Page 69 - Biennial Report 2018-20 Jun 2021

targeting hTERT promoter quadruplexes we will establish the second and third arms of the study,
developing a double whammy for telomerase repression. As of now, post synthesis of the
molecules, the cytotoxic effects of these molecules are being tested on normal cells and treated
HT1080 cells with 5uM, 10uM and 100uM of individual molecules with their respective solvent
controls. While repression in hTERT expression was observed, several other off target gene
expressions were also found to be altered. The target specificity of these ligands needs further
work.

DECODING TELOMERASE REACTIVATION IN CANCER - MOLECULAR
MECHANISMS OF G-QUADRUPLEX-MEDIATED TELOMERASE (hTERT) CONTROL

Aberrant telomerase reactivation in normal cells (otherwise known to be telomerase negative)
may confer immortality and trigger cancer. While clinically relevant point mutations on hTERT
promoter are associated with hTERT reactivation, there are also reports substantiating relevance
of G-quadruplexes (or G4 motifs) formed in tandem within hTERT core promoter in hTERT
reactivation. Based on mounting clinical data, it was proposed to study how the clinically noted
cancer-specific mutations affect G4 stability and recruitment of transcription factor(s) within the
telomerase promoter - and how this affects telomerase reactivation in cancer cells.

The study was initiated by stably cloning hTERT promoter upstream of Gaussia luciferase along
with their G-quaduplex mutant forms as controls using two approaches- lentiviral transduction
and CRISPR/Cas9. Simultaneously, there are several hTERT wild type promoter and mutant cell
lines (G-quadruplex mutant), in which testing the effect of alteration of various transcription
factors to narrow down to the important G-quadruplex binding epigenetic factors important in
telomerase regulation in both cancer and normal cells will be started.

hTERT core-promoter has been reported to have tandem G-quadruplexes. Therefore, it was
planned to focus on finding a G-quadruplex binding transcription factor for telomerase. More
importantly, two main G>A point mutations, that destabilize hTERT promoter G-quadruplex,
have been reported to be responsible for telomerase (hTERT) reactivation across several cancer
types, including >60% Glioblastomas and Melanomas. Taking cues from ChIP seq data of TRF2
from previous lab findings, it was realized that telomere repeat binding factor 2(TRF2), a
shelterin complex protein has occupancy on hTERT promoter. When it was tested using ChIP-
PCR, it was found that TRF2 occupancy was spread from +150 bp downstream to -750bp
upstream of transcription start site (TSS) on hTERT promoter. More interestingly, it was found
that TRF2 occupancy on hTERT promoter was lost in case of cell lines that carried hTERT
promoter mutations. It was tested in U87MG, LN229 (-124G>A hTERT promoter mutant,
Glioblastoma cell lines) along with T98G and BLM6 (-146G>A hTERT promoter mutant,
Glioblastoma and Melanoma cell lines, respectively). In all the three cell types, loss of TRF2
occupancy in G>A mutant cell lines was evident. Together this supported the potential role of
TRF2 in G-quadruplex-dependent hTERT regulation. This data gives promising leads towards the
probable role of TRF2 mediated G-quadruplex- dependent hTERT regulation.

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