Treatment Of DIPG Research Articles

DDDR-26. INHIBITION OF RAD52 ACTIVITY ENHANCES THE RADIOSENSITIVITY IN DIPG

Abstract Diffuse intrinsic pontine glioma (DIPG) is one of the most devastating childhood cancers that limited response to radiation therapy, resulting in median overall survival of approximately 10 months. There is a critical need for new therapeutics that enhance the radiation effect while enabling the use of lower doses of radiation to limit long-term side effects in young patients. We, therefore, tested the hypothesis that targeting RAD52 activity sensitizes the radiation response of DIPG. Genome-wide CRISPR/Cas9 screening identified RAD52 as potential therapeutic target in DIPG cells. RAD52 inhibition, using shRNA mediated RAD52 depletion and treatment with RAD52 inhibitor, D-I03, suppressed DIPG cell proliferation. Importantly, RAD52 inhibition in combination with radiation therapy further increased radiosensitivity of DIPG cells. Immunocytochemistry of DNA double-strand breaks (DSB) marker γH2AX and repair marker 53BP1 showed that RAD52 inhibition sustained DNA damage with high levels of γH2AX at 24 hours following radiation while the level of 53BPI was decreased, thereby inhibiting DNA DSB repair. Western blotting also revealed that RAD52 inhibition causes a sustained level of phosphorylated Rad50 and γH2AX in irradiated DIPG cells. DNA repair assay showed that RAD52 inhibition suppressed DNA DSB repair through a homologous recombination pathway. RNA sequencing showed that RAD52 inhibition downregulated genes associated with the DNA repair pathway. Finally, the combination therapy of D-I03 RAD52 inhibitor and radiation inhibited tumor growth and significantly increased survival of mice bearing DIPG patient-derived xenografts, outperforming either monotherapy. Together, these results highlight RAD52 inhibitor as a potential radiosensitizer and provide a rationale for developing combination therapy with radiation in the treatment of DIPG.

DIPG-24. BRD4 INHIBITION AS A RADIOSENSITIZER THROUGH BLOCKING DNA REPAIR FOR THE TREATMENT OF DIFFUSE MIDLINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG) is one of the devastating childhood cancers. Radiation therapy (RT) remains the only effective treatment yet provides a 5-year survival rate of only 1%. Several clinical trials have attempted to enhance RT efficacy by combining it with radiosensitizing agents, though none have been successful in doing so. Given this, there is a critical need to identify effective therapeutics to enhance the RT anti-tumor activity in DIPG. Here, we identified BRD4 inhibitors as candidate radiosensitizers from a high throughput drug screening. DIPG cells show increased H3 K27 acetylation (H3K27ac) levels, which bind to BET bromodomain protein 4 (BRD4) and are strongly associated with active transcription. We tested two BRD4 inhibitors (BRD4i: AZD5153 and JQ-1) as well as genetic BRD4 depletion, and observed enhancement of radiation-induced DNA damage, confirming BRD4i as potential radiosensitizers in the treatment of DIPG. We evaluated the effects of BRD4i on gene expression using RNAseq, and observed significant inhibition of DNA-repair proteins such as BRCA1 and RAD51. CUT-RUN qPCR showed decreased H3K27ac at BRCA1 and RAD51 promoters. Combination of BRD4i and RT inhibited cell viability significantly using clonogenic survival assay, apoptosis assay, and also showed cell cycle arrest. Radiation-induced DNA double-strand break (DSB) repair was prolonged with BRD4i with high levels of gH2AX and 53BP1 likely due to inhibition of DNA-repair. In vivo studies revealed increased survival of animals treated with combination therapy of RT and BRD4i in comparison to either monotherapy. Together, these results highlight BRD4i as a potential radiosensitizer and provide a rationale for developing combination therapy with radiation in the treatment of DIPG.

EXTH-29. RADIOSENSITIZATION BY BRD4 INHIBITION IN DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG), the most frequent brainstem tumor in pediatrics, is one of the devastating childhood cancers, and virtually all patients die within two years after diagnosis. Since these tumors occur in the brainstem which is vital area, there are no surgical options for providing relief to patients, and conventional chemotherapy. Radiation therapy (RT) remains the only effective treatment. Radiosensitization is a mean to improve the therapeutic balance between efficacy and toxicity of RT, but not used in clinically. Here, we found BRD4 inhibitor as a radiosensitizer from drug screening. DIPG shows increased H3 K27 acetylation (H3K27ac), which binds to BET bromodomain protein 4 (BRD4) and they are strongly associated with active transcription. We tested two BRD4 inhibiter (BRD4i : AZD5153 and JQ-1) and genetic BRD4 depletion enhances radiation-induced DNA damage, making it a potential radiosensitizer in the treatment of DIPG. We evaluated the effects of BRD4i on genes expression using RNA sequence, inhibited significantly DNA-repair proteins such as BRCA1 and RAD51. CUT-RUN qPCR showed decreased H3K27ac at BRCA1 and RAD51. Combination with BRD4i and RT inhibited cell viability significantly in clonogenic survival assay, apoptosis assay, and showed cell cycle arrest. Radiation-induced DNA double-strand break (DSB) repair was prolonged high level of rH2AHX and 53BP1 with BRD4i because of inhibited DNA-repair. In vivo studies revealed increased survival of animals treated with combination therapy of RT and BRD4i in compared to either monotherapy. Together, these results highlight BRD4i as a potential radiosensitizer and provide a rationale for developing combination therapy with radiation in the treatment of DIPG.

EXTH-58. UNRAVELING THE MECHANISMS UNDERLYING THE THERAPEUTIC EFFICACY OF TIM-3 BLOCKADE IN DIPGS

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor and the leading cause of pediatric death caused by cancer. Despite great strides in the understanding of this disease, the survival is still dismal. One of the objectives of our lab is to modulate the tumor microenvironment (TME) towards a proinflammatory phenotype to render these tumors amenable to immunotherapy. TIM-3 is a member of the TIM-family of immunoregulatory proteins expressed on multiple immune cell types, including T-cells, NK, myeloid populations, and microglia, regulating adaptive and innate immunity. In silico assessment of TIM-3 expression in DIPG mRNA and single-cell datasets showed a robust expression of this gene in tumor cells and microglia uncovering this molecule as a potential target in DIPGs. In vivo studies showed that TIM-3 blockade with an antibody significantly increased the overall survival of two DIPG immunocompetent orthotopic models, led to long-term survivors (50%), and showed immune resident memory. TIM-3 inhibition resulted in a significant increase in the number and proliferative state of microglia, NK, and CD8+ cells and higher levels of IFNγ, GrzB and TNFα corresponding to NK and T-cell activate phenotypes. Interestingly, there was a decrease in the Treg population, which causes an increase in the pro-inflammatory CD8/Treg ratio. Chemokine studies demonstrated an augmentation of CCL5, CCL2 chemotactic cytokines, and CXCL10/IL-1β/IFN-γ pro-inflammatory axis in the tumor microenvironment of treated-mice. Additionally, DCs, CD4+, and CD8+ cells were increased in treated draining lymph nodes and of functional significance, expressed higher amounts of pro-inflammatory cytokines than in control mice. Interestingly, the depletion of the different immune populations did not completely abrogate the treatment efficacy indicating a residual although significant TIM-3 effect in the tumor. In conclusion, these data uncover TIM-3 as a potential target for the treatment of DIPG and its potential as an immune regulator of DIPG TME.

Abstract CT001: Major tumor regressions in H3K27M-mutated diffuse midline glioma (DMG) following sequential intravenous (IV) and intracerebroventricular (ICV) delivery of GD2-CAR T cells

Abstract Background: H3K27M-mutated DMGs are universally lethal central nervous system tumors that express high levels of the disialoganglioside GD2. IV administered GD2-CAR T cells (GD2-CART) regress DMG in preclinical models, and locoregionally delivered CARs demonstrate enhanced activity in xenograft models of brain tumors. Methods: NCT04196413 is a 3+3 Phase I dose escalation trial testing GD2-CART in patients with H3K27M DMG, with dose-limiting toxicities (DLT) considered independently for DIPG and spinal DMG (sDMG). Arm A tested escalating doses of IV GD2-CART (DL1: 1e6 GD2-CART/kg; DL2=3e6 GD2-CART/kg) following lymphodepletion (LD). After the DLT period, patients with clinical and/or radiographic benefit were eligible for subsequent ICV GD2-CART (10-30e6 GD2-CART) administered via Ommaya catheter without LD every 4-8 weeks for a maximum of 12 doses. We previously reported early results from 4 patients treated on DL1, which demonstrated clinical activity and manageable toxicity. Here we provide updated results for DL1 and DL2. Results: Thirteen subjects were enrolled and 11 treated [n=4 DL1 (3 DIPG/1 sDMG); n=9 DL2 (7 DIPG/2 sDMG)]. Two subjects were removed prior to treatment due to rapid progression. No DLTs were observed on DL1. Three subjects experienced DLT on DL2 (2 DIPG/1 sDMG) due to grade 4 cytokine release syndrome (CRS), successfully managed with tocilizumab, anakinra, and corticosteroids. CRS occurred earlier on DL2 vs. DL1 (Day 3 vs 7). On both dose levels, all subjects exhibited transient symptoms related to on-tumor inflammation, termed Tumor Inflammation-Associated Neurotoxicity (TIAN), which was successfully managed with anakinra and, in some cases, CSF drainage and dexamethasone. No DLT due to TIAN has occurred. Ten patients have had adequate follow-up to assess benefit. Nine experienced radiographic and/or clinical benefit after IV infusion, and they received subsequent ICV GD2-CART infusions (median= 4 ICV infusions/pt, range 1-6). ICV infusions were not associated with high-grade CRS, although some subjects developed transient fever, headache, meningismus, nausea, and/or vomiting, and several subjects developed TIAN. Four patients continue to receive ICV infusions on study and have experienced continued clinical and radiographic benefit at 11+, 9.5+, 8+ and 7+ months following enrollment. A 31-year-old with sDMG has experienced a near-complete (>95%) reduction in tumor volume and a 17-year-old with DIPG experienced a near-complete (>98%) reduction in volume of a pontine tumor. Conclusions: IV treatment of DIPG and sDMG with GD2-CART is safe at a dose of 1e6/kg, but associated with unacceptable rates of high-grade CRS at 3e6/kg. ICV GD2-CART without LD, administered following a previous course of IV GD2-CART with LD, has been well tolerated and has mediated impressive sustained clinical benefit in some patients with DIPG/sDMG. Given these findings, we are launching a new arm to assess safety and activity and to define the recommended phase 2 dose for ICV delivery of GD2-CART without LD. Patients are eligible for up to 12 ICV infusions of GD2-CART administered every 4-6 weeks. Clinical benefit will be formally assessed using patient-reported outcomes. GD2-CART has the potential to transform therapy for patients with H3K27M+ DIPG/sDMG. Citation Format: Robbie G. Majzner, Jasia Mahdi, Sneha Ramakrishna, Shabnum Patel, Harshini Chinnasamy, Kristen Yeom, Liora Schultz, Valentin Barsan, Rebecca Richards, Cynthia Campen, Agnes Reschke, Angus Martin Shaw Toland, Christina Baggott, Sharon Mavroukakis, Emily Egeler, Jennifer Moon, Ashley Jacobs, Karen Yamabe-Kwong, Lindsey Rasmussen, Esther Nie, Sean Green, Michael Kunicki, Michelle Fujimoto, Zach Ehlinger, Warren Reynolds, Snehit Prabhu, Katherine E. Warren, Tim Cornell, Sonia Partap, Paul Fisher, Gerald Grant, Hannes Vogel, Bita Sahaf, Kara Davis, Steven Feldman, Michelle Monje, Crystal L. Mackall. Major tumor regressions in H3K27M-mutated diffuse midline glioma (DMG) following sequential intravenous (IV) and intracerebroventricular (ICV) delivery of GD2-CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT001.

DIPG-15. Major tumor regressions in H3K27M-mutated diffuse midline glioma (DMG) following sequential intravenous (IV) and intracerebroventricular (ICV) delivery of GD2-CAR T-cells

BACKGROUND: H3K27M-mutated DMGs express high levels of the disialoganglioside GD2 and GD2-CAR T-cells (GD2-CART) regress DMG in preclinical models. METHODS: NCT04196413 is a 3 + 3 Phase I dose escalation trial testing GD2-CART in patients with biopsy-proved H3K27M DMG, with dose-limiting toxicities (DLT) considered independently for DIPG and spinal DMG (sDMG). Arm A tested escalating doses of IV GD2-CART (DL1=1e6 GD2-CART/kg; DL2=3e6 GD2-CART/kg) following lymphodepletion (LD). After the DLT period, patients with clinical and/or radiographic benefit were eligible for subsequent ICV GD2-CART infusions (10-30e6 GD2-CART) administered via Ommaya without LD. RESULTS: Twelve subjects were treated after standard radiotherapy, 7 of whom began treatment at the time of progression [n=4 DL1 (3 DIPG/1 sDMG); n=8 DL2 (6 DIPG/2 sDMG)]. No DLTs were observed on DL1. Three subjects experienced DLT on DL2 (2 DIPG/1 sDMG) due to grade-4 cytokine release syndrome (CRS). On both dose levels, all subjects exhibited transient symptoms related to on-tumor inflammation, termed Tumor Inflammation-Associated Neurotoxicity (TIAN); no DLT due to TIAN has occurred. Ten subjects experienced radiographic and/or clinical benefit after IV infusion and received subsequent ICV infusions (median=4 ICV infusions/pt, range=1-7). ICV infusions were not associated with high-grade CRS. Four patients continue to receive ICV infusions on study and have experienced continued clinical and radiographic benefit, currently 7-11 months following enrollment. Two patients (one sDMG, one DIPG) have experienced near-complete (>95%) tumor volume reduction. CONCLUSIONS: IV treatment of DIPG and sDMG with GD2-CART is safe at a dose of 1e6/kg, but associated with frequent high-grade CRS at 3e6/kg. ICV GD2-CART has been well tolerated and has mediated impressive sustained clinical benefit in some patients with DIPG/sDMG. Given these findings, we are launching a new arm to assess safety and activity and to define the recommended phase 2 dose for ICV delivery of GD2-CART without LD.

Imaged-guided focused ultrasound in combination with various formulations of doxorubicin for the treatment of diffuse intrinsic pontine glioma

BackgroundDiffuse intrinsic pontine glioma (DIPG) is a notoriously difficult tumor to treat, with an overall survival of DIPG patients being only 11 months. One of the major obstacles for the effective treatment of DIPG is the blood–brain barrier (BBB). In order to circumvent the BBB, drug delivery methods are needed that target the pontine area. One such approach is microbubble-mediated focused ultrasound (FUS)—a non-invasive method that can temporarily and locally open the BBB. Previously, it was shown that FUS is safe with minimal side effects and rapid recovery times in preclinical animal models with different DIPG tumors. However, recent studies have shown that combining FUS with a single treatment of the chemotherapeutic drug doxorubicin did not improve survival in a DIPG xenograft model. As the duration of doxorubicin exposure might play a role in tumor response, we hypothesized that the use of a long-circulation (PEGylated) liposomal formulation of doxorubicin could lead to improved overall survival through a longer exposure time to the tumor.MethodDIPG xenograft models were established with orthotopic injections of HSJD-DIPG-07 tumor cells into the pontine area of female athymic nude-foxn1nu mice. Tumor engraftment was confirmed with bioluminescence imaging (BLI) 40 days post-inoculation. Mice were randomized into groups receiving either liposomal formulations of doxorubicin (2B3-101 or Caelyx®) or free doxorubicin in combination with or without FUS treatment. Treatment groups received 5 mg/kg 2B3-101 or Caelyx® 1 h before FUS treatment or 5 mg/kg free doxorubicin immediately after FUS.ResultsHistological analysis, however, revealed liposome extravasation in healthy controls but not in HSJD-DIPG-07 xenograft 24 h after treatment. Furthermore, BLI monitoring did not show reduced signal after treatment, which was further illustrated with a survival analysis, showing no significant difference between treated and control animals (p = 0.3).ConclusionWe did not observe a treatment effect after a single dose of free doxorubicin or the liposomal formulations 2B3-101 or Caelyx® in combination with FUS in DIPG-bearing mice.

TAMI-80. CELLULAR METABOLISM IN DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive form of brain tumor in children, comprising >10% of all pediatric brain tumors. The median survival after diagnosis is < 1 year. Since DIPG tumors infiltrate brainstem and pons, they are inoperable. Currently radiotherapy is the mainstay of treatment, and there is a great need for novel therapies for the treatment of DIPG. Cellular metabolism plays a key role in carcinogenesis, unravelling active metabolic pathways in DIPG would help in developing targeted therapies. Glucose and glutamine are the two major nutrients necessary for the growth and proliferation of cancer cells. In this study, we have investigated the glucose and glutamine metabolism in SF8628 DIPG cells using 1H/13C NMR and GC-MS based metabolic flux analysis. SF8628 cells were grown in DMEM containing 11.0 mM glucose, supplemented with 10% FBS, and 2.0 mM glutamine at 37 °C under humidified air and 5% CO2. When cells reached confluency (replicates = 4), treated with 11.0 mM [U-13C]glucose or 4.0 mM glutamine in DMEM (supplemented with 10% FBS). After 24 h, cells were harvested for NMR/GC-MS analysis. The 13C-isotopomer analysis revealed that SF8628 cells produced 25.26 ± 10.63% acetyl-CoA from [U-13C]glucose which is ~3.7 times higher than that produced from GBM cells (6.83 ± 0.76%; our previous work), suggesting that DIPGs are metabolically very active. [U-13C]glutamine metabolism showed that DIPG cells also have an active TCA cycle metabolism (citrate M+4; 40.07 ± 1.06%) and moderately active reductive carboxylation pathway (citrate M+5; 10.59 ± 1.13%). Inhibition of both glycolytic and glutaminolysis pathways will be valuable in developing treatment strategies for DIPGs and these studies are in progress.

HGG-17. FOCUSED ULTRASOUND-ENHANCED DELIVERY OF RADIOLABELED AGENTS TO DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG) arising in the brainstem is the deadliest pediatric brain cancer with nearly 100% fatality and a median survival of <1 year. The critical location in the brainstem and the often intact blood-brain barrier (BBB) pose significant challenges in the treatment of DIPG. The objective of this study was to demonstrate the potential for focused ultrasound-induced BBB disruption (FUS-BBBD) to improve DIPG treatment by enhancing the safe and efficient delivery of drugs. A genetically engineered mouse model of DIPG was generated using the RCAS (replication-competent avian sarcoma-leucosis virus long-terminal repeat with splice acceptor)/tumor virus A modeling system. A magnetic resonance-guided FUS (MRgFUS) system was used to induce BBB disruption in these mice with the FUS targeted at the center of the tumor. Two radiolabeled agents with different sizes were used to evaluate the delivery efficiency of the FUS-BBBD technique in DIPG mice: a small-molecular radiotracer, 68Ga-DOTA-ECL1i, and a radiolabeled nanoparticle, 64Cu-labeled copper nanoparticles (64Cu-CuNCs, ~ 5 nm in diameter). 68Ga-DOTA-ECL1i (half-life ~ 1 h) and 64Cu-CuNCs (half-life ~13 h) were intravenously injected into the mice after FUS sonication, and microPET/CT imaging was performed at 1 h and 24 h, respectively, to evaluate the spatial-temporal distribution of these two agents in the brain and quantify the delivery outcome. FUS treatment increased the uptake of 68Ga-DOTA-ECL1i and 64Cu-CuNCs to the DIPG tumor by 3.25 folds and 4.07 folds on average, respectively. These findings demonstrated, for the first time, that FUS can increase BBB permeability in a murine model of DIPG and significantly enhance the delivery of agents of different sizes into the DIPG tumor.

DIPG-16. COMBINATION OF ARGININE DEPLETION AND POLYAMINE INHIBITION AS AN ANTICANCER STRATEGY FOR DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

DIPG is an aggressive pediatric brainstem tumor, with a median survival below 12 months. Tumor cells are dependent upon arginine, a semi-essential amino acid, metabolised by arginase enzymes into ornithine, a pivotal precursor to the polyamine pathway. Polyamines, frequently upregulated in cancer, are intracellular polycations controlling key biological processes – the inhibition of which we have previously shown to be highly efficacious in preclinical DIPG models. Pegylated arginase (BCT-100) has recently been shown to significantly delay tumor development, prolonging survival of neuroblastoma-prone Th-MYCN mice. This study investigated the effects of arginine depletion therapy as a single agent and in combination with polyamine pathway inhibitors in DIPG. We found that ARG2, the gene encoding for arginase II, is expressed significantly more highly in DIPG tumors compared to normal brain. Arginine depletion via BCT-100 reduced DIPG cell proliferation and colony formation in patient-derived cell lines. Using orthotopic patient-derived xenograft models of DIPG, we found that frequent dosing of BCT-100 (4x/week) significantly delayed tumor development and increased the survival of the mice (p<0.0001). DFMO is an FDA-approved inhibitor of the enzyme ornithine decarboxylase, a key driver of polyamine synthesis. The combination of BCT-100 with DFMO led to significant enhancement in DIPG survival (p<0.005 compared to single agent treatments). Triple combination therapy with addition of the polyamine transport inhibitor AMXT-1501 led to a potent and profound improvement in survival. These data show that arginine depletion therapy using BCT-100 combined with dual polyamine inhibitory agents represents a potentially exciting new approach for the treatment of DIPG.

DDEL-11. CONVECTION-ENHANCED DELIVERY OF EZH2 INHIBITOR FOR THE TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMA

BACKGROUNDDiffuse intrinsic pontine glioma (DIPG) is a fatal childhood brain tumor and the majority of patients die within 2 years after initial diagnosis. Factors that contribute to the dismal prognosis of these patients include the infiltrative nature and anatomic location in an eloquent area of the brain, which precludes total surgical resection, and the presence of the blood-brain barrier (BBB), which reduces the distribution of systemically administered agents. Convection-enhanced delivery (CED) is a direct infusion technique to deliver therapeutic agents into a target site in the brain and able to deliver a high concentration drug to the infusion site without systemic toxicities.OBJECTIVEThis study aims to assess the efficacy of enhancer of zeste homolog-2 (EZH2) inhibitor by CED against human DIPG xenograft models.METHODSThe concentration of EZH2 inhibitor (EPZ-6438) in the brainstem tumor was evaluated by liquid chromatography–mass spectrometry (LC/MS). We treated mice bearing human DIPG xenografts with EPZ-6438 using systemic (intraperitoneal) or CED administration. Intracranial tumor growth was monitored by bioluminescence image and the therapeutic response was evaluated by animal survival.RESULTSLC/MS analysis showed that the concentration of EPZ-6438 in the brainstem tumor was 3.74% of serum concentration after systemic administration. CED of EPZ-6438 suppressed tumor growth and significantly extended animal survival when compared to systemic administration of EPZ-6438 (P=0.0475).CONCLUSIONOur results indicate that CED of an EZH2 inhibitor is a promising strategy to bypass the BBB and to increase the efficacy of an EZH2 inhibitor for the treatment of DIPG.

PDTM-34. RADIOSENSITIZATION BY BRD4 INHIBITION IN DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG), the most frequent brainstem tumor in pediatrics, is one of the devastating childhood cancers, and virtually all patients die within two years after diagnosis. Since these tumors occur in the brainstem which is vital area, there are no surgical options for providing relief to patients, and conventional chemotherapy as well as radiation therapy provides palliative relief at best. DIPG shows increased H3 K27 acetylation (H3K27ac), which binds to BET bromodomain protein 4 (BRD4) and they are strongly associated with active transcription. Here we tested the hypothesis that BRD4 inhibition by JQ1 enhances radiation-induced DNA damage, making it a potential radiosensitizer in the treatment of DIPG. We evaluated the effects of JQ1 on genes expression using RNA sequence. Radiation-induced DNA double-strand break (DSB) repair was analyzed by immunocytochemistry and western blotting of DSB markers γH2AX and 53BP1, and DSB repair markers pRad50 and Rad51. Clonogenic survival assay was used to determine the effect of JQ1 on radiation response of DIPG cells. In vivo response to radiation monotherapy and combination therapy of RT and JQ1 were evaluated in patient-derived DIPG xenografts. JQ1 significantly reduced the expression of DNA DSB repair genes in DIPG cells. JQ1 sustained high levels of γH2AX and 53BP1 and reduced the levels of pRad50 and Rad51 in irradiated DIPG cells. JQ1 reduced clonogenic survival and enhanced radiation effect in DIPG cells. In vivo studies revealed increased survival of animals treated with combination therapy of RT and JQ1 in compared to either monotherapy. Together, these results highlight JQ1 as a potential radiosensitizer and provide a rationale for developing combination therapy with radiation in the treatment of DIPG.

SCIDOT-48. EVALUATING THE THERAPEUTIC EFFICACY AND CONVECTION ENHANCED DELIVERY (CED) TOXICITY ANALYSIS OF CDK4/6 INHIBITOR PALBOCICLIB FOR DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Abstract DIPG is a devastating, aggressive brain tumor of childhood. No chemotherapeutic agent has so far shown significant clinical efficacy and focal radiation therapy provides only temporary relief. Recent studies on the genomic aberrations of pediatric gliomas have shown that a subset of these tumors harbor focal gains in proteins of the cyclin-dependent kinase (CDK) 4/6 cell cycle pathway. H3.3K27M mutation, associated with &gt;80% of DIPGs, has also been associated with epigenetic silencing of p16 tumor suppressor, an important effector in regulating CDK4/6 pathway. In this study, we characterized and confirmed through immunoblot analysis that proteins involved in CDK 4/6 pathway, including Rb1, are intact in multiple patient derived DIPG (SU-DIPG-IV, SU-DIPG-XIII, SU-DIPG-XVIII, SF8628) and wild type (SF9428) cell lines. Expression of p16 was decreased or undetectable in DIPG cells compared to normal astrocytes. In addition, pharmacologic inhibition of CDK4/6 with palbociclib showed dose dependent reduction in DIPG cell proliferation with IC50 ranging from 5.42uM to 21.35uM between different cell lines. However, major problems of palbociclib are that it does not readily cross the blood-brain-barrier (BBB) and has major systematic toxicities, such as myelosuppression, which limits oral dosing. One solution to overcome the BBB is convection enhanced delivery (CED) which is a form of local delivery that bypasses the BBB by placement of small diameter catheters directly into the brain tumor. With this approach, high regional drug concentration can be achieved with negligible systemic toxicity. In this study, we also observed no significant short- or long-term neurotoxicity of palbociclib delivered via CED in a mouse model. We hypothesize that the CDK4/6 cell cycle pathway has an important role in DIPG tumorigenesis and progression and that existing therapies targeting these pathways, which are already in clinical use for other cancers, can be rapidly repurposed for the treatment of DIPG through CED.

EXTH-60. CONVECTION-ENHANCED DELIVERY OF EZH2 INHIBITOR FOR THE TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood brain tumor and the majority of patients with DIPG die within 2 years after initial diagnosis. Factors that contribute to the dismal prognosis of these patients include the infiltrative nature and anatomic location in an eloquent area of the brain, which prevents total surgical resection and the presence of the blood-brain barrier (BBB), which reduces the distribution of systemically administered agents. Convection-enhanced delivery (CED) is a direct infusion technique to deliver therapeutic agents into a target site in the brain and able to deliver a high concentration drug to the infusion site without systemic toxicities. EZH2, histone methyltransferase of polycomb repressive complex 2, is a potential therapeutic target in DIPG. The study aims to assess the efficacy of EZH2 inhibitor by CED against DIPG xenograft model. The biodistribution of EZH2 inhibitor was evaluated by high-performance liquid chromatography (HPLC). HPLC analysis showed that the concentrationof EZH2 inhibitor in the brainstem tumor was only 3.74% of blood concentration by systemic administration. We treated mice bearing human DIPG xenografts with EZH2-inhibitor using intraperitoneal or CED administration. Intracranial tumor growth was monitored by bioluminescence image and therapeutic response was evaluated by animal survival. CED of EZH2 inhibitor suppressed tumor growth and significantly extended animal survival in compared to intraperitoneal administration of EZH2 inhibitor (P=0.0475). Taken together, our results indicate that CED of EZH2 inhibitor would provide an alternative strategy for bypassing the BBB and increasing efficacy of EZH2 inhibitor for the treatment of DIPG.

Diagnostics and treatment of diffuse intrinsic pontine glioma: where do we stand?

IntroductionDiffuse intrinsic pontine glioma (DIPG) is a rare clinically, neuro-radiologically, and molecularly defined malignancy of the brainstem with a median overall survival of approximately 11 months. Our aim is to evaluate the current tendency for its treatment in Europe in order to develop (inter)national consensus guidelines.MethodsHealthcare professionals specialized in DIPG were asked to fill in an online survey with questions regarding usual treatment strategies at diagnosis and at disease progression in their countries and/or their centers, respectively.ResultsSeventy-four healthcare professionals responded to the survey, of which 87.8% were pediatric oncologists. Only 13.5% of the respondents biopsy all of their patients, 41.9% biopsy their patients infrequently. More than half of the respondents (54.1%) treated their patients with radiotherapy only at diagnosis, whereas 44.6% preferred radiotherapy combined with chemotherapy. When the disease progresses, treatment strategies became even more diverse, and the tendency for no treatment increased from 1.4% at diagnosis to 77.0% after second progression. 36.5% of the healthcare professionals treat children younger than 3 years differently than older children at diagnosis. This percentage decreased, when the disease progresses. Most of the participants (51.4%) included less than 25% of their patients in clinical trials.ConclusionThis survey demonstrates a large heterogeneity of treatment regimens, especially at disease progression. We emphasize the need for international consensus guidelines for the treatment of DIPG, possible by more collaborative clinical trials.

Abstract 3192: Aptamers, antibodies and radiotherapy for the treatment of DIPG

Abstract Diffuse intrinsic pontine gliomas (DIPGs) is one of the most aggressive pediatric brain tumors. Radiotherapy (RT) constitute the standard of care of these tumors being its therapeutic effect just palliative. There is plenty of evidence that RT is able to activate immune responses through the induction of immunogenic cell death. However, it also elicits immunosuppressive effects and enforces immunological tolerance. The result is that in DIPGs the RT effect is just transitory. In this context, immune costimulatory or inhibitory molecules with the capability to boost the immune effect of RT could be amenable agents to use in combination to treat DIPGs. Agonist antibodies has been widely used in immunotherapy. Aptamers are high-affinity single-stranded nucleic acid ligands that exhibit remarkable affinity and specificity to their targets, comparable or exceeding that of antibodies. 4-1BB is a major costimulatory receptor promoting the survival and expansion of activated T cells. TIM-3 is a negative regulator of lymphocyte function that is involved in T-cell exhaustion. To this end, we examined the effect of RT in combination with either an agonist 4-1BB antibody or an aptamer against TIM-3. Importantly, both combined treatments showed a safe profile. Moreover, combination treatment of 4-1BB agonist antibody or TIM-3 aptamer with RT resulted in a significant improvement in the median survival of mice bearing DIPG orthotopic tumors when compared with single treatment in around 20 days (P=0.001 and P=0.04, for the combination of RT and 4-1BB or TIM-3,respectively). In addition, both combination led to long-term survivors (90 days). Rechallenge experiments in these animals showed the generation of memory against the tumors in both combined treatment. Mechanistic studies performed on day 16 showed an increase in CD8 effector cells, a decrease in T-regulators Foxp3+ cells and an increase in INF-gamma expression suggesting the triggering of an antitumor-immune response. Our data underscore that combination of RT with immune-boosting strategies for DIPGs are worth exploring. Citation Format: Naiara Martinez-Velez, Miguel Marigil, Javier Aristu, Luis Ramos, Fernando Pastor, Ana Patiño-García, Marc García-Moure, Juan Fueyo, Candelaria Gomez-Manzano, Ricardo Diez-Valle, Sonia Tejada, Marta M. Alonso. Aptamers, antibodies and radiotherapy for 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 3192.

Combination of EZH2 inhibitor and BET inhibitor for treatment of diffuse intrinsic pontine glioma

BackgroundDiffuse intrinsic pontine glioma is an infiltrative, often high-grade glioma of the brainstem that is not amenable to surgical resection. The current treatment of DIPG by radiation therapy showed dramatically improvement of patient’s condition, however, the tumor recurs rapidly. More and more studies are focused on the genetic and epigenetic drivers of DIPGs, which may provide more and more novel therapy target for DIPG. EZH2 has been proved to be a potential therapeutic target for H3K27M-mutant pediatric gliomas recently. Meanwhile, BET family protein is a hot target in many different types of cancers, including DIPG. In this study, we performed the treatment of both EZH2 and BET inhibitor for DIPG cells.ResultsThe combination of these two inhibitors exhibited better inhibition of the tumor growth both in vitro and in vivo compared to use the inhibitor individually. This inhibition was performed by blocking the proliferation and promoting the cell apoptosis. Meanwhile, combination treatment of these two inhibitors would also affect the epigenetic markers which were abnormal in the tumors of the certain set of genes.ConclusionThus we provided a novel therapy strategy for clinical treatment of DIPG.

Abstract LB-B06: Functionally defined therapeutic targets in diffuse intrinsic pontine glioma

Abstract Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer that has a limited response to treatment (median survival after diagnosis is only 9 months). Historically, DIPG research has been limited by a dearth of tumor tissue available for study and a lack of experimental model systems. Recently, both cell cultures derived from patients with DIPG and orthotopic xenograft models have been established. We performed a chemical screen in 16 patient-derived DIPG cultures using 83 drugs, selected by pediatric neurooncologists as either promising small-molecule compounds or traditional chemotherapeutic agents. Of these 83 drugs, 14 showed activities against three or more DIPG cultures. Notable ‘misses’ that highlight the resistance of this tumor to traditional chemotherapies included temozolomide, carboplatin, and vincristine, while DIPG cell cultures were reported to have substantial sensitivity to histone deacetylase inhibitors, consistent with the highly recurrent H3K27M driving mutations in ∼80% of DIPG. Panobinostat was one of the most effective drugs screened, with 12/16 DIPG cultures showing sensitivity to this drug. Compared to vorinostat, another HDAC inhibitor, panobinostat exhibited substantially greater potency against DIPG cells. Treatment of DIPG cultures with panobinostat yielded a time and dose-dependent decrease in viability (RNA-mediated knockdown of HDAC1 and HDAC2 confirmed the mechanism of action). Treatment of DIPG cultures with panobinostat also increased H3 acetylation and H3K27 trimethylation, and led to a partial rescue of the H3K27M induced global hypotrimethylation phenotype. Increased K27 trimethylation was an unexpected effect of the drug, but it is consistent with recent findings that acetylated H3K27 can ‘detoxify’ K27M-induced inhibition of PCR2. RNA-seq performed on panobinostat- or vehicle-treated DIPG cells revealed sweeping changes in gene expression, including normalization of the K27M gene expression signature and decrease of the oncogenic MYC target gene-expression signature. In addition, the multi-histone deacetylase inhibitor panobinostat demonstrated efficacy in DIPG orthotopic xenograft models administered using convection enhanced delivery. Combination testing of panobinostat and the histone demethylase inhibitor GSK-J4, recently shown to decrease viability of mutant DIPG cells, revealed that the two had synergistic effects. Together, these data suggest a promising FDA-approved therapeutic strategy for DIPG that could be rapidly translated for use in the clinic, further indicating that epigenetic modifying therapies are emerging as the most promising class of agents for the treatment of DIPG. Citation Format: Catherine S. Grasso. Functionally defined therapeutic targets in diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B06.

OP38 * ROBOT-ASSISTED CONVECTION-ENHANCED DELIVERY OF CARBOPLATIN FOR PROGRESSIVE DIPG - A SAFETY AND FEASIBILITY REPORT OF 5 CASES

INTRODUCTION: Convection-enhanced delivery is a method of drug delivery to the brain through implanted microcatheters. There has been significant research focus on applying CED to the treatment of DIPG, where prognosis remains poor. METHOD: 5 paediatric patients with tumour progression after radiotherapy were treated on compassionate grounds. Up to 4 catheter trajectories per patient were planned on pre-operative 3T MRI scans using custom-made planning software. Catheters were implanted using a robot-assisted technique. Infusions of carboplatin (0.18 mg/mL) were performed on 2 consecutive days. Serial T2W MRI was used to determine drug distribution. RESULTS: No adverse events related to implantation of catheters were observed. High volume (9mL/day), high flow rate infusions (10 µL/min) were performed. During infusions patients exhibited worsening of pre-existing neurological dysfunction, which was transient in all except one patient who required a prolonged period of non-invasive respiratory support. Real-time MRI indicated drug distribution throughout the majority of targeted tumour in 4 patients. Reduction in the volume of enhancing tumour was seen in 2 patients on follow-up imaging. CONCLUSION:Robot-assisted catheter insertion was safe and achieved satisfactory targeting. Satisfactory drug distribution was achieved in 4 out of 5 patients. Reduction in tumour enhancement was seen in 2 patients. Infusions were associated with only transient worsening of neurological function in all but one patient. This initial experience in 5 patients represents a strong foundation for a clinical trial of CED of carboplatin for DIPG.

ABCG2 AND MDR PROMOTE BRAINSTEM GLIOMAGENESIS AND LIMIT THE EFFICACY OF DASATINIB IN A DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) MOUSE MODEL

BACKGROUND: DIPG is an incurable tumor that arises in the brainstem of children. Our laboratory generates murine DIPGs by expressing PDGF-B and deleting p53 in nestin progenitors of the neonatal brainstem using the RCAS/tv-a system. Dasatinib, an inhibitor of PDGFR-A and SRC activation, has been evaluated in clinical trials for children with DIPG but with disappointing results. As dasatinib is a substrate of the ABC transporters ABCG2 and MDR, we hypothesized that ABCG2 and MDR may limit the efficacy of dasatinib in DIPG. METHODS: We generated murine DIPGs driven by PDGF-B and p53 loss in ABC WT (ABCG2+/+; MDRa+/+; MDRb+/+) and ABC KO (ABCG2-/-; MDRa-/-; MDRb-/-) mice. Next, we investigated whether dasatinib is efficacious in ABC KO and ABC WT DIPG-bearing mice. We treated ABC WT DIPG-bearing mice with dasatinib alone, elacridar (a dual ABCG2/MDR inhibitor) alone, and dasatinib in combination with elacridar. We also measured dasatinib levels in the tumor and plasma of all of the different tumor cohorts. Lastly, we evaluated the blood-brain-barrier (BBB) of our DIPG mouse model with Dextran Texas Red. RESULTS: Surprisingly, we observed a significant difference in tumor latency between untreated ABC KO and untreated ABC WT mice in this PDGF-B; p53 deficient DIPG model. Median survival of DIPG-bearing ABC KO was longer than DIPG-bearing ABC WT: 43 days vs. 34 days respectively (p = 0.02). We observed that dasatinib almost doubled the survival of DIPG-bearing mice in the ABC KO model: 44 days vs. 80 days (p = 0.0004). Dasatinib modestly prolonged survival in ABC WT mice: 33 days vs. 42 days (p < 0.0001). Co-treatment with elacridar significantly increased the efficacy of dasatinib in the ABC WT model 42 days vs. 59 days (p < 0.0001). However, elacridar alone did not significantly prolong survival in the ABC WT mice 33 days vs. 34 days (p = 0.38). Pharmacokinetic analysis demonstrated that genetic ablation of the ABC transporters or inhibition of ABC transporters with elacridar significantly increased dasatinib delivery into normal brain but not into tumor. Dextran Texas Red studies demonstrated a disrupted BBB in DIPG models independent of ABC transporters. CONCLUSIONS: 1. ABC transporters promote brainstem gliomagenesis 2. ABC transporters regulate dasatinib delivery into normal brain but not into the tumor as BBB is disrupted in our DIPG mouse model 3. ABC transporter activity limits the efficacy of dasatinib in vivo. Our results suggest that ABCG2 and MDR inhibitors may have a therapeutic role in the treatment of DIPG. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.