www.nature.com/npjprecisiononcology ARTICLE OPEN Synergistic activation of mutant TERT promoter by Sp1 and GABPA in BRAFV600E-driven human cancers ✉ ✉ Yongxing Wu1,2, Liang Shi1, Yuelei Zhao1, Pu Chen1, Rongrong Cui1, Meiju Ji3, Nongyue He4, Maode Wang5 , Gang Li6 and ✉ Peng Hou1 The activating TERT promoter mutations and BRAFV600E mutation are well-established oncogenic alterations in human cancers. Coexistence of BRAFV600E and TERT promoter mutations is frequently found in multiple cancer types, and is strongly associated with poor patient prognosis. Although the BRAFV600E-elicited activation of ERK has been demonstrated to contribute to TERT reactivation by maintaining an active chromatin state, it still remains to be addressed how activated ERK is selectively recruited to mutant TERT promoter. Here, we report that transcription factor GABPA mediates the regulation of BRAFV600E/MAPK signaling on TERT reactivation by selectively recruiting activated ERK to mutant TERT promoter, where activated ERK can phosphorylate Sp1, thereby resulting in HDAC1 dissociation and an active chromatin state. Meanwhile, phosphorylated Sp1 further enhances the binding of GABPA to mutant TERT promoter. Taken together, our data indicate that GABPA and Sp1 synergistically activate mutant TERT promoter, contributing to tumorigenesis and cancer progression, particularly in the BRAFV600E-driven human cancers. Thus, our findings identify a direct mechanism that bridges two frequent oncogenic alterations together in TERT reactivation. npj Precision Oncology (2021) 5:3 ; https://doi.org/10.1038/s41698-020-00140-5 1234567890():,; INTRODUCTION promoter exhibited active chromatin marks, while its wild-type 9 Telomeres are special DNA-protein structures located at both ends allele remained an inactive chromatin state , indicating that these of eukaryotic chromosomes, protecting genomic material and mutations initiated an epigenetic switch and the mono-allelic sheltering chromosome ends from DNA damage response expression of TERT. There is also a study reporting that binding of machinery1. Since telomeres shorten with every cell division, GABPA to mutant TERT promoter can mediate the long-range telomerase re-expression is required for maintaining stable chromatin interactions, facilitating the acquisition of an active telomeres and permissive for the indefinite cell growth in most chromatin state of TERT promoter and enhancing TERT transcrip- advanced malignancies2,3. Telomerase reverse transcriptase (TERT) tion10. Collectively, these studies have highlighted the importance encodes the catalytic subunit of telomerase, and TERT reactivation of TERT promoter mutations as a gate-keeper for TERT reactivation is a key step in telomerase re-expression. Recently, there are many and tumor progression. V600E studies showing that TERT potentiates the oncogenesis not only BRAF mutation is another frequent genetic alteration that by catalyzing the elongation of telomeres, which is the primary drives tumorigenesis and tumor progression, particularly in 11,12 function of telomerase, but also by conferring proliferation thyroid cancers and melanomas . This mutation results in advantages through directly regulating MYC stability and tRNA constitutively activating BRAF kinase, contributing to phosphor- transcription4,5, suggesting its critical role in tumorigenesis and ylation of the downstream mitogen-activated protein kinases 13 tumor progression. (MAPK) . The abnormal activation of BRAF signals regulates Since TERT reactivation has played a pivotal role in maintaining various pathways and has a vital role in tumorigenesis, and recent the telomeres and thereby supporting infinite cell division in studies demonstrated that inhibitors targeting BRAF pathway cancer cells, many researchers have sought to determine the showed promising clinical activities14,15. Co-occurrence of genetic basis for TERT reactivation in different types of cancer. It BRAFV600E and TERT promoter mutations has been found in should be noted that TERT promoter mutations are frequently 7.1–9.8% of papillary thyroid cancers (PTCs) and 13.6–20.7% found in multiple cancer types and predict poor patient melanomas16,17, which is closely correlated with worse prognosis prognosis6,7. In particular, two hotspot somatic mutations and more aggressive pathological characteristics, suggesting a precisely located at positions −124 or −146 bp upstream of the cooperative role between BRAFV600E signaling and TERT promoter TERT transcription start site (−124 C > T and −146 C > T, also mutations in cancer initiation and progression. A recent study has known as C228T and C250T) were documented each to create a demonstrated that GABPB1L, a catalytic subunit of GABP, de novo ETS binding site (EBS), and enhanced transcriptional positively regulates TERT expression in a TERT promoter activity of TERT promoter7. Further studies demonstrated that mutation-dependent manner18. Moreover, there is evidence these hotspot mutations selectively recruited transcription factor indicating that the BRAFV600E/MAPK cascade enhances GABPB GABPA to promote TERT transcription8. In addition, mutant TERT transcription by phosphorylating transcription factor Fos, thereby 1Key Laboratory for Tumor Precision Medicine of Shaanxi Province and Department of Endocrinology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061 Xi’an, People’s Republic of China. 2Department of Critical Care Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, 710061 Xi’an, People’s Republic of China. 3Center for Translational Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, 710061 Xi’an, People’s Republic of China. 4State Key Laboratory of Bioelectronics, Southeast University, 210096 Nanjing, People’s Republic of China. 5Department of Neurosurgery, The First Affiliated Hospital of Xi’an Jiaotong University, 710061 Xi’an, People’s Republic of China. 6Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, 710038 Xi’an, People’s Republic of China. ✉ email: [email protected]; [email protected]; [email protected] Published in partnership with The Hormel Institute, University of Minnesota YWuetal. 2 activating mutant TERT promoter through forming a hetero- abolish this effect, indicating that de novo ETS binding site is tetrameric complex with GAPBA19. Another study has also required for this novel regulatory interaction20. Taken together, revealed that the constitutively activated BRAFV600E/ERK signal our data indicate that the GABP tetramer is required for the can selectively regulate an active chromatin state on mutant TERT regulation of mutant TERT promoter by BRAFV600E/MAPK/ERK promoter by phosphorylating Sp1 and facilitating the dissociation signaling. of HDAC120. These observations indicate a direct link between V600E BRAF and TERT promoter mutations in activating TERT ERK regulates the binding of GABPA to mutant TERT promoter transcription. However, questions have been raised on how via Sp1 activated ERK selectively binds to mutant TERT promoter, while Next, we attempted to determine whether BRAFV600E-mediated not its wild-type allele. MAPK/ERK signaling can affect the binding capacity of GABP to In this study, we attempt to fill this gap by testing the hypothesis mutant TERT promoter. To address this, we treated A375, 8305C, that activated ERK may be recruited to mutant TERT promoter by BCPAP, and MDA-MB-231 cells with siRNA targeting BRAF (si-BRAF) pioneer transcription factors. We demonstrate that transcription or 100 nM MEK inhibitor trametinib for 6 or 24 h to block the ERK factor GABPA is required for recruiting activated ERK to mutant cascade. The results showed that treatment with si-BRAF or TERT promoter, thereby resulting in Sp1 phosphorylation, the trametinib markedly inhibited ERK phosphorylation, while almost consequent dissociation of HDAC1 and TERT activation. Our data did not change GABPA expression (Supplementary Fig. 1a, b). also show that activated ERK signaling can enhance the binding of However, the ChIP assay showed that inhibition of the BRAFV600E/ GABPA to mutant TERT promoter in an Sp1-dependent manner. ERK signaling by si-BRAF (Fig. 2a) or trametinib (Fig. 2b) could Altogether, our results reveal that GABPA bridges the BRAFV600E significantly reduce the binding of GABPA to mutant TERT and TERT promoter mutations together in TERT reactivation, and promoter in the indicated cells. demonstrate that GABPA and Sp1 synergistically transactivate There is evidence showing that both subunits of GABP can be mutant TERT promoter. phosphorylated by MAPK/ERK signaling; however, in vitro assay showed that DNA-binding capacity of GABP heterodimer or RESULTS heterotetramer was almost not affected by ERK-elicited phosphor- ylation24. Considering that the presence of GABPB and the Activated ERK is recruited to mutant TERT promoter by the consequent formation of GABPA/B heterodimer may stabilize GABP tetramer the GABPA–DNA interaction25, and the MAPK/ERK signaling 1234567890():,; Given that GABPA can selectively bind to de novo EBS created by upregulated GABPB expression via Fos phosphorylation19, thus C250T or C228T TERT promoter mutation8, and GABPA is a direct fi 21 we rst validated the effect of ERK cascade on GABPB expression. downstream target of MAPK/ERK signaling . Besides, there is Expectedly, using qRT-PCR assay, we found that treatment of fi evidence showing that ERK can be recruited to speci c target A375, 8305C, BCPAP, and MDA-MB-231 cells with 100 nM sequences and exert its