Fig 2: A schematic model showing the function of SATB2 and CBP in promoting FOXM1 expression and GSC‐driven tumor growthSATB2 binds to the MAR region of its target gene FOXM1 to remodel local chromatin by recruiting histone acetyltransferase CBP, which in turn promotes FOXM1 transcription, leading to GSC proliferation and GBM tumor growth. Inhibition of SATB2/CBP transcriptional activity by the CBP inhibitor C646 suppressed GSC proliferation and tumor growth. Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography © 2020. All Rights Reserved.

Fig 3: Ectopic expression of FOXM1 rescued the decreased GSC proliferation and GBM growth caused by SATB2 disruption AImmunoblot analysis of FOXM1 and SATB2 expression in T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2.BCell viability assay of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (n = 5). Ectopic expression of FOXM1 restored the cell proliferation impaired by SATB2 silencing.CTumorsphere number of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (n = 5). Ectopic expression of FOXM1 restored the tumorsphere formation of GSCs impaired by SATB2 silencing.D, E In vivo bioluminescent images (D) or quantification (E) of the tumor xenografts derived from luciferase‐labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n = 5 mice; shNT + FOXM1: n = 4 mice; shSATB2: n = 5 mice; shSATB2 + FOXM1: n = 5 mice). Representative images on day 21 posttransplantation are shown. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth.FKaplan–Meier survival curves of mice intracranially implanted with T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n = 8 mice; shNT + FOXM1: n = 7 mice; shSATB2: n = 6 mice; shSATB2 + FOXM1: n = 8 mice). Median survival: shNT, 30 days; shNT + FOXM1, 28 days; shSATB2, 50.5 days; shSATB2 + FOXM1, 34 days. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly attenuated the increased survival of mice bearing the GSC‐derived GBMs.GImmunofluorescence of Ki67 (Green) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n = 6 tumors; shNT + FOXM1: n = 5 tumors; shSATB2: n = 6 tumors; shSATB2 + FOXM1: n = 6 tumors). Scale bar: 40 μm.HQuantification of Ki67 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n = 6 tumors; shNT + FOXM1: n = 5 tumors; shSATB2: n = 6 tumors; shSATB2 + FOXM1: n = 6 tumors).IImmunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n = 6 tumors; shNT + FOXM1: n = 5 tumors; shSATB2: n = 6 tumors; shSATB2 + FOXM1: n = 6 tumors). Scale bar: 40 μm.JQuantification of SOX2 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n = 6 tumors; shNT + FOXM1: n = 5 tumors; shSATB2: n = 6 tumors; shSATB2 + FOXM1: n = 6 tumors). Data information: Data are shown as mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001, two‐way ANOVA analysis followed by Tukey’s test (B), one‐way ANOVA analysis followed by Tukey’s test (C, E, H, and J) or log‐rank test (F). Exact P values are reported in Appendix Table S3.

Fig 4: C646 treatment inhibited GSC proliferation and GSC‐driven tumor growth Cell viability of T3359 GSCs treated with indicated doses of C646 or the vehicle control (n = 5).Tumorsphere images of T3359 GSCs treated with indicated doses of C646 or the vehicle control for 6 days. Scale bar: 100 μm.qPCR analysis of SATB2, FOXM1, and FOXM1 downstream targets in T3359 GSCs treated with indicated doses of C646 or the vehicle control for 24 h (n = 3).Schematic diagram showing the treatment of mice bearing the GSC‐derived xenografts with C646. After GSC transplantation for 7 days, mice were treated with C646 or the vehicle control daily. Mice were monitored by IVIS bioluminescent imaging and maintained until neurological signs occur.Bioluminescent imaging of tumor growth in mice bearing xenografts derived from the luciferase‐labeled T3359 GSCs treated with C646 or the vehicle control at indicated days after GSC transplantation (n = 5 mice per group).Quantification of tumor growth from (E) (n = 5 mice per group).Kaplan–Meier survival curves of mice bearing T3359 GSC‐derived xenografts treated with C646 or the vehicle control (Vehicle control: n = 9 mice; C646: n = 8 mice). Median survival: Vehicle control, 27 days; C646, 33.5 days.Immunofluorescence of Ki67 (Green) in T3359 GSC‐derived xenografts from mice treated with C646 or the vehicle control (n = 6 tumors per group). Scale bar: 40 μm.Quantification of Ki67 positive cells in T3359 GSC‐derived xenografts from mice treated with C646 or the vehicle control (n = 6 tumors per group). Data information: Data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two‐way ANOVA analysis followed by Tukey’s test (A), one‐way ANOVA analysis followed by Tukey’s test (C), two‐way ANOVA analysis followed by Sidak’s test (F), log‐rank test (G), or Mann–Whitney test (I). Exact P values are reported in Appendix Table S3.

Fig 5: SATB2 is preferentially expressed by GSCs Immunofluorescence of SATB2 (green) and the GSC marker SOX2 or OLIG2 (red) on frozen tissue sections of human GBM surgical specimens. SATB2 is preferentially expressed by GSCs in human GBMs. Scale bar, 25 μm.Quantification of the fraction of SATB2+ cells in SOX2+ or OLIG2+ cells in human GBMs. More than 90% SOX2+ or OLIG2+ cells showed SATB2 staining. n = 3 GBMs.qPCR analysis of SATB2 mRNA expression in GSCs and matched non‐stem tumor cells (NSTCs) (n = 5).Immunoblot analysis of SATB2, SOX2, and OLIG2 expression in cell lysates of GSCs and matched NSTCs.Immunofluorescence of SATB2 (green) and SOX2 (red) in T3359 GSCs and matched NSTCs. Scale bar, 50 μm.qPCR analysis of SATB2 mRNA expression in GSCs and neural progenitor cells (NPCs) (n = 3).Immunoblot analysis of SATB2 and SOX2 expression in cell lysates of GSCs and NPCs.Immunoblot analysis of SATB2, GSC marker SOX2, and differentiation marker GFAP expression during serum‐induced GSC differentiation. Data information: Data are represented as mean ± SD. **P < 0.01, ***P < 0.001, Mann–Whitney test (C) or one‐way ANOVA analysis followed by Tukey’s test (F). Exact P values are reported in Appendix Table S3.