The enabler of tumor cell immortality in TERT promoter mutant cancers

About This Grant

Replicative immortality is a hallmark of cancer that underpins tumor growth. Immortality is achieved by reactivating the Telomerase Reverse Transcriptase (TERT) gene. Without TERT, telomeres erode with each cell replication, limiting cellular lifespan. Heterozygous activating mutations in the TERT promoter (TERTp) are the most common non-coding mutations in cancer, but their activation mechanism is unresolved for most cancers. Understanding how TERTp mutations lead to reactivation and immortality is a central question in cancer research with major therapeutic implications. However, a cadre of regulators of the mutant TERTp has been proposed. The two hotspot mutations generate identical de novo E26 transformation specific (ETS) transcription factor binding motifs largely shared by the 28 members of the ETS family. We were the first to discover that one unique ETS factor, the multimeric GA-binding protein (GABP), activates the two hotspot mutations in TERTp mutant glioblastoma. In our preliminary data, we analyzed cell lines representing sixteen cancer types and six recurrent mutations and found that a B1L heterotetramer is responsible for activation in all cases. Surprisingly, TERT expression is maintained after B1L tetramer depletion. Further investigation revealed an underlying network of GABP transcriptional auto-suppression, the release from which drives the upregulation of the GABPB1S dimer or the alternative GABPB2 tetramer. We hypothesize that the B1L tetramer is the pan- cancer activator of the mutant TERT promoter, but it is replaceable exclusively by the upregulated B1S dimer or B2 tetramer. Based on this new regulatory model, we anticipate that targeting the common elements of the GABP complexes will reverse tumor cell immortality phenotypes and slow or eliminate further tumor growth. We devised three specific aims to evaluate our hypothesis and develop experimental tools to subvert immortality. We will use CRISPR knockouts and a biological proteolysis-targeting chimera designed to selectively degrade GABP. In TERTp mutant and wildtype cancer cells, we will use these targeting tools to resolve the temporal relationships between GABP targeting and tumor immortality phenotypes including TERT expression, telomere loss, and cell viability associated gene expression signatures at the bulk and single cell level (Aim 1). In Aim 2, we will discover vulnerabilities in domains shared among GABP complexes via in silico mutagenesis at an unprecedented scale with Artificial Intelligence tools AlphaFold and Struct-Evo, and validation experiments. This will allow us to refine and interpret the structure predictions of complete GABP complexes bound to the mutant TERTp at atomic resolution, to develop potent GABP degraders and potentially small molecule binders or inhibitors. In Aim 3, we will determine whether an inducible GABP-degrader can subvert immortality in vivo, including sensitization and rescue experiments. Targeting this core enabler of immortality could convert oncogenes into drivers of cell death and senescence. The results of our study will be applicable to a wide spectrum of adult and pediatric cancers. The AI-based mutagenesis and experimental validations may be applicable to many other cancer targets.