## 3 Results
### GABPA Knockdown Does Not Affect TERT Expression across Thyroid Cancer Cell Lines  
_Carrying TPMs_  
Given the prior body of evidence that pointed towards GABPA being a key regulator of mutant _TERT_ promoter in glioblastoma, the effect of GABPA knockdown on _TERT_ expression was further tested across a comprehensive range of thyroid cell lines harboring wild-type _TERT_ promoter sequences and heterozygous or homozygous TPMs. In this analysis, the U251 glioblastoma cell line was also included as a positive control. GABPA was silenced using short-hairpin RNAs and cells were cultured for at least two weeks in selection media to observe the effect of sustained knockdown. Overall, GABPA knockdown did not show a consistent genotype-dependent effect on _TERT_ expression across cell lines. The wild-type _TERT_ promoter cell line Cal62 demonstrated a significant decrease in _TERT_ expression upon GABPA knockdown against the paired scrambled shRNA control, whereas MDAT41 and CUTC5, also wild-type for TPMs, did not (Figure 3A). In comparison, the cell lines that are heterozygous for a _TERT_ promoter mutation did not show a significant reduction in _TERT_ expression (Figure 3B). In TCO-1 and Hth7 cells, which are homozygous for the _TERT_ promoter mutations -124C>T and -146C>T, respectively, and which should theoretically facilitate GABPA binding, showed no effects on _TERT_ expression upon GABPA knockdown (Figure 3C). This was not a result of poor knockdown efficiency as TCO-1 and Hth7 demonstrated sustained and consistent reduction of GABPA expressions (average decreases of 61.5% and 46.8%, respectively; Figure 3C). In contrast, continued GABPA knockdown in U251 glioblastoma cells confirmed the previously shown significant reduction in _TERT_ expression (Figure 3D). Taken together, these data caution against GABPA being a universal regulator of the mutant _TERT_ promoter in thyroid cancer cells and suggest that targeting GABPA over a sustained period is unlikely to result in a consistent reduction in _TERT_ expression in this tumor type. \begin{table}
\begin{tabular}{c c c c c} \hline
**Cell Line** & **Tissue Derivation** & **MAPK Driver** & _TERT Promoter_ & **Use** \\ \hline TCO1 & Thyroid, ATC & BRAF V600E & c-124C>T, HOM & 1 st screen, 20 ETS \\ \hline SW1736 & Thyroid, ATC & BRAF V600E & c-124C>T, HET & 1 st screen, 20 ETS \\ \hline C643 & Thyroid, ATC & HRAS G13R & c-124C>T, HET & 1 st screen, 20 ETS \\ \hline Cal62 & Thyroid, ATC & KRAS G12R & Wild type & 1 st screen, 20 ETS + MAPKi experiments \\ \hline BCPAP & Thyroid, PTC & BRAF V600E & c-124C>T, HET & 2 nd screen, 10 ETS + MAPKi experiments \\ \hline BHT101 & Thyroid, ATC & BRAF V600E & c-124C>T, HET & 2 nd screen, 10 ETS + MAPKi experiments \\ \hline T238 & Thyroid, ATC & BRAF V600E & c-124C>T, HET & 2 nd screen, 10 ETS \\ \hline MDAT41 & Thyroid, PTC & BRAF V600E & Wild type & Extended screen, GABPA + MAPKi experiments \\ \hline CUTC5 & Thyroid, PTC & BRAF V600E & Wild type & Extended screen, GABPA \\ \hline K1 & Thyroid, PTC & BRAF V600E & c-124C>T, HET & Extended screen, GABPA \\ \hline Hth7 & Thyroid, ATC & NRAS Q61L & c-146C>T, HOM & Extended screen, GABPA \\ \hline U251 & Glioblastoma & Unknown & c-124C>T, HOM & Extended screen, GABPA \\ \hline \end{tabular}
\end{table} 
Table 1: Name, genetic background, and other features of the cancer cell lines used in this study. Inhibiting MAPK Signaling Primarily Affects Expression of TERT in Cells with Wild-Type TERT Promoter  
Considering the known role of MAPK signaling in regulating wild-type _TERT_ expression, we sought to explore the effect of MAPK blockade in thyroid cancer cells with or without TPMs and to assess whether it cooperates with ETS to control _TERT_ transcription. To this end, we treated cells with FDA-approved dabrafenib (RAF inhibitor) and/or tetrantinb (MEK inhibitor), depending on their driver mutation (BRAFV600E or oncogenic RAS).