Telomerase activation by genomic rearrangements in high-risk neuroblastoma

Authors

Peifer M

Hertwig F

Roels F

Dreidax D

Gartlgruber M

Menon R

Krämer A

Roncaioli JL

Sand F

Heuckmann JM

Westermann F

Thomas RK

Fischer M

Doi

10.1038/nature14980

PMID: 26466568 · DOI: 10.1038/nature14980 · Journal: Nature (2015)

TL;DR

Peifer et al. performed whole-genome sequencing of 56 neuroblastomas (39 high-risk, 17 low-risk) and identified recurrent genomic rearrangements at chromosome 5p15.33, just upstream of the TERT transcriptional start site, in 12/39 (31%) of high-risk tumours. In an extended cohort of 217 patients, these TERT rearrangements were mutually exclusive with MYCN amplifications and ATRX mutations, defined a high-risk subgroup with particularly poor outcome, and produced massive transcriptional upregulation of TERT by juxtaposing the gene to strong enhancer elements (super-enhancers). The authors propose a unifying model in which high-risk neuroblastoma is molecularly defined by telomere lengthening — via TERT rearrangement, MYCN amplification, or ALT (alternative lengthening of telomeres) downstream of ATRX mutation.

Cohort & data

Key findings

  • Recurrent 5p15.33 rearrangements upstream of TERT. Breakpoint cluster analysis across 56 WGS tumours identified four recurrent breakpoint clusters; one new cluster lay at chromosome 5p15.33, ~50 kb upstream of the TERT transcriptional start site, without directly affecting the gene body or core promoter, in 12/56 (21%) of tumours PMID:26466568.
  • High-risk specificity. TERT rearrangements occurred only in high-risk tumours: 12/39 (31%) of high-risk vs 0/17 low-risk WGS cases (P = 0.01) PMID:26466568.
  • Mutual exclusivity. TERT rearrangements, ATRX alterations, and MYCN amplifications occurred in a mutually exclusive fashion within the high-risk WGS group (P = 0.008) PMID:26466568.
  • Extended-cohort prevalence. 28/217 (13%) of the full cohort and 27/114 (24%) of high-risk cases carried TERT rearrangements; among MYCN-non-amplified high-risk tumours the rate rose to 22/65 (34%) PMID:26466568.
  • Structural diversity. Rearrangements included balanced translocations, translocations with single-copy gain, focal high-level amplifications, and chromothripsis affecting chromosome 5 (n = 2). Partner regions were scattered across chromosome 5 (7 cases) and other chromosomes (5 cases) PMID:26466568.
  • Clinical impact. TERT-rearranged patients had overall survival similar to MYCN-amplified patients and significantly worse than high-risk patients with neither alteration (OS P = 0.056; event-free survival P = 0.038). In multivariable analyses, TERT rearrangement predicted unfavourable outcome independently of stage and MYCN PMID:26466568.
  • TERT mutations absent. No mutations were detected in the TERT gene body or its core promoter (in contrast with adult cancers carrying recurrent C228T/C250T promoter SNVs) PMID:26466568.
  • Low overall mutation rate. Average 13.3 non-synonymous mutations per genome; low-risk 5.9 ± 5.5 vs high-risk 16.6 ± 9.9 mutations (P < 0.001). Only three recurrently altered, expressed genes appeared in >2 samples: MYCN (n = 10), ALK (n = 7), and ATRX (n = 7) PMID:26466568.
  • TERT mRNA upregulation. TERT expression in rearranged tumours was significantly higher than in MYCN-amplified tumours (P = 0.028) and than in tumours with neither alteration (P < 0.001); median 92-fold higher than low-risk tumours PMID:26466568.
  • Mechanism: enhancer hijacking, not copy gain. TERT copy number and mRNA did not correlate among rearranged tumours; rearranged alleles were mono-allelically expressed in five evaluable tumours. ChIP-seq showed that rearrangements juxtapose TERT to strong enhancer clusters (compatible with super-enhancer definition) marked by H3K27ac and H3K4me1 PMID:26466568.
  • Massive chromatin remodelling. In rearranged cases, active marks H3K4me3, H3K27ac, and H3K36me3 spread across the TERT locus, while the repressive H3K27me3 mark (broad in unrearranged tumours) was lost. CpG methylation increased across the TERT locus in both rearranged and MYCN-amplified tumours, strongest at a CpG near the TERT core promoter previously linked to disabled repression PMID:26466568.
  • Neighbouring genes co-activated. SLC6A18 and SLC6A19 (distal of the breakpoint) were markedly upregulated only in TERT-rearranged tumours, while CLPTM1L (proximal) was not, suggesting directional spread of active chromatin from the translocated enhancers PMID:26466568.
  • MYCN activates TERT transcriptionally. TERT was the most strongly downregulated gene upon siRNA-mediated MYCN knockdown in MYCN-amplified IMR5/75 cells, identifying MYCN as a direct transcriptional activator of TERT PMID:26466568.
  • Functional telomerase activity. Cell lines with TERT translocations or MYCN amplification showed elevated TERT mRNA and significantly increased enzymatic telomerase activity (TRAP assay) versus cell lines with neither alteration PMID:26466568.
  • ALT as the third route to telomere maintenance. ATRX-mutant tumours and high-risk tumours lacking TERT/MYCN alterations carried abundant telomere repeat sequences and ALT pathway markers (e.g., CHLA-90 cell line); telomeres were short in telomerase-activated primary tumours. Low-risk tumours showed telomere lengths similar to matched normals PMID:26466568.

Genes & alterations

  • TERT — recurrent 5p15.33 structural rearrangements ~50 kb upstream of the transcriptional start site in 12/39 (31%) high-risk WGS cases and 28/217 (13%) of the full extended cohort. Rearrangements bring strong enhancer/super-enhancer elements into proximity with TERT, causing massive transcriptional upregulation, mono-allelic expression of the rearranged allele, and enzymatic telomerase activation. No coding or promoter point mutations were observed PMID:26466568.
  • MYCN — focal amplification in 10/56 (~18%) of the WGS cohort, mutually exclusive with TERT rearrangements and ATRX mutations in high-risk neuroblastoma. MYCN amplification produces transcriptional activation of TERT (TERT is the top hit upon MYCN knockdown in IMR5/75) and is functionally interchangeable with TERT rearrangement as a route to telomerase activation PMID:26466568.
  • ATRX — inactivating mutations in 7/56 WGS cases, exclusively in tumours lacking MYCN amplification and TERT rearrangement. ATRX-mutant tumours show alternative lengthening of telomeres (ALT) — long telomere repeats without telomerase activation — as the third axis of telomere maintenance in high-risk neuroblastoma PMID:26466568.
  • ALK — mutations in 7/56 WGS cases, distributed across both high-risk and low-risk groups (i.e., not specific to high-risk biology, in contrast with TERT/MYCN/ATRX) PMID:26466568.
  • SLC6A18, SLC6A19 — distal neighbours of TERT that become markedly upregulated only in TERT-rearranged tumours, reflecting directional spread of the hijacked enhancer chromatin landscape PMID:26466568.
  • CLPTM1L — proximal-side neighbour of TERT that, in contrast to SLC6A18/SLC6A19, is not upregulated in TERT-rearranged tumours; used as the FISH probe target (BAC CTD-2191M2) to call rearrangements in the validation cohort PMID:26466568.

Clinical implications

  • Prognostic stratification. TERT rearrangement identifies a high-risk neuroblastoma subgroup with outcome comparable to MYCN-amplified disease and significantly worse than other high-risk patients, and the prognostic signal is independent of INSS stage and MYCN status in multivariable analysis PMID:26466568.
  • Diagnostic assay. Rearrangements are detectable by FISH (CTD-2191M2 / CTD-2511M20 BAC probes flanking TERT) plus hybrid-capture targeted sequencing across the TERT/CLPTM1L locus; the authors required both methods to call a rearrangement, making this approach deployable in routine pathology labs PMID:26466568.
  • Unifying biological model. Combining MYCN amplification, TERT rearrangement, and ATRX mutation/ALT covers the majority of high-risk neuroblastomas. The authors propose that high-risk neuroblastoma is molecularly defined by telomere lengthening, opening the door to telomerase inhibitors (TERT-rearranged and MYCN-amplified tumours) and ALT-targeted strategies (ATRX-mutant tumours) as biology-matched therapeutics PMID:26466568.
  • Stability across disease course. Identical rearrangements and elevated TERT expression were detected in matched diagnosis–relapse biopsies of four patients, supporting use of the marker for risk stratification at initial diagnosis PMID:26466568.

Limitations & open questions

  • Cohort source. The 217-patient extended cohort is single-country (Germany) and recruited through one trial network (GPOH), so generalisability of frequency estimates to other geographies/ethnicities is unverified PMID:26466568.
  • Borderline survival significance. The headline overall-survival comparison reaches P = 0.056 (not <0.05); the event-free survival result (P = 0.038) is the stronger of the two, so larger cohorts will be needed to firmly anchor the prognostic claim PMID:26466568.
  • No prospective validation. The validation cohort is retrospective, with no blinding during outcome assessment (“The investigators were not blinded to allocation during experiments and outcome assessment.”) PMID:26466568.
  • Mechanism of enhancer selection. Why rearrangement partners are biased to the 50 kb window immediately upstream of TERT — and what selects which enhancer landscapes “win” once the breakpoint is established — is not resolved.
  • Therapeutic translation. Telomerase inhibitors are proposed as a candidate therapeutic axis but no in vivo or clinical data are provided in this paper; ALT-directed therapies for the ATRX-mutant subset are likewise speculative.
  • Distinct mutational drivers in MYCN-non-amplified high-risk disease. Among the 65 MYCN-non-amplified high-risk tumours, 22 carry TERT rearrangements and a smaller number carry ATRX mutations; the remaining cases lack any of the three telomere-maintenance lesions described here, leaving open what defines their high-risk biology.

Citations from this paper used in the wiki

  • “we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT).” (Abstract)
  • “These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations” (Abstract)
  • “In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome.” (Abstract)
  • “27 of 114 high-risk neuroblastomas (24%) and 22 of 65 MYCN-non-amplified high-risk tumours (34%) harboured TERT rearrangements” (Results, p. 3)
  • “TERT expression was significantly higher in neuroblastomas with rearrangements than in MYCN-amplified tumours (P = 0.028) and in those without these alterations (P < 0.001)” (Results, p. 3)
  • “The median TERT expression was 92-fold higher in TERT-rearranged tumours than in low-risk tumours” (Results, p. 3)
  • “rearrangements consistently juxtapose TERT to strong enhancer elements, several of which are compatible with the recent definition of super-enhancers” (Results, p. 4)
  • “TERT was the most strongly downregulated gene upon short-interfering-RNA-mediated MYCN knockdown in a MYCN-amplified neuroblastoma cell line” (Results, p. 4)
  • “All high-throughput data have been deposited at the European Genome-phenome Archive (https://www.ebi.ac.uk/ega/) under accession number EGAS00001001308.” (Front matter)

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