Medulloblastoma is the most common solid brain tumor that develops in children, with more than five hundred new cases diagnosed in the United States every year. There are four broad types of medulloblastoma. One of these is called the “Sonic Hedgehog” subtype, named after the biological pathway that becomes re-activated in these tumors. Only about half of patients with this subtype survive for more than 10 years. Moreover, medulloblastoma treatment combines surgery, chemotherapy and radiation, which can cause severe side effects including psychiatric disorders and cognitive impairment.

Several drugs that treat medulloblastoma by targeting the Sonic Hedgehog pathway are currently being tested in clinical trials. However, these drugs are usually only effective for a limited time before the tumor evades the treatment. Therefore, there is a need to develop new treatment options for medulloblastoma, perhaps by targeting different signaling pathways in the cells.

A protein called Atoh1 is needed for proper brain development in humans, but is not normally present after the first year of life. This protein is, however, re-expressed at high levels in medulloblastoma in mice and humans and is essential for Sonic Hedgehog-type medulloblastoma to form in mice.

Klisch et al. used genetic techniques to reduce the amount of Atoh1 in mice that develop medulloblastoma. This intervention reduced the number of mice that developled tumors and increased their lifespan. Biochemical experiments showed that the tumor stem cells of the mice contain a modified version of Atoh1 where a phosphate molecule is bound to a particular region of the protein. This phosphorylation increased the amount and activity of Atoh1 in the cell, and so caused tumors to grow more quickly in mice. Phosphorylated Atoh1 was also detected in samples taken from human medulloblastoma tumors.

Klisch et al. also found that an enzyme called Jak2 phosphorylates Atoh1. Inhibiting Jak2 reduced the levels of Atoh1 in medulloblastoma cells and slowed tumor growth in mice. Future work could investigate different ways of preventing Atoh1 phosphorylation, with the hope of finding new treatments for Sonic-Hedgehog-type medulloblastomas.

Medulloblastoma (MB) is the most common malignant primary brain tumor in children (Dhall, 2009) and is thought to originate from rhombic lip-derived precursors that have failed to differentiate into their usual progeny (Hatten and Roussel, 2011). Molecular studies have allowed us to classify MB into four major subtypes: WNT, sonic hedgehog (SHH), Group C, and Group D (Taylor et al., 2012). The cell of origin differs among the subgroups, as does the clinical prognosis (Taylor et al., 2012). SHH-type tumors arise from precursors of the cerebellar granule neurons and can contain mutations in Smoothened, Patched, or other genes in this pathway (Rimkus et al., 2016). Elevated expression of Atoh1, the basic helix-loop-helix transcription factor that is crucial for the development of the cerebellar granule neurons (Ben-Arie et al., 1996), can also drive uncontrolled cell proliferation and formation of medulloblastoma (Flora et al., 2009). In addition to attempting to inhibit SHH signaling—a strategy that can be foiled by the development of secondary mutations (Yauch et al., 2009)—we hypothesized that controlling the levels of Atoh1 protein will provide another therapeutic entry point for the treatment SHH-driven medulloblastoma.

To determine the influence of Atoh1 gene dosage on MB development and prognosis, we examined the rate of tumor formation in a mouse model of Shh-dependent MB, the Nd2::SmoA1^Tg line (Hatton et al., 2008). These mice have a constitutively active Shh pathway in all cerebellar granule cells and spontaneously develop MBs (Hatton et al., 2008). Whereas almost a quarter (23.1%) of Nd2::SmoA1^Tg mice developed MB and succumbed to the disease within 300 days of life, fewer than one in ten (6.7%) Nd2::SmoA1^Tg mice – heterozygous for Atoh1 (Atoh1^+/-, Nd2::SmoA1^Tg) – developed signs of disease (Figure 1a, Figure 1—source data 1 for all statistical analysis). Atoh1 heterozygosity dropped the overall tumor incidence by over three-fold and never reached that of Nd2::SmoA1^Tg, Atoh1 wildtype mice. Importantly, while both groups expressed the tumorigenic transgene (Nd2::SmoA1) at similar levels (Figure 1—figure supplement 1a), levels of Gli2, the primary Shh mediator in medulloblastoma and direct target of Atoh1 (Read et al., 2009; Klisch et al., 2011) were reduced in the Atoh1 heterozygous mice. Hence, a 50% reduction in Atoh1 protein level has a direct, positive, effect on survival in this medulloblastoma mouse model. This suggests that Atoh1 protein dosage is critical for the development of medulloblastoma in mice.

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Figure 1—source data 1

This excel file contains all relevant statistical analyses for the manuscript.

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Figure 1—source data 2

Atoh1 tyrosine 78 coverage and Atoh1 interacting proteins.

https://doi.org/10.7554/eLife.31181.006

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The regulation of Atoh1 expression during the development of the cerebellum is highly complex, involving not only Shh signaling but also other signaling pathways required for proper development, such as BMP and WNT signaling (Butts et al., 2014). While this regulation is transcriptional in nature, another layer of regulation has been reported to influence Atoh1 protein levels: phosphorylation (Forget et al., 2014). Hence, we asked whether Atoh1 was aberrantly phosphorylated in the tumors. Using tumors isolated from Nd2::SmoA1^Tg mice, we performed IP-MS on tumor-initiating cells that express Atoh1 [marked by cell surface protein CD15 (Read et al., 2009)] and CD15-negative cells (Figure 1—figure supplement 1b). We tested the integrity of both cell populations by flank injections of sorted cells in immunosuppressed mice and found that Atoh1 expression in the tumor-initiating cells is required to drive secondary tumor formation (Figure 1—figure supplement 1b). Using Atoh1 IP-MS, we discovered that tyrosine 78 (Y78, Figure 1—source data 2) on Atoh1 is phosphorylated exclusively in tumor-initiating cells. We confirmed phosphorylation of this Atoh1 residue in MB tissue using a phospho-specific antibody (p-Atoh1-Y78, Figure 1b). We further confirmed these findings in tumorigenic and control tissues from mice and found that Atoh1 Y78 phosphorylation is specific to the tumor-initiating cells (Figure 1c). Y78 immuno-reactivity was observed mostly in the initiating cell islets of the tumor; neither the Y78-specific phospho-antibody, nor an antibody against CD15, reacted in the normal tissue, even in the presence of strong Atoh1 expression in the external granule layer (Figure 1c).

Because Atoh1 is not endogenously expressed in neuronal cell lines, and reliable primary cultures of human SHH-type medulloblastoma cells are not established, we turned to modify and study medulloblastoma cell lines that have been used in the past. We engineered cell lines expressing either doxycycline-inducible wildtype (Y78), phospho-dead (Y78F) or phospho-mimetic (Y78D) Atoh1, using three different human-derived MB parental cell lines: DAOY (marked in blue), UW228 (orange) and ONS-76 (purple) (Zanini et al., 2013). While these cell lines have been used extensively, it has been recently suggested that at least the DAOY cell line is more ‘Shh-like’ than the others (Higdon et al., 2017). We found that Y78 phosphorylation status did not alter the interaction of Atoh1 with its E-protein binding partner or its nuclear-cytoplasmic localization (Figure 2—figure supplement 1a–b). However, upon doxycycline withdrawal, Y78 phosphorylation dramatically increased Atoh1 protein stability in all three parental lines (Figure 2a, Figure 2—figure supplement 1c–d). Protein half-life of phospho-mimetic Atoh1 was triple that of wildtype Atoh1 (Figure 2—source data 1), while the RNA stability of the transgenes was comparable (Figure 2—figure supplement 1e).

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Atoh1 half-life analysis based on stably expressing cell lines.

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To test the transcriptional activity of the Atoh1 variants we used an Atoh1-specific luciferase reporter system (Klisch et al., 2011). Overexpression of phospho-mimetic Atoh1 (Y78D), but not the phospho-mutant (Y78F), increased Atoh1 transcriptional activity (Figure 2b, Figure 2—figure supplement 2b–c). In a proliferation assay using all three parental cell lines, we found that the enhanced activity of Atoh1 Y78D resulted in hyper-proliferation, whereas the phospho-dead Atoh1 resulted in a slight decrease in cell proliferation (Figure 2—figure supplement 2d–f). While the increase in transcriptional activity could be due to the increased half-life, the steady-state levels of the three Atoh1 mutants were similar in the luciferase lysates (Figure 2—figure supplement 2a). These data argue for two distinct effects of tyrosine 78 phosphorylation: increased Atoh1 stability as well as increased Atoh1 transcriptional activity.

To test whether this phosphorylation drives tumor growth in vivo, we injected DAOY cells [known to form secondary tumors (Jacobsen et al., 1985)] stably expressing doxycycline-inducible Atoh1-GFP WT, Atoh1-GFP Y78F, or Atoh1-GFP Y78D in flank xenografts. While these flank xenograft models lack the fully functional immune system, they provide a useful model for longitudinal tumor growth monitoring with minimal burden to the animal. The mice were fed doxycycline chow continuously to express the transgenes. Expression of wildtype or phospho-dead Atoh1 (Y78F) led to a similar average tumor weight, but overexpression of phospho-mimetic Atoh1 (Y78D) increased tumor weight by roughly 40% (Figure 2c). These data suggest that tyrosine 78 phosphorylation increases Atoh1 transcriptional activity, which in turn increases cellular proliferation in vitro as well as in vivo.

To identify the tyrosine kinase responsible for the aberrant phosphorylation of Atoh1 in the malignant cells, we revisited our IP-MS data. We recovered 637 Atoh1-interacting proteins from CD15-positive cells and 278 interactors from CD15-negative cells (Figure 1—source data 2). As would be expected, E-proteins were the strongest interactors present in all pull down assays, but interestingly only one tyrosine kinase, Janus kinase 2 (Jak2), was present exclusively in tumor-initiating cells. The Jak2 paralog Jak1 was present in the non-initiating cell population (Figure 1—source data 2). The Janus kinases typically function in the cytoplasm, downstream of cytokine signaling, and activate canonical cascades such as signal transducer and activator of transcription (STAT), mitogen activated protein kinase (MAPK), and/or the phosphotidylinositol 3-kinase (PI3K)–Akt pathways (Levine et al., 2007). Although one of the most common gain-of-function mutations in Jak2 (Jak2^V617F) is associated with increased proliferation in human myeloproliferative diseases (Levine et al., 2007), Jak2 function in medulloblastoma has not been investigated.

We tested Jak1-3 and Tyrosine Kinase 2 (Tyk2), as well as Protein Tyrosine Kinase 2 (Ptk2) in a bimolecular fluorescence complementation assay (BiFC) for an Atoh1 interaction (Neet and Hunter, 1996). Only Jak2 displayed a strong interaction with Atoh1 as indicated by the shift in GFP signal detected by flow cytometry (Figure 3—figure supplement 1a). We further investigated whether these kinases were able to phosphorylate Y78 using a luciferase-based kinase assay. Once again, only Jak2 was able to phosphorylate wildtype Atoh1 (Y78) in a dose dependent manner in vitro (Figure 3—figure supplement 1b). This phosphorylation was specific to Y78, as the addition of phospho-mutant Atoh1 (Y78F) abolished this effect (Figure 3—figure supplement 1b). Given that both Atoh1 and Jak2 are expressed during cerebellar development and both are highly overexpressed in MB tissue (Figure 3—figure supplement 1c), we tested whether the Jak2-Atoh1 interaction occurs in vivo. We immunoprecipitated Atoh1 or Jak2 and found a strong interaction in malignant but not normal cerebellar tissue (Figure 3a). To determine whether Jak2 is a critical kinase phosphorylating tyrosine 78, we isolated Shh-type MB cells and cultured them in the presence or absence of a strong and highly specific Jak2 inhibitor (AG490) (Kobayashi et al., 2015). One-hour treatment with the inhibitor was sufficient to drastically reduce Jak2 and Atoh1 Y78 phosphorylation (Figure 3b). These findings demonstrate that Jak2 is activated in Shh-type MB, where it interacts with Atoh1 and phosphorylates it on tyrosine 78.

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We examined whether Jak2 influences Atoh1 activity through its canonical downstream cascades by using the Atoh1 transcriptional reporter assay. Co-transfection of Atoh1 and Jak2 dramatically increased Atoh1 activity in all three MB lines (Figure 3c, Figure 3—figure supplement 1d–e). Inhibiting Jak2 with AG490 abolished this, but blocking STAT, PI3K or MAPK signaling in this assay had no effect (Figure 3c, Figure 3—figure supplement 1d–e), arguing that Jak2 influences Atoh1 directly. Prior studies have argued for non-canonical roles of Jak2, e.g. Jak2 phosphorylates tyrosine 41 on histone H3 (Dawson et al., 2009). Moreover, most of the genes regulated by Jak2 do not contain the predicted STAT DNA-binding motifs (Ghoreschi et al., 2009) suggesting the existence of unidentified transcriptional mediators. Overexpression of Jak2 pheno-copied the transcriptional gain-of-function phenotype of Atoh1 Y78D, increased the stability of wildtype Atoh1 (Y78) to Y78D levels (Figure 3—figure supplement 2, Figure 2—source data 1) and increased cellular proliferation in vitro, which was blocked by phospho-mutant Atoh1 (Y78F) and the addition of AG490 (Figure 3—figure supplement 3).

Having identified Jak2 as a critical kinase phosphorylating tyrosine 78, we asked if inhibition of Jak2 might be a viable strategy to decrease Atoh1 levels in vivo. We isolated Shh-type medulloblastoma cells, infected them with lentiviral particles harboring Jak2 shRNAs tagged with RFP, and grafted these into mice. Control shRNA-infected tumors grew rapidly, but both Jak2 shRNA-infected tumors showed dramatically reduced tumor growth (Figure 4a), in accordance with their potency to inhibit Jak2 (Figure 4b). The decrease in Jak2 resulted in a concomitant decrease in Y78 phosphorylation (Figure 4b). Moreover, Jak1, a close relative of Jak2, did not compensate for the inhibition of Jak2 as it was expressed at similar levels in control and Jak2 inhibited tumors (Figure 4b). Sections from control shRNA-infected tumors revealed large areas of infected cells (Figure 4c left panel, red) that expressed Atoh1 and had pockets of tumor-initiating cells (CD15-positive, yellow) that co-localized with phosphorylated Y78 (blue). In contrast, tumors infected with the most potent Jak2 shRNA revealed scattered initiating cell clusters, none of which contained Y78 phosphorylated Atoh1 (Figure 4c, right panel).

Figure 4

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Taken together, here we demonstrate that Atoh1, a protein not expressed in the post-natal normal brain, is expressed specifically in SHH-type medulloblastoma malignancies (Figure 4d), and that tyrosine 78 phosphorylation by Jak2 increases Atoh1 activity and stability, thus augmenting the proliferation of tumor-initiating cells in MB. Our results suggest that targeting Atoh1 by inhibiting its phosphorylation on tyrosine 78 presents a viable avenue for targeting MB cells without impinging on healthy brain tissue. Moreover, tyrosine 78 of Atoh1 can be detected in protein lysates of human samples of SHH-type medulloblastoma (Figure 4e), further supporting the clinical relevance of these findings.

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Author details

1. Tiemo J Klisch

   1. Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, United States
   2. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Writing—original draft, Writing—review and editing

   For correspondence

   klisch@bcm.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8182-384X

2. Anna Vainshtein

   1. Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, United States
   2. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0368-5044

3. Akash J Patel

   1. Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, United States
   2. Department of Neurosurgery, Baylor College of Medicine, Houston, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1280-2989

4. Huda Y Zoghbi

   1. Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, United States
   2. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
   3. Department of Neurosurgery, Baylor College of Medicine, Houston, United States
   4. Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States

   Contribution

   Conceptualization, Funding acquisition, Writing—review and editing

   Competing interests

   Senior editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0002-0700-3349

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