Medulloblastomas cause roughly 1/4 of children's malignant brain tumors. Medulloblastomas break into 4 classes, two of which harbour mutations in two well known pathways that regulate the normal development of the brain: Sonic Hedgehog/Patched (Shh) and Wnt (Thompson et al, 2006). Despite recent success in treating medulloblastomas using a combination of surgery, chemotherapy and radiation; one third remain incurable (Maleci et al. 1992; Roberts et al. 1991). Additionally, current therapies cause problems in normal brain development, including reduction of IQ, that greatly affect the quality of life of survivors (Gajjar et al. 2006). Glioblastoma multiforme (GBM), the most common and aggressive brain tumor in adults and in children, is extremely difficult to treat, with a median survival of around one year. Despite advances in chemo- and radiation therapy, the 5-year survival rate for GBM has not improved in the last 30 years and currently is still less than 3% (Ohgaki and Kleihues 2005). Clearly, new and better therapies are needed for these brain cancers.
Medulloblastomas are believed to originate in specific cells in the developing brain, granule neuron progenitors (GNPs), that fail to stop dividing when they are supposed to do so during cerebellum development. Sonic hedgehog (Shh) is the major regulator that drives proliferation of GNPs (Wechsler-Reya and Scott 2001). Mutations in Shh, or its helper proteins, occur in ~25% of patients with medulloblastoma (Thompson et al. 2006). Although an inhibitor of Shh signaling (HH antag) causes medulloblastoma-cells to stop dividing and assume their proper function and eliminates tumor formation in mice, treatment of young animals with this compound causes abnormal bone development (Kimura et al. 2008). Because patients with medulloblastoma would be treated at the time when their bone growth is at its peak, this compound is likely to severely affect the growth of children. Thus we have been interested in the identification of novel therapeutic targets that inhibit tumor development without causing such side effects.
We have developed mouse models of medulloblastoma that faithfully mimic the human tumors that have problems with the control of cell fate by Shh. We genetically engineered mice that lacked a copy of the Patched gene and two copies of the cell cycle negative regulatory protein, p18Ink4c, two proteins found mutated (Easton et al. 1998) or down regulated (Schwaller et al. 1997), respectively, in human tumors. We confirmed by
molecular analysis that mouse medulloblastoma that occur in our [Cdkn2c-/-; Ptch1+/-] and [Cdkn2c-/-; p53-/-] mice (Zindy et al. 2007) mimic human medulloblastomas with a Shh "signature". We recently found that bone morphogenic proteins (BMP2, 4 and 7) reverse Shh-dependent cell growth and cause tumor cells to convert into neurons. BMP molecules are critical in the development of all organisms throughout evolution from flies to man (See Figure 1 for a description of the BMP signaling pathway). Differentiation of GNP's is mediated in part by the rapid inactivation of the transcription factor Atonal (Atoh1) (Zhao et al., 2008) which is critical for normal GNP cell function in brain development (Ben-Arie et al. 1997). Analysis of the BMP pathway showed that it is largely shut down at the genetic level whereas Atoh1 is highly active both in mouse and human MBs (Zhao et al. 2008).
Besides their relevance in inhibiting medulloblastoma, BMP2, 4 and 7 also block the development of other cancers, including glioblastoma. BMP4 stops the proliferation of tumor-initiating cells that cause GBMs both in vitro and in vivo (Piccirillo et al. 2006). Children's tumors originating from the gonads downregulate the BMP pathway suggesting that its reactivation may be critical for the treatment of these tumors (James Amatruda, personal communication). In vivo, daily systemic administration of BMP7 significantly inhibits growth of breast cancer cells metastasized to the bones of nude mice (Buijs et al. 2007). All these results suggest that BMP2, 4, and 7 have broad therapeutic use.
However, several drawbacks are related to the use of BMPs as therapeutic agents. First, the production of recombinant BMPs is costly, probably reaching costs of greater than $50,000 per treatment course. Second, to reach the effective doses would require giving very large amounts of the BMP's (Schmidmaier et al. 2008) that could lead to severe side effects such as inflammation, edema and abnormal bone formation (Gottfried and Dailey 2008). Because Shh antagonists and BMPs can act together, BMP agonists/activators could be combined with Shh antagonists to lower doses of Shh antagonist and potentially diminish the side effects, especially the inhibition of bone development.
In summary, we have generated mouse models that faithfully mimic medulloblastoma with improperly functioning Shh and have begun to use them as preclinical models (Ayrault et al. 2009). We believe that BMP signaling is an attractive target because tumor cells will be induced to differentiate in neurons and eliminated, leaving normal neurons intact. In addition, small molecule agonists/activators of BMP signaling should be more effective and less toxic therapeutic agents than BMP itself. This grant application describes our preliminary data and experimental plan toward the identification of small molecules that activate the BMP pathway using a high throughput screen of a large library of compounds (~ 525,000) and the validation of their function in tumor cells, with a hope to use them in the future in the clinic.
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