Combination of PARP and WEE1 inhibitors in vitro: Potential for use in the treatment of SHH medulloblastoma.

Medulloblastoma (MB), grouped as either WNT­activated, Sonic hedgehog (SHH)­activated, or non-WNT/non-SHH group 3, accounts for almost 20% of all childhood brain cancers. In spite of current intensive treatments, not all patients are cured and survivors suffer from severe side­effects. The present study therefore examined the effects of the poly­ADP­ribose polymerase (PARP) and WEE1­like protein kinase (WEE1) inhibitors, BMN673 and MK­1775, respectively, alone or in combination on four MB cell lines. More specifically, the MB cell lines, DAOY, UW228­3, MED8A and D425, were tested for their sensitivity to BMN673 and MK­1775 alone or in combination, using cell viability, cell confluency and cytotoxicity assays. The effects on the cell cycle phases were also examined using FACS analysis. Monotherapy with BMN673 and MK­1775 exerted dose­dependent inhibitory effects on the viability of almost all MB cell lines. Notably, when BMN673 and MK­1775 were used in combination, synergistic effects were noted in the SHH group cell lines (DAOY and UW228­3), but not in the already WEE1­sensitive group 3 (MED8A and D425) lines. Moreover, the combination treatment decreased the percentage of cells in the G1 phase and induced the novel distribution of both DAOY and UW228­3 cells in the S and G2/M phases, with the UW228­3 cells exhibiting a greater delay. To conclude, MK­1775 was efficient in all and BMN673 in most cell lines, and their combined use exerted synergistic effects on the SHH, but not the group 3 cell lines. These data suggest that MK­1775 alone may be of interest for all MB cell lines, and that the combination of PARP/WEE1 inhibitors may provide possible therapeutic opportunities for the therapy of SHH MBs. Their use warrants further investigations in the future.

Integrated Proteomic and Transcriptomic-Based Approaches to Identifying Signature Biomarkers and Pathways for Elucidation of Daoy and UW228 Subtypes.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Patient survival has remained largely the same for the past 20 years, with therapies causing significant health, cognitive, behavioral and developmental complications for those who survive the tumor. In this study, we profiled the total transcriptome and proteome of two established MB cell lines, Daoy and UW228, using high-throughput RNA sequencing (RNA-Seq) and label-free nano-LC-MS/MS-based quantitative proteomics, coupled with advanced pathway analysis. While Daoy has been suggested to belong to the sonic hedgehog (SHH) subtype, the exact UW228 subtype is not yet clearly established. Thus, a goal of this study was to identify protein markers and pathways that would help elucidate their subtype classification. A number of differentially expressed genes and proteins, including a number of adhesion, cytoskeletal and signaling molecules, were observed between the two cell lines. While several cancer-associated genes/proteins exhibited similar expression across the two cell lines, upregulation of a number of signature proteins and enrichment of key components of SHH and WNT signaling pathways were uniquely observed in Daoy and UW228, respectively. The novel information on differentially expressed genes/proteins and enriched pathways provide insights into the biology of MB, which could help elucidate their subtype classification.