Cancer. The transcription factor GABP selectively binds and activates the mutant TERT promoter in cancer

Abstract

Reactivation of telomerase reverse transcriptase (TERT) expression enables cells to overcome replicative senescence and escape apoptosis, which are fundamental steps in the initiation of human cancer. Multiple cancer types, including up to 83% of glioblastomas (GBMs), harbor highly recurrent TERT promoter mutations of unknown function but specific to two nucleotide positions. We identified the functional consequence of these mutations in GBMs to be recruitment of the multimeric GA-binding protein (GABP) transcription factor specifically to the mutant promoter. Allelic recruitment of GABP is consistently observed across four cancer types, highlighting a shared mechanism underlying TERT reactivation. Tandem flanking native E26 transformation-specific motifs critically cooperate with these mutations to activate TERT, probably by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression in multiple cancers.

Figure 1

The de novo ETS motif is critical for mutant TERT promoter activity in GBM. (A) TERT promoter-luciferase reporter assays for wild-type, G228A, G250A, or targeted mutation sequences. * P <0.05, Student’s t-test compared to wild-type (WT) (B) TERT expression relative to siScramble (siScr) 72 hours post ETS factor siRNA knockdown. * P <0.05, Student’s t-test compared to siScr. The results are an average of at least 3 independent experiments. Values are mean ± sd.

Figure 2

GABPA selectively regulates and binds the mutant TERT promoter across multiple cancer types. (A) Wild-type, G228A, or G250A luciferase activity 72 hours post ETS siRNA knockdown in GBM1 cultured cells, scaled to WT-siScramble (siScr). The results are an average of at least 3 independent experiments. Values are mean ± sd * P <0.05, Student’s t-test compared to siScr. (B) Enrichment of mutant (CCGGAA) or wild-type (CCGGAG) hexamer sequences in ENCODE GABPA ChIP-seq peaks relative to flanking regions. (C) ENCODE GABPA ChIP-seq data at the proximal TERT promoter and around distal qPCR primers. Native ETS motifs and mutation positions are annotated by orange and black tick marks respectively. Inset shows allelic read coverage at G228A in HepG2 cells. (D) GABPA ChIP-qPCR for the TERT promoter and a nearby control locus in seven cancer cell lines. Values represent mean % input based on triplicate qPCR measurments. N=1 for each cell line. (E) Allelic variant frequency in GABPA (IP) or input control DNA.

Figure 3

G228A and G250A cooperate with the native ETS sites ETS-195 and ETS-200 and fall within spacing for GABP heterotetramer recruitment. (A) Distribution of motif separation in weak and strong GABP peaks. Vertical dotted lines denote periodicity of 10.5bp. Horizontal dashed line indicates the theoretical null distribution. (B) Native and de novo putative ETS-binding sites in the core TERT promoter. (C) Site-directed mutagenesis of the GABP heterotetramer motifs in the wild-type, G228A, G250A, or insertion TERT reporter constructs. Mutation of the ETS-195, ETS-294, or junction motif are indicated by ‘+’. The results are an average of at least 3 independent experiments. Values are mean ± sd * P <0.05, Student’s t-test. (D) Site-directed mutagenesis deleting between 2 to 16 base pairs at the G228A site. Deletions were tested for promoter activity in a G250A or G250A+G201T background. The sinusoidal fits were obtained by using the model a sin(2π(x − b)/10.5) + cx + d. The results are an average of at least 3 independent experiments. Values are mean ± sd.