## 4 Discussion
A separate study reported that ETV1, ETV4, and ETV5 preferentially bind _TERT_ in various thyroid cell lines carrying c.-124C\(>\)T mutation [11]. Taken together, our exhaustive screening of all 20 ETS proteins expressed in thyroid cells show that no unequivocal ETS factor is responsible for _TERT_ mutant control. We show that sustained knockdown of specific ETS over weeks, at most, moderately impacts _TERT_ levels. This is a key difference from other studies, which assessed _TERT_ knockdown transiently,not allowing for the likely compensatory mechanisms that other factors exert in our cells and probably in actual tumor specimens re-expressing telomerase. Another aspect of \(TERT\) promoter regulation that remains elusive is determining whether some of the reported non-canonical TPMs, which occur at very low frequencies (1-2% of cases) but also create de novo ETS sites, reactive telomerase expression through the same mechanisms operating in \(TERT\) c.-124C\(>\)T or c.-146C\(>\)T specimens [27; 28]. In this regard, it is reasonable to speculate that some of these rare TPMs might activate \(TERT\) to a different degree, while others could be passenger alterations resulting from an overdetailed dissection of this particular genomic _locus_. In any case, once the mechanisms of hotspot TPM-mediated expression of \(TERT\) are identified, it would be advisable to study these other non-coding lesions. Our work also explored the role of a MAPK pathway blockade via pharmacological inhibition by dabrafenib and/or trametinib in \(TERT\) transcription. We observed that, at low doses (low nanomolar range), MAPK inhibition was very effective at suppressing \(TERT\) mRNA levels in cells without TPMs but not in their \(TERT\)-mutant counterparts. Although we acknowledge the preliminary nature of this observation, our results point to a potential partial switch of \(TERT\) mutant regulation, which might have been missed by other works employing MAPK inhibitors in the micromolar range, which fully suppressed this pathway [16; 18]. Studying the cooperation between ETS- and MAPK-mediated regulation in \(TERT\) transcription is inherently difficult due to their roles in the absence of TPMs. This is because the \(TERT\) wildtype promoter has native ETS sites as well as binding motifs for Sp1 and c-Myc, both MAPK-activated factors that control \(TERT\) expression [20; 21; 22; 23]. In addition, MAPK signaling is able to activate discrete ETS factors either via transcription [29] or phosphorylation [30], which would feed into both wildtype and mutant \(TERT\) regulation. However, in the absence of unambiguous ETS proteins controlling \(TERT\) mutant expression, the TPM-specific role of MAPK input remains elusive. Beyond the regulation by the ETS/MAPK axis, \(TERT\) promoter has been shown to be subjected to other regulatory inputs, some of which might be modified by the presence of TPMs, whereas others likely operate as determinants of \(TERT\) baseline transcriptional repression. Although this is beyond the scope of this paper, the role of genomic insulator CTCF, which likely determines long-distance interactions [31] as well as the contribution of DNA methylation and allele-specific histone marks, which were recently characterized in thyroid cancer cell lines [18; 32; 33], are worth noting. Overall, these add to the idea of a tightly regulated, multi-faceted control of \(TERT\) transcription, which only becomes unchecked during cancer transformation. Incidentally, it is possible that some of the variability in ETS-binding is determined by specific co-factors and chromatin states that might respond to other signaling inputs. Other differences are likely attributable to the stable nature of the ETS silencing used here (vs. transient approaches in other studies), which likely allows cancer cells to enhance compensatory mechanisms.