SummaryWe collated data from 157 unpublished cases of pediatric high-grade glioma and diffuse intrinsic pontine glioma and 20 publicly available datasets in an integrated analysis of >1,000 cases. We identified co-segregating mutations in histone-mutant subgroups including loss of FBXW7 in H3.3G34R/V, TOP3A rearrangements in H3.3K27M, and BCOR mutations in H3.1K27M. Histone wild-type subgroups are refined by the presence of key oncogenic events or methylation profiles more closely resembling lower-grade tumors. Genomic aberrations increase with age, highlighting the infant population as biologically and clinically distinct. Uncommon pathway dysregulation is seen in small subsets of tumors, further defining the molecular diversity of the disease, opening up avenues for biological study and providing a basis for functionally defined future treatment stratification.

Angiocentric gliomas are pediatric low-grade gliomas (PLGGs) without known recurrent genetic drivers. We performed genomic analysis of new and published data from 249 PLGGs including 19 Angiocentric Gliomas. We identified MYB-QKI fusions as a specific and single candidate driver event in Angiocentric Gliomas. In vitro and in vivo functional studies show MYB-QKI rearrangements promote tumorigenesis through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression, and hemizygous loss of the tumor suppressor QKI. This represents the first example of a single driver rearrangement simultaneously transforming cells via three genetic and epigenetic mechanisms in a tumor.

The tet-off system has been widely used to create transgenic models of neurological disorders including Alzheimer’s, Parkinson’s, Huntington’s, and prion disease. The utility of this system lies in the assumption that the tetracycline transactivator (TTA) acts as an inert control element and does not contribute to phenotypes under study. Here we report that neuronal expression of TTA can affect hippocampal cytoarchitecture and behavior in a strain-dependent manner. While studying neurodegeneration in two tet-off Alzheimer’s disease models, we unexpectedly discovered neuronal loss within the dentate gyrus of single transgenic TTA controls. Granule neurons appeared most sensitive to TTA exposure during postnatal development, and doxycycline treatment during this period was neuroprotective. TTA-induced degeneration could be rescued by moving the transgene onto a congenic C57BL/6J background, and recurred on re-introduction of either CBA or C3H/He backgrounds. Quantitative trait analysis of B6C3 F2 TTA mice identified a region on Chromosome 14 that contains a major modifier of the neurodegenerative phenotype. Although B6 mice were resistant to degeneration, they were not ideal for cognitive testing. F1 offspring of TTA C57BL/6J and 129X1/SvJ, FVB/NJ, or DBA/1J showed improved spatial learning, but TTA expression caused subtle differences in contextual fear conditioning on two of these backgrounds indicating that strain and genotype can interact independently under different behavioral settings. All model systems have limitations that should be recognized and mitigated where possible; our findings stress the importance of mapping the effects caused by TTA alone when working with tet-off models.

Pediatric low-grade gliomas (PLGGs) are commonly associated with BRAF gene fusions that aberrantly activate the mitogen-activated protein kinase (MAPK) signaling pathway. This has led to PLGG clinical trials utilizing RAF- and MAPK pathway-targeted therapeutics. Whole-genome profiling of PLGGs has also identified rare gene fusions involving another RAF isoform, CRAF/RAF1, in PLGGs and cancers occuring in adults. Whereas BRAF fusions primarily dysregulate MAPK signaling, the CRAF fusions QKI-RAF1 and SRGAP3-RAF1 aberrantly activate both the MAPK and phosphoinositide-3 kinase/mammalian target of rapamycin (PI3K/mTOR) signaling pathways. Although ATP-competitive, first-generation RAF inhibitors (vemurafenib/PLX4720, RAFi) cause paradoxical activation of the MAPK pathway in BRAF-fusion tumors, inhibition can be achieved with ‘paradox breaker’ RAFi, such as PLX8394. Here we report that, unlike BRAF fusions, CRAF fusions are unresponsive to both generations of RAFi, vemurafenib and PLX8394, highlighting a distinct responsiveness of CRAF fusions to clinically relevant RAFi. Whereas PLX8394 decreased BRAF-fusion dimerization, CRAF-fusion dimerization is unaffected primarily because of robust protein–protein interactions mediated by the N-terminal non-kinase fusion partner, such as QKI. The pan-RAF dimer inhibitor, LY3009120, could suppress CRAF-fusion oncogenicity by inhibiting dimer-mediated signaling. In addition, as CRAF fusions activate both the MAPK and PI3K/mTOR signaling pathways, we identify combinatorial inhibition of the MAPK/mTOR pathway as a potential therapeutic strategy for CRAF-fusion-driven tumors. Overall, we define a mechanistic distinction between PLGG-associated BRAF- and CRAF/RAF1 fusions in response to RAFi, highlighting the importance of molecularly classifying PLGG patients for targeted therapy. Furthermore, our study uncovers an important contribution of the non-kinase fusion partner to oncogenesis and potential therapeutic strategies against PLGG-associated CRAF fusions and possibly pan-cancer CRAF fusions.