## 3 Results
### GABPA Knockdown Does Not Affect TERT Expression across Thyroid Cancer Cell Lines
To ensure accurate comparisons across cell lines with variable MAPK outputs, IC\({}_{50}\) values for metatinib and dabrafenib were first determined, and each cell line was treated at its IC\({}_{50}\) (Figure 4A). Cell lines were transduced with short-hairpin RNAs targeting the ETS factors--ETV1, ELK1, and GABPA--and cultured under puromycin selection media for at least two weeks prior to treatment to ensure sustained RNA knockdown (Figure 4A). Figure 3: Stable knockdown of ETS factor GABPA did not result in consistent changes to _TERT_ expression across multiple thyroid cancer cell lines. (**A**) Cell lines with wild-type _TERT_ promoter sequences. (**B**) Cell lines with heterozygous -124C\(>\)T _TERT_ promoter mutation. (**C**) Cell lines with homozygous -124C\(>\)T or -146 C\(>\)T _TERT_ promoter mutations and (**D**) U251 glioblastoma cell line, which is heterozygous for the -124C\(>\)T mutation. Across all cell lines, TERT expression was analyzed by RT-qPCR and then normalized to actin expression. Cal62, which acted as a non-_TERT_ promoter mutation control, and U251 glioblastoma cells demonstrated a significant reduction in _TERT_ expression. All other cell lines did not exhibit a significant reduction in _TERT_ expression. Cells were cultured for at least two weeks prior to RNA extraction to appraise the longer-term effects of GABPA knockdown on _TERT_ expression. All results are derived from at least triplicate experiments; ns = non-significant; * = _p\(<\)_0.05; ** = _p_\(<\)_0.005, *** = _p_\(<\)_0.001. After treatment at the IC\({}_{50}\) dose with the appropriate MAPK inhibitors, Cal62 and MDAT41 cells demonstrated a strong reduction in _TERT_ expression across all shRNA conditions. Comparatively, BHT101 and BCPAPs were refractory to this treatment and did not show significant reductions in _TERT_ expression at their IC\({}_{50}\) doses. Although this may point to a partial switch of _TERT_ regulation away from MAPK towards other transcription factors such as ETS factors in TPM-harboring cells, _TERT_ expression was not further reduced upon knockdown of either ETV1, ELK1, or GABPA in BHT101 or BCPAP cells. Functional knockdown of the MAPK pathway was confirmed after the short-term treatment, as shown by diminished phospho-ERK1/2 levels (Figure 4C). Overall, our experiments did not show cooperativity between ETS- and MAPK-mediated control of _TERT_ transcription but pointed to a shift in _TERT_ mutant promoter regulation. Figure 4: (**A**) The IC\({}_{50}\) value for Trametinib (Cal62, 0.96 nM; BHT101, 2.04 nM; and BCPAP, 0.82 nM) and Dabrafenib (BHT101, 5.55 nM; BCPAP, 13.56 nM) were first calculated by cell viability assay on parental cell lines. Knockdown cell lines were generated using shRNA targeting ETV1, ELK1, and GABPA. shRNA cell lines were treated at the IC\({}_{50}\) dose; then, protein and RNA were extracted to confirm pathway inhibition and to assess the effect on _TERT_ expression, respectively. Created with Biorender.com (**B**) RT-qPCR analysis demonstrated a significant reduction in _TERT_ expression across all Cal62 and MDAT41 ETS knockdown cell lines. BHT101 and BCPAP did not demonstrate a reduction in _TERT_ expression after treatment with Trametinib and Dabrafenib for six hours. (**C**) Western blotting confirmed functional inhibition of the MAPK pathway after treatment with Trametinib or Trametinib + Dabrafenib using phospho-ERK as a biomarker. Samples were run on one membrane per cell line, and BCPAP and BHT101 images were arranged for a consistent loading order. ns = non-significant; * = \(p\)\(<\) 0.05, *** = \(p\)\(<\) 0.001.