Background

Medulloblastoma (MB) is the most common malignant pediatric brain tumor, and can be stratified into four major subgroups^1,2. One of these subgroups, Group 3, is often characterized by MYC amplification and arises in the presence of a functional ARF/p53 suppressor pathway³. Group 3 tumors are considered very high risk (<50% survival) and primarily affect young children and infants⁴. In this publication, we set out to generate and validate a transgenic model of MYC-driven Group 3 MB (the GMYC model), and then additionally explore the intricate differences between this new model and our previously characterized MYCN-driven model⁵.

Key Findings

The GMYC model - The GMYC model is an immunocompetent, transgenic mouse model with regulatable MYC, where tumors develop in the brain spontaneously around 6 months of age. These tumors bore molecular and genetic resemblance to photoreceptor-positive Group 3 MB in patients. We show how this model system, and cell lines derived from this model, can be used to study mechanisms of tumorigenesis in addition to pharmaceutical studies.

CDKN2A/p53 suppression- Inactivation or loss of the upstream p53 regulator gene, CDKN2A, which encodes the tumor suppressor protein, ARF, is rarely seen in medulloblastoma, but rather more commonly found in pediatric high grade glioma (pHGG). In this study, we show that suppression or transient silencing of the CDKN2A gene appeared to be a driver for initial tumorigenesis in the GMYC model, and was also observed in Group 3 patients; underlying the ability for tumor cells to impact the functionality of p53 without p53-specific mutational removal. Similarly, complete ARF loss in the GMYC model led to a significant increase in leptomeningeal dissemination, suggesting its importance in metastatic spread - an event common at patient diagnosis. Early ARF loss also promoted a higher incidence of pHGGs in both MYC- and MYCN-driven mouse models, which is in line with the clinical and genetic nature underlying these tumor entities.

Confetti and clonality - Utilization of the Confetti fate-tracing system revealed that GMYC tumors arise from a dominant, monoclonal cell population – putatively indicating that acquisition of secondary genetic events, such as CDKN2A suppression, occur in a small subpopulation of cells where their survival and expansion is facilitated by such events.

Treatment options - Currently, there are no targeted drugs that can restore CDKN2A expression, but the use of global demethylation agents, such as 5-Azacytidine, are well tolerated in patients and have been used in treatments for other morbidities^6,7. While 5-Azacytidine treatment was not successful in our animal models, we show that ARF regulation and restoration is indeed possible.

HSP90 activation is known to influence and facilitate DNMT1-driven methylation and was found to be enriched in our MYC-driven model compared to the MYCN-driven model. HSP90 inhibition, through the use of the HSP90 inhibitor, Onalespib, targeted aggressive MYC-driven tumors, but not MYCN-driven tumors, in an ARF-dependent manner. In vitro HSP90 inhibition elicited an ARF-restorative response with subsequent tumor cell death in GMYC cells. Similar results were seen in vivo when tumor-bearing mice were treated with Onalespib – survival time was significantly extended when the treatment was given as a monotherapy, even more so than irradiation treatment. Onalespib therapy failed in tumors where ARF had been knocked out, suggesting a link between HSP90 and ARF, where successful HSP90 inhibitory treatment in brain cancer cells requires presence of functional ARF. These treatment outcomes were mirrored in patient cell lines, supporting the hypothesis that minor expression of CDKN2A, along with MYC-expression, is a requirement for a more sensitive response to HSP90 inhibition, at least when compared to MYCN-driven tumors.

Given that the HSP90 inhibitor, Onalespib, has radiosensitizing properties and can cross the Blood Brain Barrier⁸, it holds exciting potential for a thorough evaluation into its tumor suppressive effects alongside standard irradiation in MYC-driven Group 3 patients, where efficacious treatment options remain rare.

Take Home Message

What began as the initiative to develop a patient-accurate transgenic mouse model of Group 3 MB quickly evolved into a significant undertaking exploring the mechanistic differences underlying MYC-driven and MYCN-driven brain tumors.

The GMYC model is a valuable tool for studying the mechanisms behind MYC-driven brain cancer, as well as evaluating treatment options both in vitro and in vivo. It is an easily modifiable model that can be changed and adapted based on the research question and hypothesis, and one that we hope many other research groups will continue to expand upon. The GMYC model and cells derived from this model are available to research groups worldwide and we are excited to see what findings stem from this.

We also highlight the importance of ARF suppression driving these tumors and the necessity to evaluate how this can be leveraged in MYC-driven patient tumors, where treatments are suboptimal and patient survival is low.

References

1. Ostrom, Q. T. et al. CBTRUS Statistical Report: Primary brain and other central nervous system tumors diagnosed in the United States in 2010-2014. Neuro Oncol 19, v1-v88 (2017).1022 https://doi.org:10.1093/neuonc/nox1581023
2. Hovestadt, V. et al. Medulloblastomics revisited: biological and clinical insights from thousands of patients. Nat Rev Cancer 20, 42-56 (2020). https://doi.org:10.1038/s41568-019-0223-8
3. Northcott, P. A. et al. The whole-genome landscape of medulloblastoma subtypes. Nature 547, 311-317 (2017). https://doi.org:10.1038/nature22973
4. Ramaswamy, V. et al. Risk stratification of childhood medulloblastoma in the molecular era: the current consensus. Acta Neuropathol 131, 821-831 (2016). https://doi.org:10.1007/s00401-016-1569-6
5. Swartling, F. J. et al. Pleiotropic role for MYCN in medulloblastoma. Genes & development 24, 1059-1072 (2010). https://doi.org:10.1101/gad.1907510
6. Issa, J. P. et al. Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in hematopoietic malignancies. Blood 103, 1635-1640 (2004).1159 https://doi.org:10.1182/blood-2003-03-06871160
7. George, R. E. et al. Phase I study of decitabine with doxorubicin and cyclophosphamide in children with neuroblastoma and other solid tumors: a Children's Oncology Group study. Pediatr Blood Cancer 55, 629-1162 638 (2010). https://doi.org:10.1002/pbc.22607
8. Spiegelberg, D. et al. The novel HSP90 inhibitor AT13387 potentiates radiation effects in squamous cell carcinoma and adenocarcinoma cells. Oncotarget 6, 35652-35666 (2015) https://doi.org:10.18632/oncotarget.5363