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Paediatric high grade glioma and diffuse midline glioma (including DIPG) are comprised of multiple biological and clinical subgroups, the majority of which urgently require novel therapies. Patient-derivedin vitroprimary cell cultures represent potentially useful tools for mechanistic and preclinical investigation based upon their retention of key features of tumour subgroups under experimental conditions amenable to high-throughput approaches. We present 17 novel primary cultures derived from patients in London, Dublin and Belfast, and together with cultures established or shared from Barcelona, Brisbane, Rome and Stanford, assembled a panel of 52 models under 2D (laminin matrix) and/or 3D (neurospheres) conditions, fully credentialed by phenotypic and molecular comparison to the original tumour sample (methylation BeadArray, panel/exome sequencing, RNAseq). In screening a subset of these against a panel of ~400 approved chemotherapeutics and small molecules, we identified specific dependencies associated with tumour subgroups and/or specific molecular markers. These includedMYCN-amplified cells and ATM/DNA-PK inhibitors, and DIPGs withPPM1Dactivating truncating mutations and inhibitors of MDM2 or PARP1. Specific mutations inPDGFRAwere found to confer sensitivity to a range of RTK inhibitors, though not all such mutations conferred sensitivity to targeted agents. Notably, dual PDGFRA/FGFR and downstream pathway MEK inhibitors showed profound effects against both PDGFRA-sensitising mutant and FGFR1-dependent non-brainstem pHGG and DIPG. In total, 85% cells were found to have at least one drug screening hit in short term assays linked to the underlying biology of the patient’s tumour, providing a rational approach for individualised clinical translation.

Paediatric high-grade glioma comprise multiple biological and clinical subgroups, the majority of which urgently require novel therapies. Patient-derived models represent useful tools for mechanistic and preclinical investigations based upon their retention of key genetic/epigenetic features and their amenability to high-throughput approaches. We have collected ~100 in vitro models representing multiple subtypes (H3.3/H3.2/H3.1K27M, H3.3G34R/V, BRAF, MYCN_amp, NTRK_fusion, hypermutator, others) established under 2D (laminin) and/or 3D (neurosphere) conditions, credentialed by phenotypic (growth, invasion/migration) and molecular (methylation array, DNA sequencing, RNAseq) comparison to the original tumour sample. These were derived from patients at our local hospitals (n=29), as part of national co-clinical trials (n=19), from international collaborating centres (n=11), or shared directly by research groups worldwide (n=45). These have variously been subjected to pharmacological (approved/experimental drug libraries) and/or genetic screening (whole-genome CRISPR) to identify specific biological dependencies. Many have been established as orthotopic xenografts in vivo (PDX), with detailed pathological and radiological correlations with the clinical disease, and with tumorigenic latencies ranging from 48–435 days. This resource has allowed us to identify genotype-specific synthetic lethalities and responses to targeted inhibitors, including olaparib (PARP) with ATRX, nutlin-3 (MDM2) with PPM1D, AZD1775 (WEE1) with TP53, and CYC065 (CDK9) with MYCN-amplification. Combinatorial screening highlighted synergies in ACVR1-mutant DIPG between novel ALK2 inhibitors and ONC201 (DRD2). Rapid screening allows for feedback of drug sensitivities to treating clinicians at relapse, whilst mechanistic underpinning of these interactions and use of the models to identify specific mediators of resistance will allow for rational future trial design.

Paediatric high grade glioma (pHGG) are defined by recurrent mutations in H3 histones, as well as frequent alterations in the SWI/SNF chromatin remodelling gene ATRX (α-thalassemia mental retardation X-linked), although the precise role of ATRX in tumorigenesis remains unclear. We sought to explore this using genomic analysis of patient samples, CRISPR-Cas9 engineered isogenic ATRX knockout (KO) cell lines and primary-patient-derived cultures. In combined retrospective and prospective cohorts of pHGG samples, we found ATRX mutations in 95/510 (18.6%) cases (27% hemispheric glioblastoma, 13% diffuse midline glioma), with the majority of truncating mutations found in the ADD domain, and missense mutations almost exclusively in the helicase domain. ATRX mutations commonly co-segregate with H3.3G34 and TP53 mutations, and define a subgroup of patients with a longer overall survival, though with a greater number of somatic mutations and copy number alterations than wild-type cases. CRISPR/Cas9-mediated ATRX KO targeting the ADD domain in TP53 mutant paediatric glioblastoma cells lead to loss of imprinting at the H19 locus in concert with upregulation of a pro-invasive transcriptional programme, though a slower rate of orthotopic tumour growth in vivo. ATRX deficient cells showed an abrogated DNA damage response, with prolonged accumulation of gH2AX foci after irradiation, and an increased dependency on PARP1 through persistent parylation and stalled replication forks. Screening ATRX-deficient isogenics and patient-derived cells against a library of >400 chemotherapeutics and small molecules identified a specific dependency for ATRX loss and sensitivity to distinct PARP inhibitor chemotypes, including catalytic inhibitors (olaparib, rucaparib), and PARP trappers (talazoparib). ATRX deficiency further conferred an enhanced radiosensitization of olaparib in vitro and a prolonged survival of mice treated with combined PARP inhibition and radiotherapy in vivo. These data suggest a synthetic lethality for PARP inhibitors in ATRX-deficient pHGG, and may represent a novel therapeutic strategy for these highly aggressive tumours.

Paediatric high grade glioma and diffuse midline glioma (including DIPG) are comprised of multiple biological and clinical subgroups, the majority of which urgently require novel therapies. Patient-derived in vitro primary cell cultures represent potentially useful tools for mechanistic and preclinical investigation based upon their retention of key features of tumour subgroups under experimental conditions amenable to high-throughput approaches. We established 21 novel primary cultures derived from patients in London, Dublin and Rome, and together with cultures shared from Barcelona, Brisbane and Stanford we assembled a panel of 42 models under 2D (laminin matrix) and/or 3D (neurospheres) conditions, fully credentialed by phenotypic and molecular comparison to the original tumour sample (methylation BeadArray, panel/exome sequencing, RNAseq). Screening against a panel of ~400 approved chemotherapeutics and small molecules, we identified specific dependencies associated with tumour subgroups and/or specific molecular markers. Examples included cells with sensitizing (HSJD-GBM-001, PDGFRA_A385ins; HSJD-DIPG-008, PDGFRA_D846N) or resistance (HSJD-GBM-002, PDGFRA_D842Y) mutations to a range of PDGFRA inhibitors, and individual models showing profound sensitivity to multiple FGFR (QCTB-R006) or EGFR (HSJD-DIPG-012) inhibitors. H3.3G34R cells were differentially sensitive to agents targeting the proteasome, whilst H3.3 K27M cells were responsive to crizotinib. MAPK-dysregulated PXA-like cultures differentially responded to inhibitors of upstream signalling via PKC and CK2. In total, 85% cells were found to have at least one drug screening hit in short term assays linked to the underlying biology of the patient’s tumour, providing a rational approach for individualised clinical translation.

The embryonic epicardium is an important source of cardiovascular precursor cells and paracrine factors required for adequate heart formation. During embryonic heart formation, WT1 is mainly expressed in epicardial cells and epicardial derived cells. Its expression has been used to trace epicardial derivatives in embryos and recently it has been used to follow the reactivation of epicardial cells after myocardial infarction. Interestingly, the highest level of expression of WT1 during epicardium development correlates with the highest proliferative state, stem cell properties, and migratory capacity of epicardial cells. Here, we review the various types of tools and strategies used to study WT1 function in the embryonic epicardium and provide examples of their use.

The embryonic epicardium is an important source of cardiovascular precursor cells and paracrine factors required for adequate heart formation. During embryonic heart formation, WT1 is mainly expressed in epicardial cells and epicardial derived cells. Its expression has been used to trace epicardial derivatives in embryos and recently it has been used to follow the reactivation of epicardial cells after myocardial infarction. Interestingly, the highest level of expression of WT1 during epicardium development correlates with the highest proliferative state, stem cell properties, and migratory capacity of epicardial cells. Here, we review the various types of tools and strategies used to study WT1 function in the embryonic epicardium and provide examples of their use.

Paediatric high grade glioma and diffuse midline glioma (including DIPG) are comprised of multiple biological and clinical subgroups, the majority of which urgently require novel therapies. Patient-derived in vitro primary cell cultures represent potentially useful tools for mechanistic and preclinical investigation based upon their retention of key features of tumour subgroups under experimental conditions amenable to high-throughput approaches. We established 21 novel primary cultures derived from patients in London, Dublin and Rome, and together with cultures shared from Barcelona, Brisbane and Stanford we assembled a panel of 42 models under 2D (laminin matrix) and/or 3D (neurospheres) conditions, fully credentialed by phenotypic and molecular comparison to the original tumour sample (methylation BeadArray, panel/exome sequencing, RNAseq). Screening against a panel of ~400 approved chemotherapeutics and small molecules, we identified specific dependencies associated with tumour subgroups and/or specific molecular markers. This allowed for functional annotation of distinct variants in human tumours, for example cells with sensitizing (HSJD-GBM-001, PDGFRA_A385ins; HSJD-DIPG-008, PDGFRA_D846N) or resistance (HSJD-GBM-002, PDGFRA_D842Y) mutations to a range of PDGFRA inhibitors. We found individual models showing profound sensitivity to distinct kinase inhibitors based upon cell-specific mechanisms of activation, such as QCTB-R006 to multiple FGFR-targeted drugs, and HSJD-DIPG-012 to those directed against EGFR. Subclasses with functionally relevant pathway-based dependencies included sensitivity of DIPGs with PPM1Dmutation (HSJD-DIPG-008, HSJD-DIPG-014) to PARP and MDM2 inhibitors, and MAPK-dysregulated PXA-like cultures (ICR-CXJ-008, ICR-CXJ015) differentially responsive to inhibitors of upstream signalling viaPKC and CK2. Of note, all cultures were insensitive to temozolomide. In total, 85% cells were found to have at least one drug screening hit in short term assays linked to the underlying biology of the patient’s tumour, providing a rational approach for individualised clinical translation.

Paediatric glioblastomas (pGBM) are amongst the most common causes of cancer-related deaths in children, and are defined by highly recurrent mutations in H3 histones. Mutations affecting the chromatin remodeling protein ATRX have been reported in 30% of pGBM cases, and are strongly associated with the alternative lengthening of telomeres (ALT) phenotype, but their precise interaction with histone mutations and their role in tumorigenesis remain unclear. We collected sequence data from 262 published and 64 unpublished cases of pGBM and identified somatic ATRX mutations in 54/326 (17%) of cases. ATRX mutations are mainly loss of function mutations, with the majority of frameshift mutations (37/54, 68,5%) found upstream of the helicase domain resulting in truncation of the main functional domain of ATRX. Missense mutations (16/54, 29,6%) reside almost exclusively in the helicase domain (11/54, 20,4%), whereas nonsense mutations are a less common event (7/54, 13%) but present in both the helicase (4/7, 57,1%) and ADD domains (3/7, 42,9%). ATRX mutations commonly co-segregate with H3.3 G34 (16/54) and TP53 (42/54) mutations, and define a subgroup of patients with a longer overall survival (16 months median overall survival in mutant ATRX cases versus 11 months in wild-type ATRX cases, COXPH p = 0.079), though with a greater number of somatic mutations (MWU p = 0.023) and copy number alterations (MWU p = 0.0011) than wild-type cases. We screened a series of 21 primary patient-derived pGBM cell cultures for histone and ATRX mutation status in addition to ATRX protein expression and ALT, and subjected the panel to a high-throughput in vitro cell viability screen of >400 chemotherapeutics and small molecules. We identified a specific genetic dependency for ATRX mutation and sensitivity to distinct PARP inhibitor chemotypes, including olaparib and rucaparib (PARP catalytic inhibitors), and talazoparib (PARP trapper inhibitor). These data were validated using CRISPR-Cas9-engineered ATRX knockout, targeting either the ADD or helicase domain, in SF188 pGBM cells. Gene editing was confirmed by IonTorrent sequencing and Western blot. ATRX mutant clones were also more sensitive to drugs targeting DNA damage response pathways such as bleomycin and sapacitabine. Gene expression analysis of ATRX mutant pGBM samples confirmed an intact homologous recombination pathway and overexpression of PARP1, suggesting an underlying mechanism distinct from that observed in BRCA-mutant breast and ovarian cancers. Ongoing work is aimed at unravelling the specific pathways involved, and evaluating the utility of PARP inhibition in orthotopic pGBM xenografts in vivo. These data suggest a synthetic lethality for PARP inhibitors in ATRX-deficient pGBM cells, and may represent a novel therapeutic strategy for these highly aggressive tumours in children. Citation Format: Janat Fazal-Salom, Mara Vinci, Diana Carvalho, Helen Pemberton, Stephen J. Pettitt, Christopher J. Lord, Alan Mackay, Lynn Bjerke, Chris Jones. Mutations in ATRX increase genetic instability and sensitivity to PARP inhibitors in paediatric glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1932. doi:10.1158/1538-7445.AM2017-1932