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Purpose: PNOC003 is a multicenter precision medicine trial for children and young adults with newly diagnosed diffuse intrinsic pontine glioma (DIPG)., Patients and Methods: Patients (3-25 years) were enrolled on the basis of imaging consistent with DIPG. Biopsy tissue was collected for whole-exome and mRNA sequencing. After radiotherapy (RT), patients were assigned up to four FDA-approved drugs based on molecular tumor board recommendations. H3K27M-mutant circulating tumor DNA (ctDNA) was longitudinally measured. Tumor tissue and matched primary cell lines were characterized using whole-genome sequencing and DNA methylation profiling. When applicable, results were verified in an independent cohort from the Children's Brain Tumor Network (CBTN)., Results: Of 38 patients enrolled, 28 patients (median 6 years, 10 females) were reviewed by the molecular tumor board. Of those, 19 followed treatment recommendations. Median overall survival (OS) was 13.1 months [95% confidence interval (CI), 11.2-18.4] with no difference between patients who followed recommendations and those who did not. H3K27M-mutant ctDNA was detected at baseline in 60% of cases tested and associated with response to RT and survival. Eleven cell lines were established, showing 100% fidelity of key somatic driver gene alterations in the primary tumor. In H3K27-altered DIPGs, TP53 mutations were associated with worse OS (TP53mut 11.1 mo; 95% CI, 8.7-14; TP53wt 13.3 mo; 95% CI, 11.8-NA; P = 3.4e-2), genome instability (P = 3.1e-3), and RT resistance (P = 6.4e-4). The CBTN cohort confirmed an association between TP53 mutation status, genome instability, and clinical outcome., Conclusions: Upfront treatment-naïve biopsy provides insight into clinically relevant molecular alterations and prognostic biomarkers for H3K27-altered DIPGs., (©2022 The Authors; Published by the American Association for Cancer Research.)

Purpose: Diffuse intrinsic pontine gliomas (DIPG) are the most severe pediatric brain tumors. Although accepted as the standard therapeutic, radiotherapy is only efficient transiently and not even in every patient. The goal of the study was to identify the underlying molecular determinants of response to radiotherapy in DIPG., Experimental Design: We assessed in vitro response to ionizing radiations in 13 different DIPG cellular models derived from treatment-naïve stereotactic biopsies reflecting the genotype variability encountered in patients at diagnosis and correlated it to their principal molecular alterations. Clinical and radiologic response to radiotherapy of a large cohort of 73 DIPG was analyzed according to their genotype. Using a kinome-wide synthetic lethality RNAi screen, we further identified target genes that can sensitize DIPG cells to ionizing radiations., Results: We uncover TP53 mutation as the main driver of increased radioresistance and validated this finding in four isogenic pairs of TP53 WT DIPG cells with or without TP53 knockdown. In an integrated clinical, radiological, and molecular study, we show that TP53 MUT DIPG patients respond less to irradiation, relapse earlier after radiotherapy, and have a worse prognosis than their TP53 WT counterparts. Finally, a kinome-wide synthetic lethality RNAi screen identifies CHK1 as a potential target, whose inhibition increases response to radiation specifically in TP53 MUT cells., Conclusions: Here, we demonstrate that TP53 mutations are driving DIPG radioresistance both in patients and corresponding cellular models. We suggest alternative treatment strategies to mitigate radioresistance with CHK1 inhibitors. These findings will allow to consequently refine radiotherapy schedules in DIPG., (©2019 American Association for Cancer Research.)

Purpose: Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor, for which no effective therapeutic options currently exist. We here determined the potential of inhibition of the maternal embryonic leucine zipper kinase (MELK) for the treatment of DIPG. Experimental Design: We evaluated the antitumor efficacy of the small-molecule MELK inhibitor OTSSP167 in vitro in patient-derived DIPG cultures, and identified the mechanism of action of MELK inhibition in DIPG by RNA sequencing of treated cells. In addition, we determined the blood-brain barrier (BBB) penetration of OTSSP167 and evaluated its translational potential by treating mice bearing patient-derived DIPG xenografts. Results: This study shows that MELK is highly expressed in DIPG cells, both in patient samples and in relevant in vitro and in vivo models, and that treatment with OTSSP167 strongly decreases proliferation of patient-derived DIPG cultures. Inhibition of MELK in DIPG cells functions through reducing inhibitory phosphorylation of PPARγ, resulting in an increase in nuclear translocation and consequent transcriptional activity. Brain pharmacokinetic analyses show that OTSSP167 is a strong substrate for both MDR1 and BCRP, limiting its BBB penetration. Nonetheless, treatment of Mdr1a/b;Bcrp1 knockout mice carrying patient-derived DIPG xenografts with OTSSP167 decreased tumor growth, induced remissions, and resulted in improved survival. Conclusions: We show a strong preclinical effect of the kinase inhibitor OTSSP167 in the treatment of DIPG and identify the MELK-PPARγ signaling axis as a putative therapeutic target in this disease. Clin Cancer Res; 24(22); 5645-57. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Purpose: This study was conducted to investigate the incidence of ERBB1 amplification and overexpression in samples of diffusely infiltrative (WHO grades II-IV) pediatric brain stem glioma (BSG) and determine the relationship of these abnormalities to expression and mutation of TP53 and tumor grade., Experimental Design: After central pathology review, the incidence of ERBB1 amplification and overexpression was determined in 28 samples (18 surgical biopsy and 10 postmortem specimens) of BSG using quantitative PCR and immunohistochemistry, respectively. Mutation and expression of TP53 were also determined in these same samples by direct sequence analysis of microdissected tumor material and immunohistochemistry, respectively. All experimental procedures were performed blind to tumor grade., Results: Twelve, 9, and 7 tumors were classified as WHO grades II, III, and IV, respectively. A significant increase in ERBB1 expression was observed with increasing tumor grade (P < 0.001). Two grade IV tumors displayed intense membranous ERBB1 expression in 90% of tumor cells in association with high-level ERBB1 gene amplification. One grade III tumor also contained low-level amplification of ERBB1. Six tumors demonstrated TP53 nuclear immunoreactivity, and six contained a mutation in TP53. No correlation was observed between abnormalities in TP53 and either tumor grade or amplification and overexpression of ERBB1., Conclusions: These data suggest that ERBB1 signaling is important for the development of childhood BSG and is worthy of study as a therapeutic target in this disease. Our data also indicate that the genetics of childhood BSG are complex and include both grade-dependent amplification and overexpression of ERBB1 and grade-independent expression and mutation of TP53.