Abstract BACKGROUND Sonic hedgehog medulloblastoma (SHH-MB) is a malignant pediatric brain tumor that accounts for 30% of all MBs and is characterized by constitutive activation of the SHH signaling pathway. Besides the standard-of-care therapies, current therapies are targeting Smoothened (SMO) receptor, in order to inhibit the pathway. However, acquired or de novo resistance against SMO inhibition limits its efficacy. Therefore, our aim is to unravel novel therapeutic targets for SHH-MB, potentially beyond the SHH pathway. MATERIAL AND METHODS We performed genome-wide CRISPR/Cas9 knockout screens, in order to decipher cancer dependencies and synthetic lethal interactors for SHH-MB. The hits identified were first validated genetically and pharmacologically in in vitro assays, while further molecular analyses including RNAseq and differential methylation were also conducted. Moreover, the identified hits were evaluated in in vivo mouse models representing normal cerebellar, as well as SHH-MB development. RESULTS Functional genomics identified members of the epigenetic machinery, such as Dnmt1 and Smarca5 as context-specific dependencies for SHH-MB. We prove that DNMT1 inhibition is efficacious against SMO-resistant and sensitive SHH-MB cell lines, by inhibiting SHH pathway output, as shown via RNAseq. A further knockout drug screen unraveled novel synthetic lethal interactors for DNMT1 inhibitors. Genetic ablation and pharmacological inhibition of epigenetic regulators prolongs survival of established mouse models of SHH-MB. CONCLUSION Summarizing, our data indicate inhibitors of epigenetic regulators as novel therapeutic targets for SHH-MB irrespective of mutational drivers within the SHH pathway. We further propose combinatorial treatment approaches for this tumor entity that involve DNMT1 inhibitors.
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