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. 2018 Sep 10;34(3):513-528.e8.
doi: 10.1016/j.ccell.2018.08.003.

Disruption of the β1L Isoform of GABP Reverses Glioblastoma Replicative Immortality in a TERT Promoter Mutation-Dependent Manner

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Free PMC article

Disruption of the β1L Isoform of GABP Reverses Glioblastoma Replicative Immortality in a TERT Promoter Mutation-Dependent Manner

Andrew Mancini et al. Cancer Cell. .
Free PMC article

Abstract

TERT promoter mutations reactivate telomerase, allowing for indefinite telomere maintenance and enabling cellular immortalization. These mutations specifically recruit the multimeric ETS factor GABP, which can form two functionally independent transcription factor species: a dimer or a tetramer. We show that genetic disruption of GABPβ1L (β1L), a tetramer-forming isoform of GABP that is dispensable for normal development, results in TERT silencing in a TERT promoter mutation-dependent manner. Reducing TERT expression by disrupting β1L culminates in telomere loss and cell death exclusively in TERT promoter mutant cells. Orthotopic xenografting of β1L-reduced, TERT promoter mutant glioblastoma cells rendered lower tumor burden and longer overall survival in mice. These results highlight the critical role of GABPβ1L in enabling immortality in TERT promoter mutant glioblastoma.

Keywords: GABP; TERT promoter mutation; cancer immortality; glioblastoma; telomerase; telomeres.

Conflict of interest statement

Competing Financial Interests: R.J.A.B and J.F.C are co-founders of Telo Therapeutics Inc. and have ownership interest.

Figures

Figure 1.
Figure 1.
The GABP tetramer-forming isoform β1L positively regulates TERT expression solely in TERT promoter mutant tumor cells. (A) TERT expression following siRNA-mediated knockdown of β1 (siGABPB1) in TERT promoter mutant (left) or TERT promoter-wild-type (right) cell lines and primary cultures. *p value<0.05, **p value<0.01, two-sided Student’s t-test compared to a non-targeting siRNA control (siCTRL) in each respective line. (B) Correlation of GABPB1L (top graphs) or GABPB1S (bottom graphs) expression (log2[RSEM normalized counts]) versus TERT expression (log2[RSEM normalized counts]) from 109 TERT-expressing GBMs (left graphs) or 49 TERT promoter-mutant oligodendrogliomas (right graphs). Red line indicates trend line. Black points indicate Sanger-validated TERT promoter mutant GBM and oligodendroglioma samples, teal points are GBM samples that were not tested for TERT promoter mutation status. Spearman’s Rank-Order Correlation was used to generate Spearman rho and p values for each correlation. (C) GABPB1L expression following siRNA-mediated knockdown of β1 (siGABPB1) in TERT promoter mutant (left) and wild-type (right) lines. *p value<0.05, **p value<0.01, two-sided Student’s t-test compared to a non-targeting siRNA control (siCTRL) in each respective line. (D) TERT expression following LNA-ASO knockdown of β1L (LNA-GABPB1L) in TERT promoter mutant (left) or wild-type (right) cell lines and primary cultures compared to a control LNA-ASO (LNA-CTRL). *p value<0.05, **p value<0.01, two-sided Student’s t-test compared to LNA-CTRL in each respective line. Values are mean ± S.D. of at least three independent experiments (A, C, and D; two independent experiments for SF10417). See also Figure S1.
Figure 2.
Figure 2.
CRISPR-Cas9-mediated disruption of GABPB1L reduces GABP-mediated activation of the mutant TERT promoter. (A) Exon structure for the GABPB1 locus, depicting the GABPB1S and GABPB1L isoforms. Inset shows targeting strategy for CRISPR-Cas9 editing of GABPB1L. Red blocks indicate sgRNA target sites. Red arrows and dashed lines indicate primer locations and target amplicon for PCR validation of editing. (B) Quantification of β1L tetramerization in the wild-type (POS) or mutated (DEL1-3) state. The negative (NEG) state consists of one β1L vector and one β1S vector, the products of which are unable to form a tetramer. *p value<0.05, **p value<0.01, two-sided Student’s t-test of DEL1-3 or NEG respective to the positive control (POS). (C) GABPα or IgG control ChIP-qPCR for the TERT promoter in CRISPR control (CTRL) or β1L-reduced clones (C1 and C2). *p value<0.05, **p value<0.01, two-sided Student’s t-test compared to respective CTRL. (D) TERT expression relative to CTRL for β1L-reduced TERT promoter mutant (left) or wild-type (right) clones. *p value<0.05, **p value<0.01, two-sided Student’s t-test compared to CTRL. (E,F) TERT expression (E) or GABPα occupancy (F) in β1L-reduced clones relative to CTRL 48 hr following transfection with empty (VECTOR) or β1L expression vector. *p value<0.05, **p value<0.01, two-sided Student’s t-test compared to respective VECTOR control. Values are mean ± S.D. of at least two independent experiments (C and F) or three independent experiments (B, D, and E). See also Figures S2–S3 and Tables S1–S3.
Figure 3.
Figure 3.
β1L-mediated activation of the mutant TERT promoter is required for telomere maintenance in GBM. (A) Telomere length at days 44, 61, and 78 in TERT promoter mutant lines or days 44, 61 and 83 in TERT promoter wild-type lines post-editing relative to day 33 post-editing for CTRL or β1L-reduced clones. *p value<0.05, two-sided Student’s t-test comparing values between CTRL and β1L-reduced clones at day 78/83 for each respective line. Values are mean ± S.D. of at least three independent assays. (B) Relative telomere length after transfection of an empty (VECTOR), β1L, or TERT expression vector in TERT promoter-mutant lines 78 or 83 days post-editing. Red dotted line indicates time of transfection (at day 58 [LN229] or 61 [GBM1 and T98G] post-editing). *p value<0.05, two-sided Student’s t-test of values of β1L or TERT versus VECTOR at day 78/83. Values are mean ± S.D. of at least three independent experiments. (C) Representative DAPI images (left images) and quantification (right graphs) of chromatin bridges (arrow) in CTRL or β1L-reduced clones at days 70–75 post-editing. Scale bar = 20 μm. *p value<0.05, **p value<0.01, two-sided Student’s t-test compared to CTRL. Quantification values are weighted mean ± S.D. of at least ten independent fields of view. See also Figure S4.
Figure 4.
Figure 4.
β1L reduction induces loss of replicative immortality in TERT promoter-mutant GBM lines. (A) Cell viability of CTRL or β1L-reduced clones measured approximately every 7 days from day 33 to day 99 post-editing for TERT promoter mutant and wild-type lines. **p value<0.01, Welch’s t-test of CTRL clones versus β1L-reduced clones at day 83 post-editing. (B) Cell viability measurements following transfection with an empty (VECTOR), β1L, or TERT expression vector. Red dotted line indicates time of transfection. *p value<0.05, **p value<0.01, Welch’s t-test of vector transfected cells versus β1L and TERT transfected cells at the final recorded time-point for each line. Values are median of three independent experiments. See also Figure S5.
Figure 5.
Figure 5.
β1L regulates a subset of GABP transcription factor targets in GBM cells. (A) Expression of one GABP dimer target and four GABP tetramer targets relative to CTRL for β1L-reduced clones derived from TERT promoter mutant and wild-type lines at day 45 post-editing. *p value<0.05, **p value<0.01, two-sided Student’s t-test compared to CTRL. Values are mean ± S.D of at least three independent assays. (B) Volcano plot of expression differences between CTRL and β1L-reduced TERT promoter mutant lines (GBM1, T98G, and LN229) as determined via RNA-seq at day 45 post-editing. Maroon-colored points represent putative GABP-regulated genes that are differentially expressed (log2 Fold Change>1 & FDR<0.05). (C) GO-terms analysis of 161 genes that are commonly differentially expressed genes between CTRL and multiple β1L-reduced TERT promoter mutant lines. See also Tables S4 and S5.
Figure 6.
Figure 6.
β1L-reduced GBM lines accrue DNA damage and undergo mitotic cell death in a TERT promoter mutation-dependent manner. (A) Representative images (left images) and quantification (right graphs) of γ-H2AX staining in CTRL or β1L-reduced clones at day 70–75 post-editing. Scale bar = 200μm. **p value<0.01, two-sided Student’s t-test compared to CTRL. Quantification values are sums of at least ten independent fields of view. (B) Representative DAPI images (left images) and quantification (right graphs) of giant cell micronucleation (GCM) in CTRL or β1L-reduced clones at day 70–75 post-editing. Scale bar = 20 μm. *p value<0.05, **p value<0.01, two-sided Student’s t-test compared to CTRL. Quantification values are weighted mean ± S.D. of at least ten independent fields of view. (C,D) Histograms (C) and quantification (D) for cell cycle analysis of CTRL or β1L-reduced LN229 (top graphs) and NHAPC5 (bottom graphs) lines at day 75 post-editing. See also Figures S6–S7 and Table S6.
Figure 7.
Figure 7.
Reduction of β1L impairs tumor growth and extends mouse survival in vivo. (A) Representative IVIS bioluminescent images of CTRL or β1L-reduced LN229-derived tumors at 7 time points post-intracranial injection (injected on cellular day 51 post-editing). DPI = days post-injection. (B) Relative tumor bioluminescence quantified twice per week for each group (CTRL: n=12, C1: n=12, C2: n=10) until first recorded mortality. **p value<0.01, two-sided Student’s t-test compared to CTRL peak luminescence. Values are mean ± S.D of all mice in each group. (C) Kaplan-Meier survival curve displaying disease-specific survival of mice (Simonsen Labs, see STAR Methods) injected with LN229 CTRL or C1 and C2 β1L-reduced cells over time. **p value<0.01, log-rank test compared to CTRL. (D) TERT expression 4 days post-transduction of CTRL or β1L-reduced LN229 clones (41 days post-editing) with either a control (V) or TERT (T) lentiviral expression vector. **p value<0.01, two-sided Student’s t-test relative to respective vector (V) control. Values are mean ± S.D of three independent experiments. (E) Relative tumor bioluminescence quantified twice per week for each group (n=7 mice per group) following stable transduction with a control (V) or TERT (T) lentiviral expression vector. **p value<0.01, two-sided Student’s t-test compared to vector control peak luminescence for each respective line. Values are mean ± S.D of all mice in each group. (F) Kaplan-Meier survival curve displaying disease-specific survival of mice (Envigo, see STAR Methods) injected with LN229 CTRL or C1 and C2 β1L-reduced cells following stable transduction with a control (V) or TERT (T) lentiviral expression vector. **p value<0.01, log-rank test compared to CTRL.

Comment in

  • GABPβ1L Wakes Up TERT.
    Rahme GJ, Gaskell E, Bernstein BE. Rahme GJ, et al. Cancer Cell. 2018 Sep 10;34(3):358-360. doi: 10.1016/j.ccell.2018.08.011. Cancer Cell. 2018. PMID: 30205042

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