Molecular and in vivo characterization of cancer-propagating cells derived from MYCN-dependent medulloblastoma

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. While the pathways that are deregulated in MB remain to be fully characterized, amplification and/or overexpression of theMYCNgene, which is has a critical role in cerebellar development as a regulator of neural progenitor cell fate, has been identified in several MB subgroups. Phenotypically, aberrant expression of MYCN is associated with the large-cell/anaplastic MB variant, which accounts for 5-15% of cases and is associated with aggressive disease and poor clinical outcome. To better understand the role of MYCN in MBin vitroandin vivoand to aid the development of MYCN-targeted therapeutics we established tumor-derived neurosphere cell lines from the GTML (Glt1-tTA/TRE-MYCN-Luc) genetically engineered mouse model. A fraction of GTML neurospheres were found to be growth factor independent, expressed CD133 (a marker of neural stem cells), failed to differentiate upon MYCN withdrawal and were highly tumorigenic when orthotopically implanted into the cerebellum. Principal component analyzes using single cell RNA assay data suggested that the clinical candidate aurora-A kinase inhibitor MLN8237 converts GTML neurospheres to resemble non-MYCN expressors. Correlating with this, MLN8237 significantly extended the survival of mice bearing GTML MB allografts. In summary, our results demonstrate that MYCN plays a critical role in expansion and survival of aggressive MB-propagating cells, and establish GTML neurospheres as an important resource for the development of novel therapeutic strategies

Supplementary Data Only: Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network.

The Myc/Max/Mad transcription factor network is critically involved in cell behavior; however, there is relatively little information on its genomic binding sites. We have employed the DamID method to carry out global genomic mapping of the Drosophila Myc, Max, and Mad/Mnt proteins. Each protein was tethered to Escherichia coli DNA adenine-methyltransferase (Dam) permitting methylation proximal to in vivo binding sites in Kc cells. Microarray analyses of methylated DNA fragments reveals binding to multiple loci on all major Drosophila chromosomes. This approach also reveals dynamic interactions among network members as we find that increased levels of dMax influence the extent of dMyc, but not dMnt, binding. Computer analysis using the REDUCE algorithm demonstrates that binding regions correlate with the presence of E-boxes, CG repeats, and other sequence motifs. The surprisingly large number of directly bound loci ( approximately 15% of coding regions) suggests that the network interacts widely with the genome. Furthermore, we employ microarray expression analysis to demonstrate that hundreds of DamID-binding loci correspond to genes whose expression is directly regulated by dMyc in larvae. These results suggest that a fundamental aspect of Max network function involves widespread binding and regulation of gene expression

Characterization of GTML orthotopic tumors.

<p>(<b>A</b>) M10519 GTML spheres (passage 10–27) were orthotopically implanted into the cerebellum of FVB/N mice, and tumor development was monitored by bioluminescence. Once bioluminescence signal were reached at 5x10⁹ photons/s the tumors were either treated with dox to induce MYCN withdrawal or left untreated (n = 6 per treatment arm) and then the level of luciferase expression was monitored until day 26. (<b>B</b>) H&E staining of orthotopic tumors. Mice were treated with dox on day 1 (left panels) on tumors with 5x10⁹ photons/s. Tumor regression at day 26 is shown (right panels). Bars, 500μm (upper panels) and 20μm (lower panels). (<b>C</b>) Immunoperoxidase staining of paraffin-embedded orthotopic tumors. Tumors generated after the implantation of M10519 cells were harvested, paraffin-embedded, sectioned, and labeled with antibodies shown. Sections were counter-stained with hematoxylin. Bar, 20μm.</p

Effect of MLN8237 on GTML spheres and mice.

<p>(<b>A</b>) PC projections of 96 cells from WT1, WT2, M10519 cells treated with and without dox, and M10519 cells with 100nM MLN8237. PC1 and PC2 captured 21% and 13% of variances in the dataset. (<b>B</b>) MYCN stability in MLN8237-treated M10519 cells. Cells were incubated with dox or MLN8237, and cell extracts were analyzed by western blotting. (<b>C</b>) Kaplan-Meier curves for overall survival of mice having orthotopic tumors treated with (red) or without MLN8237 (blue). 250 cells were transplanted in to the cerebellum of FBVN mice, and once the tumor burden reached at a level of 1x10⁹ photon/s, mice were daily treated twice with (n = 8) or without (n = 9) MLN8237 (daily oral dosing at 60mg/kg). Mice were treated with MLN8237 for 5 days and then untreated for 2 days, and then the repeat this schedule twice more.</p

Tumor-propagating potential of CD133+ cells.

<p>(<b>A</b>) Purification of CD133+ and CD133- cells. M10519 cells (passage 13) were stained with anti-IgG1 or anti-Prominin-1 (CD133) conjugated with PE prior to purification. Stained cells were first separated into bead-bound and bead-unbound fractions by magnetic beads conjugated with anti-PE (middle panels). Then each fraction was further purified by FACS sorting. <b>(B</b>) Orthotopic tumors generated after implantation of CD133+ cells (10 cells per mouse). Sections were stained with hematoxylin and eosin. Bars, 1000μm (upper), 50μm (lower). (<b>C</b>) Kaplan-Meier curves for overall survival of mice implanted with 10 CD133+ (red, n = 10) or CD133- (blue, n = 10) M10519 cells per site.</p