Neuroblastoma is malignant pediatric tumor with diverse clinical courses, ranging from spontaneous regression to fatal progression. We discovered that activation of telomere maintenance mechanisms discriminate high-risk neuroblastoma from spontaneously regressing disease. We also found out that telomerase is an actionable target in a large fraction of high-risk tumors. Our findings establish a novel mechanistic classification of neuroblastoma that may benefit the clinical management of patients.
Neuroblastoma is a malignant pediatric tumor of the developing sympathetic nervous system. Clinical courses of neuroblastoma patients are remarkably heterogeneous: Spontaneous regression occurs in up to 50% of the patients, resulting in excellent outcome. By contrast, survival of the high-risk patients is still below 50%, despite intensive multimodal treatment. The molecular mechanisms driving the divergent phenotypes have remained largely unknown until recently. Early massively parallel sequencing studies revealed a low rate of somatic mutations in neuroblastoma in general, and few recurrently mutated genes. In a recent study, we discovered genomic rearrangements of the TERT locus in roughly 12% of primary tumors, encoding the catalytic subunit of the enzyme telomerase (Peifer et al., Nature 2015). We observed that TERT rearrangements caused massive induction of TERT expression and telomerase activity. TERT expression was also upregulated by MYCN amplification, which occurred in mutually exclusive fashion with TERT rearrangements, and both TERT and MYCN alterations were associated with poor patient outcome. These findings prompted us to investigate the pathogenetic and clinical role of telomere maintenance mechanisms and the potential clinical implications of dysregulated telomerase.
We integrated information on telomere maintenance mechanisms in a cohort of 208 primary human neuroblastomas with information on genomic alterations in genes of the RAS and the p53 pathway, which are recurrently mutated in neuroblastoma (Ackermann et al., 2018). We found that patients whose tumors lacked telomere maintenance mechanisms had an excellent prognosis, whereas the prognosis of patients whose tumors harbored telomere maintenance mechanisms was substantially worse. Survival rates were lowest for neuroblastoma patients whose tumors harbored telomere maintenance mechanisms in combination with RAS and/or p53 pathway mutations. By contrast, spontaneous tumor regression occurred both in the presence and absence of these mutations in patients with telomere maintenance-negative tumors. We also observed that patients whose tumors were telomerase-positive had worse outcome than those whose tumors were positive for the alternative lengthening of telomeres (ALT) pathway (Roderwieser et al., in revision). On the basis of these data, we propose a mechanistic classification of neuroblastoma that may benefit the clinical management of patients (Fig. 1).
Our data suggest that telomerase is playing a key role in the pathogenesis of a large fraction of high-risk tumors. To test this hypothesis, we set out to modulate telomerase activity in genetically engineered mouse models of neuroblastoma using two complementary approaches. First, we generated Rosa26LSL.Tert mice in collaboration with Dr. Thomas Wunderlich (MPI for Metabolism Research, Cologne) to examine whether telomerase activation is able to drive neuroblastoma development. Chimeras are currently bred to other transgenic mice to generate the final models, i.e., Rosa26LSL.Tert;Th-IRES-Cre and Rosa26LSL.Tert;Th-IRES-Cre;Th-ALKF1174L mice. Second, we generated Th-MYCN;Th-ALKF1174LTERT-/- mice to evaluate whether telomerase inactivation impairs initiation and growth of established murine neuroblastoma models. Mice bearing the correct genotype have recently been born and are currently being observed for neuroblastoma development.
We also evaluated growth-inhibitory effects of telomerase-interacting compounds in preclinical neuroblastoma models, both in vitro and in vivo (Roderwieser et al., in revision). We found that the nucleoside analogue 6-thio-2’-deoxyguanosine inhibited viability and proliferation of neuro-blastoma cells bearing activated telomerase, whereas ALT-positive neuroblastoma cells were significantly less affected. Similarly, tumor growth was strongly impaired upon 6-thio-2’-deoxyguanosine treatment in telomerase-positive neuroblastoma xenografts in mice (Fig. 2). We next examined whether 6-thio-2’-deoxyguanosine in combination with genotoxic or targeted drugs synergistically impair proliferation of telomerase-positive neuroblastoma cell lines in vitro (Fischer, Otte et al., in preparation). We observed strong synergistic effects across a panel of cell lines when 6-thio-2’-deoxyguanosine was combined with cytotoxic drugs that are currently being used in neuroblastoma treatment (etoposide, doxorubicin). This finding was validated in mouse xenografts, in which growth was inhibited much more effectively by the combination treatment than by single compounds. By contrast, 6-thio-2’-deoxyguanosine did not synergize with the ALK inhibitor ceritinib in ALK-mutated cell lines, arguing against the hypo-thesis that specific targeting of telomerase and constitutively active ALK represents a promising concept for neuroblastoma treatment.
Our data provide a strong rationale to evaluate the clinical value of telomere maintenance mechanisms for risk assessment of neuroblastoma patients and the therapeutic effect of 6-thio-2’-deoxyguanosine in prospective trials. In-depth evaluation of the genetically engineered mouse models generated in this project may contribute to elucidate the pathogenetic role of telomerase in neuroblastoma development and other human malignancies.
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Dr. Sandra Ackermann (PostDoc)
Dr. Justus Ackermann (PostDoc)
Dr. Christoph Bartenhagen (PostDoc)
Dr. Janina Fischer (PostDoc)
Dr. Andrea Roderwieser (PostDoc)
Dr. Carolina Rosswog (PostDoc)
Yvonne Kahlert (technician)
Anne Welte (technician)
Frederik Sand (student)
Esther Walter (student)
Felix Otte (student)