Patient-derived Diffuse Intrinsic Pontine Glioma Cell Lines Research Articles

AB005. Development of tumour specific therapy for treatment of diffuse intrinsic pontine glioma (DIPG).

Diffuse intrinsic pontine glioma (DIPG), is an aggressive form of paediatric high-grade glioma (pHGG) that affects children below the age of 10 months. The survival period for a child suffering from DIPG has not changed in decades (approximately 10 months). This pattern is similar for most pHGG; even though the survival period is more extended, tumour recurrence and death are almost inevitable. This is primarily due to the presence of the blood-brain barrier (BBB), which blocks the entry of most therapeutics into the brain, and also due to tumour heterogeneity associated with central nervous system (CNS) tumours that blunt the efficacy of targeted therapy. The development of a meaningful cure for paediatric brain cancer hinges on discovering chemotherapy agents that (I) can cross the BBB; (II) accumulates explicitly in tumour tissues; and (III) can block pathways leading to the escape of cancer stem cells, promoting recurrence. This project aims to develop therapeutics that can cross the BBB, a significant hindrance to delivering medicines across the brain, and specifically target cancer cells without affecting normal brain cells. We will accomplish this by attaching novel dyes possessing tumour specificity to various classes of chemotherapy agents. The compounds will be tested on patient-derived paediatric brain cancer cell lines and the most potent compounds will be progressed to an animal model of DIPG. Several drug-dye conjugates were designed and synthesized to target various aberrant pathways involved in disease initiation and progression of DIPG. These were tested first in patient-derived DIPG cell lines. Several of these drug-dye conjugates showed potent antiproliferative effect in various DIPG cell lines. One of these conjugates is currently undergoing maximum tolerated dose study in an animal model of DIPG. The present work details an effort to develop BBB crossing tumour specific therapeutic agents for the treatment of DIPG. The work has resulted in several promising drug-dye conjugates showing antiproliferative activity in various patient-derived DIPG cell lines, enabling the progression of such conjugates into animal models of DIPG. Such studies will inform the utility of such drug-dye conjugates for application in difficult to treat pHGGs such as DIPG.

DIPG-69. IN-VITRO DETERMINATION OF 5-AMINOLEVULINIC-ACID-TREATMENT EFFICACY IN PATIENT-DERIVED DIPG MODELS

Abstract BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) presents a significant clinical challenge due to its inoperable nature and poor response to current treatments. Sonodynamic Therapy (SDT) is a non-invasive therapy that combines low-intensity ultrasound stimulation with a sonosensitizing agent. 5-Aminolevulinic Acid (5-ALA) as a sonosensitizer has yielded positive outcomes in in-vivo glioblastoma models, and a clinical trial (NCT05123534) for DIPG patients is currently ongoing. 5-ALA is a precursor of Protoporphyrin IX (PpIX), which preferentially accumulates in cancer cells and generates cytotoxic reactive oxygen species upon activation by ultrasound or light. Our work is aimed at investigating the accumulation of PpIX in in-vitro DIPG patient-derived cell models to predict the efficacy of 5-ALA SDT treatment. METHODS Patient derived DIPG cell lines (H3K27M, P53 mutant and wild type) were used to create three-dimensional spheroids. The spheroids were dosed with 5-ALA for 4 hours, and PpIX accumulation was quantified by measuring its fluorescence intensity within the cells. The spheroids were then exposed to controlled light stimulation as an in-vitro proxy for SDT. RESULTS We confirmed the uptake of 5-ALA and subsequent PpIX accumulation in all patient-derived DIPG spheroids. Light exposure of 5-ALA treated DIPG spheroids resulted in reduced viability of the tumor models, while 5-ALA itself did not show cytotoxic effects. The extent of PpIX accumulation and cytotoxicity upon PpIX light activation varied among DIPG models with higher sensitivity of p53 wild type cells. Further studies incorporating a larger number of DMG cells with p53 alterations are underway. CONCLUSIONS These findings indicate that SDT using 5-ALA as sonosensitizer may offer clinical benefits for treatment of DIPG, however treatment efficacy could vary considerably between patients. In vitro evaluation of PpIX accumulation and sensitivity to activation could potentially provide a means to stratify patients and to identify those most likely to benefit from 5-ALA-mediated SDT.

A combined immunopeptidomics, proteomics, and cell surface proteomics approach to identify immunotherapy targets for diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG), recently reclassified as a subtype of diffuse midline glioma, is a highly aggressive brainstem tumor affecting children and young adults, with no cure and a median survival of only 9 months. Conventional treatments are ineffective, highlighting the need for alternative therapeutic strategies such as cellular immunotherapy. However, identifying unique and tumor-specific cell surface antigens to target with chimeric antigen receptor (CAR) or T-cell receptor (TCR) therapies is challenging. In this study, a multi-omics approach was used to interrogate patient-derived DIPG cell lines and to identify potential targets for immunotherapy. Through immunopeptidomics, a range of targetable peptide antigens from cancer testis and tumor-associated antigens as well as peptides derived from human endogenous retroviral elements were identified. Proteomics analysis also revealed upregulation of potential drug targets and cell surface proteins such as Cluster of differentiation 27 (CD276) B7 homolog 3 protein (B7H3), Interleukin 13 alpha receptor 2 (IL-13Rα2), Human Epidermal Growth Factor Receptor 3 (HER2), Ephrin Type-A Receptor 2 (EphA2), and Ephrin Type-A Receptor 3 (EphA3). The results of this study provide a valuable resource for the scientific community to accelerate immunotherapeutic approaches for DIPG. Identifying potential targets for CAR and TCR therapies could open up new avenues for treating this devastating disease.

Reversal of cancer gene expression identifies repurposed drugs for diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is an aggressive incurable brainstem tumor that targets young children. Complete resection is not possible, and chemotherapy and radiotherapy are currently only palliative. This study aimed to identify potential therapeutic agents using a computational pipeline to perform an in silico screen for novel drugs. We then tested the identified drugs against a panel of patient-derived DIPG cell lines. Using a systematic computational approach with publicly available databases of gene signature in DIPG patients and cancer cell lines treated with a library of clinically available drugs, we identified drug hits with the ability to reverse a DIPG gene signature to one that matches normal tissue background. The biological and molecular effects of drug treatment was analyzed by cell viability assay and RNA sequence. In vivo DIPG mouse model survival studies were also conducted. As a result, two of three identified drugs showed potency against the DIPG cell lines Triptolide and mycophenolate mofetil (MMF) demonstrated significant inhibition of cell viability in DIPG cell lines. Guanosine rescued reduced cell viability induced by MMF. In vivo, MMF treatment significantly inhibited tumor growth in subcutaneous xenograft mice models. In conclusion, we identified clinically available drugs with the ability to reverse DIPG gene signatures and anti-DIPG activity in vitro and in vivo. This novel approach can repurpose drugs and significantly decrease the cost and time normally required in drug discovery.

Abstract 318: Multi-omics to edge into precision medicine for DIPG

Abstract Effective targeted cancer therapies are limited due to lack of our understanding of disease pathogenesis at the molecular level. Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brain tumor with 80% of DIPGs harboring H3F3A (H3.3) mutation that lead to a substitution of methionine for lysine at position 27 (K27M). The tumors bearing H3.3K27M mutation arise throughout the midline structures with global depletion of H3.3K27 me3 (trimethylation). These histone mutations modify the epigenome and alter oncogenic transcription, causing oncogenic insults to progenitor cells in early neurodevelopment (PMID: 30453529). To determine the reprogramming pathways in the cell context of H3.3K27M tumors, we conducted liquid chromatography-mass spectrometry-based proteomic and phosphoproteomic analysis on seven patient-derived DIPG cell lines that can be stably passaged in serum-free neural stem cell media and displayed distinct morphologies, growth rates and chromosome abnormalities (PMID: 21368213). Three normal neuronal stem cell lines were included as non-tumor brain cells for comparison. Pathway analysis identified 29 pathways that are significantly altered in DIPG compared to normal brain cells at both the protein abundance and phosphosite level. Notably, AKT and MAPK associated PI3K signaling, VEGF signaling, mTOR signaling, and HIF1a signaling were differentially active in H3.3K27M tumors compared to healthy control cell lines. We saw significantly higher activity of multiple kinases involved in axon guidance and cytoskeletal remodeling in DIPG, such as PTK2B, DYRK2, TTBK2 and MARK2. This is the first time to report an increased abundance and kinase activity of Pyk2 protein (coded by PTK2B), a close homologue of FAK and its associated signaling in DIPG. Overexpression and autophosphorylation of Pyk2 are required to stimulate glioma cell migration (PMIDs: 15967096, 18648907). Pyk2 has also been proposed to act in concert with Src to link Gi- or Gq-coupled receptors with the mitogen-activated protein (MAP) kinase signaling pathway (PMID:12960403). Because of the shared signaling across kinase pathways, targeting activated Pyk2 in DIPG may complement inhibitors of other dysregulated signaling networks in DIPG such as MAPK2, VEGFR, PI3K and Src. Our data also found that IL13RA2 was upregulated in DIPG; others have shown that increased expression of IL13RA2 is associated with poor prognosis in GBM (PMIDs: 32913543, 30366424). We conclude that for H3 K27M DIPG tumors, campaigns to target Pyk2, MAPK2, VEGFR, PI3K, Src and IL13Ra2 using small molecules that traverse the blood brain barrier loom as promising opportunities for drug development. Citation Format: Nanyun Tang, Kristin Leskoske, Krystine Garcia-Mansfield, Ritin Sharma, Hannah Tolson, Patrick Pirrotte, Michael E. Berens. Multi-omics to edge into precision medicine for DIPG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 318.

HGG-42. PEDIATRIC H3K27M MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) SHOWS ROBUST RESPONSE TO IMIPRIDONE BASED COMBINATION THERAPY

ONC201 is a first-in-class small molecule imipridone therapy, which is known to selectively induce apoptosis of cancer cells independent of p53. This novel chemotherapeutic, as well as its analogs ONC206 and ONC212, has been shown to have potent preclinical efficacy against H3K27M mutant diffuse intrinsic pontine glioma (DIPG). We sought to identify synergy between imipridones and other FDA-approved chemotherapeutics. Seven patient-derived DIPG cell lines, six H3.3K27M mutant (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SUDIPG-27, SU-DIPG-29, SF8628), and one H3.1K27M mutant (SU-DIPG-36) were grown in culture and exposed to first and second generation imipridones, both as monotherapies and in combination with histone de-acetylase inhibitors [HDACi], Marizomib, Etoposide, and Temozolomide. A dose dependent response was demonstrated across all cell lines, with increased potency of ONC206 and ONC212 as compared to ONC201, with half maximal inhibitory concentration (IC50) of 0.11 µM, 0.03 µM, and 1.46 µM respectively. Strong synergy is demonstrated between ONC201 and Panobinostat with best combination index (CI) of 0.01. ONC201 similarly shows strong synergy with Romidepsin with best CI of 0.02, and Marizomib with best CI of 0.18. Combination of ONC201 and Etoposide or Entinostat shows some synergy, with best CI of 0.53 and 0.71 respectively. When combined with Temozolomide, some synergy is evident, however, there is overall poor efficacy, with lack of cell death even at the highest doses of Temozolomide. Second generation imipridones show a similar pattern of strong synergy with Panobinostat, Romidepsin, and Marizomib. Immunoblotting showed evidence of apoptosis, as measured by the induction of PARP cleavage, with a combination of imipridones and Panobinostat, as well as induction of integrated stress response with a combination of imipridones and Romidepsin. These results are indicative of promising synergy between imipridones and Panobinostat, Romidepsin, or Marizomib against H3K27M mutant DIPG, combinations which should be considered for future clinical trials.

Synergistic imipridone‐based drug combinations for treatment of pediatric H3K27M mutant diffuse intrinsic pontine glioma (DIPG)

The H3K27M oncohistone mutation has been identified in approximately 80% of diffuse intrinsic pontine gliomas (DIPG), and now represents a potential target for urgently needed new therapies. ONC201, a first-in-class imipridone therapy, is known to selectively induce apoptosis of cancer cells independent of p53, and has shown efficacy against H3K27M mutant DIPG. We sought to identify synergy between ONC201 or second generation imipridones (ONC206 and ONC212), and other chemotherapeutics with known preclinical activity against DIPG. We hypothesized that second generation imipridones would demonstrate superior activity to ONC201, and that the combination of imipridones and other chemotherapeutics may be superior to single agent treatment in H3K27M mutant DIPG. Seven patient derived DIPG cell lines (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SUDIPG-27, SU-DIPG-29, SU-DIPG-36, SF8628) were grown in culture and exposed to imipridones as a monotherapy and in combination with other FDA approved chemotherapeutics (histone de-acetylase inhibitors [HDACi], marizomib, etoposide, and temozolomide). Cell viability was measured by CellTiter-Glo. Dose dependent response to imipridone therapy was noted in all cell lines with half maximal inhibitory concentration (IC50) of 1.46 µM, 0.11 µM, and 0.03 µM, in ONC201, ONC206, and ONC212 respectively. Synergy was identified via combination analyses, with combination indices of <1 indicating some degree of synergy, and <0.5 indicating very strong synergy. Strong synergy was identified between ONC201 and HDACi Panobinostat (CI 0.01), Romidepsin (CI 0.08) and protease inhibitor Marizomib (CI 0.19). Lesser synergy was demonstrated between ONC201 and Entinostat (CI 0.71) or Etoposide (CI 0.54). Importantly, temozolomide and ONC201 showed a lack of combinational efficacy against H3K27M mutant DIPG, consistent with the literature to date. Second generation imipridones showed similar synergistic effects with Panobinostat, Romidepsin, and Marizomib. Additionally, induction of apoptosis as measured by the induction of poly (ADP-ribose) polymerase (PARP) cleavage via immunoblotting, was demonstrated with a combination of imipridones and Panobinostat. These results suggest increased sensitivity of H3K27M mutant DIPG cell lines to second generation imipridone therapies, as compared to ONC201. Additionally, there is evidence of promising synergy between imipridones and Panobinostat, Romidepsin, or Marizomib, which should be taken into consideration for future clinical trials.

EXTH-67. PHARMACOLOGIC INHIBITION OF LYSINE SPECIFIC DEMETHYLASE-1 (LSD1) AS AN ADJUVANT IMMUNE-SENSITIZATION STRATEGY IN DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Abstract Diffuse midline gliomas (DMG), including brainstem DIPG (diffuse intrinsic pontine glioma), are incurable pediatric brain tumors. Mutations in the H3 histone tail (H3.1/3.3-K27M) are a feature of DIPG, highlighting epigenetic aberrations in these tumors. Lysine specific demethylase-1 (LSD1), is an enzyme that acts upon mono and di-methylated histone H3K4/K9, and pharmacological inhibitors of its enzymatic and scaffolding domains are clinically viable. Patient-derived DIPG cell lines, orthotopic mouse models, and pediatric high-grade glioma (pHGG) datasets were used to evaluate effects of LSD1 inhibitors on cytotoxicity and immune gene expression. Because an unique immune signature was sen with enzymatic LSD1 inhibitors in DIPG cells in vitro, immune cell cytotoxicity was assessed in DIPG cells pre-treated with LSD1 inhibitors and informatics platforms were used to determine immune infiltration of pHGG. Selective cytotoxicity and an immunogenic gene expression signature was established in DIPG cell lines and in brainstem tumor-bearing mice using clinically-relevant LSD1 inhibitors. Pediatric HGG patient sequencing data demonstrated survival benefit using this LSD1-dependent gene expression signature. On-target binding of catalytic LSD1 inhibitors was confirmed in DIPG and pre-treatment of DIPG with these inhibitors increased lysis by natural killer (NK) cells. CIBERSORT analysis of patient data confirmed NK infiltration is beneficial to patient survival while CD8 T-cells are negatively prognostic. Catalytic LSD1 inhibitors are non-perturbing to NK cells while scaffolding LSD1 inhibitors are toxic to NK cells and do not induce the gene expression signature in DIPG cells. Treatment of orthotopic xenograft bearing DIPG mouse models with catalytic LSD1 inhibitors and NK cells showed a significant reduction in tumor burden. In summary, we find that LSD1 inhibition using catalytic inhibitors is both selectively cytotoxic and promotes a functional immune gene expression signature that is associated with NK cell killing, representing a therapeutic opportunity for pHGG.

Targeting Mutant PPM1D Sensitizes Diffuse Intrinsic Pontine Glioma Cells to the PARP Inhibitor Olaparib.

Diffuse intrinsic pontine glioma (DIPG) is an invariably fatal brain tumor occurring predominantly in children. Up to 90% of pediatric DIPGs harbor a somatic heterozygous mutation resulting in the replacement of lysine 27 with methionine (K27M) in genes encoding histone H3.3 (H3F3A, 65%) or H3.1 (HIST1H3B, 25%). Several studies have also identified recurrent truncating mutations in the gene encoding protein phosphatase 1D, PPM1D, in 9%-23% of DIPGs. Here, we sought to investigate the therapeutic potential of targeting PPM1D, alone or in combination with inhibitors targeting specific components of DNA damage response pathways in patient-derived DIPG cell lines. We found that GSK2830371, an allosteric PPM1D inhibitor, suppressed the proliferation of PPM1D-mutant, but not PPM1D wild-type DIPG cells. We further observed that PPM1D inhibition sensitized PPM1D-mutant DIPG cells to PARP inhibitor (PARPi) treatment. Mechanistically, combined PPM1D and PARP inhibition show synergistic effects on suppressing a p53-dependent RAD51 expression and the formation of RAD51 nuclear foci, possibly leading to impaired homologous recombination (HR)-mediated DNA repair in PPM1D-mutant DIPG cells. Collectively, our findings reveal the potential role of the PPM1D-p53 signaling axis in the regulation of HR-mediated DNA repair and provide preclinical evidence demonstrating that combined inhibition of PPM1D and PARP1/2 may be a promising therapeutic combination for targeting PPM1D-mutant DIPG tumors. IMPLICATIONS: The findings support the use of PARPi in combination with PPM1D inhibition against PPM1D-mutant DIPGs.

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.

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.

Patient-derived DIPG cells preserve stem-like characteristics and generate orthotopic tumors.

Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor, with a median survival of less than one year. Due to enormous difficulties in the acquisition of DIPG specimens and the sophisticated technique required to perform brainstem orthotopic injection, only a handful of DIPG pre-clinical models are available. In this study, we successfully established eight patient-derived DIPG cell lines, mostly derived from treatment-naïve surgery or biopsy specimens. These patient-derived cell lines can be stably passaged in serum-free neural stem cell media and displayed distinct morphologies, growth rates and chromosome abnormalities. In addition, these cells retained genomic hallmarks identical to original human DIPG tumors. Notably, expression of several neural stem cell lineage markers was observed in DIPG cell lines. Moreover, three out of eight cell lines can form orthotopic tumors in mouse brainstem by stereotactic injection and these tumors faithfully represented the characteristics of human DIPG by magnetic resonance imaging (MRI) and histopathological staining. Taken together, we established DIPG pre-clinical models resembling human DIPG and they provided a valuable resource for future biological and therapeutic studies.

TRTH-18. COMBINED TOPOTECAN AND CARBOPLATIN THERAPY FOR DIPG SUPPRESSES CELL GROWTH IN VITRO AND IS WELL TOLERATED WHEN DELIVERED BY CONVECTION ENHANCED DELIVERY IN VIVO

AbstractDespite clinical advances, diffuse intrinsic pontine glioma (DIPG) remains universally fatal and urgently requires new treatment modalities to improve prognosis. Topotecan and carboplatin have formed treatment regimes for pediatric cancers as single agents with limited success. Both drugs have poor central nervous system penetration which may explain the lack of efficacy in the clinic when delivered systemically, furthermore, as single agents they have been administered to children with DIPG by convection enhanced delivery (CED) in early phase clinical trials and compassionate use programmes. Here, we describe the use of topotecan and carboplatin as combinatorial therapy for the treatment of DIPG in vitro and its tolerance when administered in vivo in a small animal model of intermittent CED. In this study, we assessed the in vitro cytotoxicity of combined topotecan and carboplatin in two ex vivo patient derived DIPG cell lines (SF7761 and SF8628). Combination therapy in vitro showed an additive effect on cell viability when compared to each single agent. Next, we determined the tolerance and toxicity of both drugs administered by CED using an implantable catheter system in juvenile wistar rats by clinical and neuropathological examination of infused brain 21 days after treatment. Results show that topotecan and carboplatin have an enhanced effect on DIPG cell viability in vitro when used in combination. This combination is currently being explored in a wider cohort of DIPG cell lines. In vivo assessment of sequential drug therapy in normal rat brain, shows this combination to be well tolerated and exhibited minimal CNS toxicity. Further work is required to see if this combination is effective in pre-clinical models of DIPG when administered by CED.

DIPG-35. A SIRNA APPROACH FOR TARGETING IMMUNOSUPPRESSIVE IDO1 IN PEDIATRIC DIFFUSE INTRINSIC PONTINE GLIOMA

OBJECTIVE/GOALS: The purpose of our project is to delineate the role of indoleamine 2,3 dioxygenase 1 (IDO1) in pediatric diffuse intrinsic pontine glioma (DIPG), and develop therapeutic potential of IDO1 inhibition through small inhibitory (si)RNA oligonucleotides and spherical nucleic acids (SNAs). Our specific aims are: (1) confirm the gene expression, protein levels and enzymatic activity of IDO1 in different human DIPG cell lines; (2) generate and characterize siRNA oligonucleotides specifically targeting human IDO1 for an in vitro targeted approach to validate knockdown of IDO1 expression; and (3) generate and characterize gold nanoparticles for further targeted approach to inhibit IDO1. METHODS/STUDY: Unique patient-derived DIPG cell lines were grown in culture, stimulated with increasing concentrations of IFN-γ, and analyzed by levels of mRNA, protein, enzymatic activity and cell growth. IDO1 small inhibitory (si)RNA were created to target IDO1 specifically, and transfected into cells. SNA generation is currently underway. RESULTS: ANTICIPATED RESULTS: IDO1 is expressed in different human pediatric DIPG cells at the mRNA and protein levels. We created siRNA that specifically targets IDO1 and preliminary results show overall decrease of IDO1 at the mRNA and protein level. Generation of SNA is ongoing for targeted approach and improved blood brain barrier penetration & stability, with anticipated results that siIDO1-SNAs inhibit IDO1 expression and reduce tumorigenesis within brain tumor cells. DISCUSSION/SIGNIFICANCE OF IMPACT: The overall poor prognosis of children with DIPG combined with the lack of effective therapies, emphasizes the importance of understanding the IDO1 pathway and the potential effects of IDO1 inhibition for therapeutically-modulating these devastating tumors. Our research confirms IDO1 presence in DIPG, and the use of nanotechnology and development of siRNA-SNAs for IDO1 inhibition has the capability of direct translatability of therapeutically-valuable data.

EZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas.

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 p16INK4A. 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.