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  • Medulloblastoma Growth is Halted by Inhibiting Jak2-mediated Phosphorylation of Atoh1

    Final Number:
    1123

    Authors:
    Akash J. Patel MD; Tiemo Klisch PhD; Anna Vainshtein PhD; Huda Y Zoghbi MD

    Study Design:
    Laboratory Investigation

    Subject Category:

    Meeting: Congress of Neurological Surgeons 2017 Annual Meeting

    Introduction: Treatment for medulloblastoma, the most common malignant primary brain tumor in children, remains limited to surgical resection, radiation, and traditional chemotherapy; the 10-year survival rate is less than 50%. Seeking to identify key factors that drive tumor development in order to establish better therapies, we found that the transcription factor Atonal homologue 1 (Atoh1) is required for the development of Sonic Hedgehog (SHH)-type medulloblastoma in mice.

    Methods: Using the Nd2::SmoA1 transgenic mice, harboring a constitutively active sonic hedgehog pathway, we explored how Atoh1 is involved in medulloblastoma development. We used immunoprecipitation-mass spectrometry for Atoh1 in the tumor initiating cells to identify phosphorylation events that occur in the tumors. The interaction of Jak2 and Atoh1 was confirmed using the bimolecular fluorescence complementation assay. Atoh1 phosphomimetic and phospho-dead mutants were cloned for studies and the Y78 phosphorylation site was investigated using inducible medulloblastoma cell lines. Proliferation was measured using an MTT proliferation assay. Xenograft transplants of inducible cell-lines of wild-type atoh1, phosphor-mimetic and phospho-dead atoh1 were used to compare tumor formation as it relates to this phosphorylation event.

    Results: Heterozygosity of Atoh1 reduced tumor occurrence and prolonged survival. More importantly, we discovered that tyrosine 78 of Atoh1 is phosphorylated by a novel Jak2-mediated pathway only in tumor- initiating cells and in human SHH-type medulloblastoma. We show that phosphorylation of this site stabilizes Atoh1 protein, increases its transcriptional activity, and is independent of the canonical Jak2 signaling cascades, whereas inhibiting Jak2 impairs tumor growth in vivo.

    Conclusions: We found that the transcription factor Atoh1 is required for the development of Sonic Hedgehog (SHH)-type medulloblastoma in mice. Atoh1 is a transcription factor critical in hindbrain development, that is no longer expressed post-natally, making it an ideal therapeutic target. We have identified a Jak2-mediated phosphorylation event that occurs only in medulloblastoma initiating cells and is necessary for tumor formation. Inhibition of Jak2-mediated Atoh1 phosphorylation could provide a viable therapy for medulloblastoma.

    Patient Care: We have identified a critical phosphorylation event that is required for the formation of medulloblastoma in mice. This provides an exciting therapeutic avenue for a deadly childhood brain tumor.

    Learning Objectives: Understand how a novel Jak2-Atoh1 signaling cascade promotes medulloblastoma formation

    References: 1. G. Dhall, Medulloblastoma. J. Child Neurol. 24, 1418–1430 (2009). 2. S. Pfister, C. Hartmann, A. Korshunov, Histology and molecular pathology of pediatric brain tumors. J. Child Neurol. 24, 1375–1386 (2009). 3. M. F. Roussel, G. Robinson, Medulloblastoma: advances and challenges. F1000 Biol Rep. 3, 5 (2011). 4. R. L. Yauch et al., Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma. Science. 326, 572–574 (2009). 5. C. M. Rudin et al., Treatment of medulloblastoma with hedgehog pathway inhibitor GDC-0449. N. Engl. J. Med. 361, 1173–1178 (2009). 6. N. Ben-Arie et al., Evolutionary conservation of sequence and expression of the bHLH protein Atonal suggests a conserved role in neurogenesis. Hum Mol Genet. 5, 1207–1216 (1996). 7. A. Flora, T. J. Klisch, G. Schuster, H. Y. Zoghbi, Deletion of Atoh1 disrupts Sonic Hedgehog signaling in the developing cerebellum and prevents medulloblastoma. Science. 326, 1424–1427 (2009). 8. B. A. Hatton et al., The Smo/Smo model: hedgehog-induced medulloblastoma with 90% incidence and leptomeningeal spread. Cancer Res. 68, 1768–1776 (2008). 9. A. Forget et al., Shh signaling protects Atoh1 from degradation mediated by the E3 ubiquitin ligase Huwe1 in neural precursors. Dev Cell. 29, 649–661 (2014). 10. T.-A. Read et al., Identification of CD15 as a marker for tumor-propagating cells in a mouse model of medulloblastoma. Cancer Cell. 15, 135–147 (2009). 11. R. C. Castellino et al., Overexpressed TP73 induces apoptosis in medulloblastoma. BMC Cancer. 7, 127 (2007). 12. A. O. von Bueren et al., RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells. BMC Cancer. 9, 10 (2009). 13. T. J. Klisch et al., In vivo Atoh1 targetome reveals how a proneural transcription factor regulates cerebellar development. Proc Natl Acad Sci USA. 108, 3288–3293 (2011). 14. P. F. Jacobsen, D. J. Jenkyn, J. M. Papadimitriou, Establishment of a human medulloblastoma cell line and its heterotransplantation into nude mice. J. Neuropathol. Exp. Neurol. 44, 472–485 (1985). 15. R. L. Levine, A. Pardanani, A. Tefferi, D. G. Gilliland, Role of JAK2 in the pathogenesis and therapy of myeloproliferative disorders. Nat. Rev. Cancer. 7, 673–683 (2007). 16. S. Pusch, N. Dissmeyer, A. Schnittger, Bimolecular-fluorescence complementation assay to monitor kinase-substrate interactions in vivo. Methods Mol. Biol. 779, 245–257 (2011). 17. M. W. Rousseaux et al., TRIM28 regulates the nuclear accumulation and toxicity of both alpha-synuclein and tau. Elife. 5, e19809 (2016). 18. K. Neet, T. Hunter, Vertebrate non-receptor protein-tyrosine kinase families. Genes Cells. 1, 147–169 (1996). 19. R. F. Ohana et al., HaloTag-based purification of functional human kinases from mammalian cells. Protein Expr. Purif. 76, 154–164 (2011). 20. A. Kobayashi et al., AG490, a Jak2 inhibitor, suppressed the progression of murine ovarian cancer. Eur. J. Pharmacol. (2015), doi:10.1016/j.ejphar.2015.09.039. 21. A. Quintás-Cardama, H. Kantarjian, J. Cortes, S. Verstovsek, Janus kinase inhibitors for the treatment of myeloproliferative neoplasias and beyond. Nat Rev Drug Discov. 10, 127–140 (2011). 22. Y. H. Joung et al., Combination of AG490, a Jak2 inhibitor, and methylsulfonylmethane synergistically suppresses bladder tumor growth via the Jak2/STAT3 pathway. Int J Oncol. 44, 883–895 (2014). 23. S. Haftchenary et al., Potent Targeting of the STAT3 Protein in Brain Cancer Stem Cells: A Promising Route for Treating Glioblastoma. ACS Med Chem Lett. 4, 1102–1107 (2013). 24. S.-M. Maira, F. Stauffer, C. Schnell, C. García-Echeverría, PI3K inhibitors for cancer treatment: where do we stand? Biochem Soc Trans. 37, 265–272 (2009). 25. N. Mody, J. Leitch, C. Armstrong, J. Dixon, P. Cohen, Effects of MAP kinase cascade inhibitors on the MKK5/ERK5 pathway. FEBS Lett. 502, 21–24 (2001). 26. M. A. Dawson et al., JAK2 phosphorylates histone H3Y41 and excludes HP1alpha from chromatin. Nature. 461, 819–822 (2009). 27. K. Ghoreschi, A. Laurence, J. J. O'Shea, Janus kinases in immune cell signaling. Immunol. Rev. 228, 273–287 (2009).

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