CK2 substrates. Data are n = 3 biological replicates per experimental time point (14 to 40 mice per replicate at each time point).

Fig. 3.. CK2 is required for cerebellar development.

(A) Experimental scheme for CK2 inhibitor (TBB) studies in early postnatal mice. (B and C) Immunofluorescent imaging of whole brains and sagittal cerebellar sections from control (DMSO) and CK2 inhibitor-treated Math1/nGFP transgenic mice. Green, Math1 (proliferating GNP marker); red, Tag1 (postmitotic GNP marker). Empty arrow: Folia length. Solid arrow: Folia width. (D) Ratio of Math1-expressing GNPs, early differentiating GNPs (Tag1), proliferation [5-ethynyl-2′-deoxyuridine (EdU)], and apoptosis [terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)] in TBB-treated mice relative to control mice. Data are means ± SD from n = 10 to 11 mice, as indicated. ns = P > 0.05, **P < 0.01, ***P < 0.001, two-tailed t test. Scale bars, 1 mm (left), 0.1 mm (middle), and 0.05 mm (right).

Fig. 4.. CK2 is required for Hh signal transduction.

(A) Canonical Hh signaling pathway, for reference in subsequent panels. (B and C) Fold change in Gli1 mRNA expression in NIH3T3 cells exposed to murine SHH (3 μg/ml) for 6 hours after knockdown of CSNK2A1, CSNK2A2, or CSNK2B (B) or when treated with the CK2 inhibitor TBB (50 μM) or CX-4945 (10 μM) or SMO inhibitor GDC-0449 (100 nM) (C) relative to controls. (D and E) Gli1 mRNA expression after exposure to TBB (as indicated) or GDC-0449 (100 nM) in NIH3T3 cells pretreated with SMO agonist SAG (100 nM) (D) and in Sufu^−/− MEFs (E). (F) Immunoblotting for protein abundance of endogenous GLI2 (GLI2FL, full length) and exogenous constitutively active GLI2 (GLI2ΔN) in NIH3T3 cells treated with various concentrations of TBB for 6 hours. Blots are from a representative of three experiments. (G) Gli1 mRNA expression after exposure to TBB in NIH3T3 cells expressing GLI2ΔN. Data in (A) to (D) and (G) were obtained by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and are shown as means ± SD from n = 3 experiments. ns = P > 0.05, *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t test.

Fig. 5.. Efficacy of CK2 inhibitors against MB in vitro.

(A and B) Cell viability after 72 hours of exposure to CX-4945 (10 μΜ), assessed by luminescent cell viability assays, in murine Ptch^+/− (MB21 and MB55), Ptch^+/−;Tpr53^−/− (MB53) MB lines in culture (A) and in human MB PDX lines (RCMB32, BT084, ICb-984, and ST01) assayed in culture (B). (C and D) Cell viability and cell cycle stage of human SHH MB cells cultured in DMSO, CX-4945, or vismodegib for 24 hours, as assessed by high-throughput single-cell imaging. (E) Cell viability after 72 hours of exposure to CX-4945 (10 μM), assessed by luminescent cell viability assays, in pediatric glioma (DIPG) cells. Data are means ± SD from n = 3 experiments.

Fig. 6.. Efficacy of CK2 inhibitors against MB in vivo.

(A) Experimental scheme for flank and cerebellar tumor trials. Blue triangle, treatment. (B and C) Relative tumor growth of Ptch^+/−;Tpr53^−/− (B) and Ptch^+/−;Tpr53^−/−;SmoD477G (C) MB flank allografts. Data are means ± SD; P values by a two-tailed t test. (D) Representative picture of mice treated with TBB versus GDC-0449. Dashed line demarcates the tumor. (E) Kaplan-Meier survival analysis of mice with Ptch^+/−;Tpr53^−/−;SmoD477G MB cerebellar allografts treated with CX-4945 or vehicle control. n = 7 mice in each treatment group. (F) Kaplan-Meier analysis of CK2 gene expression and overall survival in human patients (n = 179) with SHH-subtype MB. P values in (E) and (F) were determined by a log-rank Mantel-Cox test.

Fig. 7.. TBB resistance results from mutation in Csnk2a1.

(A) Gli1 transcript abundance in parental, TBB-sensitive MB55 cells and serial passage/drug-induced, TBB-resistant MB55 cells treated with CX-4945 or TBB relative to that in control (DMSO-treated) cells. Data are means ± SD from n = 3 experiments. ns = P > 0.05, *P < 0.05, ****P < 0.0001, two-tailed t test. (B and C) MD simulation and docking analysis of TBB (B) and CX-4945 (C) with wild-type (WT) CK2 and mutant (D175N) CK2 in MB55 cells.