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OBJECTIVE Diffuse midline gliomas, including diffuse intrinsic pontine gliomas (DIPGs), are among the most malignant and devastating childhood brain cancers. Despite aggressive treatment, nearly all children with these tumors succumb to their disease within 2 years of diagnosis. Due to the anatomical location of the tumors within the pons, surgery is not a treatment option, and distribution of most systematically administered drugs is limited by the blood-brain barrier (BBB). New drug delivery systems that bypass the BBB are desperately needed to improve outcomes of DIPG patients. Intranasal delivery (IND) is a practical and noninvasive drug delivery system that bypasses the BBB and delivers the drugs to the brain through the olfactory and trigeminal neural pathways. In this study, the authors evaluated the efficacy of nanoliposomal (LS) irinotecan (CPT-11) and an active metabolite of CPT-11, 7-ethyl-10-hydroxycamptothecin (SN-38), using IND in DIPG patient-derived xenograft models. METHODS In vitro responses to LS-CPT-11 and LS-SN-38 in DIPG cells were evaluated with cell viability, colony formation, and apoptosis assays. The cellular uptakes of rhodamine-PE (Rhod)–labeled LS-CPT-11 and LS-SN-38 were analyzed with fluorescence microscopy. Mice bearing DIPG patient-derived xenografts were treated with IND of LS-control (empty liposome), LS-CPT-11, or LS-SN-38 by IND for 4 weeks. In vivo responses were measured for tumor growth by serial bioluminescence imaging and animal subject survival. The concentration of SN-38 in the brainstem tumor administered by IND was determined by liquid chromatography–mass spectrometry (LC-MS). Immunohistochemical analyses of the proliferative and apoptotic responses of in vivo tumor cells were performed with Ki-67 and TUNEL staining. RESULTS LS-SN-38 inhibited DIPG cell growth and colony formation and increased apoptosis, outperforming LS-CPT-11. Rhod-labeled LS-SN-38 showed intracellular fluorescence signals beginning at 30 minutes and peaking at 24 hours following treatment. LC-MS analysis revealed an SN-38 concentration in the brainstem tumor of 0.66 ± 0.25 ng/ml (5.43% ± 0.31% of serum concentration). IND of LS-SN-38 delayed tumor growth and significantly prolonged animal survival compared with IND of LS-control (p < 0.0001) and LS-CPT-11 (p = 0.003). IND of LS-SN-38 increased the number of TUNEL-positive cells and decreased the Ki-67–positive cells in the brainstem tumor. CONCLUSIONS This study demonstrates that IND of LS-SN-38 bypasses the BBB and enables efficient and noninvasive drug delivery to the brainstem tumor, providing a promising therapeutic approach for treating DIPG.

Aberrant activity of the H3K27 modifiers EZH2 and BRD4 is an important oncogenic driver for atypical teratoid/rhabdoid tumor (AT/RT), and each is potentially a possible therapeutic target for treating AT/RT. We, therefore, determined whether targeting distinct histone modifier activities was an effective approach for treating AT/RT. The effects of EZH2 and BRD4 inhibition on histone modification, cell proliferation, and cell invasion were analyzed by immunoblotting, MTS assay, colony formation assay, and cell invasion assay. RNA- and chromatin immunoprecipitation-sequencing were used to determine transcriptional and epigenetic changes in AT/RT cells treated with EZH2 and BRD4 inhibitors. We treated mice bearing human AT/RT xenografts with EZH2 and BRD4 inhibitors. Intracranial tumor growth was monitored by bioluminescence imaging, and the therapeutic response was evaluated by animal survival. AT/RT cells showed elevated levels of H3K27 trimethylation (H3K27me3) and H3K27 acetylation (H3K27ac), with expression of EZH2 and BRD4, and lack of SMARCB1 proteins. Targeted inhibition of EZH2 and BRD4 activities reduced cell proliferation and invasiveness of AT/RT in association with decreasing H3K27me3 and H3K27ac. Differential genomic occupancy of H3K27me3 and H3K27ac regulated specific gene expression in response to EZH2 and BRD4 inhibitions. A combination of EZH2 and BRD4 inhibition increased the therapeutic benefit in vitro and in vivo, outperforming either monotherapy. Overall, histones H3K27me3 and H3K27ac were elevated in AT/RT cells and distributed in distinct chromatin regions to regulate specific gene expression and to promote AT/RT growth. Targeting EZH2 and BRD4 activity is, therefore, a potential combination therapy for AT/RT.

Supplementary Figure from Therapeutic Targeting of EZH2 and BET BRD4 in Pediatric Rhabdoid Tumors

Supplementary Data from Therapeutic Targeting of EZH2 and BET BRD4 in Pediatric Rhabdoid Tumors

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Supplementary Table S2

Purpose:Radiotherapy (RT) has long been and remains the only treatment option for diffuse intrinsic pontine glioma (DIPG). However, all patients show evidence of disease progression within months of completing RT. No further clinical benefit has been achieved using alternative radiation strategies. Here, we tested the hypothesis that histone demethylase inhibition by GSK-J4 enhances radiation-induced DNA damage, making it a potential radiosensitizer in the treatment of DIPG.Experimental Design: We evaluated the effects of GSK-J4 on genes associated with DNA double-strand break (DSB) repair in DIPG cells by RNA sequence, ATAC sequence, and quantitative real-time PCR. Radiation-induced DNA DSB repair was analyzed by immunocytochemistry of DSB markers γH2AX and 53BP1, DNA-repair assay, and cell-cycle distribution. Clonogenic survival assay was used to determine the effect of GSK-J4 on radiation response of DIPG cells. In vivo response to radiation monotherapy and combination therapy of RT and GSK-J4 was evaluated in patient-derived DIPG xenografts.Results:GSK-J4 significantly reduced the expression of DNA DSB repair genes and DNA accessibility in DIPG cells. GSK-J4 sustained high levels of γH2AX and 53BP1 in irradiated DIPG cells, thereby inhibiting DNA DSB repair through homologous recombination pathway. GSK-J4 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 GSK-J4 compared with either monotherapy.Conclusions:Together, these results highlight GSK-J4 as a potential radiosensitizer and provide a rationale for developing combination therapy with radiation in the treatment of DIPG.

Methods, Figures, legends and Tables

Purpose:Diffuse intrinsic pontine glioma (DIPG) is among the deadliest of pediatric brain tumors. Radiotherapy is the standard-of-care treatment for DIPG, but offers only transient relief of symptoms for patients with DIPG without providing significant survival benefit. Oncolytic virotherapy is an anticancer treatment that has been investigated for treating various types of brain tumors.Experimental Design:Here, we have explored the use of mesenchymal stem cells (MSC) for oncolytic virus (OV) delivery and evaluated treatment efficacy using preclinical models of DIPG. The survivin promoter drives the conditional replication of OV used in our studies. The efficiency of OV entry into the cells is mediated by fiber modification with seven lysine residues (CRAd.S.pK7). Patients' samples and cell lines were analyzed for the expression of viral entry proteins and survivin. The ability of MSCs to deliver OV to DIPG was studied in the context of a low dose of irradiation.Results:Our results show that DIPG cells and tumors exhibit robust expression of cell surface proteins and survivin that enable efficient OV entry and replication in DIPG cells. MSCs loaded with OV disseminate within a tumor and release OV throughout the DIPG brainstem xenografts in mice. Administration of OV-loaded MSCs with radiotherapy to mice bearing brainstem DIPG xenografts results in more prolonged survival relative to that conferred by either therapy alone (P < 0.01).Conclusions:Our study supports OV, CRAd.S.pK7, encapsulated within MSCs as a therapeutic strategy that merits further investigation and potential translation for DIPG treatment.

Aberrant activity of the H3K27 modifiers EZH2 and BRD4 is an important oncogenic driver for atypical teratoid/rhabdoid tumor (AT/RT), and each is potentially a possible therapeutic target for treating AT/RT. We, therefore, determined whether targeting distinct histone modifier activities was an effective approach for treating AT/RT. The effects of EZH2 and BRD4 inhibition on histone modification, cell proliferation, and cell invasion were analyzed by immunoblotting, MTS assay, colony formation assay, and cell invasion assay. RNA- and chromatin immunoprecipitation-sequencing were used to determine transcriptional and epigenetic changes in AT/RT cells treated with EZH2 and BRD4 inhibitors. We treated mice bearing human AT/RT xenografts with EZH2 and BRD4 inhibitors. Intracranial tumor growth was monitored by bioluminescence imaging, and the therapeutic response was evaluated by animal survival. AT/RT cells showed elevated levels of H3K27 trimethylation (H3K27me3) and H3K27 acetylation (H3K27ac), with expression of EZH2 and BRD4, and lack of SMARCB1 proteins. Targeted inhibition of EZH2 and BRD4 activities reduced cell proliferation and invasiveness of AT/RT in association with decreasing H3K27me3 and H3K27ac. Differential genomic occupancy of H3K27me3 and H3K27ac regulated specific gene expression in response to EZH2 and BRD4 inhibitions. A combination of EZH2 and BRD4 inhibition increased the therapeutic benefit in vitro and in vivo, outperforming either monotherapy. Overall, histones H3K27me3 and H3K27ac were elevated in AT/RT cells and distributed in distinct chromatin regions to regulate specific gene expression and to promote AT/RT growth. Targeting EZH2 and BRD4 activity is, therefore, a potential combination therapy for AT/RT.

BackgroundDiffuse intrinsic pontine glioma (DIPG) is associated with transcriptional dysregulation driven by H3K27 mutation. The super elongation complex (SEC) is required for transcriptional elongation through release of RNA polymerase II (Pol II). Inhibition of transcription elongation by SEC disruption can be an effective therapeutic strategy of H3K27M-mutant DIPG. Here, we tested the effect of pharmacological disruption of the SEC in H3K27M-mutant DIPG to advance understanding of the molecular mechanism and as a new therapeutic strategy for DIPG.MethodsShort hairpin RNAs (shRNAs) were used to suppress the expression of AF4/FMR2 4 (AFF4), a central SEC component, in H3K27M-mutant DIPG cells. A peptidomimetic lead compound KL-1 was used to disrupt a functional component of SEC. Cell viability assay, colony formation assay, and apoptosis assay were utilized to analyze the effects of KL-1 treatment. RNA- and ChIP-sequencing were used to determine the effects of KL-1 on gene expression and chromatin occupancy. We treated mice bearing H3K27M-mutant DIPG patient-derived xenografts (PDXs) with KL-1. Intracranial tumor growth was monitored by bioluminescence image and therapeutic response was evaluated by animal survival.ResultsDepletion of AFF4 significantly reduced the cell growth of H3K27M-mutant DIPG. KL-1 increased genome-wide Pol II occupancy and suppressed transcription involving multiple cellular processes that promote cell proliferation and differentiation of DIPG. KL-1 treatment suppressed DIPG cell growth, increased apoptosis, and prolonged animal survival with H3K27M-mutant DIPG PDXs.ConclusionsSEC disruption by KL-1 increased therapeutic benefit in vitro and in vivo, supporting a potential therapeutic activity of KL-1 in H3K27M-mutant DIPG.

Background Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood brain tumor and the majority of patients die within 2 yr 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. Objective To assess the efficacy of enhancer of zeste homolog-2 (EZH2) inhibitor by CED against human DIPG xenograft models. Methods The 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. Results LC/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 = .0475). Conclusion Our 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.

Purpose: Diffuse intrinsic pontine glioma (DIPG) is among the deadliest of pediatric brain tumors. Radiotherapy is the standard-of-care treatment for DIPG, but offers only transient relief of symptoms for patients with DIPG without providing significant survival benefit. Oncolytic virotherapy is an anticancer treatment that has been investigated for treating various types of brain tumors. Experimental Design: Here, we have explored the use of mesenchymal stem cells (MSC) for oncolytic virus (OV) delivery and evaluated treatment efficacy using preclinical models of DIPG. The survivin promoter drives the conditional replication of OV used in our studies. The efficiency of OV entry into the cells is mediated by fiber modification with seven lysine residues (CRAd.S.pK7). Patients' samples and cell lines were analyzed for the expression of viral entry proteins and survivin. The ability of MSCs to deliver OV to DIPG was studied in the context of a low dose of irradiation. Results: Our results show that DIPG cells and tumors exhibit robust expression of cell surface proteins and survivin that enable efficient OV entry and replication in DIPG cells. MSCs loaded with OV disseminate within a tumor and release OV throughout the DIPG brainstem xenografts in mice. Administration of OV-loaded MSCs with radiotherapy to mice bearing brainstem DIPG xenografts results in more prolonged survival relative to that conferred by either therapy alone (P < 0.01). Conclusions: Our study supports OV, CRAd.S.pK7, encapsulated within MSCs as a therapeutic strategy that merits further investigation and potential translation for DIPG treatment.

Diffuse intrinsic pontine glioma (DIPG) is among the deadliest of pediatric brain tumors. Radiation therapy is the standard of care treatment for DIPG, but offers only transient relief of symptoms for DIPG patients without providing significant survival benefit. Oncolytic virotherapy (OV) is an anticancer treatment that has been investigated for treating various types of brain tumors. Here, we have explored the use of mesenchymal stem cells (MSC) for OV delivery and evaluated treatment efficacy using preclinical models of DIPG. Our results show that DIPG cells and tumors exhibit robust expression of cell surface proteins that are important for OV entry, and that MSCs loaded with OV disseminate within and release OV throughout the tumor in mice bearing DIPG brainstem xenografts. When combining administration of OV-loaded MSCs with radiotherapy, mice bearing brainstem DIPG xenografts experience a significant survival benefit, relative to that conferred by either therapy alone (p

Diffuse Intrinsic pontine glioma is an incurable brain tumor of childhood characterized by histone mutations at lysine 27 which results in epigenomic dysregulation. There has been a failure to develop effective treatment for this tumor. We performed a combined RNAi and chemical screen targeting epigenetic regulatory genes and identified the polycomb (PcG) repressive complex 1, component BMI1 as a critical factor for DIPG tumor maintenance in vivo. BMI1 chromatin occupancy was enriched at genes associated with differentiation and tumor suppressors in DIPG cells. Inhibition of BMI1 results in decreased cell self-renewal and attenuation of tumor growth due to induction of senescence. Prolong BMI1 inhibition induces a senescence associated secretory phenotype which promotes tumor recurrence. Clearance of senescent cells using BH3 protein mimetics co-operates with BMI1 inhibition to synergistically attenuate tumor growth in vivo.

Background: Diffuse midline glioma (DMG) 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. Objective: This study aims to assess the efficacy of enhancer of zeste homolog-2 (EZH2) inhibitor by CED against human DMG xenograft models. Methods: The concentration of EZH2 inhibitor (EPZ-6438) in the brainstem tumor was evaluated by liquid chromatography mass spectrometry (LC/MS). We treated mice bearing human DMG 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. Results: LC/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). Conclusion: Our 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 DMG.

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.

Purpose: Radiotherapy (RT) has long been and remains the only treatment option for diffuse intrinsic pontine glioma (DIPG). However, all patients show evidence of disease progression within months of completing RT. No further clinical benefit has been achieved using alternative radiation strategies. Here, we tested the hypothesis that histone demethylase inhibition by GSK-J4 enhances radiation-induced DNA damage, making it a potential radiosensitizer in the treatment of DIPG. Experimental Design: We evaluated the effects of GSK-J4 on genes associated with DNA double-strand break (DSB) repair in DIPG cells by RNA sequence, ATAC sequence, and quantitative real-time PCR. Radiation-induced DNA DSB repair was analyzed by immunocytochemistry of DSB markers γH2AX and 53BP1, DNA-repair assay, and cell-cycle distribution. Clonogenic survival assay was used to determine the effect of GSK-J4 on radiation response of DIPG cells. In vivo response to radiation monotherapy and combination therapy of RT and GSK-J4 was evaluated in patient-derived DIPG xenografts. Results: GSK-J4 significantly reduced the expression of DNA DSB repair genes and DNA accessibility in DIPG cells. GSK-J4 sustained high levels of γH2AX and 53BP1 in irradiated DIPG cells, thereby inhibiting DNA DSB repair through homologous recombination pathway. GSK-J4 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 GSK-J4 compared with either monotherapy. Conclusions: Together, these results highlight GSK-J4 as a potential radiosensitizer and provide a rationale for developing combination therapy with radiation in the treatment of DIPG.

BACKGROUND: An impermeable blood–brain barrier and drug efflux via ATP-binding cassette (ABC) transporters such as p-glycoprotein may contribute to underwhelming efficacy of peripherally delivered agents to treat diffuse intrinsic pontine glioma (DIPG). OBJECTIVE: To explore the pharmacological augmentation of convection-enhanced delivery (CED) infusate for DIPG. METHODS: The efficacy of CED dasatinib, a tyrosine kinase inhibitor, in a transgenic H3.3K27M mutant murine model was assessed. mRNA expression of ABCB1 (p-glycoprotein) was analyzed in 14 tumor types in 274 children. In Vitro viability studies of dasatinib, the p-glycoprotein inhibitor, tariquidar, and dexamethasone were performed in 2 H3.3K27M mutant cell lines. Magnetic resonance imaging (MRI) was used to evaluate CED infusate (gadolinium/dasatinib) distribution in animals pretreated with tariquidar and dexamethasone. Histological assessment of apoptosis was performed. RESULTS: Continuous delivery CED dasatinib improved median overall survival (OS) of animals harboring DIPG in comparison to vehicle (39.5 and 28.5 d, respectively; P = .0139). Mean ABCB1 expression was highest in K27M gliomas. In Vitro, the addition of tariquidar and dexamethasone further enhanced the efficacy of dasatinib (P

BACKGROUND Diffuse intrinsic pontine gliomas (DIPGs) driven by mutations in the histone 3 (H3) gene (H3K27M) are aggressive pediatric brain tumors for which there is no curative therapy. METHODS To identify novel therapeutic targets we performed a high throughput drug screen combined with an epigenetically targeted RNAi screen using H3K27M and H3.3 WT DIPG cells. RESULTS Chemical and genetic depletion of BMI1 in vitro resulted in inhibition of clonogenicity and cell self-renewal consistent with previous studies. We show for the first time that clinically relevant BMI1 inhibitors attenuates growth of orthotopic DIPG xenografts as measured by MRI and prolong survival in vivo. We found that BMI1 inhibition drives phenotypic cellular senescence and that the senescent cells were able reactivate to form new neurospheres in vitro and tumor growth in vivo. RNA-seq, ChIP-Seq and immuno-proteomic analysis revealed that the senescent cells induced the expression of the Senescence Associated Secretory Phenotype (SASP) cytokines by increasing occupancy of activated histone marks at SASP factor promoters. The SASP results in increased expression of anti-apoptotic BH3 proteins including BCLxl, and BCL2. Treatment of the PTC028 treated senescent DIPG cells with BH3 mimetics induces apoptosis and clears the senescent cells. Combining BH3 mimetics with BMI1 inhibition attenuates tumor growth in vivo synergistically and significantly prolongs survival of DIPG bearing mice compared to BMI1 inhibition alone. CONCLUSION These data inform the current trial of BMI1 inhibition as a monotherapy and predict the need for adding BH3 mimetics to achieve efficacy.

Diffuse intrinsic pontine glioma (DIPG) is highly aggressive brain stem tumor and needed to develop novel therapeutic agents for the treatment. The super elongation complex (SEC) is essential for transcription elongation through release of RNA polymerase II (Pol II). We found that AFF4, a scaffold protein of the SEC, is required for the growth of H3K27M-mutant DIPG cells. In addition, the small molecule SEC inhibitor, KL-1, increased promoter-proximal pausing of Pol II, and reduced transcription elongation, resulting in down-regulate cell cycle, transcription and DNA repair genes. KL-1 treatment decreased cell growth and increased apoptosis in H3K27M-mutant DIPG cells, and prolonged animal survival in our human H3K27M-mutant DIPG xenograft model. Our results demonstrate that the SEC disruption by KL-1 is a novel therapeutic strategy for H3K27M-mutant DIPG.

INTRODUCTION: Children with diffuse intrinsic pontine gliomas (DIPGs) die within 2 years after initial diagnosis. The infiltrative nature and anatomic location of DIPGs preclude surgical resection, and the blood-brain barrier (BBB) reduces the availability of systemically administered agents. New drug delivery approaches circumventing the BBB are greatly needed. Intranasal delivery (IND) is a practical, noninvasive method to deliver therapeutic agents into the brain along with the olfactory and trigeminal nerves pathway. With the advantages of reducing systemic side effects and convenient self-administration for patients, IND is an alternative to systemic and direct invasive drug deliveries. METHODS: Human DIPG cell lines were treated with hydrophobic fluorophore (DiI)–labeledliposomes containing SN38 (LS-SN38). Cell viability was determined by MTS assay and intracellular localization was imaged by confocal microscopy. Mice bearing human brainstem gliomas were randomly assigned to 2 groups: empty liposomes (LS-empty) and LS-SN38, administrated IND for 3 weeks. Tumor growth and response to therapy were quantitatively measured by bioluminescence imaging, and efficacy was assessed by survival analysis. Ex vivo distribution of fluorescent liposomes were confirmed with fluorescent microscopy. Pharmacokinetics of LS-SN38 was determined in DIPG tumor by HPLC method. RESULTS: Intracellular fluorescence signals were detected at 30 minutes and peaked at 24 hours. LS-SN38 showed greater inhibition than LS-CPT11 of DIPG cell growth. IND of LS-SN38 showed significant reduction of growth rate and prolongation of survival in compared to control group. Ex vivo fluorescent signals were detected throughout different brain regions, indicates diffuse distribution in brainstem. Results from pharmacokinetics will be reported at the meeting.

INTRODUCTION: Focal radiation therapy has long been and remains the only treatment option for diffuse intrinsic pontine glioma (DIPG). However, all patients show evidence of disease progression within months of completing radiation therapy (RT). Since chemotherapy does not provide significant outcome improvement, it is crucial to find a suitable radiosensitizer. Our research has shown that the JMJD3 demethylase inhibitor, GSK-J4, exerts potent anti-tumor activity on DIPG cells while restoring methylation. Our aim is to We hypothesized that GSK-J4 may inhibit radiation-induced DNA repair, making it a potential radiosensitizer. METHODS: RNA sequencing (RNA-Seq) was used to analyze gene expression changes by GSK-J4 in DIPG cells. ATAC-seq was conducted to determine chromatin accessibility in DIPG cells treated with GSK-J4. We evaluated DNA damage repair using DNA repair assay and immunocytochemistry of DSB markers γH2AX and 53BP1. Western blotting and quantitative PCR (qPCR) were conducted to evaluate differential expression of mRNA and proteins involved in DNA DSB repair. In vivo response to radiation monotherapy and combination of RT + GSK-J4 were measured by animal survival studies. RESULTS: RNA-Seq and qPCR show that GSK-J4 significantly reduces DNA DSB repair genes in DIPG cells. ATAC-seq results reveal that GSK-J4 modifies DNA accessibility to regulate expression of DNA repair genes. Immunocytochemistry results support that GSK- J4 sustains high levels of γH2AX and 53BP1 in irradiated DIPG cells, thereby inhibiting DNA DSB repair. DNA repair assay demonstrate that GSK-J4 inhibits DNA damage repair via the homologous recombination pathway. Western blotting revealed that GSK-J4 causes a sustained level of phosphorylated Rad50 and gH2AX in irradiated DIPG cells. In vivo studies revealed increased survival of animals treated with combination therapy compared to monotherapy. These results highlight GSK-J4 as a potential radiosensitizer in DIPG treatment.

Recent discovery of somatic histone gene mutations, resulting in replacement of lysine 27 by methionine (K27M) in the encoded histone H3.3 proteins, in diffuse intrinsic pontine glioma (DIPG) has dramatically improved our understanding of disease pathogenesis, and stimulated the development of novel therapeutic approaches targeting epigenetic regulators for disease treatment. K27M mutant DIPG shows a dramatic reduction in global methylation at K27 residues. We have shown that the JMJD3 demethylase inhibitor, GSKJ4, acted to restore K27 methylation in DIPG cells, while demonstrating potent anti-tumor activity, in vitro and in vivo. In addition to H3K27 methylation, H3K27 can also be acetylated (K27ac), which requires bromo- and extra-terminal domain (BET) protein activity. Increase level of H3K27 acetylation and bromodomain proteins in K27M-containing nucleosomes suggests that inhibitor of BET bromodomain protein 4 (BRD4), JQ1, could be useful for the treatment of K27M DIPG. Our aim is to investigate the hypothesis that the combined JQ1 + GSKJ4 have greater anti-tumor activity in vitro and in vivo than either monotherapy and reduce the likelihood of drug resistance. The level of H3K27 methylation and acetylation in DIPG cells with treated JQ1 + GSKJ4 were studied by western blotting. JQ1 + GSKJ4 increased H3K27 methylation and reduced K27acetylation in DIPG cells. MTS assay showed that combination treatment of JQ1 + GSKJ4 significantly increased growth inhibition, compared with either therapy alone. Colony formation assay showed that combination treatment of JQ1 + GSKJ4 significantly reduced the number of colonies. Boyden chamber assay showed that combination treatment of JQ1 + GSKJ4 significantly reduced cell invasion. Annexin V assay showed that combination treatment of JQ1 + GSKJ4 significantly promoted cell apoptosis. In vivo response to monotherapy and combination of JQ1 + GSKJ4 will be measured by bioluminescence imaging and animal survival studies in our human DIPG xenograft model.

INTRODUCTION: Children with diffuse intrinsic pontine gliomas (DIPGs) die within 2 years after initial diagnosis. The infiltrative nature and anatomic location of DIPGs preclude surgical resection, and the blood-brain barrier (BBB) reduces the availability of systemically administered agents. New drug delivery approaches circumventing the BBB are greatly needed. Intranasal delivery (IND) is a practical, noninvasive method to deliver therapeutic agents into the brain along with the olfactory and trigeminal nerves pathway. With the advantages of reducing systemic side effects and convenient self-administration for patients, IND is an alternative to systemic and direct invasive drug deliveries. METHODS: Human DIPG cell lines were treated with hydrophobic fluorophore (DiI)–labeled nanoparticle liposomes containing CPT-11 (LS-CPT-11) and SN-38 (LS-SN-38). Cell viability was determined by MTS assay and intracellular localization was imaged by confocal microscopy. In vivo toxicity was determined by administrating empty nanoliposomes (LS-empty), LS-CPT11 and LS-SN38 via IND and intravenous routes for 5 days, with body weights monitoring for 20 days. Mice bearing human brainstem gliomas were randomly assigned to 3 groups: LS-empty, LS-CPT-11, LS-SN-38, administrated IND for 3 weeks. Tumor growth and response to therapy were quantitatively measured by bioluminescence imaging, and efficacy was assessed by survival analysis. RESULTS: Intracellular DiI-fluorescence signal were detected at 30 minutes and peaked at 24 hours. LS-SN-38 showed greater inhibition than LS-CPT-11 of DIPG cell growth. Animals administrated by IND showed no effect on body weight. IND of LS-SN-38 showed significant reduction of the growth rate and prolongation of survival in compared to control group.

The super elongation complex (SEC) is required for robust and productive transcription through release of RNA polymerase II (Pol II) with its P-TEFb module and promoting transcriptional processivity with its ELL2 subunit. Malfunction of SEC contributes to multiple human diseases including cancer. Here, we identify peptidomimetic lead compounds, KL-1 and its structural homolog KL-2, which disrupt the interaction between the SEC scaffolding protein AFF4 and P-TEFb, resulting in impaired release of Pol II from promoter-proximal pause sites and a reduced average rate of processive transcription elongation. SEC is required for induction of heat-shock genes and treating cells with KL-1 and KL-2 attenuates the heat-shock response from Drosophila to human. SEC inhibition downregulates MYC and MYC-dependent transcriptional programs in mammalian cells and delays tumor progression in a mouse xenograft model of MYC-driven cancer, indicating that small-molecule disruptors of SEC could be used for targeted therapy of MYC-induced cancer.

Key Clinical Message PTTM (Pulmonary tumor thrombotic microangiopathy) is very difficult to diagnose before death. We report a case of urothelial carcinoma of the urinary bladder associated with PTTM in which an antemortem diagnosis by PMC (pulmonary microvascular cytology). PMC may represent the only chance for diagnosis and achievement of remission in PTTM.

Objective. The aim of this study was to review HIV-negative patients with pulmonary cryptococcosis to analyze the correlations between clinical characteristics and chest computed tomography (CT) findings.Methods. We retrospectively analyzed medical records of 16 HIV-negative patients with pulmonary cryptococcosis diagnosed at our institution, and clinical characteristics of the patients with nodules or masses without ground-glass attenuation (GGA)/consolidation type were compared with those of patients with inclusive GGA or consolidation type.Results. Host status was immunocompromised (81.2%) in most of the patients, and 6 (37.5%) were asymptomatic. The most frequent radiologic abnormalities on chest CT scans were one or more nodules (87.5%), GGA (37.5%), and consolidations (18.8%). Most lesions were located in the lower lung. Levels of hemoglobin and platelets were significantly lower in patients with inclusive GGA or consolidation type. Although the differences were not significant, patients with inclusive GGA or consolidation type tended to have a C-reactive protein level of ≥1.0 mg/dL.Conclusion. If a patient with anemia and thrombocytopenia shows GGA or consolidation in the lung, pulmonary cryptococcosis should be given careful consideration.

A 78-year-old man had fatigue and appetite loss for 5 months. He had been receiving low-dose methotrexate for rheumatoid arthritis. Computed tomography revealed multiple pulmonary infiltrations and muddiness of the fatty tissue surrounding the right kidney, ureter wall thickening, and hydroureter/nephrosis, which were suspected retroperitoneal fibrosis. Lung biopsy revealed polymorphic/lymphoplasmacytic lymphoproliferative disorder. Methotrexate withdrawal resulted in spontaneous regression. Therefore, retroperitoneal lesion may account for the diagnosis as having retroperitoneal lymphoproliferative disorder, not retroperitoneal fibrosis.

Atypical teratoid/rhabdoid tumors (AT/RTs) are lethal childhood cancers and defined by inactivation of the SMARCB1 tumor suppressor gene encoding a chromatin remodeling complex which suppresses activity of EZH2 methyltransferase. The absence of SMARCB1 protein promotes increased EZH2 activity, results in increased methylation of histone H3 lysine 27 (H3K27), which is generally associated with transcriptional repression. H3K27 can also be acetylated, which requires bromo- and extra-terminal domain (BET) protein activity to activate transcription. Recent genome-wide ChIP-sequence analyses identified high occupancy of H3K27ac in association with BET bromodomain-containing protein 4 (BRD4) at super-enhancer elements in AT/RT. The aberrant activity of EZH2 and BRD4 histone modifiers is of fundamental importance to the occurrence and biology of AT/RT and actionable targets for treating AT/RT. We tested the effects of targeting inhibition of EZH2 and BRD4 in AT/RT. BRD4 inhibitor JQ1 induced dose-dependent growth inhibition of AT/RT cells with decreased expression of H3K27ac, and combination treatment of EZH2 inhibitor (GSK126) and JQ1 resulted in further suppressed the cell growth and cell invasion relative to either monotherapy. RNA sequence analysis showed differentially expressed genes in response to each treatment and slightly overlap genes associated with JQ1 and GSK126 treatment. We treated the mice with GSK-126 and JQ1 for 3 weeks. Combination therapy suppressed the tumor growth and prolonged animal survival in compared to monotherapy in the mice with intracranial AT/RT xenografts. Collectively, these findings suggest that therapeutic combination of EZH2 and BRD4 inhibitor provides a rational epigenetic targeted therapy for AT/RT.

We describe a 7-year-old girl with angiomatoid fibrous histiocytoma (AFH) presenting severe inflammatory symptoms. The cytokine/chemokine profile of serum samples before and after surgery demonstrated that interleukin (IL)-6 had decreased by the greatest percentage. The AFH cells were immunopathologically positive for IL-6 and Tyr705-phosphorylation of signal transducer and activator of transcription 3. The EWSR1-CREB1 fusion gene detected in the tumor leads to continuous activation of CREB1 and IL-6 production, because the promoter region of IL-6 has a CREB binding site. Thus, IL-6 plays pivotal roles in both paraneoplastic syndrome and the oncogenesis of AFH.

Intravascular large B-cell lymphoma (IVLBCL) is a rare extranodal lymphoma characterized by the presence of tumor cells within blood vessels, and it is considered to be a subtype of diffuse large B-cell lymphoma. We report a case of IVLBCL presenting as progressive hypoxemia. In this case, a definitive diagnosis could not be achieved by repeated transbronchial lung biopsy, a bone marrow biopsy, and a random skin biopsy, and the ultimate diagnosis was made on the basis of a pulmonary microvascular cytology (PMC) examination. Therefore, PMC is considered to be a useful strategy for the diagnosis of IVLBCL, particularly in this critically ill patient suffering from hypoxemia.

The therapeutic strategy against hepatocellular carcinoma (HCC) is determined by tumor stage and liver function. Improvements of stratification contribute to extending the survival of patients. However, stratification has been attributed little attention in animal models largely due to the lack of suitable models. Herein we showed that the recently-reported, non-alcoholic steatohepatitis-derived HCC model (STAM model) is the first murine model in which the concept of human stratification is applicable by demonstrating the following features: (i) at least 4 detectable tumor nodules; (ii) average tumor growth rate of 150 % from 16 to 20 weeks of age; (iii) no visible metastasis; and (iv) relatively preserved liver function. These observations suggested that HCC in STAM mice is equivalent to stages B to C of the Barcelona Clinic Liver Cancer (BCLC) staging system for humans. Application of the stratification concept to experimental animals will create new avenues to establish pharmacological intervention against HCC.

A 39-year-old man was admitted for spontaneous pneumothorax. He underwent pulmonary resection to correct the lesion causing the air leakage, and a pathological diagnosis of pulmonary pleomorphic carcinoma was made because we thought that the pneumothorax developed due to the direct rupture of necrotic neoplastic tissue into the pleural cavity. After the operation, the patient received chemotherapy, during which multiple cystic metastases gradually developed in the lung that caused repeated occurrences of pneumothorax. Clinicians must be careful to recognize that pneumothorax can also be a complication of primary and various metastatic pulmonary malignancies.

We manufactured a highly sensitive oligonucleotide microarray system comprised entirely of transcription regulatory factors (a TF oligo microarray) in order to comprehensively analyze the expression profiles of transcription factors in mice. We compared the expression profiles of transcription regulatory factors in mouse embryonic stem (ES) cells and ES-differentiated cells by using this TF oligo microarray, a cDNA microarray, a GeneChip system, and quantitative RT-PCR. The TF oligo microarray was able to comprehensively analyze the expression profile of transcription regulatory factors. In addition, we used the manufactured TF oligo microarray to analyze the expression patterns of transcriptional regulatory factors during the formation of embryoid bodies. The TF array was able to reveal the chronologic expression profile of transcription regulatory factors involved in embryogenesis or the maintenance of pluripotency in ES cells.

Two rapid and simple in planta transformation methods have been developed for the model legume Medicago truncatula. The first approach is based on a method developed for transformation of Arabidopsis thaliana and involves infiltration of flowering plants with a suspension of Agrobacterium. The second method involves infiltration of young seedlings with Agrobacterium. In both cases a proportion of the progeny of the infiltrated plants is transformed. The transformation frequency ranges from 4.7 to 76% for the flower infiltration method, and from 2.9 to 27.6% for the seedling infiltration method. Both procedures resulted in a mixture of independent transformants and sibling transformants. The transformants were genetically stable, and analysis of the T2 generation indicates that the transgenes are inherited in a Mendelian fashion. These transformation systems will increase the utility of M. truncatula as a model system and enable large-scale insertional mutagenesis. T-DNA tagging and the many adaptations of this approach provide a wide range of opportunities for the analysis of the unique aspects of legumes.

We were able to develop a shoot-forming culture of Pelargonium graveolens in jar fermentors by a modification of Linsmaier-Skoog's medium with 10-5M indoleacetic acid and 10-6M 6-benzyl-adenine. Young adventitious shoots (10g fresh weight) which had been cultured on agar media were inoculated into 5-liter jar fermentors containing 3-liters of medium and cultured under continuous illumination at 25°C with agitation of 3 liters/min (1vvm) aeration through sintered glassfilter. After 3 weeks, the yield was 145g fresh weight. The GC-pattern of volatile compounds in the extract of the shoot-forming culture was similar to that of natural Pelargonium leaf oil, and these volatile compounds in the shoot-forming culture were identified with standard compounds. But the content of citronellol (a major constituent of natural Pelargonium leaf oil) in the culture was less than that in intact leaves. With the use of 10-5M indolebutyric acid as auxin and reduced concentration of NH4+, the weight of the shoot-forming culture increased to 37.5-fold during 3 weeks of jar culture.

A possible role of Ca2+ -activated potassium efflux in brain ischemia was studied using a rat focal cortical infarction model. Three days after ischemic insult, tissue contents of water, sodium, potassium and calcium ions were measured. Charybdotoxin, a specific inhibitor of Ca2+ -activated potassium efflux, was found to reduce the formation of ischemic brain edema when a dosage of 0.15 mg/kg was given by i.v. 20-30 min prior to the onset of ischemic insult.

The relation between the composition of the phospholipid molecular species in a cell membrane and the velocity of protoplast fusion was studied using cells cultured at a low temperature (10 C). Cells cultured at a low temperature contained larger proportions of phospholipids of low phase transition point, the 1,2-dilinoleoyl-type, than those cultured at a normal temperature (25 C). When treated with polyethylene glycol 6000, protoplasts from cells cultured at 10 C fused and progressed to the fused sphere stage more rapidly than did those from cells cultured at 25 C.

Charybdotoxin, a specific inhibitor of the calcium-activated potassium channel, was found to inhibit the in vitro formation of irreversibly dehydrated cells and of irreversibly sickled cells, which occur as a result of repeated cycles of sickling and unsickling of sickle red blood cells. The degree of formation of dense cells was measured by Percoll-renografin density gradient centrifugation. 50% inhibition of the formation was achieved at a concentration of 30 nM of charybdotoxin. The approximate half-life of this compound in the circulation of the guinea pig was determined to be 4 h. Charybdotoxin did not inhibit the sickling of sickle cells under deoxygenation. The effects of charybdotoxin in preventing the irreversible changes of sickle cell membranes may be related to the inhibition of calcium-activated potassium efflux in sickle red blood cells.