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Supplementary Data from DIPG Harbors Alterations Targetable by MEK Inhibitors, with Acquired Resistance Mechanisms Overcome by Combinatorial Inhibition

The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling and drug screening in newly established patient-derived models in vitro and in vivo. We identified in vitro sensitivity to MEK inhibitors in DIPGs harboring MAPK pathway alterations, but treatment of patient-derived xenograft models and a patient at relapse failed to elicit a significant response. We generated trametinib-resistant clones in a BRAFG469V model through continuous drug exposure and identified acquired mutations in MEK1/2 with sustained pathway upregulation. These cells showed hallmarks of mesenchymal transition and expression signatures overlapping with inherently trametinib-insensitive patient-derived cells, predicting sensitivity to dasatinib. Combined trametinib and dasatinib showed highly synergistic effects in vitro and on ex vivo brain slices. We highlight the MAPK pathway as a therapeutic target in DIPG and show the importance of parallel resistance modeling and combinatorial treatments for meaningful clinical translation.Significance:We report alterations in the MAPK pathway in DIPGs to confer initial sensitivity to targeted MEK inhibition. We further identify for the first time the mechanism of resistance to single-agent targeted therapy in these tumors and suggest a novel combinatorial treatment strategy to overcome it in the clinic.This article is highlighted in the In This Issue feature, p. 587

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Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an “intrinsic” spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n = 31), NTRK1/2/3 (n = 21), ROS1 (n = 9), and MET (n = 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management.Significance:Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of ALK, NTRK1/2/3, ROS1, or MET gene fusions. Kinase fusion–positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype.See related video: https://vimeo.com/438254885See related commentary by Szulzewsky and Cimino, p. 904.This article is highlighted in the In This Issue feature, p. 890

Somatic mutations in ACVR1 are found in a quarter of children with diffuse intrinsic pontine glioma (DIPG), but there are no ACVR1 inhibitors licensed for the disease. Using an artificial intelligence–based platform to search for approved compounds for ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (Kd = 150 nmol/L) and reduce DIPG cell viability in vitro but has limited ability to cross the blood–brain barrier. In addition to mTOR, everolimus inhibited ABCG2 (BCRP) and ABCB1 (P-gp) transporters and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination was well tolerated in vivo and significantly extended survival and reduced tumor burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Four patients with ACVR1-mutant DIPG were treated with vandetanib plus an mTOR inhibitor, informing the dosing and toxicity profile of this combination for future clinical studies.Significance:Twenty-five percent of patients with the incurable brainstem tumor DIPG harbor somatic activating mutations in ACVR1, but there are no approved drugs targeting the receptor. Using artificial intelligence, we identify and validate, both experimentally and clinically, the novel combination of vandetanib and everolimus in these children based on both signaling and pharmacokinetic synergies.This article is highlighted in the In This Issue feature, p. 275

Supplementary Figures S1-S3

Somatic mutations in ACVR1 are found in a quarter of children with diffuse intrinsic pontine glioma (DIPG), but there are no ACVR1 inhibitors licensed for the disease. Using an artificial intelligence–based platform to search for approved compounds for ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (Kd = 150 nmol/L) and reduce DIPG cell viability in vitro but has limited ability to cross the blood–brain barrier. In addition to mTOR, everolimus inhibited ABCG2 (BCRP) and ABCB1 (P-gp) transporters and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination was well tolerated in vivo and significantly extended survival and reduced tumor burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Four patients with ACVR1-mutant DIPG were treated with vandetanib plus an mTOR inhibitor, informing the dosing and toxicity profile of this combination for future clinical studies. Significance: Twenty-five percent of patients with the incurable brainstem tumor DIPG harbor somatic activating mutations in ACVR1, but there are no approved drugs targeting the receptor. Using artificial intelligence, we identify and validate, both experimentally and clinically, the novel combination of vandetanib and everolimus in these children based on both signaling and pharmacokinetic synergies. This article is highlighted in the In This Issue feature, p. 275

Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an “intrinsic” spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n = 31), NTRK1/2/3 (n = 21), ROS1 (n = 9), and MET (n = 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management. Significance: Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of ALK, NTRK1/2/3, ROS1, or MET gene fusions. Kinase fusion–positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype. See related video: https://vimeo.com/438254885 See related commentary by Szulzewsky and Cimino, p. 904. This article is highlighted in the In This Issue feature, p. 890

Paediatric-type diffuse high-grade gliomas are classified into distinct subgroups based upon their location and defining molecular alterations, with very poor clinical outcomes in patients >3yrs. This extensive inter-tumour heterogeneity is further complicated by a wide diversity of genotypically- and phenotypically-distinct subclonal populations within individual tumours, providing a substantial barrier to developing effective treatments. We have sought to understand the dynamic cellular make-up of PDHGGs such that novel strategies aimed at targeting specific subpopulations based upon their contribution to disease progression as whole may be employed. Two complementary approaches have been undertaken to address this – first by carrying out single-cell profiling of bulk specimens, and the second isolation and propagation of single-cell-derived stem cell-like cultures in vitro. To-date we have studied 10 cases and a total of 218 subclonal colonies from both DMG-H3K27 and DHG-WT. In a spinal metastatic case of DMG-H3K27, lpWGS-FISH highlighted subpopulations driven by mis-segregation of amplified oncogenic ecDNA, and mutually exclusive subpopulations defined by MYCN, PDGFRA and CCND1. Through integrated analysis of scRNA-seq and scATAC-seq, we show distinct chromatin accessibility profiles to underlie gene expression signatures defining unique subpopulations of cells. In addition to cycling populations and those associated with lineage-specificity, we identified ‘aggressive’ subpopulations defined by significant upregulation of immediate early response genes such as FOS/FOSL1/JUN, those associated with promotion of invasion-migration such as SERPINs and MMPs. These subpopulations could be mapped to isolated single-cell-derived subclones with highly proliferative or motile phenotypes, defined by comprehensive profiling of expressed and secreted proteins. Differential cis-regulation driving cell identity-tumorigenesis was found in one example to occur via a trans-histone mechanism mediated by an H4-lysine-methyltransferase, KMT5B. Application of functionally-defined interventional strategies aimed at disrupting the interactions between these subpopulations based upon evolutionary biology principles may offer a novel approach to treat these otherwise incurable tumours in children and young adults.

BACKGROUND: Gliomatosis cerebri (GC), a radiologically defined diffusely infiltrating glioma, is no longer considered a distinct entity since the 2016 WHO classification for tumors of the CNS. Due to its rarity and dismal prognosis treatment recommendations in children remain ambiguous. Using central neuroradiological review, we performed a multi-institutional, retrospective study of GC providing comprehensive radiological, clinical, and (epi)genetic characterization. RESULTS: We included 104 patients between 1-19 years. Within a median follow-up of 15.5 months (range, 2.3-138.8), 93 patients (89.4 %) had died, 4 (3.8 %) were lost to follow-up and 7 (6.8 %) were alive with stable/progressive disease. Median progression-free- (PFS) and overall survival (OS) were 8.6 months (interquartile range, 4.3-14.0) and 15.5 months (10.9-27.7), respectively. Former WHO grading correlated significantly with median OS: WHO °II: 47.8 months (25.2-55.7); WHO °III: 15.9 months (11.4-26.3); WHO °IV: 10.4 months (8.8-14.4) (p

The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling and drug screening in newly established patient-derived models in vitro and in vivo. We identified in vitro sensitivity to MEK inhibitors in DIPGs harboring MAPK pathway alterations, but treatment of patient-derived xenograft models and a patient at relapse failed to elicit a significant response. We generated trametinib-resistant clones in a BRAFG469V model through continuous drug exposure and identified acquired mutations in MEK1/2 with sustained pathway upregulation. These cells showed hallmarks of mesenchymal transition and expression signatures overlapping with inherently trametinib-insensitive patient-derived cells, predicting sensitivity to dasatinib. Combined trametinib and dasatinib showed highly synergistic effects in vitro and on ex vivo brain slices. We highlight the MAPK pathway as a therapeutic target in DIPG and show the importance of parallel resistance modeling and combinatorial treatments for meaningful clinical translation. Significance: We report alterations in the MAPK pathway in DIPGs to confer initial sensitivity to targeted MEK inhibition. We further identify for the first time the mechanism of resistance to single-agent targeted therapy in these tumors and suggest a novel combinatorial treatment strategy to overcome it in the clinic. This article is highlighted in the In This Issue feature, p. 587

Paediatric-type diffuse high-grade gliomas are classified into distinct subgroups based upon their location and defining molecular alterations, with very poor clinical outcomes in patients >3yrs. This extensive inter-tumour heterogeneity is further complicated by a wide diversity of genotypically- and phenotypically-distinct subclonal populations within individual tumours, providing a substantial barrier to developing effective treatments. We aimed to understand the dynamic cellular make-up of PDHGGs such that novel strategies aimed at targeting specific subpopulations based upon their contribution to disease progression as whole may be employed. Two complementary approaches have been undertaken to address this – first by carrying out single-cell profiling of bulk specimens, and the second isolation and propagation of single-cell-derived stem cell-like cultures in vitro. To-date we have studied 10 cases and a total of 218 subclonal colonies from both DMG-H3K27 and DHG-WT. In a spinal metastatic case of DMG-H3K27, lpWGS/FISH highlighted subpopulations driven by mis-segregation of amplified oncogenic ecDNA, and mutually exclusive subpopulations defined by MYCN, PDGFRA and CCND1. Through integrated analysis of scRNA-seq and scATAC-seq, we show distinct chromatin accessibility profiles to underlie gene expression signatures defining unique subpopulations of cells. In addition to cycling populations and those associated with lineage-specificity, we identified ‘aggressive’ subpopulations defined by significant upregulation of immediate early response genes such as FOS/FOSL1/JUN, those associated with promotion of invasion/migration such as SERPINs and MMPs. These subpopulations could be mapped to isolated single-cell-derived subclones with highly proliferative and/or motile phenotypes, defined by comprehensive profiling of expressed and secreted proteins. Differential cis-regulation driving cell identity/tumorigenesis was found in one example to occur via a trans-histone mechanism mediated by an H4-lysine-methyltransferase, KMT5B. Application of functionally-defined interventional strategies aimed at disrupting the interactions between these subpopulations based upon evolutionary biology principles may offer a novel approach to treat these otherwise incurable tumours in children and young adults.

SUMMARYDIPG are characterised by histone H3K27M mutations, resulting in global loss of the repressive mark H3K27me3, although certain key loci are retained. We recently identified subclonal loss-of-function mutations in the H4 lysine methyltransferase KMT5B to be associated with enhanced invasion/migration, but the mechanism by which this occurred was unclear. Here we use integrated ChIP-seq, ATAC-seq and RNA-seq on patient-derived, subclonal and CRISPR-Cas9-KD DIPG cells to show that loss of KMT5B/C causes depletion of these retained H3K27me3 loci via changes in chromatin accessibility, causing a raft of transcriptional changes which promote tumorigenesis. De-repression occurred at bivalent loci marked by H3K4me3, driving increased transcriptional heterogeneity and elevated gene expression associated with increased invasion, abrogated DNA repair and mesenchymal transition, along with a markedly altered secretome. These data suggest a previously unrecognised trans-histone (H4/H3) interaction in DIPG cells with a potentially profound effect on their diffusely infiltrating phenotype.

PDHGG are a diverse group of childhood brain tumours comprising multiple subgroups carrying distinct molecular drivers. Patient-derived models accurately recapitulating this underlying biology are critical for mechanistic/preclinical studies aimed at improving patient outcome, however their behaviour over time in the environments in which they are propagated, and how this relates to the human disease, is largely unknown. To explore this, we collected 94 models of PDHGG established as 2D/3D stem cell cultures in vitro, and generated patient-derived xenografts (PDX) in 33/62 specimens implanted orthotopically in vivo. We carried out exome/targeted sequencing, methylation profiling and RNAseq to profile cells through their first 25 passages in culture, and sequential implantation from p0-p2 in mice. In 15/83 cultures, we observed enrichment of gene expression signatures of non-malignant cells over the first 5 passages, with concurrent depletion of somatic mutations/CNAs, excluding them from further study. Validated models retained tumour-matched genotypes, CNAs and driver alterations including H3.3G34R, H3.3/H3.1K27M, BRAF and ACVR1 over time, however subclonal alterations underwent selection in culture which profoundly altered their response to targeted drug treatment. In 6/7 PDGFRA-mutant models, activating mutations were selected against between p5-20 in 2D and/or 3D, whilst MAPK pathway mutations in NF1/PIK3R1 similarly diverged over 15 passages under different growth conditions, resulting in isogenic models with differential signalling, in vivo tumorigenicity, and in vitro sensitivity to multiple MEK inhibitors. In PDXs, serial xenografting reduced the time to tumour formation by up to half, with a concomitant shift in clonal architecture. Multi-region sequencing of diffusely-infiltrating tumours showed selection for alterations such as PIK3CA/NF1 at distant sites, with evidence for convergent evolution of subclonal mutations, as in human tumours. Understanding the evolutionary dynamics of targetable/predictive alterations in PDHGG model systems is key to developing new and effective therapeutic interventions in this highly heterogenous disease.

Background The use of liquid biopsy is of potential high importance for children with high grade (HGG) and diffuse midline gliomas (DMG), particularly where surgical procedures are limited, and invasive biopsy sampling not without risk. To date, however, the evidence that detection of cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA) could provide useful information for these patients has been limited, or contradictory. Methods We optimized droplet digital PCR (ddPCR) assays for the detection of common somatic mutations observed in pediatric HGG/DMG, and applied them to liquid biopsies from plasma, serum, cerebrospinal fluid (CSF), and cystic fluid collected from 32 patients. Results Although detectable in all biomaterial types, ctDNA presented at significantly higher levels in CSF compared to plasma and/or serum. When applied to a cohort of 127 plasma specimens from 41 patients collected from 2011 to 2018 as part of a randomized clinical trial in pediatric non-brainstem HGG/DMG, ctDNA profiling by ddPCR was of limited use due to the small volumes (mean = 0.49 mL) available. In anecdotal cases where sufficient material was available, cfDNA concentration correlated with disease progression in two examples each of poor response in H3F3A_K27M-mutant DMG, and longer survival times in hemispheric BRAF_V600E-mutant cases. Conclusion Tumor-specific DNA alterations are more readily detected in CSF than plasma. Although we demonstrate the potential of the approach to assessing tumor burden, our results highlight the necessity for adequate sample collection and approach to improve detection if plasma samples are to be used.

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.

Somatic mutations in ACVR1 are found in a quarter of children with diffuse intrinsic pontine glioma (DIPG), however there are no ACVR1 inhibitors licensed for the disease. Using an Artificial Intelligence-based platform to search for approved compounds for ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (Kd=150nM) and reduce DIPG cell viability in vitro, but has limited ability to cross the blood-brain-barrier. In addition to mTOR, everolimus inhibits ABCG2 (BCRP) and ABCB1 (P-gp) transporters, and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination is well-tolerated in vivo, and significantly extended survival and reduced tumor burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Four patients with ACVR1-mutant DIPG were treated with vandetanib plus mTOR inhibitor, informing the dosing and toxicity profile of this combination for future clinical studies.

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.

Methylation array-based molecular profiling has redefined the classification of brain tumours and now forms an important part of their integrated diagnosis, providing both subgroup assignment and genome wide DNA copy number profiles. These latter data can be used to identify intragenic breakpoints which are frequently associated with structural variations resulting in therapeutically targetable oncogenic fusion genes. To systematically assess the landscape of these alterations, we combined publicly available methylation datasets resulting in a total of 5660 CNS tumours, around half paediatric, and including >1000 high grade glioma and DIPG. These were analysed by standard methodology (MNP, conumee), and intragenic breakpoint enrichment was compared within methylation subgroups, superfamilies, and tumours with no high-scoring classification. Benchmarking included sequence-verified cases such as infant hemispheric gliomas (IHG) with ALK(15%) and ROS1(7%) fusions, and pathognomic alterations associated with specific entities such as RELA-EPN, MYB-LGG and HGNET-MN1. We identified previously unreported enrichments of well-recognised fusion targets such as NTRK2in GBM_MID and NTRK3in DMG_K27 (both 5%), METin A_IDH / A_IDH_HG (3–5%), and FGFR1/3in GBM_G34 (8–9%). Novel recurrent kinase gene candidates to be verified and explored further include IGF1Rin 2–12% cases spanning glioma subgroups, and TIE1in poorly classified tumours. This latter ‘NOS’ group were also enriched in various transcription factor targets of breakpoints, including TCF4and PLAGL2. Despite limitations due to sample quality, resolution or balanced translocations, breakpoint analysis of methylation copy number profiles provides simple screening for structural rearrangements which may directly influence targeted therapy in paediatric CNS tumours.

High-grade gliomas harboring H3 G34R/V mutations exclusively occur in the cerebral hemispheres of adolescents and young adults, suggesting a distinct neurodevelopmental origin. Combining multimodal bulk and single-cell genomics with unbiased genome-scale CRISPR/Cas9 approaches, we here describe a GABAergic interneuron progenitor lineage as the most likely context from which these H3 G34R/V mutations drive gliomagenesis, conferring unique and tumor-selective gene targets essential for glioma cell survival, as validated genetically and pharmacologically. Phenotypically, we demonstrate that while H3 G34R/V glioma cells harbor the neurotransmitter GABA, they are developmentally stalled, and do not induce the neuronal hyperexcitability described in other glioma subtypes. These findings offer a striking counter-example to the prevailing view of glioma origins in glial precursor cells, resulting in distinct cellular, microenvironmental, and therapeutic consequences.

Diffuse intrinsic pontine glioma (DIPG) are incurable childhood brain tumours marked by alterations in histone 3 in the form of somatic K27M mutations and loss of H3K27 trimethylation (H3K27me3). These highly aggressive tumours display extensive intratumoral heterogeneity, despite their relatively modest mutational burden. We recently proposed a model whereby co-operative subclonal interactions may exist to promote tumorigenesis in DIPG, and identified rare cancer stem cell populations to harbour mutations in the histone H4 lysine methyltransferase KMT5B. Although not previously described in glioma, loss of H4K20me2/3 caused by abrogated H4 methyltransferase function has been linked to defects in the DNA damage response and enhanced invasion in breast and pancreatic cancer. In order to explore whether dysregulation of H4 methylation may be directly responsible for tumorigenic phenotypes in DIPG, we targeted the methyltransferases by genetically engineering numerous isogenic cells from patient-derived in vitro models using CRISPR/Cas9 to knock out KMT5B and/or KMT5C, alongside pharmacological treatment with the small molecule KMT5B/C inhibitor A-196. We observed the expected reduction in H4K20me2 and H4K20me3 in response to inhibition of KMT5B/C, with an attendant increase in H3K27me3. Knock-out of KMT5B caused a significant increase in tumour cell migration and invasion in vitro, whilst KMT5C-deficient cells had reduced motility. Double knock-outs presented an intermediate phenotype. With H4K20me2/3 playing an important role in DNA repair via 53BP1 recruitment, we further observed a significantly enhanced efficacy of the PARP inhibitors olaparib and talazoparib in knock-out DIPG cells in vitro. Ongoing analysis of differential binding of post-translational modifications by ChIP-seq will provide important insights into the underlying mechanisms of epigenetic dysregulation, particularly in respect of the cross-talk between H3 and H4 marks. Although often present only in small tumour cell populations, H4K20me2/3 and KMT5B/C may represent an important novel aspect of DIPG tumorigenesis.

The survival of children with DIPG remains dismal, and new treatments are desperately needed. The development of patient-specific in vitro and in vivo models represents one such opportunity, however the time taken to establish such models and the rapid disease progression has been thought to limit the utility of such an approach. We sought to explore a co-clinical trial model for DIPG patients enrolled in an ongoing biopsy-stratified study in order to identify rational therapeutic options with individualised preclinical evidence as to their efficacy. To date we have established novel patient-derived in vitro cultures from biopsy specimens of 11 patients, in both 2D (laminin matrix) and 3D (neurosphere) conditions, as well as orthotopic xenografts in vivo, with a high concordance in their molecular profile compared to the original tumour specimen (methylation BeadArray, exome, RNA sequencing). Cells were screened against a series of common and bespoke FDA-approved drugs based upon previous evidence in DIPG and/or the specific molecular alterations found in the patient sample. We identified a high degree of in vitro sensitivity to the MEK inhibitor trametinib (GI50 23-312nM) in samples which harboured genetic alterations targeting the MAPK pathway, specifically the non-canonical BRAF_G469V mutation and those affecting PIK3R1(N564D, H450E_VF_ins), with assessment of tumour volume in vivo by MRI. Allelic imbalance of PIK3R1_N564D by stochastic selection was observed in two independent cultures from the same patient showing a greater response to trametinib (6.4-fold) as well as a decreased sensitivity to dasatinib in the mutant-enriched culture (38-fold). RNAseq of the cultures revealed differential gene expression associated with hypoxia and interferon signalling linked to drug sensitivity. These data show the feasibility in generating patient-specific, testable hypotheses that may be clinically translated in a subset of patients, and we are currently exploring parallel resistance modelling to further inform novel treatment strategies at tumour progression.

The survival of children with DIPG remains dismal, with new treatments desperately needed. In the era of precision medicine, targeted therapies represent an exciting treatment opportunity, yet resistance can rapidly emerge, playing an important role in treatment failure. In a prospective biopsy-stratified clinical trial (BIOMEDE), we combined detailed molecular profiling (methylation BeadArray, exome, RNAseq, phospho-proteomics) linked to drug screening in newly-established patient-derived models of DIPG in vitro and in vivo. We identified a high degree of in vitro sensitivity to the MEK inhibitor trametinib (GI50 16-50nM) in samples which harboured genetic alterations targeting the MAPK pathway, including the non-canonical BRAF_G469V mutation, and those affecting PIK3R1. Treatment of PDX models and the patient with trametinib at relapse, however, failed to elicit a significant response. We generated trametinib-resistant clones (62-188-fold, GI50 2.4–5.2µM) in the BRAF_G469V model through continuous drug exposure, and identified acquired mutations in MEK1/2 (MEK1_K57N, MEK1_I141S and MEK2_I115N) with sustained pathway up-regulation. These cells showed the hallmarks of mesenchymal transition, with overexpression of key proteins involved in invasion/migration, such as collagen-family proteins, integrins, MMPs and AHNAK2, amongst others. Resistant clones were conversely sensitive to the upstream receptor tyrosine kinase inhibitor dasatinib (GI50 36-93nM), and combinations of trametinib with dasatinib and the downstream ERK inhibitor ulixertinib showed synergistic effects in vitro. These data highlight the MAPK pathway as a therapeutic target in DIPG, and show the importance of parallel resistance modelling and rational combinatorial treatments likely to be required for meaningful clinical translation.

Approximately 15% of paediatric/young adult cerebral hemispheric glioblastomas (pGBM) harbour G34R/V mutations in H3F3A, encoding the histone H3.3 variant. Development of novel therapeutic interventions demands models that accurately recapitulate this subset of disease and sensitive imaging methods with which to study tumours in situ. Three H3F3A_G34R primary-patient-derived cultures, alongside established cell-line KNS42 (H3F3A_G34V), were implanted orthotopically in immunocompromised mice. KNS42 (TP53_R342*) tumours were clearly detectable using T2-weighted (T2w)-MRI, enhanced following contrast agent administration, indicating impaired blood-brain barrier (BBB) integrity, and demonstrated minimal invasion. OPBG_GBM_001 cells (TP53_89-90X,ATRX_II2133-2144X) formed infiltrative tumours that were hyperintense on T2w-MRI and demonstrated contrast-enhancement suggestive of heterogeneous BBB integrity. HSJD_GBM_002 cells (TP53_P278T,ATRX_R666*) spread diffusely throughout the brain with their full extent typically not discernible by T2w-MRI, the BBB also remaining intact. No evidence of CHOP_GBM_001 tumour was detected by MRI 11months post-implantation. Immunocompetent syngeneic models using tumour cells induced by mutations modelling hemispheric pGBM (NRAS/shP53/shATRX±H3.3G34R) are being explored. Fast growing heterogeneous lesions with variable contrast-enhancement were identified; the H3.3G34R mutation conferred longer median survival (2 clones:25/28days, control:14days). These models have the advantage of an intact immune system and short latency for initial efficacy studies. Primary pGBM cells yield tumours that are more representative of the spectrum of clinical disease; variable hyperintensity on T2w-MRI corresponding to cellular density, with diffusely infiltrative disease less clearly definable, a paucity of oedema and a range of contrast-enhancement. Pathological features including giant multinucleated cells, and mitotic figures were also evident.

H3.3G34R/V mutations are restricted to glioblastomas of the cerebral hemispheres, and occur predominantly in adolescents and young adults. We had previously shown these mutations to result in a global re-organisation of the activating mark H3K36me3 to drive transcription of key developmental transcription factors and oncogenes such as MYCN, however the precise mechanism was unclear. Using multiple H3G34R/V samples and ChIP-seq with antibodies specific to both wild-type and mutant histone H3.3, we show a high degree of incorporation of mutant histone into nucleosomes, with only a minority (

The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In the era of precision medicine, targeted therapies represent an exciting treatment opportunity, yet resistance can rapidly emerge, playing an important role in treatment failure. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling (methylation BeadArray, exome, RNAseq, phospho-proteomics) linked to drug screening in newly-established patient-derived models of DIPG in vitro and in vivo. We identified a high degree of in vitro sensitivity to the MEK inhibitor trametinib (GI50 16-50nM) in samples, which harboured genetic alterations targeting the MAPK pathway, including the non-canonical BRAF_G469V mutation, and those affecting PIK3R1 and NF1. However, treatment of PDX models and of a patient with trametinib at relapse failed to elicit a significant response. We generated trametinib-resistant clones (62-188-fold, GI50 2.4–5.2µM) in the BRAF_G469V model through continuous drug exposure, and identified acquired mutations in MEK1/2 (MEK1_K57N, MEK1_I141S and MEK2_I115N) with sustained pathway up-regulation. These cells showed the hallmarks of mesenchymal transition, and expression signatures overlapping with inherently trametinib-insensitive primary patient-derived cells that predicted an observed sensitivity to dasatinib. Combinations of trametinib with dasatinib and the downstream ERK inhibitor ulixertinib showed highly synergistic effects in vitro. These data highlight the MAPK pathway as a therapeutic target in DIPG, and show the importance of parallel resistance modelling and rational combinatorial treatments likely to be required for meaningful clinical translation.

Infant gliomas have been considered as early-onset examples of tumour types found throughout paediatric populations. Interestingly, infant high-grade gliomas (HGG) have a better overall survival compared to older children, indicating that grading may not accurately reflect the biology of these tumours. We have to-date collected 181 cases of diffuse glioma (WHO II-IV) occurring in children aged

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.

We developed an integrated chip for real-time amplification and detection of nucleic acid using pH-sensing complementary metal-oxide semiconductor (CMOS) technology. Here we show an amplification-coupled detection method for directly measuring released hydrogen ions during nucleotide incorporation rather than relying on indirect measurements such as fluorescent dyes. This is a label-free, non-optical, real-time method for detecting and quantifying target sequences by monitoring pH signatures of native amplification chemistries. The chip has ion-sensitive field effect transistor (ISFET) sensors, temperature sensors, resistive heating, signal processing and control circuitry all integrated to create a full system-on-chip platform. We evaluated the platform using two amplification strategies: PCR and isothermal amplification. Using this platform, we genotyped and discriminated unique single-nucleotide polymorphism (SNP) variants of the cytochrome P450 family from crude human saliva. We anticipate this semiconductor technology will enable the creation of devices for cost-effective, portable and scalable real-time nucleic acid analysis.

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.