Hotspot mutations in the TERT (telomerase reverse transcriptase) gene are key determinants of thyroid cancer progression. TERT promoter mutations (TPM) create de novo consensus binding sites for the ETS (“E26 transformation specific”) family of transcription factors. In this study, we systematically knocked down each of the 20 ETS factors expressed in thyroid tumors and screened their effects on TERT expression in seven thyroid cancer cell lines with defined TPM status. We observed that, unlike in other TPM-carrying cancers such as glioblastomas, ETS factor GABPA does not unambiguously regulate transcription from the TERT mutant promoter in thyroid specimens. In fact, multiple members of the ETS family impact TERT expression, and they typically do so in a mutation-independent manner. In addition, we observe that partial inhibition of MAPK, a central pathway in thyroid cancer transformation, is more effective at suppressing TERT transcription in the absence of TPMs. Taken together, our results show a more complex scenario of TERT regulation in thyroid cancers compared with other lineages and suggest that compensatory mechanisms by ETS and other regulators likely exist and advocate for the need for a more comprehensive understanding of the mechanisms of TERT deregulation in thyroid tumors before eventually exploring TPM-specific therapeutic strategies.

Mutations in the promoter of the telomerase reverse transcriptase (TERT) gene are the paradigm of a cross-cancer alteration in a non-coding region. TERT promoter mutations (TPMs) are biomarkers of poor prognosis in several tumors, including thyroid cancers. TPMs enhance TERT transcription, which is otherwise silenced in adult tissues, thus reactivating a bona fide oncoprotein. To study TERT deregulation and its downstream consequences, we generated a Tert mutant promoter mouse model via CRISPR/Cas9 engineering of the murine equivalent locus (Tert-123C>T) and crossed it with thyroid-specific BrafV600E-mutant mice. We also employed an alternative model of Tert overexpression (K5-Tert). Whereas all BrafV600E animals developed well-differentiated papillary thyroid tumors, 29% and 36% of BrafV600E+Tert-123C>T and BrafV600E+K5-Tert mice progressed to poorly differentiated thyroid cancers at week 20, respectively. Braf+Tert tumors showed increased mitosis and necrosis in areas of solid growth, and older animals from these cohorts displayed anaplastic-like features, i.e., spindle cells and macrophage infiltration. Murine Tert promoter mutation increased Tert transcription in vitro and in vivo, but temporal and intra-tumoral heterogeneity was observed. RNA-sequencing of thyroid tumor cells showed that processes other than the canonical Tert-mediated telomere maintenance role operate in these specimens. Pathway analysis showed that MAPK and PI3K/AKT signaling, as well as processes not previously associated with this tumor etiology, involving cytokine and chemokine signaling, were overactivated. Braf+Tert animals remained responsive to MAPK pathway inhibitors. These models constitute useful pre-clinical tools to understand the cell-autonomous and microenvironment-related consequences of Tert-mediated progression in advanced thyroid cancers and other aggressive tumors carrying TPMs.

Hotspot mutations in the proximal promoter of the telomerase reverse transcriptase (TERT) gene are the first cross-cancer alterations lying in a gene regulatory region. TERT promoter mutations (TPMs) are enriched in advanced thyroid tumors and constitute markers of disease severity. TPMs enhance TERT transcription, which is otherwise silenced in adult tissues, thus reactivating a bone fide oncoprotein. To study TERT deregulation and its downstream consequences in a biologically accurate model, we generated a Tert-mutant promoter mouse model via CRISPR/Cas9 editing of the equivalent murine locus and crossed these animals with thyroid-specific BrafV600E-mutant mice. BrafV600E animals develop highly penetrant papillary thyroid tumors (PTC) by week 5, but do not progress. In contrast, BrafV600E+TertMUT animals showed an increased incidence of poorly differentiated thyroid cancers (PDTC) by 20 weeks (30% vs. 0% in BrafV600E; chi-squared P= 0.03), mimicking those exhibited by an alternative transgenic model of Tert overexpression (BrafV600E+K5-Tert; 36% PDTCs). Mouse Tert promoter mutation increased Tert transcription in vitro and in vivo, as reported in patients’ tumors carrying TPMs. Braf+Tert animals partially responded to MAPK pathway inhibition (dabrafenib plus trametinib), showing that MAPK signaling remains relevant in these specimens. Interestingly, RNA sequencing of Tert-reactivated murine thyroid tumors showed unique transcriptomic profiles (compared to BrafV600E alone), suggesting that downstream effects, other than the canonical Tert-mediated telomere maintenance, operate in cancers harboring TPMs. These cancer models of telomerase reactivation provide excellent pre-clinical settings to understand the regulatory mechanisms and biological underpinnings of TPM-induced progression of thyroid and other tumors, and to explore novel therapeutic strategies. Citation Format: Inigo Landa, Jingzhu Hao, Bin Xu, Joseph Giacalone, Zach Herbert, Maria A. Blasco, Jeffrey A. Knauf, Ronald Ghossein, James A. Fagin. Tert mutant promoter mouse model induces cancer progression in BrafV600E-driven thyroid tumors: A novel tool to understand the biology of telomerase-reactivated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 913.