Abstract Activating mutations in the Telomerase Reverse Transcriptase (TERT) promoter are the single most common non-coding mutation in cancer and enable limitless cell division characteristic of immortal cells1–12. Solving the immortality mechanism represents a major step towards selectively reversing it in cancer cells. TERT promoter mutations create a de novo E26 transformation specific (ETS) transcription factor binding motif, however most of the 28 ETS factors and many other transcriptional regulators have been implicated13–22. Cancer type and mutation specific mechanisms have also been proposed. Here, we uniformly and robustly analyzed fifty-three cell lines representing sixteen cancer types and six recurrent mutations and found that a tetramer of the GA-binding protein (GABP) is specifically responsible for mutant TERT promoter activation in all cases, with no such role in TERT promoter wild type cells. Strikingly, TERT expression is maintained in tetramer depleted tumor cells. We show how and why the tetramer is serially replaced, not by other transcription factors, but by GABP dimers and then weakly by a paralogous tetramer complex. Elimination of the tetramer and dimer reinstates epigenetic repression of TERT, activates checkpoint programs and prevents cancer cell division. We conclude that unique features of the GABP tetramer must therefore determine positive selection of nearly all TERT promoter mutations in human cancer. Furthermore, domains shared among the three GABP complexes present pan-cancer vulnerabilities.

Abstract Activating mutations in the Telomerase Reverse Transcriptase ( TERT ) promoter are the single most common non-coding mutation in cancer and enable limitless cell division characteristic of immortal cells 1–12 . Solving the immortality mechanism represents a major step towards selectively reversing it in cancer cells. TERT promoter mutations create a de novo E26 transformation specific (ETS) transcription factor binding motif, however most of the 28 ETS factors and many other transcriptional regulators have been implicated 13–22 . Cancer type and mutation specific mechanisms have also been proposed. Here, we uniformly and robustly analyzed fifty-three cell lines representing sixteen cancer types and six recurrent mutations and found that a tetramer of the GA-binding protein (GABP) is specifically responsible for mutant TERT promoter activation in all cases, with no such role in TERT promoter wild type cells. Strikingly, TERT expression is maintained in tetramer depleted tumor cells. We show how and why the tetramer is serially replaced, not by other transcription factors, but by GABP dimers and then weakly by a paralogous tetramer complex. Elimination of the tetramer and dimer reinstates epigenetic repression of TERT , activates checkpoint programs and prevents cancer cell division. We conclude that unique features of the GABP tetramer must therefore determine positive selection of nearly all TERT promoter mutations in human cancer. Furthermore, domains shared among the three GABP complexes present pan-cancer vulnerabilities.

Abstract Telomerase Reverse Transcriptase promoter mutations enable tumor cell immortality in millions of cancer patients annually. While prior therapies targeting telomerase lacked tumor selectivity and were poorly tolerated, TERT promoter mutations and their regulation present a unique opportunity for tumor specific reversal of cellular immortality. The two hotspot mutations, G228A and G250A, generate identical de novo E26 transformation specific (ETS) transcription factor binding sites. However, the 28 ETS factors share a similar binding site preference, raising the question of whether all ETS factors can reactivate TERT. In glioblastoma and a few other cancers, we have shown the de novo ETS site along with a nearby native ETS site (ETS-195/200) recruit one ETS factor, the GA-binding protein (GABP) tetramer, to reactivate TERT. This GABP-mediated mechanism may be restricted to specific cancer types and the two hotspot mutations or could be widely relevant to any cancer with a de novo ETS TERT promoter mutation. We have found that the GABP tetramer activates 6 distinct mutant TERT promoters across 16 different cancer types. However, TERT expression is maintained in most tetramer depleted tumor cells as the tetramer is serially replaced by GABP dimers and weakly by a paralogous tetramer complex that increase following disengagement of GABP tetramer-mediated negative feedback loops. Elimination of both the tetramer and dimer reinstates epigenetic repression of TERT, shortens telomeres, and prevents cancer cell division. However, some knockout cancer cells avoid this fate through weak activation of TERT by the paralogous GABP tetramer complex. The compensatory maintenance of TERT expression can be overcome with a dominant negative GABPB1, leading to TERT silencing, evidenced by gene expression and non-invasive metabolic imaging correlates, and telomere shortening in TERT promoter mutant glioblastoma. We are currently investigating the delivery of this dominant negative to tumor cells via clinically approved retroviral replicating vectors.

Ninety percent of human tumors reactivate telomerase reverse transcriptase (TERT) to achieve cellular immortality. The route to immortality found in over 50 cancer types is mutation of the TERT promoter (TERTp) mutations. Studies in glioblastoma have shown the two most common TERTp mutations, G228A and G250A, reactivate TERT expression through de novo E26 transformation specific (ETS) site generation that, in tandem with a native TERTp ETS site, recruits the tetrameric GA-binding protein (GABP) complex formed by two GABP alpha (GABPA) subunits and two tetramer specific GABPB1 (B1) subunits, GABPB1L (B1L). While ten additional TERTp mutations have been described across cancer types, functional studies have been limited to select mutations and cancer types. We found that each de novo ETS motif generating TERTp mutation increases TERTp transcriptional activity in a GABP-dependent manner. Furthermore, our pan-cancer analysis demonstrated selective enrichment of GABPA at the mutant TERTp across 14 cancer types and further determined that this recruitment is necessary for TERT expression. Prior investigations found that CRISPR-cas9 mediated mutagenesis of the B1 ninth exon, specific to B1L, reduces TERT transcriptional activity in a TERTp-mutation dependent manner. We explored B1L targeting in the reversal of tumor cell immortality in several cancers. We found that the B1 dimer restricted isoform, GABPB1S (B1S), is consistently and significantly increased following B1L reduction, which we determined to be the consequence of a disengagement of a GABP tetramer mediated negative feedback loop acting on the B1 promoter. Interestingly, this upregulated B1S expression enables GABP dimer binding and activation of the mutant TERTp thereby compensating for B1L loss. Indeed, knockout of B1L and B1S together resulted in a near complete elimination of GABPA TERTp recruitment and a striking reduction in TERT expression, leading to telomere shortening and ultimately resulting in tumor cell death and senescence. This data identifies GABP as the master regulator of the mutant TERTp across cancer type and de novo ETS mutation type and suggests a new model of the GABP-TERT axis involving both the GABP tetramer, and the GABP dimer. Citation Format: Nicholas O. Stevers, Carter Barger, Olivia Lenzo, Chibo Hong, Katarzyna Soczek, Samuel H. Wu, Andrew M. McKinney, Abigail Suwala, Jennifer A. Doudna, Joseph F. Costello. The pan-cancer regulator of the mutant TERT promoter and a new model of the GABP-TERT axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2302.

Tumor cell immortality is a fundamental hallmark of human cancers. Normally silenced during somatic cell differentiation, 90% of human tumors reactivate Telomerase Reverse Transcriptase (TERT) expression to achieve cellular immortality. TERT, the catalytic subunit of telomerase, complexes with the RNA template molecule TERC to maintain telomeres. Mutations in the TERT promoter (TERTp) are the most common non-coding mutation across all cancer types and the most frequent mutation within many cancers, such as IDH wildtype glioblastoma (GBM), Melanoma, and Bladder Cancer. TERTp mutations generate de novo E26 Transformation Specific (ETS) binding motifs that are spaced full helical turns from TERTp native ETS sites. Together the de novo and native ETS motifs specifically recruit the GABP tetrameric complex but not the GABP dimer. CRISPR-cas9 mediated insertion/deletion mutagenesis of the unique exon of GABP tetramer forming subunit, GABPB1L (B1L), reduces TERT transcriptional activity in a TERT promoter-mutation dependent manner. Here we show that GABPB1S (B1S), the GABP dimer restricted alternative isoform of GABPB1, is consistently and significantly increased following B1L reduction, a process we have determined to be driven by a conserved homeostatic mechanism whereby the GABP tetramer suppresses expression of one of its own components, GABPB1. In contrast to the native setting, in the absence of B1L the elevated B1S expression leads to dimer binding to the mutant TERTp and maintenance of TERT expression. Indeed, co-targeting B1L and B1S together, but not B1L alone, via CRISPR-cas9 knockout resulted in a near complete elimination of GABP recruitment to the TERTp and TERT expression, and lead to tumor cell death and eventual senescence in a telomere length dependent manner. Together, this data suggests a new model of the TERT-GABP axis involving the tetramer and dimer and highlights a new and potentially more potent therapeutic strategy to eliminate TERT expression and reverse tumor cell immortality.