Diffuse Intrinsic Pontine Glioma Cells Research Articles

MELK Inhibition in Diffuse Intrinsic Pontine Glioma.

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.

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.

Abstract PR04: GD2-directed chimeric antigen receptor T cells mediate potent antitumor effect and cure in xenograft models of diffuse intrinsic pontine glioma

Abstract Introduction: Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brainstem tumor with a median survival of less than one year. Despite advances in the understanding of the molecular origins of DIPG, improvement in clinical outcomes has yet to materialize. To date, there has been little target exploration for immunotherapy applications in DIPG. Methods: Patient-derived DIPG cell cultures were screened for expression of more than 350 surface antigens as potential immunotherapeutic targets. The disialoganglioside GD2 was found to have the highest expression across cell cultures and was verified by IHC on post-mortem samples. Chimeric antigen receptor (CAR) T-cell therapy against this target was explored both in vitro and in vivo. Results: We found high levels of the disialoganglioside GD2 expressed on cell cultures derived from post-mortem samples of DIPG. Quantification of the number of GD2 molecules per cell demonstrated higher GD2 expression on DIPG than any other tumors, including neuroblastoma, for which GD2 targeted immunotherapy is part of the standard of care. Most cases of DIPG are caused by a mutation in Histone 3.3 (H3K27M). GD2 is highly and uniformly expressed in patient-derived H3K27M DIPG cultures, whereas H3 wild-type pediatric high-grade gliomas, including those diagnosed as DIPG, do not express significant levels of GD2. The H3K27M mutation is associated with increased levels of enzymes in the ganglioside synthesis pathway, suggesting that expression of the target antigen is driven by H3K27M-induced transcriptional dysregulation. Anti-GD2 CAR T cells with a 4-1BB costimulatory domain demonstrate remarkable preclinical activity against H3K27M DIPG. GD2 CAR T cells specifically kill DIPG cells and produce cytokines IL-2 and IFN- upon coculture with tumor. Systemic administration of anti-GD2 CAR T cells achieves potent and durable cure compared to control T cells in multiple orthotopic xenograft models of DIPG. Using a CAR fluorescent protein fusion construct, we demonstrate significant T-cell trafficking to the brainstem where the antitumor effect is mediated. Universal response was observed across multiple cohorts, and treatment-associated toxicity was transient and tolerated during the period of peak antitumor activity. Conclusion: We have previously demonstrated that antigen density drives CAR efficacy. Extremely high expression of GD2 on DIPG makes this a particularly good disease for CAR T-cell therapy. If these results are predictive of human response, CAR T cells could have a transformative impact upon DIPG outcomes. A clinical trial of second generation anti-GD2 CAR T cells in relapsed and progressive DIPG is planned. Citation Format: Robbie G. Majzner, Christopher Mount, Shree Sundaresh, Evan Arnold, Meena Kadapakkam, Louai Labanieh, Pamelyn Woo, Michelle Monje, Crystal L. Mackall. GD2-directed chimeric antigen receptor T cells mediate potent antitumor effect and cure in xenograft models of diffuse intrinsic pontine glioma [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 PR04.

Abstract B30: Dianhydrogalactitol (VAL-083) has the potential to overcome major challenges in the treatment of DIPG

Abstract Despite several decades of clinical trial, diffuse intrinsic pontine gliomas (DIPG) continue to have a dismal outcome and survival remains dismal. DIPG is inoperable and standard treatment is radiation alone, as the addition of chemotherapy has not improved survival. Major obstacles to the successful treatment of DIPG include an intact blood-brain barrier impeding drug penetration and inherent tumor-cell resistance mechanisms to chemotherapeutics. Dianhydrogalactitol (VAL-083) is a structurally unique bifunctional DNA targeting agent that readily crosses the blood-brain barrier. VAL-083 forms interstrand DNA crosslinks at the N7 position of guanine, leading to persistent and irreversible DNA double-strand breaks, cell cycle arrest, and ultimately cancer cell death. VAL-083 has cytotoxic activity in several pediatric brain tumors as assessed in historical NCI-sponsored clinical trials, both as a single agent and in combination with other chemotherapeutics. We have previously demonstrated that VAL-083 is able to overcome chemoresistance mediated by DNA repair protein O6-methylguanine DNA methyltransferase (MGMT). Expression of MGMT is strongly correlated with resistance to temozolomide (TMZ), which is commonly used in combination with radiation for the treatment of adult brain tumors. VAL-083 activity is also independent of DNA mismatch repair (MMR) system in vitro, a secondary mechanism of resistance to TMZ. VAL-083 potentiates the effect of radiation in TMZ-resistant adult glioblastoma (GBM) cells in vitro and overcomes TMZ resistance in GBM cancer stem cells (CSCs) and non-CSCs. Additionally, VAL-083 demonstrated synergistic efficacy with inhibitors of topoisomerase 1 (camptothecin) and topoisomerase 2 (etoposide) against multiple cancer cell lines. VAL-083’s ability to cross the blood-brain barrier and its ability to overcome common resistance mechanisms, combined with its radiotherapy-potentiating effects, suggest that VAL-083 may provide a new treatment option for DIPG and other pediatric CNS tumors as a single agent, in combination with radiotherapy, or as part of a combination regimen with topoisomerase inhibitors. We recently completed a phase I/II clinical trial in refractory GBM and established a well-tolerated dosing regimen of VAL-083 in adult brain tumor patients. In the present study, we investigated the effects of VAL-083 in combination with radiation or irinotecan (topoisomerase 1 inhibitor) in a panel of DIPG cell lines as well as patient-derived xenografts models. The results will guide a potential clinical trial of VAL-083 in treatment of DIPG, either as part of a chemo-radiation regimen or in combination with topoisomerase inhibitors. Citation Format: Anne Steino, Beibei Zhai, Beibei Zhai, Jeffrey Bacha, Dennis Brown, Shaun Fouse, Joe Costello, Mads Daugaard, Mads Daugaard, Sabine Mueller. Dianhydrogalactitol (VAL-083) has the potential to overcome major challenges in the treatment of 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 B30.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Combined use of CDK4/6 and mTOR inhibitors induce synergistic growth arrest of diffuse intrinsic pontine glioma cells via mutual downregulation of mTORC1 activity.

BackgroundDiffuse intrinsic pontine glioma (DIPG) is a lethal type of pediatric brain tumor that is resistant to conventional chemotherapies. Palbociclib is a putative novel DIPG treatment that restricts the proliferation of rapidly dividing cancer cells via selective inhibition of cyclin-dependent kinase (CDK) 4 and CDK6. However, implementing palbociclib as a monotherapy for DIPG is unfeasible, as CDK4/6 inhibitor resistance is commonplace and palbociclib does not readily cross the blood–brain barrier (BBB) or persist in the central nervous system. To inhibit the growth of DIPG cells, we aimed to use palbociclib in combination with the rapamycin analog temsirolimus, which is known to ameliorate resistance to CDK4/6 inhibitors and inhibit BBB efflux.Materials and methodsWe tested palbociclib and temsirolimus in three patient-derived DIPG cell lines. The expression profiles of key proteins in the CDK4/6 and mammalian target of rapamycin (mTOR) signaling pathways were assessed, respectively, to determine feasibility against DIPG. Moreover, we investigated effects on cell viability and examined in vivo drug toxicity.ResultsImmunoblot analyses revealed palbociclib and temsirolimus inhibited CDK4/6 and mTOR signaling through canonical perturbation of phosphorylation of the retinoblastoma (RB) and mTOR proteins, respectively; however, we observed noncanonical downregulation of mTOR by palbociclib. We demonstrated that palbociclib and temsirolimus inhibited cell proliferation in all three DIPG cell lines, acting synergistically in combination to further restrict cell growth. Flow cytometric analyses revealed both drugs caused G1 cell cycle arrest, and clonogenic assays showed irreversible effects on cell proliferation. Palbociclib did not elicit neurotoxicity in primary cultures of normal rat hippocampi or when infused into rat brains.ConclusionThese data illustrate the in vitro antiproliferative effects of CDK4/6 and mTOR inhibitors in DIPG cells. Direct infusion of palbociclib into the brain, in combination with systemic delivery of temsirolimus, represents a promising new approach to developing a much-needed treatment for DIPG.

Characterization of the immune microenvironment of diffuse intrinsic pontine glioma: implications for development of immunotherapy.

Diffuse intrinsic pontine glioma (DIPG) is a uniformly fatal CNS tumor diagnosed in 300 American children per year. Radiation is the only effective treatment and extends overall survival to a median of 11 months. Due to its location in the brainstem, DIPG cannot be surgically resected. Immunotherapy has the ability to target tumor cells specifically; however, little is known about the tumor microenvironment in DIPGs. We sought to characterize infiltrating immune cells and immunosuppressive factor expression in pediatric low- and high-grade gliomas and DIPG. Tumor microarrays were stained for infiltrating immune cells. RNA was isolated from snap-frozen tumor tissue and Nanostring analysis performed. DIPG and glioblastoma cells were co-cultured with healthy donor macrophages, T cells, or natural killer (NK) cells, and flow cytometry and cytotoxicity assays performed to characterize the phenotype and function, respectively, of the immune cells. DIPG tumors do not have increased macrophage or T-cell infiltration relative to nontumor control, nor do they overexpress immunosuppressive factors such as programmed death ligand 1 and/or transforming growth factor β1. H3.3-K27M DIPG cells do not repolarize macrophages, but are not effectively targeted by activated allogeneic T cells. NK cells lysed all DIPG cultures. DIPG tumors have neither a highly immunosuppressive nor inflammatory microenvironment. Therefore, major considerations for the development of immunotherapy will be the recruitment, activation, and retention of tumor-specific effector immune cells.

Measuring Tumor Metabolism in Pediatric Diffuse Intrinsic Pontine Glioma Using Hyperpolarized Carbon-13 MR Metabolic Imaging.

Objective The purpose of this study was to demonstrate the feasibility of using hyperpolarized carbon-13 (13C) metabolic imaging with [1-13C]-labeled pyruvate for evaluating real-time in vivo metabolism of orthotopic diffuse intrinsic pontine glioma (DIPG) xenografts. Materials and Methods 3D 13C magnetic resonance spectroscopic imaging (MRSI) data were acquired on a 3T scanner from 8 rats that had been implanted with human-derived DIPG cells in the brainstem and 5 healthy controls, following injection of 2.5 mL (100 mM) hyperpolarized [1-13C]-pyruvate. Results Anatomical images from DIPG-bearing rats characteristically exhibited T2-hyperintensity throughout the cerebellum and pons that was not accompanied by contrast enhancement. Evaluation of real-time in vivo13C spectroscopic data revealed ratios of lactate-to-pyruvate (p < 0.002), lactate-to-total carbon (p < 0.002), and normalized lactate (p < 0.002) that were significantly higher in T2 lesions harboring tumor relative to corresponding values of healthy normal brain. Elevated levels of lactate in lesions demonstrated a distinct metabolic profile that was associated with infiltrative, viable tumor recapitulating the histopathology of pediatric DIPG. Conclusions Results from this study characterized pyruvate and lactate metabolism in orthotopic DIPG xenografts and suggest that hyperpolarized 13C MRSI may serve as a noninvasive imaging technique for in vivo monitoring of biochemical processes in patients with DIPG.

Functional diversity and cooperativity between subclonal populations of pediatric glioblastoma and diffuse intrinsic pontine glioma cells.

The failure to develop effective therapies for paediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is in part due to their intrinsic heterogeneity. We aimed to quantitatively map assess the extent to which this was present in these tumours through subclonal genomic analyses, and to determine whether distinct tumour subpopulations may interact to promote tumorigenesis by generating novel subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced cases revealed multiple tumour subclones, spatially and temporally co-existing in a stable manner as observed by multiple sampling strategies. We isolated genotypically and phenotypically distinct subpopulations which we propose co-operate to enhance tumorigenicity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present in <1% of cells, confer an abrogated DNA repair pathway and increased invasion/migration of neighbouring cells, in vitro and in vivo, through chemokine signalling and modulation of integrins. These data indicate that even rare tumour subpopulations may exert profound effects on tumorigenesis as a whole, and may represent a novel avenue for therapeutic development. Unravelling the mechanisms of subclonal diversity and communication in pGBM/DIPG will be an important step to overcoming barriers to effective treatments.

Abstract 2073: Transcriptome profiles of cancer stem-like cells in patient-derived diffuse intrinsic pontine glioma (DIPG)

Abstract Diffuse intrinsic pontine glioma (DIPG) is a rare, but lethal childhood cancer with a 5-year survival less than 1 %. Genetic profiling of DIPG biopsies and post-mortem tissue have recently identified mutations in PI3KCA, PTEN, TP53, ATM/MPL, histones and PDGF receptor overexpression. PI3KCA and PTEN mutations as well as PDGF receptor overexpression indicate upregulation of the PI3K/AKT/mTOR signaling axis, representing druggable targets. We recently developed a multifunctional kinase inhibitor (ST-182), which targets the PI3K/AKT/mTOR and MAPK pathways which is often upregulated in various malignancies as a compensatory mechanism when PI3K is inhibited. We evaluated ST-182's efficacy for targeting these pathways in patient derived DIPG by western blotting and reverse phase protein array analysis (RPPA). Phosphorylation changes of ERK and AKT, downstream signaling inhibition as well as diminished proliferation was shown in two DIPG cell lines (SU-DIPGIV and XIII) when treated with ST-182, indicating efficacy of co-targeting these pathways as a new therapeutic advance for DIPG. FACS analysis of DIPG cells (SU-DIPGXIII) identified a large percentage (&amp;gt;10%) of DIPG cells as ALDH positive indicating aggressive stem like features. Characterization of these distinct DIPG populations (ALDH+,-) at the transcriptome level was performed to understand differences in pathway signaling and to identify potential drug resistance mechanisms to ST-182. Utilizing an innovative transcriptome analysis approach, we identified elevated levels of MYC, E2F and DNA repair genes in ALDH+ cells, supporting stem like phenotype of ALDH+ DIPG cells. MYC has long been identified as a crucial player in maintaining embryonic stem cell pluripotency and self-renewal, whereas E2F provide transcriptional control of stem cell fate and DNA repair mechanisms maintain and regulate cancer stem cells. Pharmacological targeting of MAPK/PI3K/mTOR by ST-182 demonstrated up regulation of NFkB, apoptosis, hypoxia, p53 and inflammatory response in ALDH+ and ALDH- cells and down-regulation of MYC, E2F and DNA replication indicating efficacy of targeting these pathways in preventing/reversing stem-like phenotypes in the ALDH+ cell population. Our findings indicate efficacy of ST-182 for the treatment of ALDH+ cancer stem cells providing impetus for evaluation of molecularly targeted MAPK/PI3K/mTOR therapy. Our comprehensive transcriptome studies provide a new direction for the treatment of DIPG through novel insights into the underlying transcriptomic basis of drug resistant cancer stem cells. Development of new compounds such as ST-182 provides opportunities to implement precision medicine to improve treatment outcomes for DIPG patients. Citation Format: Stefanie Galbán, Carlos Espinoza, Karan Bedi, Uday B. Maachani, Mark M. Souweidane, Mats Ljungman, Marcian Van Dort, Brian D. Ross. Transcriptome profiles of cancer stem-like cells in patient-derived diffuse intrinsic pontine glioma (DIPG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2073.

Abstract 900: Dianhydrogalactitol (VAL-083) has the potential to overcome major challenges in the treatment of DIPG

Abstract Despite decades of clinical trials, children with diffuse intrinsic pontine gliomas (DIPG) continue to have a very poor prognosis and dismal survival. DIPG is inoperable and standard treatment is radiation alone. Major obstacles to the successful treatment of DIPG include 1) an intact blood brain barrier impeding drug penetration, 2) inherent tumor cell resistance mechanisms to conventional chemotherapeutics, and 3) a lack of drug-induced potentiation of radiotherapy. VAL-083 is a structurally unique bi-functional DNA targeting agent that has the potential to overcome these barriers. VAL-083 readily crosses the blood-brain barrier and accumulates in brain tumor tissue where it rapidly forms interstrand DNA crosslinks at guanine-N7, leading to persistent DNA double strand breaks, cell cycle arrest, and cancer cell death. VAL-083 has cytotoxic activity in several pediatric brain tumors as assessed in NCI-sponsored clinical trials, both as a single agent and in combination with other chemotherapeutics. We recently completed a Phase I/II clinical trial in refractory GBM and established a well-tolerated dosing regimen of VAL-083 in adult brain tumor patients. We have previously demonstrated that VAL-083 cytotoxicity is independent of both the primary, DNA repair protein O6-methylguanine DNA methyltransferase (MGMT), and the secondary, DNA mismatch repair (MMR) system, mechanisms of resistance to temozolomide (TMZ) in vitro. TMZ is commonly used in combination with radiation for the treatment of adult brain tumors but has failed to improve the effect of radiation in DIPG. Importantly, VAL-083 potentiates the effect of radiation and overcomes TMZ resistance in TMZ-resistant adult glioblastoma cancer stem cells (CSCs) and non-CSCs at concentrations that are physiologically achievable in the CNS. Additionally, VAL-083 demonstrated synergistic efficacy in prostate and lung cancer cells with topoisomerase (Topo) inhibitors camptothecin and etoposide, commonly used in the treatment of pediatric brain tumors. In conclusion, VAL-083 is able to 1) cross the blood brain barrier, 2) overcome resistance mechanisms to chemotherapeutics commonly used in the treatment of adult gliomas, and 3) potentiate the effects of radiation in adult glioma cells at physiologically achievable concentrations. Thus, VAL-083 may have the potential to overcome major challenges in DIPG treatment as a single agent, in combination with radiotherapy, or as part of a combination regimen with Topo inhibitors. In the present study, we investigated the effects of VAL-083 in combination with radiation, irinotecan (Topo 1 inhibitor), or Wee1 inhibitor AZD1775 in a panel of DIPG cell lines with varying genetic profiles. The results will provide guidance for the design of animal models studying VAL-083 treatment of DIPG, either as part of a chemo-radiation regimen or in combination with Topo or Wee1 inhibitors. Citation Format: Anne Steino, Beibei Zhai, Jeffrey A. Bacha, Dennis M. Brown, Mads Daugaard, Sabine Mueller. Dianhydrogalactitol (VAL-083) has the potential to overcome major challenges in the treatment of DIPG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 900.

Non-inflammatory tumor microenvironment of diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal malignancy of the childhood central nervous system, with a median overall survival of 9–11 months. We have previously shown that primary DIPG tissue contains numerous tumor-associated macrophages, and substantial work has demonstrated a significant pathological role for adult glioma-associated macrophages. However, work over the past decade has highlighted many molecular and genomic differences between pediatric and adult high-grade gliomas. Thus, we directly compared inflammatory characteristics of DIPG and adult glioblastoma (GBM). We found that the leukocyte (CD45+) compartment in primary DIPG tissue samples is predominantly composed of CD11b + macrophages, with very few CD3+ T-lymphocytes. In contrast, T-lymphocytes are more abundant in adult GBM tissue samples. RNA sequencing of macrophages isolated from primary tumor samples revealed that DIPG- and adult GBM-associated macrophages both express gene programs related to ECM remodeling and angiogenesis, but DIPG-associated macrophages express substantially fewer inflammatory factors than their adult GBM counterparts. Examining the secretome of glioma cells, we found that patient-derived DIPG cell cultures secrete markedly fewer cytokines and chemokines than patient-derived adult GBM cultures. Concordantly, bulk and single-cell RNA sequencing data indicates low to absent expression of chemokines and cytokines in DIPG. Together, these observations suggest that the inflammatory milieu of the DIPG tumor microenvironment is fundamentally different than adult GBM. The low intrinsic inflammatory signature of DIPG cells may contribute to the lack of lymphocytes and non-inflammatory phenotype of DIPG-associated microglia/macrophages. Understanding the glioma subtype-specific inflammatory milieu may inform the design and application of immunotherapy-based treatments.

H3.3K27M mutant proteins reprogram epigenome by sequestering the PRC2 complex to poised enhancers

Expression of histone H3.3K27M mutant proteins in human diffuse intrinsic pontine glioma (DIPG) results in a global reduction of tri-methylation of H3K27 (H3K27me3), and paradoxically, H3K27me3 peaks remain at hundreds of genomic loci, a dichotomous change that lacks mechanistic insights. Here, we show that the PRC2 complex is sequestered at poised enhancers, but not at active promoters with high levels of H3.3K27M proteins, thereby contributing to the global reduction of H3K27me3. Moreover, the levels of H3.3K27M proteins are low at the retained H3K27me3 peaks and consequently having minimal effects on the PRC2 activity at these loci. H3K27me3-mediated silencing at specific tumor suppressor genes, including Wilms Tumor 1, promotes proliferation of DIPG cells. These results support a model in which the PRC2 complex is redistributed to poised enhancers in H3.3K27M mutant cells and contributes to tumorigenesis in part by locally enhancing H3K27me3, and hence silencing of tumor suppressor genes.

DIPG-65. PRELIMINARY EXPERIENCE OF CHRONIC INTERMITTENT CONVECTION ENHANCED DELIVERY OF CARBOPLATIN AND VALPROIC ACID FOR THE TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMA FOLLOWING RADIATION THERAPY

Diffuse intrinsic pontine glioma (DIPG) is a lethal disease of middle childhood with few treatment options. The failure of chemotherapy is in part due to poor blood-brain barrier penetration. Convection enhanced delivery (CED) uses direct intraparenchymal infusion to distribute drug through target brain volumes. Valproic acid and carboplatin are readily compatible with CED and kill DIPG cells as monotherapies and with synergy when combined. These drugs were combined and translated into a two-day CED treatment schedule for treatment of DIPG on compassionate grounds using a long-term, implantable, drug delivery system (undergoing approval, Renishaw Plc). This device utilises a bone-anchored transcutaneous port for intermittent drug administration without the need for repeated surgery. 9 patients who had previously received radiotherapy were selected for treatment. Surgical implantation of the device was well-tolerated. A total of 27 cycles (54 infusions) of sodium valproate and carboplatin were delivered achieving infusion volumes between 3.6–5.6 mL per infusion. The most common acute infusion-related side-effects included headache and mild limb weakness and all patients were discharged within 24 hours of finishing treatment. We describe our preliminary experience as a prelude to a forthcoming clinical trial.

DIPG-09. HDAC INHIBITION IN THALAMIC AND SPINAL CORD H3K27M+ DIFFUSE MIDLINE GLIOMA

BACKGROUND Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the leading cause of brain tumor-related deaths in children. There are no effective treatments and median survival remains dismal. Genomics identified a mutation in the majority of DMGs, a lysine to methionine substitution (K27M) in histones 3.1 and 3.3, which causes changes in gene expression that promote gliomagenesis. Panobinostat, a multiple histone deacetylase (HDAC) inhibitor, was one of the most effective agents against patient-derived DIPG cell cultures and xenograft models in previous studies and is presently in clinical trial for DIPG. HDAC inhibition with panobinostat may also exhibit activity against H3K27M+ DMG of the thalamus and spinal cord.

09 Could DLX2 regulation of neural progenitor cell fate contribute to differentiation of diffuse intrinsic pontine glioma (DIPG)?

Introduction: Diffuse intrinsic pontine glioma (DIPG) is refractory to therapy. The identification of histone H3.1/H3.3 K27M mutations in most DIPG has provided new insights. The DLX homeobox genes are expressed in the developing forebrain. The Dlx1/Dlx2 double knockout (DKO) mouse loses tangential GABAergic interneuron migration to the neocortex. We have identified genes that encode glutamic acid decarboxylase (GAD) enzymes as direct targets of DLX1/DLX2. In DIPG patients with H3.3 K27M mutations there is decreased Dlx2 and increased expression of the myelin transcription factor, Myt1. Methods and Results: We used bioinformatics approaches and chromatin immunoprecipitation (ChIP) assays to identify Olig2, Nkx2.2 and Myt1 promoter sequences as candidate DLX2 targets in vivo. DNA binding specificity was confirmed. The functional consequences of Dlx2 co-expression with reporter constructs of ChIP-isolated promoter fragments of Olig2 and Nkx2.2 demonstrated repression of gene targets in vitro. qPCR showed increased Olig2 and Nkx2.2 expression in the DKO forebrain. Stable transfection of a murine DIPG cell line with Dlx2 resulted in increased Gad1 and Gad2 and decreased Olig2 and Nkx2.2 expression. Of significance, we demonstrated decreased expression of H3.3 K27M and restoration of H3.3 K27 tri-methylation (me3). Conclusions: DLX transcription factors promote GABAergic interneuron and concomitant inhibition of oligodendroglial differentiation in neural progenitors by repression of a suite of genes including Olig2 and Nkx2.2. Restoration of H3 K27me3 expression in DIPG provides a promising lead towards exploration of differentiation as a therapeutic strategy for DIPG.

Live-cell single-molecule dynamics of PcG proteins imposed by the DIPG H3.3K27M mutation

Over 80% of diffuse intrinsic pontine gliomas (DIPGs) harbor a point mutation in histone H3.3 where lysine 27 is substituted with methionine (H3.3K27M); however, how the mutation affects kinetics and function of PcG proteins remains elusive. We demonstrate that H3.3K27M prolongs the residence time and search time of Ezh2, but has no effect on its fraction bound to chromatin. In contrast, H3.3K27M has no effect on the residence time of Cbx7, but prolongs its search time and decreases its fraction bound to chromatin. We show that increasing expression of Cbx7 inhibits the proliferation of DIPG cells and prolongs its residence time. Our results highlight that the residence time of PcG proteins directly correlates with their functions and the search time of PcG proteins is critical for regulating their genomic occupancy. Together, our data provide mechanisms in which the cancer-causing histone mutation alters the binding and search dynamics of epigenetic complexes.

Brainstem blood brain barrier disruption using focused ultrasound: A demonstration of feasibility and enhanced doxorubicin delivery

Magnetic Resonance Image-guided Focused Ultrasound (MRgFUS) has been used to achieve transient blood brain barrier (BBB) opening without tissue injury. Delivery of a targeted ultrasonic wave causes an interaction between administered microbubbles and the capillary bed resulting in enhanced vessel permeability. The use of MRgFUS in the brainstem has not previously been shown but could provide value in the treatment of tumours such as Diffuse Intrinsic Pontine Glioma (DIPG) where the intact BBB has contributed to the limited success of chemotherapy. Our primary objective was to determine whether the use of MRgFUS in this eloquent brain region could be performed without histological injury and functional deficits. Our secondary objective was to select an effective chemotherapeutic against patient derived DIPG cell lines and demonstrate enhanced brainstem delivery when combined with MRgFUS in vivo.Female Sprague Dawley rats were randomised to one of four groups: 1) Microbubble administration but no MRgFUS treatment; 2) MRgFUS only; 3) MRgFUS + microbubbles; and 4) MRgFUS + microbubbles + cisplatin. Physiological assessment was performed by monitoring of heart and respiratory rates. Motor function and co-ordination were evaluated by Rotarod and grip strength testing. Histological analysis for haemorrhage (H&E), neuronal nuclei (NeuN) and apoptosis (cleaved Caspase-3) was also performed. A drug screen of eight chemotherapy agents was conducted in three patient-derived DIPG cell lines (SU-DIPG IV, SU-DIPG XIII and SU-DIPG XVII). Doxorubicin was identified as an effective agent. NOD/SCID/GAMMA (NSG) mice were subsequently administered with 5 mg/kg of intravenous doxorubicin at the time of one of the following: 1) Microbubbles but no MRgFUS; 2) MRgFUS only; 3) MRgFUS + microbubbles and 4) no intervention. Brain specimens were extracted at 2 h and doxorubicin quantification was conducted using liquid chromatography mass spectrometry (LC/MS).BBB opening was confirmed by contrast enhancement on T1-weighted MR imaging and positive Evans blue staining of the brainstem. Normal cardiorespiratory parameters were preserved. Grip strength and Rotarod testing demonstrating no decline in performance across all groups. Histological analysis showed no evidence of haemorrhage, neuronal loss or increased apoptosis.Doxorubicin demonstrated cytotoxicity against all three cell lines and is known to have poor BBB permeability. Quantities measured in the brainstem of NSG mice were highest in the group receiving MRgFUS and microbubbles (431.5 ng/g). This was significantly higher than in mice who received no intervention (7.6 ng/g).Our data demonstrates both the preservation of histological and functional integrity of the brainstem following MRgFUS for BBB opening and the ability to significantly enhance drug delivery to the region, giving promise to the treatment of brainstem-specific conditions.

An efficient method for the transduction of primary pediatric glioma neurospheres

Pediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are rare, but rapidly fatal malignancies of the central nervous system (CNS), and the leading cause of cancer-related death in children. Besides the scarcity of available biological material for research, the study of these diseases has been hampered by methodological problems. One of the major obstacles is the difficulty with which these cells can be genetically modified by conventional laboratory methods, such as lentiviral transduction. As a result, only very few successful stable modifications have been reported. As pHGG and DIPG cells are most often cultured as neurospheres, and therefore retain stem cell-like characteristics, we hypothesized that this culture method is also responsible for their resistance to transduction. We therefore developed a protocol in which pHGG and DIPG cells are temporarily forced to form an adherent monolayer by exposure to serum, to create a window-of-opportunity for lentiviral transduction. We here demonstrate that this protocol reliably and reproducibly introduces stable genetic modifications in primary DIPG and pHGG cells.•Short-term serum exposure enables lentiviral transduction of primary pediatric glioma neurospheres.

Abstract PL05-03: Histone-3K27M mutant diffuse midline gliomas of childhood: Epigenetic pathogenesis and therapeutic opportunities

Abstract High-grade gliomas of childhood occur in a striking spatiotemporal pattern, with tumors of the midline occurring chiefly in the first decade of life and tumors of the cerebral lobes occurring later in adolescence and young adulthood. This pattern of gliomagenesis bespeaks dysregulation of a developmental process, and maps well onto discrete waves of developmental myelination of the nervous system, a process that spans over 3 decades in humans. Increasing data from our group and from others implicate myelin-forming precursor cells as putative cells of origin for the most common and best-studied diffuse midline glioma, diffuse intrinsic pontine glioma (DIPG). DIPG is a fatal pediatric cancer peaking in incidence at age 6-7 years with a median survival of only 9 months and 5-year survival of less than 1%. DIPG affects 300-400 children each year in the US and represents the leading cause of brain tumor-related death in children. Therapeutic options are limited, and effective treatment is direly needed. During the early part of this decade, next-generation sequencing technologies enabled a comprehensive view of the DIPG genome. DIPG and other diffuse gliomas of the midline structures of the central nervous system, in particular the thalamus and spinal cord, exhibit a specific and highly recurrent mutation in histone-3 (H3K27M). The H3K27M mutation, occurring in genes encoding histone H3.3 and H3.1, affect ~80% of DIPG cases and the majority of thalamic and spinal cord gliomas as well. The H3K27M mutation causes perturbation of Polycomb repressive complex 2 (PRC2) function, resulting in global hypomethylation of K27 in all H3 variants. The broad epigenetic dysregulation and characteristic gene expression changes that ensue are believed to be central to DIPG oncogenesis. We have generated a panel of patient-derived DIPG cultures and correlate orthotopic xenograft models and have used these new resources for drug screening efforts. DIPG cells are vulnerable to HDAC inhibition, which we found restores H3 trimethyl mark and normalizes gene expression, an unexpected finding that may be explained by actetyl groups on nearby residues sterically interfering with the interaction between PRC2 and the aberrant methionine. We have also found that DIPG is vulnerable to transcriptional disruption using bromodomain inhibition or CDK7 blockade. Targeting oncogenic transcription through either of these methods synergizes with HDAC inhibition, and DIPG cells resistant to HDAC inhibitor therapy retain sensitivity to CDK7 blockade. A clinical trial of panobinostat for DIPG is ongoing, and follow-on trials of combination therapy hold promise. Other forms of diffuse midline gliomas, including H3K27M-mutant gliomas of the spinal cord and thalamus, may respond similarly to therapeutic strategies targeting the regulation of gene expression. Identification of super-enhancers in DIPG highlights oligodendroglial lineage genes, supporting the hypothesis that precursor cells in the oligodendroglial lineage are the cell of origin, and reveals unexpected mechanisms mediating tumor viability and invasion, including potassium channel function and EPH receptor signaling. Also highlighted by super-enhancer analysis are genes traditionally involved in neuronal communication. Normal oligodendroglial lineage precursor cell function is critically regulated by interactions with active neurons during development and as part of ongoing neural plasticity. We have recently shown that neuronal activity robustly promotes the proliferation of both normal and neoplastic glial cells. A key mechanism mediating neuronal influences on glioma growth is the activity-regulated secretion of the synaptic protein neuroligin-3 into the glioma microenvironment. Blocking neuroligin-3 secretion by inhibiting the enzyme responsible for its cleavage and release from normal synapses has proven robustly effective in slowing the growth of DIPG and other gliomas in preclinical models. Targeting neuron-glioma interactions represents a promising and largely unexplored avenue for glioma therapy. A therapeutic strategy is thus emerging that involves a multipronged approach, targeting the cell-intrinsic vulnerabilities of histone-3 mutant gliomas together with key microenvironmental targets central to glioma growth and progression. Increasingly effective therapy for these deadly brain cancers of childhood will be enabled by answers to outstanding basic questions, such as why H3K27M-mutant gliomas occur only in midline structures, how the tumors hijack mechanisms of normal neural development and plasticity, and what mechanisms the tumor utilizes to evolve resistance to therapy. Citation Format: Michelle Monje. Histone-3K27M mutant diffuse midline gliomas of childhood: Epigenetic pathogenesis and therapeutic opportunities [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr PL05-03.