Targeting the mutant promoter of Telomerase Reverse Transcriptase (TERT) - PROJECT SUMMARY
TElomerase Reverse Transcriptase (TERT) is rate limiting in maintenance of telomere length by telomerase and
also has telomere independent functions such as regulating cell growth. We showed TERT promoter mutations
(mutTERT) occur in ~70% of Bladder Cancers (BC), most commonly at -146bp and −124bp which generate an
identical 11 bp sequence that is recognized by a common upstream signaling mechanism. These mutations drive
TERT overexpression, maintain BC growth and are associated with poor BC patient prognosis. These data led
us to develop a panel of innovative assays specifically designed to identify, through a chemical library screening
approach, small molecules that reduce TERT expression from mutTERT but not wtTERT. Discovery of such
compounds is a first step in attaining our overall goal of developing a drug that is selectively toxic for BC cells,
while having minimal toxicity on normal stem cells, which require TERT for self-renewal. Preliminary Data:
Using CRISPR, we constructed BC cells expressing HiBiT or EGFP reporters downstream of mutant or wt TERT
promoters. We validated these assays by showing that siRNA-mediated depletion of GABPA, a regulator of
mutant promoter expression, selectively reduced mutTERT as monitored by HiBiT or EGFP. We deployed our
mutTERT-HiBiT assay in an High-Throughput Screen (HTS) pilot screen of 605 kinase inhibitors and found 23
hits that reduced mutTERT-HiBiT. Among these were inhibitors to known drivers of TERT expression such as
Aurora Kinase A, as well as inhibitors that were mutTERT selective, all of which targeted mTOR. This data leads
us to the Hypothesis that small molecules that specifically suppress TERT expression driven by a mutant
promoter can be identified by a phenotypic screen. Three Specific Aims test this hypothesis. In Aim 1, an HTS
screen of a 350K chemical library will be conducted using a mutTERT-HiBiT reporter assay to identify
compounds that decrease TERT expression driven off the mutant promoter at 16hrs. Hits will then be confirmed,
and counter screened to remove those with unwanted activities. In Aim 2, priority hits will be purchased,
reconfirmed in the primary assay and their selectivity to effect mutant over wt TERT evaluated head to head
deploying EGFP reporter assays in BC cells and by allele-specific qPCR. An iterative analog-by-catalog (ABC)
approach will be used to establish nascent structure-activity relationship of hit scaffolds and improve potency
and selectivity. In Aim 3, the best hit from each scaffold that meet a set of rigorous potency and selectivity criteria
will be selected as a probe and characterized in multiple assays to map their activity on downstream effects of
TERT. These assays use a panel of BC and non-BC cell lines with and w/o TERT promoter mutations to
determine the probes’ effects on: 1) downstream TERT-related gene transcription via evaluation of our TERT
Expression Signature (TES); 2) telomere length quantitation; 3) Telomerase-dependent and independent TERT
functions including cell growth. Future Directions: We set the stage for novel drugs targeting cancer specific,
mutTERT driven, telomerase activity in patients with BC or other TERT-driven malignancies.
