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Ibrutinib inactivates BMX-STAT3 in glioma stem cells to impair malignant growth and radioresistance

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Science Translational Medicine  30 May 2018:
Vol. 10, Issue 443, eaah6816
DOI: 10.1126/scitranslmed.aah6816
  • Fig. 1 Ibrutinib is more effective than TMZ at inhibiting GSC-derived tumor growth to extend animal survival.

    (A and B) In vivo bioluminescent images (A) and the quantification (B) of human GSC-derived xenografts in the brains of mice treated with ibrutinib or the vehicle control at the indicated time points. (C) Kaplan-Meier survival analysis of mice bearing GSC-derived xenografts treated with ibrutinib or the vehicle control. (D and E) In vivo bioluminescent images (D) and the quantification (E) of GSC-derived xenografts in mice treated with ibrutinib or TMZ at the indicated time points. (F) Kaplan-Meier survival analysis of mice bearing GSC-derived xenografts treated with the vehicle control, ibrutinib, or TMZ. (G) Survival extension of mice bearing GSC-derived GBM tumors treated with ibrutinib or TMZ relative to those treated with the vehicle control. Statistical analysis was performed using unpaired Student’s t test for two-group comparison, one-way analysis of variance (ANOVA) for multigroup comparison, or Kaplan-Meier method for survival analyses. Data are means ± SEM (B and E) or means ± SD (G). ns, not significant. *P < 0.05 and **P < 0.01. n = 5 for each group. p, photons; sr, steradian.

  • Fig. 2 Ibrutinib treatment combines with radiation therapy to disrupt GBM tumor growth.

    (A and B) In vivo bioluminescent images (A) and the quantifications (B) of GSC-derived xenografts in mice treated with the vehicle control, ibrutinib, and/or radiation at the indicated time points. (C) Kaplan-Meier survival analysis of mice bearing GSC-derived xenografts with indicated treatments. (D) Survival extension of mice bearing GSC-derived GBM tumors with indicated treatments. (E and F) Immunofluorescent staining (E) and the quantification (F) of cleaved caspase-3 (green) in GSC-derived xenografts from mice with indicated treatments. (G and H) Immunofluorescent staining (G) and the quantification (H) of Ki67 (green) in GSC-derived xenografts from mice with indicated treatments. Statistical analysis was performed using unpaired Student’s t test for two-group comparison, one-way ANOVA for multigroup comparison, or Kaplan-Meier method for survival analyses. Data are means ± SEM (B) or means ± SD (D, F, and H). *P < 0.05 and **P < 0.01. n = 5 for each group. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 3 Ibrutinib disrupts the maintenance of GSCs but not of NPCs in vitro.

    (A) In vitro limiting dilution assay of three individual human GSCs (D456, T4121, and T387) treated with indicated doses of ibrutinib or the vehicle control. (B and C) Representative images of GSC tumor spheres (B) and the quantification of sphere diameter [(C), left] and numbers [(C), right] of D456 GSCs treated with indicated doses of ibrutinib or the vehicle control. (D) Cell apoptosis assay of D456 GSCs treated with indicated doses of ibrutinib or the vehicle control. (E) Immunoblot analyses of cleaved caspase-3, cleaved PARP, PARP, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in D456 GSCs treated with indicated doses of ibrutinib or the vehicle control. (F and G) Dose-response curves of ibrutinib treatment in GSCs (F) and NPCs (G). Human GSCs and NPCs were exposed to increasing concentrations of ibrutinib (0 to 25.6 μM) for 72 hours, followed by in vitro cell viability analysis. EC50 of ibrutinib for GSCs and NPCs was measured using nonlinear regression analysis of the dose-response curves. (H to J) Representative images of neurospheres (H) and the quantification of sphere diameter (I) and numbers (J) of 15167 NPCs with indicated treatments. Statistical analysis was performed using unpaired Student’s t test for two-group comparison or one-way ANOVA for multigroup comparison. Data are means ± SD. *P < 0.05 and **P < 0.01. Scale bars, 100 μm. DMSO, dimethyl sulfoxide.

  • Fig. 4 Ibrutinib inhibits the BMX-mediated STAT3 activation in GSCs.

    (A) Immunoblot analyses of pBMX-Y40, total BMX, pSTAT3-Y705, total STAT3, and GAPDH in D456 GSCs treated with indicated doses of ibrutinib or the vehicle control. (B) Immunoblot analyses of pBMX-Y40, BMX, pSTAT3-Y705, STAT3, and GAPDH in D456 GSCs treated with ibrutinib (1 μM) at the indicated time points. (C) Immunoblot analyses of pSTAT3-Y705, STAT3, and GAPDH showing the effects of ibrutinib (1 μM) on STAT3 phosphorylation in D456 GSCs and 15167 NPCs. (D) Immunoblot analyses of pSTAT3-Y705, STAT3, and GAPDH in D456 GSC-derived xenografts with indicated treatments. n = 3 for each group. (E and F) Representative IHC images (E) and the quantification (F) of pSTAT3-Y705 in the SVZ of mouse brains with indicated treatments. n = 5 for each group. Scale bars, 50 μm. Statistical analysis was performed using unpaired Student’s t test. Data are means ± SD. LV, lateral ventricle.

  • Fig. 5 Constitutively active STAT3 largely reverses the suppressive effect of ibrutinib on GSCs.

    (A) Immunoblot analyses of STAT3-C–Flag, STAT3, and GAPDH in D456 and T387 GSCs transduced with STAT3-C–Flag or control vector. (B) In vitro cell viability assay of STAT3-C–expressing D456 GSCs treated with ibrutinib or the vehicle control. (C) In vitro limiting dilution assay of STAT3-C–expressing D456 GSCs treated with ibrutinib or the vehicle control. (D) Immunoblot analyses of cleaved caspase-3, caspase-3, cleaved PARP, PARP, and tubulin in STAT3-C–expressing D456 GSCs treated with ibrutinib or the vehicle control. (E) Fluorescence-activated cell sorting analyses of apoptosis of the STAT3-C–expressing D456 GSCs treated with ibrutinib or the vehicle control. Statistical analysis was performed using unpaired Student’s t test for two-group comparison or one-way ANOVA for multigroup comparison. Data are means ± SD. **P < 0.01.

  • Fig. 6 JAK2-mediated STAT3 activation is blocked in GSCs, and SOCS3 is responsible for JAK2 inactivation in GSCs.

    (A) Immunoblot analyses of pJAK2-Y1007/1008, JAK2, pSTAT3-Y705, STAT3, and tubulin in human GSCs (D456) and NPCs (15167) upon IL-6 stimulation. (B) Coimmunoprecipitation analyses of JAK2, STAT3, and pSTAT3-Y705 with anti-JAK2 antibody in IL-6–stimulated human GSCs (D456) and NPCs (15167). IgG, immunoglobulin G; IP, immunoprecipitation. (C) Immunofluorescent staining of pSTAT3-Y705 (green) in human GSCs (D456; top) and NPCs (15167; bottom) treated with JAK2 inhibitor ruxolitinib (100 nM) or the vehicle control. Scale bars, 25 μm. (D) Immunoblot analyses of SOCS3 and GAPDH in human GSCs and NPCs. (E) Coimmunoprecipitation analysis of SOCS3 with anti-JAK2 antibody in human GSCs (D456) and NPCs (ENSA). (F) Coimmunoprecipitation analyses of STAT3 with anti-JAK2 antibody in D456 GSCs expressing short hairpin RNA (shRNA) against SOCS3 (shSOCS3) or nontargeting shRNA (shNT). (G) Immunoblot analyses of pJAK2-Y1007/1008, JAK2, pSTAT3-Y705, STAT3, SOSC3, and GAPDH in D456 GSCs expressing shSOCS3 (sh1 or sh2) or shNT with or without IL-6 stimulation. The symbol (‘) means minutes.

  • Fig. 7 BMX interacts with STAT3 and gp130 to mediate STAT3 activation in GSCs.

    (A) Coimmunoprecipitation of gp130 and pBMX with anti-BMX antibody in D456 GSCs upon IL-6 stimulation. (B) Coimmunoprecipitation of STAT3 and pSTAT3-Y705 with anti-BMX antibody in D456 GSCs upon IL-6 stimulation. (C) Immunoblot analyses of pSTAT3-Y705, STAT3, and tubulin in D456 GSCs expressing shBMX-1, shBMX-2, or shNT upon IL-6 stimulation. (D) Immunoblot analyses of pSTAT3-Y705, STAT3, and GAPDH in GSCs expressing BMX-DN, BMX-WT, or BMX-C upon IL-6 stimulation (10 ng/ml). The symbol (‘) means minutes.

  • Fig. 8 BMX bypasses SOCS3 negative regulation on JAK2 to sustainably activate STAT3 in GSCs.

    (A) Immunoblot analyses of pBMX-Y40, BMX, pSTAT3-Y705, STAT3, SOCS3, and tubulin in D456 GSCs transduced with SOCS3 or the control vector upon IL-6 stimulation. (B) Coimmunoprecipitation of BMX with anti-SOCS3 antibody in D456 GSCs upon IL-6 stimulation. Precipitation with normal mouse IgG was used as a negative control. Total cell lysates (input) were immunoblotted with antibodies against SOCS3, BMX, and GAPDH. (C) In vitro cell viability analyses of human GSCs (D456) and NPCs (15167) expressing SOCS3 or the control vector. (D) Schematic diagram of targeting GSCs through BMX inhibition by ibrutinib and the underlying molecular mechanisms. BMX bypasses the SOCS3-mediated inhibition of JAK2 to sustain activation of STAT3 in GSCs (left), whereas JAK2-mediated STAT3 activation in NPCs (right) is negatively regulated by SOCS3, providing a molecular basis for targeting BMX by ibrutinib to specifically inhibit STAT3 activation in GSCs but not in NPCs. The symbol (‘) means minutes. Statistical analysis was performed using unpaired Student’s t test. Data are means ± SD. **P < 0.01.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/443/eaah6816/DC1

    Materials and Methods

    Fig. S1. Ibrutinib treatment inhibits GBM growth and prolongs animal survival.

    Fig. S2. Ibrutinib penetrates into mouse brains and has no severe systemic side effects.

    Fig. S3. Ibrutinib treatment is effective for most GBMs tested.

    Fig. S4. Ibrutinib treatment targets GSCs but not NPCs in vivo.

    Fig. S5. Ibrutinib is more effective at targeting GSCs than NSTCs.

    Fig. S6. Ibrutinib treatment inhibits BMX-mediated STAT3 activation in GSCs.

    Fig. S7. Ectopic expression of constitutively active STAT3 largely rescues GSC maintenance disrupted by ibrutinib.

    Fig. S8. JAK2 mediates STAT3 activation in NPCs, whereas BMX interacts with gp130 to mediate STAT3 activation in GSCs.

    Fig. S9. Forced expression of SOCS3 inhibits JAK2-mediated STAT3 activation in NPCs.

    Table S1. Pathological and molecular features of patient-derived GSCs used in this study.

    Table S2. Expression of BMX and pBMX-Y40 and the pathological characteristics of human GBMs used in this study.

    Table S3. Sequences of shRNAs used in this study.

    References (3840)

  • Supplementary Material for:

    Ibrutinib inactivates BMX-STAT3 in glioma stem cells to impair malignant growth and radioresistance

    Yu Shi, Olga A. Guryanova, Wenchao Zhou, Chong Liu, Zhi Huang, Xiaoguang Fang, Xiuxing Wang, Cong Chen, Qiulian Wu, Zhicheng He, Wei Wang, Wei Zhang, Tao Jiang, Qing Liu, Yaping Chen, Wenying Wang, Jingjing Wu, Leo Kim, Ryan C. Gimple, Hua Feng, Hsiang-Fu Kung, Jennifer S. Yu, Jeremy N. Rich, Yi-Fang Ping,* Xiu-Wu Bian,* Shideng Bao*

    *Corresponding author. Email: baos{at}ccf.org (S.B.); bianxiuwu{at}263.net (X.-W.B.); pingyifang{at}126.com (Y.-F.P.)

    Published 30 May 2018, Sci. Transl. Med. 10, eaah6816 (2018)
    DOI: 10.1126/scitranslmed.aah6816

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Ibrutinib treatment inhibits GBM growth and prolongs animal survival.
    • Fig. S2. Ibrutinib penetrates into mouse brains and has no severe systemic side effects.
    • Fig. S3. Ibrutinib treatment is effective for most GBMs tested.
    • Fig. S4. Ibrutinib treatment targets GSCs but not NPCs in vivo.
    • Fig. S5. Ibrutinib is more effective at targeting GSCs than NSTCs.
    • Fig. S6. Ibrutinib treatment inhibits BMX-mediated STAT3 activation in GSCs.
    • Fig. S7. Ectopic expression of constitutively active STAT3 largely rescues GSC maintenance disrupted by ibrutinib.
    • Fig. S8. JAK2 mediates STAT3 activation in NPCs, whereas BMX interacts with gp130 to mediate STAT3 activation in GSCs.
    • Fig. S9. Forced expression of SOCS3 inhibits JAK2-mediated STAT3 activation in NPCs.
    • Table S1. Pathological and molecular features of patient-derived GSCs used in this study.
    • Table S2. Expression of BMX and pBMX-Y40 and the pathological characteristics of human GBMs used in this study.
    • Table S3. Sequences of shRNAs used in this study.
    • References (3840)

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