Medulloblastomas are the most common malignant brain tumors in childhood. Emerging evidence suggests that medulloblastoma comprises at least four distinct diseases (WNT, SHH, Group 3 and 4) with different biology, clinical presentation, and outcome, with especially poor prognosis in Group 3. The tight connection of biology and clinical behavior in patients emphasizes the need for subgroup-specific preclinical models in order to develop treatments tailored to each subgroup. Herein we report on the novel cell line HD-MB03, isolated from tumor material of a patient with metastasized Group 3 medulloblastoma, and preclinical testing of different histone deacetylase inhibitors (HDACis) in this model. HD-MB03 cells grow long term in vitro and form metastatic tumors in vivo upon orthotopic transplantation. HD-MB03 cells reflect the original Group 3 medulloblastoma at the histological and molecular level, showing large cell morphology, similar expression patterns for markers Ki67, p53, and glial fibrillary acidic protein (GFAP), a gene expression profile most closely matching Group 3 medulloblastomas, and persistence of typical molecular alterations, i.e., isochromosome 17q [i(17q)] and MYC amplification. Protein expression analysis of HDACs 2, 5, 8, and 9 as well as the predictive marker HR23B showed intermediate to strong expression, suggesting sensitivity to HDACis. Indeed, treatment with HDACis Helminthosporium carbonum (HC)-toxin, vorinostat, and panobinostat revealed high sensitivity to this novel drug class, as well as a radiation-sensitizing effect with significantly increased cell death upon concomitant treatment. In summary, our data indicate that HD-MB03 is a suitable preclinical model for Group 3 medulloblastoma, and HDACis could represent a therapeutic option for this subgroup.

Relapse with drug-resistant disease is the main cause of death in MYCN-amplified neuroblastoma patients. MYCN-amplified neuroblastoma cells in vitro are characterized by a failure to arrest at the G(1)-S checkpoint after irradiation- or drug-induced DNA damage. We show that several MYCN-amplified cell lines harbor additional chromosomal aberrations targeting p53 and/or pRB pathway components, including CDK4/CCND1/MDM2 amplifications, p16INK4A/p14ARF deletions or TP53 mutations. Cells with these additional aberrations undergo significantly lower levels of cell death after doxorubicin treatment compared with MYCN-amplified cells, with no additional mutations in these pathways. In MYCN-amplified cells CDK4 expression is elevated, increasing the competition between CDK4 and CDK2 for binding p21. This results in insufficient p21 to inhibit CDK2, leading to high CDK4 and CDK2 kinase activity upon doxorubicin treatment. CDK4 inhibition by siRNAs, selective small compounds or p19(INK4D) overexpression partly restored G(1)-S arrest, delayed S-phase progression and reduced cell viability upon doxorubicin treatment. Our results suggest a specific function of p19(INK4D), but not p16(INK4A), in sensitizing MYCN-amplified cells with a functional p53 pathway to doxorubicin-induced cell death. In summary, the CDK4/cyclin D-pRB axis is altered in MYCN-amplified cells to evade a G(1)-S arrest after doxorubicin-induced DNA damage. Additional chromosomal aberrations affecting the p53-p21 and CDK4-pRB axes compound the effects of MYCN on the G(1) checkpoint and reduce sensitivity to cell death after doxorubicin treatment. CDK4 inhibition partly restores G(1)-S arrest and sensitizes cells to doxorubicin-mediated cell death in MYCN-amplified cells with an intact p53 pathway.

Common fragile sites (cFS) represent chromosomal regions that are prone to breakage after partial inhibition of DNA synthesis. Activation of cFS is associated with various forms of DNA instability in cancer cells, and is thought to be an initiating event in the generation of DNA damage in early-stage tumorigenesis. Only a few cFS have been fully characterized despite the growing interest in cFS instability in cancer genomes. In this study, six-color fluorescence in situ hybridization revealed that FRA2C consists of two cFS spanning 747 kb FRA2Ctel and 746 kb FRA2Ccen at 2p24.3 and 2p24.2, respectively. Both cFS are separated by a 2.8 Mb non-fragile region containing MYCN. Fine-tiling array comparative genomic hybridization of MYCN amplicons from neuroblastoma (NB) cell lines and primary tumors revealed that 56.5% of the amplicons cluster in FRA2C. MYCN amplicons are either organized as double minutes or as homogeneously stained regions in addition to the single copy of MYCN retained at 2p24. We suggest that MYCN amplicons arise from extra replication rounds of unbroken DNA secondary structures that accumulate at FRA2C. This hypothesis implicates cFS in high-level gene amplification in cancer cells. Complex genomic rearrangements, including deletions, duplications and translocations, which originate from double-strand breaks, were detected at FRA2C in different cancers. These data propose a dual role for cFS in the generation of gross chromosomal rearrangements either after DNA breakage or by inducing extra replication rounds, and provide new insights into the highly recombinogenic nature of cFS in the human cancer genome.