Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor that is located in the pons and primarily affects children. Nearly 80% of DIPGs harbor mutations in histone H3 genes, wherein lysine 27 is substituted with methionine (H3K27M). H3K27M has been shown to inhibit polycomb repressive complex 2 (PRC2), a multiprotein complex responsible for the methylation of H3 at lysine 27 (H3K27me), by binding to its catalytic subunit EZH2. Although DIPGs with the H3K27M mutation show global loss of H3K27me3, several genes retain H3K27me3. Here we describe a mouse model of DIPG in which H3K27M potentiates tumorigenesis. Using this model and primary patient-derived DIPG cell lines, we show that H3K27M-expressing tumors require PRC2 for proliferation. Furthermore, we demonstrate that small-molecule EZH2 inhibitors abolish tumor cell growth through a mechanism that is dependent on the induction of the tumor-suppressor protein p16. Genome-wide enrichment analyses show that the genes that retain H3K27me3 in H3K27M cells are strong polycomb targets. Furthermore, we find a highly significant overlap between genes that retain H3K27me3 in the DIPG mouse model and in human primary DIPGs expressing H3K27M. Taken together, these results show that residual PRC2 activity is required for the proliferation of H3K27M-expressing DIPGs, and that inhibition of EZH2 is a potential therapeutic strategy for the treatment of these tumors.

BackgroundDiffuse intrinsic pontine glioma (DIPG), or high-grade brainstem glioma (BSG), is one of the major causes of brain tumor-related deaths in children. Its prognosis has remained poor despite numerous efforts to improve survival. Panobinostat, a histone deacetylase inhibitor, is a targeted agent that has recently shown pre-clinical efficacy and entered a phase I clinical trial for the treatment of children with recurrent or progressive DIPG.MethodsA collaborative pre-clinical study was conducted using both a genetic BSG mouse model driven by PDGF-B signaling, p53 loss, and ectopic H3.3-K27M or H3.3-WT expression and an H3.3-K27M orthotopic DIPG xenograft model to confirm and extend previously published findings regarding the efficacy of panobinostat in vitro and in vivo.ResultsIn vitro, panobinostat potently inhibited cell proliferation, viability, and clonogenicity and induced apoptosis of human and murine DIPG cells. In vivo analyses of tissue after short-term systemic administration of panobinostat to genetically engineered tumor-bearing mice indicated that the drug reached brainstem tumor tissue to a greater extent than normal brain tissue, reduced proliferation of tumor cells and increased levels of H3 acetylation, demonstrating target inhibition. Extended consecutive daily treatment of both genetic and orthotopic xenograft models with 10 or 20 mg/kg panobinostat consistently led to significant toxicity. Reduced, well-tolerated doses of panobinostat, however, did not prolong overall survival compared to vehicle-treated mice.ConclusionOur collaborative pre-clinical study confirms that panobinostat is an effective targeted agent against DIPG human and murine tumor cells in vitro and in short-term in vivo efficacy studies in mice but does not significantly impact survival of mice bearing H3.3-K27M-mutant tumors. We suggest this may be due to toxicity associated with systemic administration of panobinostat that necessitated dose de-escalation.

Diffuse intrinsic pontine glioma (DIPG) is a lethal childhood brainstem tumour, with a quarter of patients harbouring somatic mutations in ACVR1 , encoding the serine/threonine kinase ALK2. Despite being an amenable drug target, little has been done to-date to systematically evaluate the role of ACVR1 in DIPG, nor to screen currently available inhibitors in patient-derived tumour models. Here we show the dependence of DIPG cells on the mutant receptor, and the preclinical efficacy of two distinct chemotypes of ALK2 inhibitor in vitro and in vivo. We demonstrate the pyrazolo[1,5-a]pyrimidine LDN-193189 and the pyridine LDN-214117 to be orally bioavailable and well-tolerated, with good brain penetration. Treatment of immunodeprived mice bearing orthotopic xenografts of H3.3K27M, ACVR1 R206H mutant HSJD-DIPG-007 cells with 25 mg/kg LDN-193189 or LDN-214117 for 28 days extended survival compared with vehicle controls. Development of ALK2 inhibitors with improved potency, selectivity and advantageous pharmacokinetic properties may play an important role in therapy for DIPG patients.

Glioblastoma multiforme (GBM) is the most lethal primary intracranial malignant neoplasm in adults and most resistant to treatment. Integration of gene therapy and chemotherapy, chemovirotherapy, has the potential to improve treatment. We have introduced an intravenous bacteriophage (phage) vector for dual targeting of therapeutic genes to glioblastoma. It is a hybrid AAV/phage, AAVP, designed to deliver a recombinant adeno‐associated virus genome (rAAV) by the capsid of M13 phage. In this vector, dual tumor targeting is first achieved by phage capsid display of the RGD4C ligand that binds the α v β 3 integrin receptor. Second, genes are expressed from a tumor‐activated and temozolomide (TMZ)‐induced promoter of the glucose‐regulated protein, Grp78 . Here, we investigated systemic combination therapy using TMZ and targeted suicide gene therapy by the RGD4C/AAVP‐ Grp78 . Firstly, in vitro we showed that TMZ increases endogenous Grp78 gene expression and boosts transgene expression from the RGD4C/AAVP‐ Grp78 in human GBM cells. Next, RGD4C/AAVP‐ Grp78 targets intracranial tumors in mice following intravenous administration. Finally, combination of TMZ and RGD4C/AAVP‐ Grp78 targeted gene therapy exerts a synergistic effect to suppress growth of orthotopic glioblastoma.

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Retinoblastoma is a pediatric solid tumor of the retina activated upon homozygous inactivation of the tumor suppressorRB1. VCN-01 is an oncolytic adenovirus designed to replicate selectively in tumor cells with high abundance of free E2F-1, a consequence of a dysfunctional RB1 pathway. Thus, we reasoned that VCN-01 could provide targeted therapeutic activity against even chemoresistant retinoblastoma. In vitro, VCN-01 effectively killed patient-derived retinoblastoma models. In mice, intravitreous administration of VCN-01 in retinoblastoma xenografts induced tumor necrosis, improved ocular survival compared with standard-of-care chemotherapy, and prevented micrometastatic dissemination into the brain. In juvenile immunocompetent rabbits, VCN-01 did not replicate in retinas, induced minor local side effects, and only leaked slightly and for a short time into the blood. Initial phase 1 data in patients showed the feasibility of the administration of intravitreous VCN-01 and resulted in antitumor activity in retinoblastoma vitreous seeds and evidence of viral replication markers in tumor cells. The treatment caused local vitreous inflammation but no systemic complications. Thus, oncolytic adenoviruses targeting RB1 might provide a tumor-selective and chemotherapy-independent treatment option for retinoblastoma.

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Low-flow vascular malformations are congenital overgrowths composed of abnormal blood vessels potentially causing pain, bleeding and obstruction of different organs. These diseases are caused by oncogenic mutations in the endothelium, which result in overactivation of the PI3K/AKT pathway. Lack of robust in vivo preclinical data has prevented the development and translation into clinical trials of specific molecular therapies for these diseases. Here, we demonstrate that the Pik3ca H1047R activating mutation in endothelial cells triggers a transcriptome rewiring that leads to enhanced cell proliferation. We describe a new reproducible preclinical in vivo model of PI3K-driven vascular malformations using the postnatal mouse retina. We show that active angiogenesis is required for the pathogenesis of vascular malformations caused by activating Pik3ca mutations. Using this model, we demonstrate that the AKT inhibitor miransertib both prevents and induces the regression of PI3K-driven vascular malformations. We confirmed the efficacy of miransertib in isolated human endothelial cells with genotypes spanning most of human lowflow vascular malformations.