Rearrangements of the TERT locus were shown to occur in a region upstream of TERT termed the breakpoint region (Peifer, 2015; Valentijn, 2015). In a cohort of neuroblastoma patients, the corresponding breakpoint was identified to occur not in the gene body or the promotor of TERT, but in a region of about 50 kb upstream of TERT transcriptional start site (TSS) (Peifer, 2015).
To obtain the individual profile of the GI-ME-N cell line and to identify the DNA breakpoint and rearrangement partner of the TERT region, whole genome sequencing (WGS) was performed. Cells were harvested and each 10 x106 cells were snap frozen in three replicates.
Sample preparation and Nanopore sequencing were performed by Rocío Chamorro González and data analysis was performed by Kerstin Haase and Konstantin Helmsauer at the Experimental and Clinical Research Center (ECRC), Berlin.
Figure 48: Low-coverage long-read sequencing localizes a candidate TERT-chr19 rearrangement.
A, Two of three long reads overlapping the TERT promotor indicate a rearrangement of the TERT promotor and a region in chr19q13.43. Read A (red) aligns to the TERT locus with an alignment block of length 1992 bp and mapping quality 60 and to chr19q13.43 with an alignment block of length 9625 bp and mapping quality 60. Read B (green) aligns to the TERT locus with an alignment block of length 488 bp and mapping quality 60 and to chr19q13.43 with an alignment block of length 2208 bp and mapping quality 0. B, Alignments of reads A and B to the TERT locus and chr19q13.43 both localize a breakpoint within the first 2-15 bases of the TERT 5’-untranslated region and the other breakpoint within a TAR1 repeat in chr19q13.43 with limited sequence homology near the breakpoints. Whole-genome sequencing reads aligned to hg38 genome and visualized with the Integrative Genomics Viewer (IGV). TAR1: telomere associated repeat 1; hg38: human genome 38. From K. Haase/K. Helmsauer.
Using low-coverage long-read Nanopore sequencing, the DNA breakpoint in GI-ME-N cells was located within the 5’-untranslated region (UTR) of TERT, fusing TERT to a subtelomeric locus on chromosome 19, truncating the native gene promotor from its gene (Figure 48 A). The analysis of the DNA breakpoint was sustained by re-analysis of published targeted DNA sequencing data of GI-ME-N cells (Peifer, 2015). The breakpoint for the hg38 annotation was identified as follows:
chr 19 …58607050, 58607051, 58607052 -breakpoint- 1295066, 1295065, 1295064, … chr 5 Published circular chromosome conformation capture sequencing (4C-seq) and FISH data suggest chromosome 19 as the TERT rearrangement partner (Gartlgruber, 2018). Low-coverage sequencing revealed two long reads, spanning the breakpoint in the repetitive area (Figure 48 B). Sequencing reveals evidence for a chr5-chr19 junction, the likely rearrangement partner was chr19:58607052(+) which joins the TERT promotor chr5:1295066 (-).
6.2 Modulation of TERT levels in models of high-risk neuroblastoma
6.2.1 TERT expression is not decreased by BET inhibitor treatment
TERT is a downstream target of MYCN and TERT levels are high in MYCN-amplified neuroblastoma, whereas MYCN is expressed at comparatively low levels in TERT-rearranged GI-ME-N and CLB-GA cell lines (Peifer, 2015; Huang, 2020; Zhao, 2014). Bromodomain and extra-terminal motif (BET) inhibitors indirectly reduce expression of MYCN and MYCN target genes such as TERT, and are promising preclinical candidates for treating MYCN-amplified neuroblastoma (Henssen, 2016). To investigate if BET inhibitors also reduce TERT expression in TERT-rearranged neuroblastoma cells, GI-ME-N and CLB-GA cells were treated with BET inhibitors JQ1, OTX015 or I-BET762 for 48 h. Additionally, a concentration series of panobinostat was included for treatment.
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Figure 49: TERT mRNA expression is reduced by panobinostat treatment but not by BET inhibitor treatment.
A, GI-ME-N and (B) CLB-GA cells were treated for 48 h with JQ1 (0.25, 0.5, 1 µM), OTX015 (0.1, 0.5, 1 µM), I-BET762 (0.25, 0.5, 1 µM) or panobinostat (5, 10, 15, 30 nM). TERT expression was analyzed by qRT-PCR (mean fold change over solvent ± SD; n≥3). Not significance if not indicated differently. Dotted lines indicate control value. *P≤0.05, **P≤0.01.
Due to the strong antitumoral effect of panobinostat, CLB-GA cells could not be treated with 30 nM panobinostat for 48 h. Indirect targeting of MYCN using BET inhibitors JQ1, OTX015 and I-BET762 had no influence on TERT mRNA expression in GI-ME-N (Figure 49 A) and CLB-GA cells (Figure 49 B). Panobinostat reduced TERT mRNA expression in a concentration-dependent manner in GI-ME-N cells (Figure 49 A), whereas TERT levels in CLB-GA cells showed a 2-fold decrease (Figure 49 B). Concentration of 30 nM panobinostat resulted in reduced TERT expression to 21% in GI-ME-N cells and of 15 nM panobinostat to 58% in CLB-GA cells.
6.2.2 TERT expression is increased and telomerase activity is induced after enforced TERT expression
To estimate the efficacy of the TERT plasmid after transfection, TERT expression was analyzed by qRT-PCR. GI-ME-N and CLB-GA cells were transfected with the TERT plasmid or empty vector and cells were harvested after 24-120 h.
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Figure 50: TERT mRNA expression is increased after transfection with TERT plasmid.
GI-ME-N and CLB-GA cells were transfected with either empty vector or TERT plasmid and harvested 24-120 h after transfection. TERT mRNA expression in (A) GI-ME-N and (B) CLB-GA cells. TERT expression was analyzed by qRT-PCR (mean fold change over solvent ± SD; n≥1).
The TERT plasmid increases TERT mRNA expression to a maximum of 356-fold 48 h after transfection in GI-ME-N cells (Figure 50 A) and to 969-fold after 48 h in CLB-GA cells (Figure 50 B).
Subsequently, telomerase activity was quantified by the telomerase activity assay. GI-ME-N and CLB-GA cells were transfected with the TERT plasmid or empty vector and cells were harvested after 24-120 h.
Figure 51: Telomerase activity is induced after transfection with TERT plasmid.
GI-ME-N and CLB-GA cells were transfected with either empty vector or TERT plasmid and harvested 24-120 h after transfection. Telomerase activity in (A) GI-ME-N and (B) CLB-GA cells. Telomerase activity was measured by ELISA (mean % over solvent ± SD; n≥1).
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Telomerase activity increased to a maximum of 153% after 24 h in transfected GI-ME-N cells (Figure 51 A) and to 163% after 72 h in CLB-GA cells (Figure 51 B).
6.2.3 Panobinostat treatment reduces TERT protein levels
Treatment with panobinostat results in decreased TERT mRNA levels and reduced telomerase activity in TERT-rearranged cell lines. To investigate the effect of panobinostat on TERT at protein level, western blot analysis was performed. GI-ME-N cells were treated for 72 h with solvent or panobinostat or cells were transfected with a TERT overexpressing plasmid or empty vector and treated with solvent (DMSO) or panobinostat for 72 h.
Figure 52: Panobinostat treatment reduces TERT protein levels.
Representative western blot analysis of GI-ME-N cells 72 h after panobinostat (5, 15, 30 nM) treatment (lanes 1-4). GI-ME-N cells were transfected with either empty vector or TERT plasmid and treated with panobinostat (5 nM) for 72 h (lanes 5-8). GAPDH served as a loading control.
Panobinostat treatment resulted in reduction of TERT in a concentration dependent manner (Figure 52). Enforced expression of TERT resulted in high TERT protein levels, that were not decreased by panobinostat treatment. This data suggests that not only TERT mRNA levels but also TERT protein levels decrease after panobinostat treatment, and that enforced expression of TERT remains high TERT protein levels after panobinostat treatment.
GAPDH TERTplasmid
- - - - - + - +
- 5 15 30 - - + + Panobinostat [nM]
TERT