Research ArticleCancer therapy

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

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Science Signaling  11 Sep 2018:
Vol. 11, Issue 547, eaau5147
DOI: 10.1126/scisignal.aau5147
  • 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 μM), 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.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/547/eaau5147/DC1

    Fig. S1. Relative phosphopeptide abundance between P14 biological replicates.

    Fig. S2. Relative proliferation in the P1, P7, and P14 cerebellum.

    Fig. S3. GNP proliferation after CX-4945 treatment in culture.

    Fig. S4. Knockdown specificity of pooled siRNA constructs targeting Csnk2a1, Csnk2a2, Csnk2b, and Smo.

    Fig. S5. Gli1 and Hh-associated transcript expression in NIH3T3 cells after CK2 inhibition.

    Fig. S6. Gli1 transcript expression in cultured SHH MB cell lines.

    Fig. S7. Weight of control or CX-4945–treated mice with cerebellar SHH MB.

    Fig. S8. CSNK2A1 expression and 5-year survival of patients with SHH MB.

    Fig. S9. Sequence conservation of Csnk2a1 mutations in TBB-resistant SHH MB cells.

    Table S1. Phosphoproteome of GNPs isolated at P1, P7, and P14.

    Table S2. Phosphopeptides of enriched kinase motifs.

    Table S3. Primer and siRNA sequences.

    Table S4. Pathological report for the primary human MB sample (ST01).

  • The PDF file includes:

    • Fig. S1. Relative phosphopeptide abundance between P14 biological replicates.
    • Fig. S2. Relative proliferation in the P1, P7, and P14 cerebellum.
    • Fig. S3. GNP proliferation after CX-4945 treatment in culture.
    • Fig. S4. Knockdown specificity of pooled siRNA constructs targeting Csnk2a1, Csnk2a2, Csnk2b, and Smo.
    • Fig. S5. Gli1 and Hh-associated transcript expression in NIH3T3 cells after CK2 inhibition.
    • Fig. S6. Gli1 transcript expression in cultured SHH MB cell lines.
    • Fig. S7. Weight of control or CX-4945–treated mice with cerebellar SHH MB.
    • Fig. S8. CSNK2A1 expression and 5-year survival of patients with SHH MB.
    • Fig. S9. Sequence conservation of Csnk2a1 mutations in TBB-resistant SHH MB cells.
    • Legends for tables S1 and S2
    • Table S3. Primer and siRNA sequences.
    • Table S4. Pathological report for the primary human MB sample (ST01).

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Phosphoproteome of GNPs isolated at P1, P7, and P14.
    • Table S2 (Microsoft Excel format). Phosphopeptides of enriched kinase motifs.

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