Since c-MYC was previously shown to activate TERT transcription by direct binding to TERT promoter30, we investigated whether MYC played a role in mediating BRAF V600E-regulated TERT expression. Inhibition of BRAF V600E by PLX4032 suppressed MYC expression, associated with decreased TERT expression in all the cells harboring BRAF mutation (Fig. 3a), suggesting that MYC indeed played a role, albeit moderately, in BRAF V600E-regulated TERT expression. MYC knockdown inhibited TERT expression to a similar moderate level as PLX4032 did in RKO and SK-MEL-3 cells which harbored wild-type TERT promoter (Fig. 3a). PLX4032 could further inhibit TERT expression after siRNA knockdown of MYC in cells harboring TERT promoter mutations, but not in cells harboring wild-type TERT promoter (Fig. 3a). These data suggest that the TERT promoter mutation-dependent activation of the TERT promoter by the BRAF V600E/MAPK pathway was a more effective mechanism for the regulation of TERT than that by the MYC component and the latter was TERT promoter mutation-independent. BRAF V600E knock-in in wild-type TERT promoter cells increased MYC expression, which was associated with an increase in TERT expression; the latter increase was abolished by siRNA knockdown of MYC (Fig. 3b). These results provided further evidence that the TERT promoter mutation-independent component in the regulation of TERT by the BRAF V600E/MAPK pathway was MYC-mediated. We further showed that BRAF V600E knock-in in cells resulted in increased TERT promoter activities in a far more robust manner in the promoter harboring the C228T or C250T mutation than the wild-type TERT promoter (Fig. 3c). Mutant TERT promoter activities were still significantly increased by BRAF V600E knock-in in the presence of MYC knockdown, but the wild-type TERT promoter activity was not increased by BRAF V600E knock-in in the presence of MYC knockdown (Fig. 3c). These results again suggested that the MYC component in the regulation of TERT by the BRAF V600E/MAPK pathway was TERT promoter mutation-independent and was minor while the TERT promoter mutation-dependent component was dominant.Fig. 3MYC-regulated TERT expression in a TERT promoter mutation-independent manner. a The four indicated cancer cell lines harboring BRAF V600E mutation were treated with 0.5 μm PLX4032 and/or 10 nm MYC-specific siRNA (siMYC) for 24 h, which were then subjected to western blotting (upper panel) and qRT-PCR (lower panel). The relative TERT mRNA expression was normalized to the control group. b, c Parental and BRAF V600E knock-in SW48 cells were treated with control siRNA (siControl) or MYC-specific siRNA (siMYC, 10 nm) for 48 h, followed by western blotting (b) and luciferase reporter assays (c). *P < 0.05, **P < 0.01, ***P < 0.001, by two-tailed Student’s t test. n.s. not significant. All the values represent the average ± standard deviation (SD) of triplicate samples from a typical experiment. Similar results were obtained in two additional independent experiments