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Mutations in the promoter of the human Telomerase Reverse Transcriptase (hTERT) gene are common and associated with its elevated expression in bladder cancer, melanoma, and glioblastoma. Though these mutations and TERT overexpression are associated with aggressive disease and poor outcome, an incomplete understanding of mutant TERT regulation limits treatment options directed at this gene. Herein, we unravel a signaling pathway that leads to upregulated hTERT expression resulting from the −124 bp promoter mutation, the most frequent variant across human cancer. We employed engineered bladder cancer cells that harbor a GFP insertion at the TSS region on −124 hTERT promoter for high-content screening drug discovery using a focused library of ~800 kinase inhibitors. Studies using in vitro and in vivo models prioritized AST-487, an inhibitor of the wild-type, and mutant RET (rearranged during transfection) proto-oncogene as a novel drug inhibitor of both wild-type and mutant promoter-driven hTERT expression. We also identified the RET kinase pathway, targeted by AST-487, as a novel regulator of mutant hTERT promoter-driven transcription in bladder cancer cells. Collectively, our work provides new potential precision medicine approaches for cancer patients with upregulated hTERT expression, perhaps, especially those harboring mutations in both the RET gene and the hTERT promoter, such as in thyroid cancer. [ABSTRACT FROM AUTHOR]

Molecular subtypes of muscle-invasive bladder cancer (MIBC) display differential survival and drug sensitivities in clinical trials. To date, they have not been used as a paradigm for phenotypic drug discovery. This study aimed to discover novel subtype-stratified therapy approaches based on high-content screening (HCS) drug discovery. Transcriptome expression data of CCLE and BLA-40 cell lines were used for molecular subtype assignment in basal, luminal, and mesenchymal-like cell lines. Two independent HCSs, using focused compound libraries, were conducted to identify subtype-specific drug leads. We correlated lead drug sensitivity data with functional genomics, regulon analysis, and in-vitro drug response-based enrichment analysis. The basal MIBC subtype displayed sensitivity to HDAC and CHK inhibitors, while the luminal subtype was sensitive to MDM2 inhibitors. The mesenchymal-like cell lines were exclusively sensitive to the ITGAV inhibitor SB273005. The role of integrins within this mesenchymal-like MIBC subtype was confirmed via its regulon activity and gene essentiality based on CRISPR–Cas9 knock-out data. Patients with high ITGAV expression showed a significant decrease in the median overall survival. Phenotypic high-content drug screens based on bladder cancer cell lines provide rationales for novel stratified therapeutic approaches as a framework for further prospective validation in clinical trials. [ABSTRACT FROM AUTHOR]

Bladder cancer (BC) has a 70% telomerase reverse transcriptase (TERT or hTERT in humans) promoter mutation prevalence, commonly at -124 base pairs, and this is associated with increased hTERT expression and poor patient prognosis. We inserted a green fluorescent protein (GFP) tag in the mutant hTERT promoter allele to create BC cells expressing an hTERT-GFP fusion protein. These cells were used in a fluorescence-activated cell sorting-based pooled CRISPR-Cas9 Kinome knockout genetic screen to identify tripartite motif containing 28 (TRIM28) and TRIM24 as regulators of hTERT expression. TRIM28 activates, while TRIM24 suppresses, hTERT transcription from the mutated promoter allele. TRIM28 is recruited to the mutant promoter where it interacts with TRIM24, which inhibits its activity. Phosphorylation of TRIM28 through the mTOR complex 1 (mTORC1) releases it from TRIM24 and induces hTERT transcription. TRIM28 expression promotes in vitro and in vivo BC cell growth and stratifies BC patient outcome. mTORC1 inhibition with rapamycin analog Ridaforolimus suppresses TRIM28 phosphorylation, hTERT expression, and cell viability. This study may lead to hTERT-directed cancer therapies with reduced effects on normal progenitor cells. [ABSTRACT FROM AUTHOR]