Telomerase upregulation induces progression of mouse BrafV600E-driven thyroid cancers and triggers non-telomeric effects

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 cancer, including thyroid tumors. 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 cancers at week 20, respectively. Tert-upregulated tumors showed increased mitosis and necrosis in areas of solid growth, and older animals 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. 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. Implications: Telomerase-driven cancer progression activates pathways that can be dissected and perhaps therapeutically exploited. Total RNA was isolated from up to 20,000 YFP+ cells sorted into TRIzol (range= 9,000-20,000 cells). Phase separation in cells lysed in TRIzol reagent was achieved with chloroform. RNA was precipitated with isopropanol, washed with 75% ethanol, and resuspended in RNase-free water following the manufacturer’s protocol. RNAseq was performed at the Dana-Farber Cancer Institute Molecular Biology Core Facility. RNA libraries were prepared following a low input mRNAseq protocol. RNA samples were fragmented at 94°C for 8 min with 14 cycles of PCR post-adapter ligation, according to manufacturer’s recommendation. The finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 2200. Libraries were pooled in equimolar ratios. Final sequencing was performed on an Illumina NovaSeq with paired-end 100 bp reads.