Matthias Fischer - A 2

The functional and therapeutic significance of activated telomerase in neuroblastoma

Summary

Our research focusses on the impact of activated telomerase in the etiology and progression of neuroblastoma. We aim to elucidate both the role of telomerase activation in tumor development, as well as its potential value as a specific therapeutic target. To that end, we utilize bioinformatics analyses of patient data, cell culture experiments and murine cancer models.

Introduction 

Neuroblastoma is a malignant pediatric tumor of the sympathetic nervous system. While half of the patients have adverse clinical courses despite intensive treatment, spontaneous regression occurs frequently in the remaining cases. The genetic etiology and pathogenesis of spontaneous regression and tumor progression in neuroblastoma have remained largely elusive to date.

We recently discovered that activation of telomere maintenance mechanisms is a hallmark of high-risk neuroblastoma (Peifer et al., Nature 2015, Fig. 1).

In the most aggressive tumors, telomerase is activated by genomic TERT rearrangements or MYCN amplification. In the remaining high-risk cases, telomeres are stabilized by the Alternative Lengthening of Telomeres pathway. By contrast, low-risk tumors lack telomere maintenance mechanisms, presumably explaining their inability to gain immortal proliferation capacity.

Based on these results, we investigated the anti-tumor effect of preclinical telomerase inhibitors in vitro and in vivo, and found that telomerase may represent a suitable molecular target in neuroblastoma cell lines bearing activated telomerase. (Fig. 2)

Aim 1: Elucidate the role of telomerase activation in the development of neuroblastoma in vivo.

In a first approach, we will examine the role of TERT activation in neuroblastoma initiation and progression (Fig. 3A). For this purpose, we will generate a conditional ROSA26-Tert-transgenic mouse model utilizing the Cre/loxP system. To direct Tert expression to cells of the sympathetic nervous system, which are supposed to be the progenitor cells of neuroblastoma, we will crossbreed R26-fl-mTERT mice with Dbh-Cre transgenic mice. In order to accurately model observations from the clinic, we will combine TERT-activation with genetic tumor mouse models like KRasG12D or ALKF1174L mice.

In a complementary approach, we will determine the relevance of telomerase activation in a MYCN-driven neuroblastoma model (Fig. 3B). To evaluate whether telomerase inactivation impairs initiation and growth of neuroblastoma, we will crossbreed Th-MYCN;Th-ALKF1174L double transgenic mice (Weiss et al., 1997; Berry et al., 2012) with Tert-/- mice (Farazi et al., 2006). We will compare spontaneous tumor formation and progression in these telomerase-deficient mice with that of the well established Th-MYCN;Th-ALKF1174L Tert-wildtype model.

Aim 2: Evaluate telomerase as a potential therapeutic target in neuroblastoma in vitro.

In a second set of analyses, we will determine the anti-tumor effect of the telomerase inhibitor imetelstat (Geron Corporation), which has already advanced to clinical trials. We will select cell lines reflecting the different types of high-risk neuroblastoma (Fig. 1). Cell viability will be determined upon imetelstat treatment using the CellTiter-Glo® reagent. Results will be validated by trypan blue exclusion assay in vitro, and by in vivo experiments using both mouse xenograft and transgenic models (see Aim 1). Finally, we will evaluate the efficacy of rational combinations of imetelstat with genotoxic and targeted drugs.

In an additional approach, we will modify neuroblastoma cell lines in a CRISPR/Cas9 guided assay (Andersson-Rolf et al., 2017) to generate Cre/Flp conditional Tert genetrap cell lines (Fig. 4). Cre recombination will result in Tert gene disruption and ZsGreen expression.

Perspectives

Our approach will provide suitable in vitro and in vivo model systems for specifically determining the role of telomerase in neuroblastoma. Results of our studies will thus contribute to understand the molecular mechanisms of the distinct clinical courses observed in this malignancy, to establish genetically guided risk estimation systems of neuroblastoma patients, and to assess the potential value of telomerase as a therapeutic target. Since telomerase is activated in the vast majority of cancers, our model systems may also represent valuable tools for analogous analyses in other malignancies.

Selected publications

Weiss WA, Aldape K, Mohapatra G, et al. (1997). Targeted expression of MYCN causes neuroblastoma in transgenic mice. EMBO J 16(11):2985-95.

Berry T, Luther W, Bhatnagar N, et al. (2012). The ALK(F1174L) mutation potentiates the oncogenic activity of MYCN in neuroblastoma. Cancer Cell 22(1):117-30.

Farazi PA, Glickman J, Horner J, et al. (2006). Cooperative interactions of p53 mutation, telomere dysfunction, and chronic liver damage in hepatocellular carcinoma progression. Cancer Res 66(9):4766-73.

Peifer M, Hertwig F, Roels F, et al. (2015). Telomerase activation by genomic rearrangements in high-risk neuroblastoma. Nature 526(7575):700-4.

Andersson-Rolf A, Mustata RC, Merenda A, et al. (2017). One-step generation of conditional and reversible gene knockouts. Nat Methods 14(3):287-289.


Prof. Dr. Matthias Fischer

Dept. of Children and Adolescent Medicine

Prof. Dr. Matthias Fischer

Principal Investigator A 2

matthias.fischer@uk-koeln.de

Work +49 221 478 6816

University Children's Hospital
Kerpener Str. 62
50937 Cologne

Publications - Matthias Fischer

Link to PubMed

Group Members

P. Justus Ackermann (PostDoc)
Yvonne Kahlert (technician)

Figure 1

CMMC Fischer
A model for neuroblastoma pathogenesis based on genomic alterations, telomere lengthening pathways, and clinical courses. HR, high-risk tumors; LR, low-risk tumors; TERT, TERT rearrangement; MNA, MYCN amplification; 2N/4N, near-diploid/-tetraploid karyotype, 3N; near-triploid karyotype.

Figure 2

CMMC Fischer
Anti-tumor effect of the telomerase inhibitor 6-thio-2’-deoxyguanosine on neuroblastoma cells in vitro (A) and in mouse xenograft models in vivo (B). TERT, cell lines with TERT rearrangement; MNA, cell lines with MYCN amplification (MNA); ALT, cell lines with Alternative Lengthening of Telomeres.

Figure 3

CMMC Fischer
Schematic overview of the experimental approaches for determining the role of telomerase activation in neuroblastoma initiation and progression.

Figure 4

CMMC Fischer
Strategy for CRISPR/Cas9 guided gene targeting of neuroblastoma cell lines.