Telomerase reactivation induces progression of mouse Braf V600E -driven thyroid cancers without telomere lengthening

Abstract

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 Braf ^V600E -mutant mice. We also employed an alternative model of Tert overexpression (K5-Tert). Whereas all Braf ^V600E animals developed well-differentiated papillary thyroid tumors, 29% and 36% of Braf ^V600E +Tert ^-123C>T and Braf ^V600E +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.

Figure 1.. Modeling of Tert reactivation in genetically engineered mouse models with endogenous Braf^V600E expression.

A. Schematic representation of the sequence alignment of human TERT (top) and mouse Tert promoter (bottom) sequences. A single C>T transition at the conserved human c.−124C and mouse c.−123C loci generates a consensus binding motif for Ets transcription factors (“Ets cons”); B. Tert promoter-driven luciferase expression, normalized by renilla (“Luc/Ren”), in mouse cell lines using Tert wildtype (mTert −123C, green) and mutant (mTert −123T, red) constructs; C. Genetic schema of the mouse models: Top: Thyroid-specific expression of Cre recombinase (driven by thyroid peroxidase (TPO) in TPO-Cre constructs) substitutes exon 15 of WT Braf by V600E mutant allele, resulting in endogenous expression of Braf oncoprotein (66). Cre-mediated excision of stop cassette also enables YFP (“yellow fluorescent protein”) expression in thyroid cells; Bottom: Tert^−123C>T is knocked-in in the germline via CRISPR/Cas9 editing of mouse zygotes, as described (left); mouse Tert is driven by the keratin 5 (K5) promoter (middle) (17); breeding of LsL-Braf, TPO-Cre/eYFP and Tert alleles generates the three genotype combinations used in this study (right).

Figure 2.. Histological analysis of the Braf+Tert mouse models.

Representative H&E-stained thyroid sections of Braf^V600E, Braf^V600E+Tert^−123C>T and Braf^V600E+K5-Tert mice at A. 6–12 weeks; and B. 20–30 weeks. C. Percentage of animals with the indicated genotypes showing papillary (PTC) or poorly differentiated thyroid cancers (PDTC) at 6–12 weeks (left) and 20–30 weeks (right). Abbreviations: “Braf+Tert” and “Braf+K5-Tert” labels denote “Braf^V600E+Tert^−123C>T” and “Braf^V600E+keratin 5-driven Tert^” genotypes, respectively.

Figure 3.. Characteristics of Tert re-expression in telomerase-reactivated thyroid cancers.

A. Relative levels of Tert transcription in YFP-sorted cells isolated from Braf^V600E+Tert^−123C>T mouse tumors, showing an increase in Tert mRNA levels for specimens collected at 20 weeks vs. 10 weeks. Each red point represents a tumor collected from a different animal at the indicated age. Results are expressed as fold change compared to the Tert baseline expression of Braf^V600E tumors from the same ages (dotted blue line). B. Representative examples of tumors from 20-week animals with the indicated genotypes subjected to RNAscope to detect Tert single mRNA molecules (green dots). DAPI (blue) is used for contrast. C. Quantification of number of Tert transcripts at single-cell resolution from RNAscope data on mouse tumors with the indicated genotypes. Data is represented as percentage of cells within each tumor expressing 1, 2, 3, 4, or 5+ transcripts. Tumor boundaries were defined manually and cell detection and quantification of green fluorescent dots was performed employing the built-in automated analysis tools on QuPath, using the same detection parameters across specimens. D. Relative mRNA levels in cell lines derived from Braf^V600E (n=4), Braf^V600E+Tert^−123C>T (n=5) and from Braf^V600E+K5-Tert (n=3) mouse tumors, showing that genotype-dependent increases in Tert transcription are maintained in vitro. E. Median telomere length, expressed in relative units, of cell lines derived from mouse thyroid tumors with the indicated genotypes. F. Median telomere length, expressed in relative units, of YFP-sorted cells isolated from mouse thyroid tumors with the indicated genotypes. Abbreviations: “Braf+Tert” and “Braf+K5-Tert” labels denote “Braf^V600E+Tert^−123C>T” and “Braf^V600E+keratin 5-driven Tert^” genotypes, respectively.

Figure 4.. Transcriptomic characterization of telomerase-reactivated mouse thyroid tumors.

A. Schematic representation of the isolation of thyroid tumor cells for RNA sequencing (RNAseq). B. Comparison of RNAseq normalized counts for Tert in pooled thyroid glands with the indicated genotypes. Values represent average expression levels for pooled WT (n=25), Tert^−123C>T (n=17) and K5-Tert (n=14) animals. C. Comparison of RNAseq normalized counts for Tert in thyroid tumors from Braf^V600E (n=4), Braf^V600E+Tert^−123C>T (n=7) and Braf^V600E+K5-Tert (n=4) animals. Each dot represents an individual mouse. D. Volcano plot showing significantly under- and overexpressed genes in Braf^V600E+K5-Tert tumors compared to Braf^V600E. Specific genes are indicated in red. E. Top 10 upregulated terms from the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database analysis for the Braf^V600E vs. Braf^V600E+K5-Tert comparison. F. Top 10 upregulated terms from the GSEA (Gene Set Enrichment Analysis) hallmarks of cancer analysis for the Braf^V600E vs. Braf^V600E+K5-Tert comparison.

Figure 5.. Validation of telomerase-reactivated pathways and consequences for treatment.

A. Western blot using protein extracts from thyroid tumors from 20-week-old animals with the indicated genotypes for phospho-NFkB p65 and vinculin (VCL, loading control). B. Representative images from phospho-Erk (pErk) immunohistochemistry performed in mouse tumors from 20-week-old animals with the indicated genotypes. C. Quantification of the percentage of tumor cells staining positive for pErk in mice with the indicated genotypes. D. Western blot using protein extracts from thyroid tumors from 20-week-old animals with the indicated genotypes for phospho- and total Erk (MAPK pathway) and Akt (PI3K effector), as well as p85 (loading control). E. Schematic representation of the in vivo treatment of mouse models. Twenty-four 20-week animals from the Braf^V600E, Braf^V600E+Tert^−123C>T and Braf^V600E+K5-Tert groups were randomly assigned and treated with either vehicle or dabrafenib plus tramentinib for 12 days. Thyroid volume was assessed by ultrasound on days 1 and 12. All mice were sacrificed on day 12, their thyroids were harvested, and each thyroid lobe was processed for either hematoxylin & eosin (H&E) staining and immunohistochemistry (IHC) or digested and preserved in Trizol for RNA extraction and subsequent real-time quantitative PCR (qPCR) assays. F. Thyroid tumor volumes, measured by ultrasound, in animals with the indicated genotypes and treatment groups. Results are expressed as the delta of normalized thyroid volumes between day 12 (last day of the experiment) and day 1 (first day). Abbreviations: “Braf+Tert” and “Braf+K5-Tert” labels denote “Braf^V600E+Tert^−123C>T” and “Braf^V600E+keratin 5-driven Tert^” genotypes, respectively; WT= wildtype; YFP= yellow fluorescent protein; D+T= Dabrafenib plus Trametinib.