Abstract
BACKGROUNDL Diffuse intrinsic pontine gliomas (DIPGs) are fatal childhood gliomas and radiotherapy is the only modality that prolongs survival; however, all patients progress with no long term survivors, indicating an urgent need to develop new treatment approaches. METHODS: Genome wide CRISPR screen was used to identify the cellular pathway(s) critical for DIPG cell survival and doxycycline inducible shRNA were used for temporal knockdown of gene expression. Colony formation assays were performed to monitor effects on cell proliferation, comet assay and immunoflourescence based gamma-H2AX foci detection were used for DNA damage assessment, and caspase3/7 activity measured apoptosis. RESULTS: Our preliminary studies to understand radio- and chemo-resistance mechanisms in DIPGs uncovered high levels of persistent basal DNA double strand breaks (DSBs) in the majority of analyzed DIPG cell lines compared to neural stem cells and astrocytes, a phenomenon that promotes oncogenic potential. The basal increase in DSBs was independent of H3.3K27M mutational status. To pinpoint the molecular mechanism(s) that support proliferation and survival of DIPG cells with highly damaged DNA, we performed unbiased CRISPR screens in unperturbed cells and preliminary results suggest an addiction of DIPG cells to specific DSB repair factors. Further analyses confirmed this intrinsic reliance of DIPG cells on specific DSB repair pathways and the key role of DNA repair mechanisms in suppressing DNA damage induced apoptotic cell death. CONCLUSION: DIPG cells exhibit persistent DNA damage. To evade catastrophic genomic instability and cell death, DIPG cells hijack DNA repair pathways. Therefore, a promising therapy against DIPG would include inhibition of specific DNA damage response pathways that allow DIPGs to survive persistent DNA damage.