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High throughput omics approaches provide an unprecedented opportunity for dissecting molecular mechanisms in cancer biology. Here we present deep profiling of whole proteome, phosphoproteome and transcriptome in two high-grade glioma (HGG) mouse models driven by mutated RTK oncogenes, PDGFRA and NTRK1, analyzing 13,860 proteins and 30,431 phosphosites by mass spectrometry. Systems biology approaches identify numerous master regulators, including 41 kinases and 23 transcription factors. Pathway activity computation and mouse survival indicate the NTRK1 mutation induces a higher activation of AKT downstream targets including MYC and JUN, drives a positive feedback loop to up-regulate multiple other RTKs, and confers higher oncogenic potency than the PDGFRA mutation. A mini-gRNA library CRISPR-Cas9 validation screening shows 56% of tested master regulators are important for the viability of NTRK-driven HGG cells, including TFs (Myc and Jun) and metabolic kinases (AMPKa1 and AMPKa2), confirming the validity of the multiomics integrative approaches, and providing novel tumor vulnerabilities., Multi-omic profiling is a powerful approach to dissecting molecular mechanisms in disease. Here the authors generate whole proteome, phosphoproteome and transcriptome profiles from two mouse models of high-grade glioma driven by different oncogenes, and validate identified master regulators with a CRISPR screen.

Diffuse intrinsic pontine glioma (DIPG) is predominantly diagnosed clinically, with biopsy often reserved for cases with atypical imaging features and/or symptoms. Outcome data and molecular profiling of DIPG have been limited almost entirely to tumors with typical features. We examined the clinicopathologic characteristics and outcomes of 28 patients with pontine-centered lesions who underwent diagnostic biopsy at our institution from 2003 to 2018 because of atypical MRI findings. The median age at diagnosis was 5.2 years (range, 0.7–17.1), and the median symptom duration pre-diagnosis was 1 month (range, 0–24). Common atypical features included pontine asymmetry, tumor extension into the medulla and cerebellar peduncles, and exophytism. Four patients (14.3%) developed transient neurologic symptoms; one (3.6%) experienced a new, permanent cranial neuropathy; and no biopsy-related deaths occurred. Sixteen of the 28 tumors (57%) were confirmed histologically as DIPGs (WHO grade II–IV gliomas; “atypical DIPG”). Of these 16 cases, H3 K27M status was assessed through immunohistochemistry in 14 (88%) and nine were immunopositive (64.3%). The remainder (“atypical non-DIPGs”) were pilocytic astrocytomas (58.3%), gangliogliomas (8.3%), CNS embryonal tumors, NOS (16.7%), or C19MC-altered embryonal tumors with multilayered rosettes (16.7%). With a median follow-up of 28.6 months (range, 3.0 – 173.2), 2-year overall survival (OS) was 72.4%, 83.3%, and 62.2% for the total cohort, atypical non-DIPGs, and atypical DIPGs, respectively (P=NS for all). However, OS with H3 K27M–mutant atypical DIPG was inferior to that with H3 WT atypical DIPG (P=0.003). Compared to OS in a contemporary typical DIPG cohort (N=100), OS was significantly longer for the total atypical cohort, atypical non-DIPGs, and atypical DIPGs (P

A greater understanding of the unique drivers of pediatric high-grade gliomas (pHGG) is needed as the two-year survival rate is only 10–30%. We recently discovered the presence of structural variants in 47% of pHGG. Recurrent fusions were found involving neurotrophin receptors (NTRK) in 7% of all pHGG and 40% of non-brainstem HGG in infants. One chimeric gene we identified encoded the ETS transcription factor, ETV6, fused to the kinase domain of NTRK3 causing ligand-independent kinase activity. ETV6-NTRK3 has been identified in other neoplasms including congenital-infantile fibrosarcoma and AML. To examine the role of ETV6-NTRK3 in glioma formation in vivo, we developed an implantation model. Cortical astrocytes with conditional loss of p53 in neural stem cells were harvested from mice at P2, transduced with a vector expressing ETV6-NTRK3, and were implanted into the cortex of adult mice. Even with a transduction efficiency of only 1.5–2%, all mice implanted with transduced astrocytes developed HGG with a median survival of 36 days. Next, to study ETV6-NTRK3 in the development of pediatric gliomas, we generated a spontaneous tumor model in which mice expressed a conditional knock-in ETV6-NTRK3 allele and conditional loss of p53 in primitive neural stem cells. Mice expressing ETV6-NTRK3 and null p53 showed rapid development and complete penetrance of high-grade glioma with a median survival of 18 days. Evidence of HGG development was readily detectable by P5, accurately recapitulating onset of pediatric gliomagenesis. Analysis showed ETV6-NTRK3 primary astrocytes had increased proliferation compared to control astrocytes and HGGs driven by ETV6-NTRK3 showed a high proliferative index, MAPK, PI3K and PLCγ activity. In vitro and in vivo models were employed to identify oncogenic targets downstream of NTRK fusions and for preclinical testing of TRK selective inhibitors.

High-grade Brainstem glioma (BSG), also known as Diffuse Intrinsic Pontine Glioma (DIPG), is an incurable pediatric brain cancer. Increasing evidence supports the existence of regional differences in gliomagenesis such that BSG is thought to be a distinct disease from glioma of the cerebral cortex (CG). In an effort to elucidate unique characteristics of BSG, we conducted expression analysis of mouse PDGF-B-driven BSG and CG initiated in Nestin progenitor cells and identified a short list of expression changes specific to the brainstem gliomagenesis process, including upregulation of paired box 3 (Pax3). In the neonatal mouse brain, while Pax3 expression is absent from the cerebral cortex, its expression marks a subset of brainstem progenitor cells, mirroring its regional expression in glioma. Ectopic expression of Pax3 in normal brainstem progenitors in vitro shows that Pax3 inhibits apoptosis. In vivo, Pax3 enhances PDGF-B-driven gliomagenesis by significantly shortening tumor latency (p = 0.02 by log rank test), and increasing tumor penetrance (from 25% to 80%, p = 0.0003 by Fisher's exact test), in a region-specific manner, while loss of Pax3 function in PDGF-B-driven;p53-deficient BSG significantly delays glioma formation (median survival of 38 versus 28.5 days, p = 0.0007 by log rank test). Importantly, Pax3 is regionally expressed in human glioma as well, with high PAX3 mRNA characterizing 40% of human BSG, revealing a subset of tumors that significantly associates with PDGFRA alterations and amplifications of cell cycle regulatory genes, and is exclusive of ACVR1 mutations. Collectively, these data suggest that regional Pax3 expression not only marks a novel subset of BSG but also contributes to PDGF-B-induced brainstem gliomagenesis.