Diffuse Intrinsic Pontine Glioma Growth Research Articles

The relationship between imaging features, therapeutic response, and overall survival in pediatric diffuse intrinsic pontine glioma

We aimed to evaluate the relationship between imaging features, therapeutic responses (comparative cross-product and volumetric measurements), and overall survival (OS) in pediatric diffuse intrinsic pontine glioma (DIPG). A total of 134 patients (≤ 18 years) diagnosed with DIPG were included. Univariate and multivariate analyses were performed to evaluate correlations of clinical and imaging features and therapeutic responses with OS. The correlation between cross-product (CP) and volume thresholds in partial response (PR) was evaluated by linear regression. The log-rank test was used to compare OS patients with discordant therapeutic response classifications and those with concordant classifications. In univariate analysis, characteristics related to worse OS included lower Karnofsky, larger extrapontine extension, ring-enhancement, necrosis, non-PR, and increased ring enhancement post-radiotherapy. In the multivariate analysis, Karnofsky, necrosis, extrapontine extension, and therapeutic response can predict OS. A 25% CP reduction (PR) correlated with a 32% volume reduction (R2 = 0.888). Eight patients had discordant therapeutic response classifications according to CP (25%) and volume (32%). This eight patients’ median survival time was 13.0 months, significantly higher than that in the non-PR group (8.9 months), in which responses were consistently classified as non-PR based on CP (25%) and volume (32%). We identified correlations between imaging features, therapeutic responses, and OS; this information is crucial for future clinical trials. Tumor volume may represent the DIPG growth pattern more accurately than CP measurement and can be used to evaluate therapeutic response.

OTME-22. Bioinformatic evaluation of ECM molecules and angiogenic associate genes in diffuse midline glioma (DMG): mapping the tumour microenvironment

Paediatric Diffuse Intrinsic Pontine Glioma (DIPG) is a devastating cancer of an extremely aggressive nature, located in the pontine area of the brain. DIPG primarily affects children, with the average age of diagnosis between 6 and 7 years. Unfortunately, the outlook and overall survival remains bleak. While there has been impressive progress in identifying genes that are central to and drive DIPG growth; there remains several gaps in documenting the DIPG microenvironment landscape. The focus of this study is to begin to examine mRNA expression of genes associated with blood vessel development, angiogenesis, and extracellular matrix molecules (ECM) in normal brain development and DIPG by utilizing publicly available genomic datasets. In-depth bioinformatics from GSE26576 dataset included differential expression and gene ontology (GO) with KEGG pathway analyses using Gene Expression Omnibus (GEO) and DAVID, which have revealed a number of significantly upregulated genes that may affect DIPG angiogenic processes (p<0.05). 38 of such genes from 9 different GO terms were then included in a protein-to-protein interaction network that revealed a surprising connection between MMP16, CSPG4 and COL11A1. Subsequently, using R2 genomic visualisation platform from publicly available single cell RNAseq data we showcased the difference in their individual expression based on the molecular subtypes of DIPG histone 3 (H3) mutation (K27M, wild type and G34R) with a strong statistical significance (p<0.05). Interestingly, during normal paediatric development such genes showed consistent expression, suggesting their potential complications in DIPG angiogenesis. Overall, this bioinformatic approach has led to the identification of a set of interacting genes that will inform our in vitro and in vivo studies. This information will add to the documentation of the host/tumour microenvironment landscape and our plan is to continue to explore this area to map the spatial and temporal expression of these genes.

OLIG2 maintenance is not essential for diffuse intrinsic pontine glioma cell line growth but regulates tumor phenotypes.

Diffuse intrinsic pontine glioma (DIPG) is a pediatric lethal high-grade brainstem glioma with no effective therapies. OLIG2 (oligodendrocyte transcription factor 2) was reported to be critical for the growth of a DIPG cell line CCHMC-DIPG-1. Surprisingly, we found that the CCHMC-DIPG-1 cells express little OLIG2 and exhibit a mesenchymal phenotype, which raised a question regarding the role of OLIG2 in the growth of DIPG cells. We evaluated the function of OLIG2 in different DIPG cell lines through molecular and genetic approaches and performed transcriptomic and genomic landscape profiling including whole-genome bisulfite sequencing, RNA-seq, ATAC-seq, and ChIP-seq. shRNA-mediated knockdown and CRISPR-Cas9-mediated knockout approaches were utilized to assess OLIG2 functions in DIPG cell growth. We found that DIPG cells are phenotypically heterogeneous and exhibit the characteristics of distinct malignant gliomas including proneural, classical, and mesenchymal subtypes. OLIG2 knockdown did not impact the growth of CCHMC-DIPG-1 cells, wherein OLIG2 is epigenetically silenced. Moreover, OLIG2 deletion did not substantially impair OLIG2-expressing proneural-like DIPG growth but led to an upregulation of HIPPO-YAP1 and epidermal growth factor receptor (EGFR) signaling and a tumor phenotype shift. Targeting HIPPO-YAP1 and EGFR signaling in OLIG2-deficient DIPG cells inhibited tumor cell growth. Our data indicate that OLIG2 is dispensable for DIPG growth but regulates the phenotypic switch of DIPG tumor cells. OLIG2 downregulation leads to deregulation of adaptive YAP1 and EGFR signaling. Targeting YAP1 and EGFR pathways inhibits the growth of OLIG2-deficient DIPG cells, pointing to a therapeutic potential by targeting adaptive signaling to treat DIPG tumors with nominal OLIG2 expression.

Diffuse intrinsic pontine glioma: molecular landscape and emerging therapeutic targets.

Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood brainstem malignancy. Despite advances in understanding of the molecular underpinnings of the tumor in the past decade, the dismal prognosis of DIPG has thus far remained unchanged. This review seeks to highlight promising therapeutic targets within three arenas: DIPG cell-intrinsic vulnerabilities, immunotherapeutic approaches to tumor clearance, and microenvironmental dependencies that promote tumor growth. Promising therapeutic strategies from recent studies include epigenetic modifying agents such as histone deacetylase inhibitors, bromodomain and extra-terminal motif (BET) protein inhibitors, and CDK7 inhibitors. Tumor-specific immunotherapies are emerging. Key interactions between DIPG and normal brain cells are coming to light, and targeting critical microenvironmental mechanisms driving DIPG growth in the developing childhood brain represents a new direction for therapy. Several DIPG treatment strategies are being evaluated in early clinical trials. Ultimately, we suspect that a multifaceted therapeutic approach utilizing cell-intrinsic, microenvironmental, and immunotherapeutic targets will be necessary for eradicating DIPG.

Re-programing Chromatin with a Bifunctional LSD1/HDAC Inhibitor Induces Therapeutic Differentiation in DIPG

H3K27M mutations resulting in epigenetic dysfunction are frequently observed in diffuse intrinsic pontine glioma (DIPGs), an incurable pediatric cancer. We conduct a CRISPR screen revealing that knockout of KDM1A encoding lysine-specific demethylase 1 (LSD1) sensitizes DIPG cells to histone deacetylase (HDAC) inhibitors. Consistently, Corin, a bifunctional inhibitor of HDACs and LSD1, potently inhibits DIPG growth invitro and in xenografts. Mechanistically, Corin increases H3K27me3 levels suppressed by H3K27M histones, and simultaneously increases HDAC-targeted H3K27ac and LSD1-targeted H3K4me1 at differentiation-associated genes. Corin treatment induces cell death, cell-cycle arrest, and a cellular differentiation phenotype and drives transcriptional changes correlating with increased survival time in DIPG patients. These data suggest a strategy for treating DIPG by simultaneously inhibiting LSD1 and HDACs.

Abstract 2888: Targeting the fatal pediatric brain tumors AT/RT and DIPG with the DNA binding agent quinacrine

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) and diffuse intrinsic pontine glioma (DIPG) are incurable pediatric brain tumors. Developing new targets and novel therapeutics are urgently needed. We have previously shown that multiple primary brain tumors and cell lines express increased amounts of the epigenetic modifier high mobility group AT-hook 2 (HMGA2). HMGA2 is a DNA-binding oncoprotein that regulates transcription during normal embryogenesis and in cancer stem cells. Targeting HMGA2 using short hairpins significantly decreased AT/RT and glioma growth and increased survival of xenografted mice. We hypothesized that pharmacological inhibition of HMGA proteins using DNA minor-groove binding drugs like quinacrine will decrease AT/RT and DIPG growth due to displacement of HMGA proteins from the DNA. We used quinacrine in ten patient-derived cell lines: five AT/RT (BT37, CHLA-05, CHLA-06, BT-12, CHLA-266) and five DIPG (JHHDIPG1, SUDIPGXIII, JHHDIPG16A, SF7761, HSJD-007). Quinacrine has been used in millions of humans to treat malaria and parasitic infections, has a well-known safety profile and can penetrate the brain. Using quinacrine fluorescence as a surrogate, we can achieve therapeutically efficacious micromolar concentration of quinacrine in the mouse and zebrafish brain after oral administration without overt toxicity. In both tumor cell lines, quinacrine causes a dose-dependent reduction in growth (MTS) and proliferation (BrdU) compared to vehicle-treated cells (P&amp;lt;0.01). Treatment of both tumor lines with quinacrine significantly increased apoptosis (cleaved caspase-3 and cleaved PARP) compared to control cells (P&amp;lt;0.01). Quinacrine had no effect on growth of normal hindbrain cells. Our results suggest that minor groove binding drugs like quinacrine are a viable potential treatment strategy for these lethal tumors. Ongoing studies include validating the in vivo efficacy of quinacrine using zebrafish and mouse models of AT/RT and DIPG. Future studies are aimed at investigating the mechanism of quinacrine in these devastating tumors. Citation Format: Harpreet Kaur, Huizi Guo, Peter Green, Sepehr Akhtarkhavari, Smit Shah, Charles G. Eberhart, Eric H. Raabe. Targeting the fatal pediatric brain tumors AT/RT and DIPG with the DNA binding agent quinacrine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2888.

Inhibition of mutant PPM1D enhances DNA damage response and growth suppressive effects of ionizing radiation in diffuse intrinsic pontine glioma

Children with diffuse intrinsic pontine glioma (DIPG) succumb to disease within 2 years of diagnosis despite treatment with ionizing radiation (IR) and/or chemotherapy. Our aim was to determine the role of protein phosphatase, magnesium-dependent 1, delta (PPM1D) mutation, present in up to 25% of cases, in DIPG pathogenesis and treatment. Using genetic and pharmacologic approaches, we assayed effects of PPM1D mutation on DIPG growth and murine survival. We assayed effects of targeting mutated PPM1D alone or with IR on signaling, cell cycle, proliferation, and apoptosis in patient-derived DIPG cells in vitro, in organotypic brain slices, and in vivo. PPM1D-mutated DIPG cell lines exhibited increased proliferation in vitro and in vivo, conferring reduced survival in orthotopically xenografted mice, through stabilization of truncated PPM1D protein and inactivation of DNA damage response (DDR) effectors p53 and H2A.X. PPM1D knockdown or treatment with PPM1D inhibitors suppressed growth of PPM1D-mutated DIPGs in vitro. Orthotopic xenografting of PPM1D short hairpin RNA-transduced or PPM1D inhibitor-treated, PPM1D-mutated DIPG cells into immunodeficient mice resulted in reduced tumor proliferation, increased apoptosis, and extended mouse survival. PPM1D inhibition had similar effects to IR alone on DIPG growth inhibition and augmented the anti-proliferative and pro-apoptotic effects of IR in PPM1D-mutated DIPG models. PPM1D mutations inactivate DDR and promote DIPG growth. Treatment with PPM1D inhibitors activated DDR pathways and enhanced the anti-proliferative and pro-apoptotic effects of IR in DIPG models. Our results support continued development of PPM1D inhibitors for phase I/II trials in children with DIPG.

H3.3 K27M depletion increases differentiation and extends latency of diffuse intrinsic pontine glioma growth in vivo.

Histone H3 K27M mutation is the defining molecular feature of the devastating pediatric brain tumor, diffuse intrinsic pontine glioma (DIPG). The prevalence of histone H3 K27M mutations indicates a critical role in DIPGs, but the contribution of the mutation to disease pathogenesis remains unclear. We show that knockdown of this mutation in DIPG xenografts restores K27M-dependent loss of H3K27me3 and delays tumor growth. Comparisons of matched DIPG xenografts with and without K27M knockdown allowed identification of mutation-specific effects on the transcriptome and epigenome. The resulting transcriptional changes recapitulate expression signatures from K27M primary DIPG tumors and are strongly enriched for genes associated with nervous system development. Integrated analysis of ChIP-seq and expression data showed that genes upregulated by the mutation are overrepresented in apparently bivalent promoters. Many of these targets are associated with more immature differentiation states. Expression profiles indicate K27M knockdown decreases proliferation and increases differentiation within lineages represented in DIPG. These data suggest that K27M-mediated loss of H3K27me3 directly regulates a subset of genes by releasing poised promoters, and contributes to tumor phenotype and growth by limiting differentiation. The delayed tumor growth associated with knockdown of H3 K27M provides evidence that this highly recurrent mutation is a relevant therapeutic target.

Intersection of Brain Development and Paediatric Diffuse Midline Gliomas: Potential Role of Microenvironment in Tumour Growth.

Diffuse intrinsic pontine glioma (DIPG) is a devastating and incurable paediatric brain tumour with a median overall survival of 9 months. Until recently, DIPGs were treated similarly to adult gliomas, but due to the advancement in molecular and imaging technologies, our understanding of these tumours has increased dramatically. While extensive research is being undertaken to determine the function of the molecular aberrations in DIPG, there are significant gaps in understanding the biology and the influence of the tumour microenvironment on DIPG growth, specifically in regards to the developing pons. The precise orchestration and co-ordination of the development of the brain, the most complex organ in the body, is still not fully understood. Herein, we present a brief overview of brainstem development, discuss the developing microenvironment in terms of DIPG growth, and provide a basis for the need for studies focused on bridging pontine development and DIPG microenvironment. Conducting investigations in the context of a developing brain will lead to a better understanding of the role of the tumour microenvironment and will help lead to identification of drivers of tumour growth and therapeutic resistance.

Abstract B05: Mechanisms of PPM1D-mediated tumorigenesis in diffuse intrinsic pontine glioma (DIPG)

Abstract Diffuse intrinsic pontine glioma (DIPG), the most common pediatric brainstem tumor, is associated with a dismal prognosis with almost no survivors within two years of diagnosis. It is unamenable to surgical resection, is not responsive to conventional chemotherapy, and responds only temporarily to radiation therapy (XRT). Recent publications identified carboxy (C)-terminal truncating mutations of PPM1D, which encodes the protein WIP1, in up to 25% of DIPGs. WIP1 is a serine/threonine protein phosphatase that plays an important role in DNA damage response (DDR). Prior publications show that most, but not all, PPM1D C-terminal mutants function in a dominant negative fashion to promote tumor growth in cancer models. We hypothesized that PPM1D mutation promotes DIPG growth, and WIP1 inhibition suppresses tumor proliferation. We further hypothesized that, due to its key role in DDR, WIP1 inhibition enhances the antitumor effects of XRT. Using Sanger sequencing, we identified de novo mutation of PPM1D in the C-terminus of multiple patient-derived DIPG cell lines. We also stably introduced clinically relevant PPM1D mutations, which we identified in DIPGs resected from patients, into a PPM1D wild-type DIPG cell line, DIPG VI, as well as into DIPG cells derived from a DIPG mouse model. Using trypan blue exclusion, bioluminescence, and immunofluorescence detection of markers of proliferation and cell death, in the presence or absence of PPM1D knockdown or pharmacologic WIP1 inhibition, and/or following XRT exposure, we measured proliferation of DIPG VI, DIPG VI L513X, a cell line with stable expression of a mutant PPM1D, and DIPG7, a cell line with a de novo PPM1D mutation. We also assayed proliferation in organotypic brain slice cultures derived from symptomatic immunocompromised mice orthotopically xenografted with DIPG cells and of DIPG cells embedded within the brainstem of organotypic brain slices derived from immunocompetent mice. Finally, we orthotopically xenografted DIPG cells into the early post-natal (P0-2) brainstem of immunodeficient mice, treated mice with vehicle or with a small-molecule WIP1 inhibitor, and measured effects on survival, as well as proliferation in tumor cells post-necropsy. Under cell culture conditions, expression of PPM1D mutants promoted tumor cell proliferation in wild-type PPM1D murine and human DIPG cells. Conversely, PPM1D knockdown significantly suppressed DIPG cell proliferation, especially in PPM1D mutated DIPG cells. Similarly, treatment with a small-molecule WIP1 inhibitor suppressed the growth of PPM1D-mutated, but not PPM1D wild-type DIPG cells, in neurosphere cultures, on immunocompetent organotypic brain slices, and on organotypic brain slices derived from symptomatic, orthotopic, xenografted mice. WIP1 inhibition had similar effects on growth inhibition to XRT alone and augmented the antiproliferative and proapoptotic effects of XRT in vitro. Mice bearing an orthotopic xenograft of PPM1D-mutated human DIPG cells, DIPG7, died of disease earlier than mice xenografted with PPM1D wild-type human DIPG cells, DIPG VI. Treatment of mice bearing orthotopically, xenografted DIPG7 cells with a WIP1 inhibitor via a continuous 14-day infusion into the lateral ventricles resulted in a significant reduction in proliferation of tumor cells. In summary, our results show that C-terminal mutated PPM1D is oncogenic in DIPG, promoting proliferation and reduced survival in vivo. WIP1 inhibition suppresses growth and increases the radiosensitivity of PPM1D-mutated DIPG. These results suggest that WIP1 is a druggable target alone and in combination with XRT in DIPG. Further studies will examine the PPM1D-mediated mechanisms of tumorigenesis and treatment responsiveness in DIPG in order to develop clinically viable treatment paradigms that target PPM1D-mutated tumors. Citation Format: Mwangala P. Akamandisa, Kai Nie, Jing Wen, Rita Nahta, Dolores Hambardzumyan, Robert C. Castellino. Mechanisms of PPM1D-mediated tumorigenesis in diffuse intrinsic pontine glioma (DIPG) [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B05.

DIPG-67. HYPOXIA-INDUCIBLE FACTORS REGULATE DIFFUSE INTRINSIC PONTINE GLIOMA GROWTH IN NORMOXIC CULTURE

Diffuse intrinsic pontine glioma (DIPG) are incurable tumors and the leading cause of pediatric brain tumor deaths. They exhibit low blood perfusion and regions of necrosis, indicative of a low-oxygen environment that supports activation of hypoxia-inducible factors (HIF) that are associated with increased proliferation, invasion, and therapy resistance. However, previous reports suggest that HIF2-alpha slows growth in some glioma models. We therefore sought to test the hypothesis that HIFs regulate DIPG growth. We cultured the human DIPG tumors SU-DIPG-IV, VUMC-DIPG-X, and SU-DIPG-XIII at ambient oxygen tension and 5% carbon dioxide. We measured protein expression by Western blot and growth by trypan blue exclusion or tetrazolium reduction following exposure to the hypoxia-mimetic (HM) compounds, cobalt (II) chloride or deferoxamine, or selective HIF inhibitors. All three DIPG cultures retained stable expression of HIF1-alpha and HIF2-alpha protein at ambient oxygen tension, unchanged by HM treatment. Selective inhibition of HIF2-alpha by TC-S 7009 increased apparent growth, whereas selective inhibition of HIF1-alpha by CAY10585 did not. We conclude that hypoxia-independent HIF expression unchanged by either HM treatment or HIF inhibition suggests impaired HIF degradation, in which hypoxia-induced activation of HIF target genes more likely depends on transcriptional co-activators rather than blocked proteasomal degradation. In both ambient and hypoxic conditions, HIF2-alpha activity may oppose DIPG growth. Future experiments will investigate whether the effects of HIF2-alpha inhibition on tumor growth can be explained by enhanced HIF1-alpha activity through desequestration of common binding partners, or through direct action of HIF2-alpha on previously reported apoptotic pathways.

Disrupting NOTCH Slows Diffuse Intrinsic Pontine Glioma Growth, Enhances Radiation Sensitivity, and Shows Combinatorial Efficacy With Bromodomain Inhibition.

NOTCH regulates stem cells during normal development and stemlike cells in cancer, but the roles of NOTCH in the lethal pediatric brain tumor diffuse intrinsic pontine glioma (DIPG) remain unknown. Because DIPGs express stem cell factors such as SOX2 and MYCN, we hypothesized that NOTCH activity would be critical for DIPG growth. We determined that primary DIPGs expressed high levels of NOTCH receptors, ligands, and downstream effectors. Treatment of the DIPG cell lines JHH-DIPG1 and SF7761 with the γ-secretase inhibitor MRK003 suppressed the level of the NOTCH effectors HES1, HES4, and HES5; inhibited DIPG growth by 75%; and caused a 3-fold induction of apoptosis. Short hairpin RNAs targeting the canonical NOTCH pathway caused similar effects. Pretreatment of DIPG cells with MRK003 suppressed clonogenic growth by more than 90% and enhanced the efficacy of radiation therapy. The high level of MYCN in DIPG led us to test sequential therapy with the bromodomain inhibitor JQ1 and MRK003, and we found that JQ1 and MRK003 inhibited DIPG growth and induced apoptosis. Together, these results suggest that dual targeting of NOTCH and MYCN in DIPG may be an effective therapeutic strategy in DIPG and that adding a γ-secretase inhibitor during radiation therapy may be efficacious initially or during reirradiation.