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To evaluate a potential relationship between BRAF V600E mutation and PD-L1 expression, we examined the expression of PD-L1 in pediatric high- and low-grade glioma cell lines as well as a cohort of pediatric low-grade glioma patient samples. Half of the tumors in our patient cohort were V600-wildtype and half were V600E mutant. All tumors expressed PD-L1. In most tumors, PD-L1 expression was low (

DIPG has elevated baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that enables cells to tolerate various forms of stress. Activation of the ISR is indicated by increased expression of activating transcription factor 4 (ATF4). Intermediate levels of ATF4 protect cells from stress, while persistent high levels result in cell death. The imipridone drug ONC201 increases mitochondrial stress and ATF4 expression by binding to and activating the mitochondrial protease ClpP. Sal003 inhibits dephosphorylation of ATF4 upstream regulator, eIF2α. The retinoic acid derivative fenretinide induces ATF4, increases reactive oxygen species, and has clinical activity in pediatric patients with neuroblastoma. Because DIPG has a high baseline level of ATF4, we hypothesized that the ISR activators Sal003, ONC201, and fenretinide would synergize to kill DIPG. After determining the IC50 of Sal003, fenretinide, and ONC201, we treated patient-derived DIPG cell lines with low micromolar doses. The combination of Sal003 and ONC201 significantly increased apoptosis as measured by CC3 immunofluorescence in comparison to DMSO and single treatment (p< 0.0001, ANOVA). Western blots for cleaved PARP expression detected induction of apoptosis in DIPG treated with both Sal003 and ONC201 over DMSO and monotherapy treated cells. The combination also increased ATF4 expression. Since Sal003 is not clinically available, we treated DIPG cells with ONC201 and fenretinide. Synergy testing in JHH-DIPG1 showed an overall ZIP synergy score of 14 with inhibitory concentrations of both drugs in the nanomolar range. CC3 immunofluorescence indicated elevated apoptosis in the combination vs. DMSO (p< 0.0001, ANOVA). Western blots showed increased cleaved PARP, ATF4, p-eIF2α, and CHOP expression in DIPG treated with ONC201 and fenretinide. We are currently testing the efficacy of this combination in brain organoids and orthotopic DIPG xenografts. Our results suggest the combination of ONC201 with fenretinide could potentially serve as a therapy for DIPG.

The mitogen-activated protein kinase pathway is one of the most frequently altered pathways in cancer. It is involved in the control of cell proliferation, invasion, and metabolism, and can cause resistance to therapy. A number of aggressive malignancies, including melanoma, colon cancer, and glioma, are driven by a constitutively activating missense mutation (V600E) in the v-Raf murine sarcoma viral oncogene homolog B (BRAF) component of the pathway. Mitogen-activated protein kinase kinase (MEK) inhibition is initially effective in targeting these cancers, but reflexive activation of mammalian target of rapamycin (mTOR) signaling contributes to frequent therapy resistance. We have previously demonstrated that combination treatment with the MEK inhibitor trametinib and the dual mammalian target of rapamycin complex 1/2 inhibitor TAK228 improves survival and decreases vascularization in a BRAFV600E mutant glioma model. To elucidate the mechanism of action of this combination therapy and understand the ensuing tumor response, we performed comprehensive unbiased proteomic and phosphoproteomic characterization of BRAFV600E mutant glioma xenografts after short-course treatment with trametinib and TAK228. We identified 13,313 proteins and 30,928 localized phosphosites, of which 12,526 proteins and 17,444 phosphosites were quantified across all samples (data available via ProteomeXchange; identifier PXD022329). We identified distinct response signatures for each monotherapy and combination therapy and validated that combination treatment inhibited activation of the mitogen-activated protein kinase and mTOR pathways. Combination therapy also increased apoptotic signaling, suppressed angiogenesis signaling, and broadly suppressed the activity of the cyclin-dependent kinases. In response to combination therapy, both epidermal growth factor receptor and class 1 histone deacetylase proteins were activated. This study reports a detailed (phospho)proteomic analysis of the response of BRAFV600E mutant glioma to combined MEK and mTOR pathway inhibition and identifies new targets for the development of rational combination therapies for BRAF-driven tumors., Graphical Abstract, Highlights • Large-scale proteomic and phosphoproteomic analysis of an in vivo BRAFV600E glioma model. • In-depth analysis of tumor and stromal cellular response to protein kinase inhibitors. • Insights into acquired resistance and alternative therapeutic approaches., In Brief BRAFV600E is a key oncogenic driver in glioma, melanoma, and colon cancer. These tumors escape mitogen-activated protein kinase pathway inhibition by upregulating mammalian target of rapamycin signaling. Using comprehensive unbiased proteomic and phosphoproteomic analysis of an in vivo BRAFV600E mutant glioma model treated with inhibitors of both these key pathways, we characterize the tumor and stromal response and suggest additional therapeutic targets for BRAF-driven cancers, including epidermal growth factor receptor and class 1 histone deacetylases.

Although most commonly benign, neurofibromas (NFs) can have devastating functional and cosmetic effects in addition to the possibility of malignant transformation. Orbitofacial NFs, in particular, may cause progressive, disfiguring tumors of the lid, brow, temple, face, and orbit, and clinical evidence suggests that they may have increased local aggressiveness compared to NFs developing at other sites. The purpose of this study was to identify biological differences between orbitofacial NFs and those occurring at other anatomic sites. We performed RNA‐sequencing in orbitofacial (n = 10) and non‐orbitofacial (n = 9) NFs. Differential gene expression analysis demonstrated that a variety of gene sets including genes involved in cell proliferation, interferon, and immune‐related pathways were enriched in orbitofacial NF. Comparisons with publicly available databases of various Schwann cell tumors and malignant peripheral nerve sheath tumor (MPNST) revealed a significant overlap of differentially expressed genes between orbitofacial versus non‐orbitofacial NF and plexiform NF versus MPNST. In summary, we identified gene expression differences between orbitofacial NF and NFs occurring at other locations. Further investigation may be warranted, given that orbitofacial NF are notoriously difficult to treat and associated with disproportionate morbidity., Global gene expression differences between orbitofacial neurofibromas and neurofibromas occurring at other anatomic locations were deteted through RNA sequencing analysis. Differences in pathways involved in cell cycle and inflammation were specifically detected through gene enrichment analyses, supporting prior clinical observations suggesting that orbitofacial neurofibromas are biologically distinct compared to neurofibromas developing in other anatomical sites.

Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor. Radiation, the standard of care, extends life for 6-18 months but has never cured a patient. Therefore, the discovery of novel treatments is imperative. DIPG tumors have elevated baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that allows cells to tolerate various forms of stress. Increased expression of activating transcription factor 4 (ATF4) indicates activation of the ISR. Low levels of ATF4 protect cells from stress, while sustained high-levels of ATF4 result in cell death. Because DIPG has a high baseline level of ATF4, we hypothesized that the ISR activators Sal003 and ONC201 would synergize and kill DIPG cells. After determining the IC25 of Sal003 and ONC201, we treated three patient-derived cell lines: JHH-DIPG1, SF-7761, and JHH-DIPG16A with low micromolar doses. To measure proliferation, we performed immunofluorescence staining for bromodeoxyuridine (BrdU) incorporation. The combination treatment significantly reduced BrdU incorporation (JHH-DIPG1 p=0.0026, SF-7761 p=0.0002, JHH-DIPG16A p Citation Format: Orlandi V. Novak, Antje Arnold, Charles Eberhart, Eric H. Raabe. Combination therapy activating the integrated stress response synergistically suppresses proliferation and induces apoptosis in diffuse intrinsic pontine glioma [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 5227.

The H3K27M mutation, observed in 70%-90% of DIPG, causes global hypomethylation. The Ten-Eleven-Translocation enzymes, TETs, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) regulate the methylation patterns in the genome. Previously, we observed an increase in the 5hmC levels and altered TET activity in DIPG. We hypothesize that targeting the TETs in DIPG could restore the epigenetic balance. In this study, we target the TET pathway with cell-permeable 2-hydroxyglutarate (2HG) and Bobcat339, a cytosine-based TET inhibitor. We see a dose-dependent decrease in 5hmC (46-96% reduction) and increase in 5mC (~5fold increase) in multiple DIPG cell lines treated with 2HG. We see a corresponding increase in apoptosis and reduction in proliferation (~8fold increase in cleaved-Caspase3 signal (P=0.0001) and ~55% reduction in BrdU-incorporation measured by IF (P=0.0036) in JHH DIPG16A with similar results in JHH DIPG1, HSJD007, and SUDIPG6). We see a 54-90% reduction in 5hmC and ~3fold increase in 5mC with Bobcat 339 treatment in DIPG cell lines JHH DIPG1, JHH DIPG16A, HSJD007, and SUDIPG6. We also see a corresponding increase in apoptosis as measured by cleaved PARP western blot and decrease in proliferation as measured by BrdU incorporation (approximately 32-53% decrease in proliferation). Combined treatment with 2HG and Bobcat339 lead to greater decreases in 5hmC and increases in 5mC compared to single treatment, with synergistic suppression of cell growth as measured by SynergyFinder in HSJD007, JHH DIPG1, and JHH DIPG16A. In vivo studies targeting DIPG orthotopic xenografts are currently underway. These results suggest that TETs contribute to the hypomethylated state observed in DIPG. Thus, inhibiting the TETs can be explored as a potential therapeutic approach to treat DIPG.

BACKGROUND: Pediatric low-grade glioma (pLGG) often initially responds to front-line therapies such as carboplatin, but more than 50% of treated tumors eventually progress and require additional therapy. With the discovery that pLGG often contains mammalian target of rapamycin (mTOR) activation, new treatment modalities and combinations are now possible for patients. The purpose of this study was to determine if carboplatin is synergistic with the mTOR complex 1 inhibitor everolimus in pLGG. METHODS: We treated 4 pLGG cell lines and 1 patient-derived xenograft line representing various pLGG genotypes, including neurofibromatosis type 1 loss, proto-oncogene B-Raf (BRAF)-KIAA1549 fusion, and BRAF(V600E) mutation, with carboplatin and/or everolimus and performed assays for growth, cell proliferation, and cell death. Immunohistochemistry as well as in vivo and in vitro metabolomics studies were also performed. RESULTS: Carboplatin synergized with everolimus in all of our 4 pLGG cell lines (combination index

Background Pediatric low-grade glioma (pLGG) is the most common childhood brain tumor. Many patients with unresectable or recurrent/refractory tumors have significant lifelong disability. The majority of pLGG have mutations increasing the activity of the Ras/mitogen-activated protein kinase (MAPK) pathway. Activation of mammalian target of rapamycin (mTOR) is also a hallmark of pLGG. We therefore hypothesized that the dual target of rapamycin complexes 1 and 2 (TORC1/2) kinase inhibitor TAK228 would synergize with the mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor trametinib in pLGG. Methods We tested TAK228 and trametinib in patient-derived pLGG cell lines harboring drivers of pLGG including BRAFV600E and neurofibromatosis type 1 loss. We measured cell proliferation, pathway inhibition, cell death, and senescence. Synergy was analyzed via MTS assay using the Chou–Talalay method. In vivo, we tested for overall survival and pathway inhibition and performed immunohistochemistry for proliferation and vascularization. We performed a scratch assay and measured angiogenesis protein activation in human umbilical vein endothelial cells (HUVECs). Results TAK228 synergized with trametinib in pLGG at clinically relevant doses in all tested cell lines, suppressing proliferation, inducing apoptosis, and causing senescence in a cell line–dependent manner. Combination treatment increased median survival by 70% and reduced tumor volume compared with monotreatment and control cohorts. Vascularization of tumors decreased as measured by CD31 and CD34. Combination treatment blocked activation of focal adhesion kinase (FAK) and sarcoma proto-oncogene non-receptor tyrosine kinase (SRC) in HUVEC cells and reduced HUVEC migration compared with each drug alone. Conclusions The combination of TAK228 and trametinib synergized to suppress the growth of pLGG. These agents synergized to reduce tumor vascularity and endothelial cell growth and migration by blocking activation of FAK and SRC.

Members of the miR-125 family are strongly expressed in several tissues, particularly brain, but may be dysregulated in cancer including adult and pediatric glioma. In this study, miR-125 members were downregulated in pilocytic astrocytoma (PA) as a group compared to non-neoplastic brain in the Agilent platform. In the Nanostring platform, miR-125 members were downregulated primarily in pleomorphic xanthoastrocytomas and gangliogliomas. Using CISH for miR-125b, highest levels of expression were present in grade II tumors (11/33, 33% grade II tumors with 3+ expression compared to 3/70, 4% grade I tumors) (p

Diffuse Intrinsic Pontine Glioma (DIPG) is one of the worst forms of pediatric brain tumors, with a 100% mortality-rate. A prominent mutation observed in them is a lysine to methionine change in histone3 which causes trapping of the repressive-chromatin-complex, leading to genome-level hypomethylation, affecting global epigenetic-regulation. Ten-Eleven Translocation (TET) proteins are alpha-ketoglutarate (α-KG) dependent dioxygenases that mediate the conversion of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), a key-step in removing DNA methylation-marks. We previously identified increased levels of 5hmC and TETs in DIPG. In IDH mutant glioma, the R132H-IDH produces an oncometabolite, 2-hydroxyglutarate (2HG), instead of α-KG. 2HG competitively inhibits TET function, resulting in a hypermethylated genome. Bobcat339, a cytosine-analog targets TETs by blocking the cytosine binding-site on TETs. We hypothesized that cell-permeable 2HG and Bobcat339 would synergize to block TET activity in DIPG, restore epigenetic-balance by increasing genomic methylation, and induce cell-death. 2HG induced apoptosis in DIPG cells at concentrations ranging from 100-300uM, as measured by cleaved-PARP western and cleaved-caspase3 immunofluorescence (2-7fold increase in CC3, depending on cell line). Cell-permeable 2HG also decreased proliferation, measured by phospho-RB western in DIPG cells (30-50% reduction in pRB band intensity, depending on cell line). Similarly, Bobcat339 suppressed proliferation at concentrations from 10-50uM, measured by BrdU incorporation (25% reduction in BrdU+ at 50uM, p= 0.02 compared to control). Bobcat339 induced apoptosis, measured by cPARP western and CC3 immunofluorescence (4-10fold more of CC3+ compared to control p< 0.0004). In combination, cell-permeable 2HG (100-200uM) and Bobcat339 (10-20uM) combined to suppress cell-viability, measured by CellTiterBlue assay, producing ZIP scores of ~20 in DIPG cells (indicating high-level synergy). In combination, 2HG and Bobcat339 increased apoptosis in DIPG (3-fold increase in cleaved-PARP band intensity in JHH-DIPG1 cells treated with 20uM Bobcat339 + 100uM 2HG, compared to single-treated cells). Our results may lead to new approaches that target TET pathway in DIPG tumors.

Although most commonly benign, neurofibromas (NFs) can have devastating functional and cosmetic effects in addition to the possibility of malignant transformation. Orbitofacial NFs in particular may cause progressive, disfiguring tumors of the lid, brow, temple, face and orbit, and anecdotal evidence suggest that they may have increased local aggressiveness compared to NFs developing at other sites. The purpose of this study was to identify biological differences between orbitofacial NFs and those occurring at other anatomic sites. We performed global RNA-sequencing in orbitofacial (n=10) and non-orbitofacial (n=9) NFs. A total of 880 mRNA transcripts were differentially expressed between the two groups (adjusted p>0.05), The top 10 genes relatively overexpressed in orbitofacial NF included NEFL, TREM2, CST1, GAP43, ADORA3, MIA, SYT6, FCGR3A, SPP1, and FCGR1A. The top 10 genes relatively underexpressed in orbitofacial NF included XG, WISP2, MMP27, CXCL14, MFAP5, APLNR, MYOC, SLITRK6, STMN2, and TDRD1. Gene enrichment analyses demonstrated a variety of gene sets differentially affected including pathways involved in cell proliferation, interferon and immune related pathways. Comparisons with publicly available databases of various Schwann cell tumors and models using CAT plots demonstrated the highest overlap with differentially expressed genes in plexiform NF vs MPNST (>10%). In summary, we identified gene expression differences between orbitofacial NF and NFs occurring at other anatomic locations. Further investigation may be warranted, given that orbitofacial NF are notoriously difficult to treat and associated with disproportionate morbidity. Citation Format: Eddie L. Imada, Deepak P. Edward, Antje Arnold, Hailah Al-Hussain, Diego Strianese, Luigi Marchionni, Fausto J. Rodriguez. Gene expression analysis by RNA-sequencing highlights differential regulated pathways involved in cell cycle and inflammation in orbitofacial neurofibromas [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 2251.

The mitogen-activated protein kinase (MAPK) pathway is one of the most frequently altered pathways in cancer. It is involved in the control of cell proliferation, invasion, metabolism, and can cause resistance to therapy. A number of aggressive malignancies including melanoma, colon cancer, and glioma, are driven by a constitutively activating missense mutation (V600E) in the BRAF component of the pathway. MEK inhibition is initially effective in targeting these cancers, but reflexive activation of mTOR signaling contributes to frequent therapy resistance. We have previously demonstrated that combination treatment with the MEK inhibitor trametinib and the dual mTORC1/2 inhibitor TAK228 improves survival and decreases vascularization in a BRAFV600E mutant glioma model. To elucidate the mechanism of action of this combination therapy and understand the ensuing tumor response, we performed comprehensive unbiased proteomic and phosphoproteomic characterization of BRAFV600E mutant glioma xenografts after short-course treatment with trametinib and TAK228. We identified 13,313 proteins and 30,928 localized phosphosites, of which 12,526 proteins and 17,444 phosphosites were quantified across all samples (data available via ProteomeXchange; identifier PXD022329). We identified distinct response signatures for each monotherapy and combination therapy and validated that combination treatment inhibited activation of the MAPK and mTOR pathways. Combination therapy also increased apoptotic signaling, suppressed angiogenesis signaling, and broadly suppressed the activity of the cyclin-dependent kinases. In addition, combination therapy had a profound impact on cancer cell metabolic pathways, increasing the expression of proteins (and their activating phosphorylations) involved in glycolysis, the tricarboxylic acid (TCA) cycle, nucleotide biosynthesis, and DNA replication. In response to combination therapy, both receptor tyrosine kinase and histone deacetylase proteins were activated. This study reports a detailed (phospho)proteomic analysis of the response of BRAFV600E mutant glioma to combined MEK and mTOR pathway inhibition and identifies new targets for the development of rational combination therapies for aggressive BRAF-driven tumors. Citation Format: Micah J. Maxwell, Antje Arnold, Heather Sweeney, Ljun Chen, Tung-Shing M. Lih, Michael Schnaubelt, Charles G. Eberhart, Jeffrey A. Rubens, Hui Zhang, David J. Clark, Eric H. Raabe. Unbiased proteomic and phosphoproteomic analysis identifies response signatures and novel susceptibilities after combined MEK and mTOR inhibition in BRAFV600E mutant glioma [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 324.

Orthotopic xenotransplantation studies represent the final stage in preclinical cancer research and could facilitate the implementation of precision medicine. To date, these xenografts have been tested in immunodeficient animals, but complete elimination of the adaptive immunity is a significant drawback. We present a method of efficient human glioblastoma (GBM) cell engraftment in adult mice with intact immune systems, mediated by a transient blockade of T-cell co-stimulation. Compared to transplants grown in immunodeficient hosts, the resulting tumors more accurately resemble the clinical pathophysiology of patient GBMs, which are characterized by blood-brain-barrier leakage and strong neo-vascularization. We expect our method to have great utility for studying human tumor cell biology, particularly in the field of cancer immunotherapy and in studies on microenvironmental interactions. Given the straightforward approach, the method may also be applicable to other tumor types and additional model organisms.

Pediatric low-grade glioma (pLGG) is the most common brain tumor in children. We and others have identified constitutive activation of the mTOR and MEK-pathway in pLGG. This led us to investigate TAK228, a mTORC1/2-inhibitor, and the FDA approved MEK-inhibitor, trametinib, in mono- and combination therapy in pLGG. We examined antitumor activity in four patient-derived pLGG cell models treated with TAK228, trametinib, and combination: JHH_NF1_PA1(NF1mut), BT40(BRAFV600E), Res186(PTEN-/-) and Res259(CDKN2A-/-). Treatment with TAK228 or trametinib reduced cell proliferation in a dose and time dependent manner (MTS-assay). The combination treatment exerted a synergistic effect at 5-20nM in JHH_NF1_PA1, Res186, and Res259 cells (Chou-Talay method). The combination of TAK228 and trametinib increased apoptosis by up to 127% (p< 0.001 by 1-way ANOVA) in Res186 and Res259 cells as measured by cleaved caspase 3 immunocytochemistry. Trametinib and combination treatment induces senescence in JHH_NF1_PA1 cell line verified through increase in p27 protein expression (Western Blot) and positive b-galactosidase staining. Mono-treatment with TAK228 or vehicle showed no signs of senescence. We tested trametinib and TAK228 against the mutant BT40BRAFV600E patient-derived cell line in immunodeficient mice. The combination treatment showed greater antitumor activity than that of either mono-treatment in vivo. BT40 tumor growth was significantly decreased in combination compared to vehicle or either agent alone (p< 0.01). Combination treatment strikingly reduced the vascularization in the tumor tissue after combination treatment analyzed by CD31 and CD34 protein expression (Western Blot), compared to vehicle and mono-treatment. Our study provides evidence that pLGG-derived cell lines in vitro and in vivo are sensitive to mTORC1/2 kinase inhibition and MEK inhibition. Combination treatment with TAK228 and trametinib had a significant anti-tumor activity in vivo shown in survival rate, decreased tumor size, and reduced vascularity. These results provide the first strong rationale for combination therapy with TAK228 and trametinib for clinical consideration in pLGG.

Atypical teratoid/rhabdoid tumors are aggressive infantile tumors highly resistant to intensive therapies. We aim to identify and target critical factors driving this therapy resistance to improve AT/RT survival. Analysis of publically available RNASeq on 32 AT/RT identified elevated expression of NRF2 (median expression 40.78, normal brain 18.81). NRF2 is a master regulator of cell’s stress response whose expression is correlated with therapy resistance and poor survival. NRF2 activation is sensitive to mTOR activity and is a biomarker predicting response to the dual mTORC1/2 inhibitor, Sapanisertib (TAK228, INK128). We performed RNASeq on 4 human-derived AT/RT cell models after Sapanisertib treatment. Pathway analysis reveals disruption of the NRF2-mediated stress response (-log p value 0.39, Z-score 1.0). As a result, Sapanisertib decreases ROS scavengers like glutathione (Metabolite analysis UHPLC-MS, t-test p

DIPG is a universally fatal pediatric brainstem tumor with no effective therapy. Recent work has shown that over 80% of DIPG cases harbor the H3K27M mutation leading to global loss of the repressive H3K27 trimethylation mark, global DNA hypomethylation, and a distinct gene expression signature. We sought to exploit epigenetic vulnerabilities in DIPG through the use of DNA methyltransferase inhibitors and histone deacetylase (HDAC) inhibitors. We find that treatment with low-dose 5-aza-2’-deoxycytidine (decitabine), alone and in combination with HDAC inhibitors, elicits profound genome-wide demethylation in DIPG patient-derived neurosphere cell lines, impairs proliferation, and induces apoptosis. We show that this treatment induces immune activation, with induction of type I interferon signaling, increased expression of major histocompatibility complexes, and expression of tumor antigens. These results suggest that the immunogenicity of DIPG may be modulated by epigenetic therapies, suggesting the possibility of novel combination approaches to immunotherapy of DIPG in the future.

‘Entertainment’ in the Early Modern Age is preferably situated in so called literary ‘salons’. The article aims to develop a theory of ‘Unterhaltung’ from a study that traces the spatial conditions and effects of entertainment. It sheds light on the case of Madeleine de Scudéry, who embodied conversational theory and practice and therefore became a role-model for early 18th century Germany. Her “carte de tendre” distributed the popular game of pre-sensitive conversation. This is embedded in Scudérys novel Clélie that influenced both poetics and ‘entertainment’ theories.

Diffuse Intrinsic Pontine Gliomas (DIPGs) are universally fatal tumors, resistant to the cytotoxic effects of all known therapies. We aim to better understand the mechanisms contributing to this resistance to design new combination therapies that will improve DIPG survival. DIPG's aggressive phenotype is driven by the H3K27M oncohistone mutation that leads to global genomic DNA hypomethylation and abnormal gene expression. To identify patterns of gene expression driven by the H3K27M mutation, we introduced the H3K27M mutation into murine neural stem cells and performed whole genome bisulfite sequencing (WGBS) to evaluate for genes with the greatest differential methylation in their promoter region. These studies identified an abundance of pro-survival genes such a BCL-2, BCL-xL, MCL-1, Caspase-3, and XIAP. Western blot confirmed elevated expression of these pro-survival factors and RNA-Seq of primary human DIPG confirmed increased expression compared to normal brain. PAC-1 is a compound that cleaves protective zinc bonds to activate procaspase-3, directly inducing cell death. ONC201 is a small molecule that inhibits the anti-apoptotic protein XIAP, which prevents activated caspase-3 from translocating to the nucleus to induce apoptosis. We hypothesized that PAC-1 combined with ONC201 will circumvent these protective abnormalities in the DIPG apoptotic signaling cascade to induce tumor cell death. PAC-1 induces high rates of apoptosis in DIPG (25µM concentrations increases the percent of cells undergoing apoptosis; cleaved caspase 3 immunofluorescence assay 5% to 30% (t-test p40% compared to DMSO control (ANOVA p Citation Format: Ashlyn Parkhurst, Youngran Park, Antje Arnold, Michael Koldobskiy, Charles Eberhart, Eric Raabe, Jeffery Rubens. The novel combination of PAC-1 and ONC201 circumvent H3K27M-driven pro-survival gene expression to induce DIPG cell death [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3904.

Atypical teratoid rhabdoid tumors (AT/RT) are the most common malignant brain tumors of infancy, highly resistant to all therapies, and responsible for one of the greatest numbers of life years lost due to cancer. We have previously demonstrated that Sapanisertib combines synergistically with cisplatin to extend survival in AT/RT. To understand the mechanisms underlying this synergy and to identify further novel combination therapies to improve AT/RT survival, we performed RNASeq on AT/RT cell models 4 hours after Sapanisertib treatment. Pathway analysis revealed that Sapanisertib disrupts the NRF2-mediated stress response and apoptotic signaling pathway leading to a pro-death phenotype. Activating mutations in the NRF2 coding gene, NFE2L2, have been identified as a possible biomarker predicting Sapanisertib efficacy in lung cancer clinical trials. Short hairpin knockdown of NRF2 led to decreased expression of the anti-apoptotic proteins MCL-1, BCL-2, and BCL-xL suggesting that interfering with NRF2 may drive this pro-death phenotype in AT/RT. Obatoclax is a synthetic small molecule inhibitor of the BCL-2 family of proteins that was well tolerated in phase II clinical trials and readily crosses the blood brain barrier. Obatoclax treatment of NRF2 knockdown AT/RT cell models slows cell growth, induces high rates of apoptosis, and leads to increased oxidative stress compared to short hairpin scramble controls (cell viability assay, t-test p Citation Format: Ashlyn Parkhurst, Sabrina Wang, Harpreet Kaur, Antje Arnold, Charles Eberhart, Eric Raabe, Jeffrey Rubens. Sapanisertib interferes with the NRF2-mediated stress response to synergize with Obatoclax and extend AT/RT survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6226.

Pediatric low-grade glioma (PLGG) is the most common brain tumor of childhood. We and others have identified mTOR and MEK-pathway activation in PLGG. The dual mTORC1/2-inhibitor, TAK228, and the FDA approved MEK-inhibitor, trametinib, are promising candidates for targeted PLGG therapy. We treated five different patient-derived PLGG cell models with TAK228 and/or trametinib: JHH_NF1_PA1 (NF1 mutation), BT66_SV40/hTERT (BRAF:KIAA1549 fusion), BT40 (BRAFV600E) Res186 (PTEN deletion) and Res259 (PDGFRα amplification and CDKN2A deletion). Treatment with TAK228 or trametinib reduces cell proliferation in a dose and time depended manner investigated via MTS-assay. Both drugs exert a synergistic effect at 5-20nM in JHH_NF1_PA1, Res186, and Res259 cells as calculated by the method of Chou-Talay. BT66_SV40/hTERT cells have a 70% reduction in cell growth with 10nM TAK228 (p < 0.001 by ANOVA) but not in combination with trametinib. In all cell lines trametinib treatment leads to a pERK inactivation at low nM levels. TAK228 treatment leads to inactivation of mTORC1 and mTORC2. Apoptosis induction was examined through cleaved PARP via western blot and CC-3 via immunocytochemistry. The combination of TAK228 and trametinib increased apoptosis by up to 127% (p < 0.001) in Res186, Res259, and JHH_NF1_PA1 cells. We tested trametinib and TAK228 against the BRAFV600E mutant BT40 patient derived xenograft in immunodeficient mice. In combination TAK228 and trametinib decreased significantly BT40 tumor growth compared to vehicle or either agent alone, (p < 0.01 by ANOVA). Striking was the reduced vascularization of the tumor tissue after combination treatment compare the vehicle control and single agent treatment. Vascularization differences will be evaluated with immunohistochemistry staining for PDGFα and PECAM-1. Our results show that PLGG-derived cell lines are sensitive to TORC1/2 kinase inhibition and MEK inhibition. Further, our in vivo experimentation provides the first strong rationale for combination therapy of these agents in aggressive PLGG.

The immunological barrier currently precludes the clinical utilization of allogeneic stem cells. Although glial-restricted progenitors have become attractive candidates to treat a wide variety of neurological diseases, their survival in immunocompetent recipients is limited. In this study, we adopted a short-term, systemically applicable co-stimulation blockade-based strategy using CTLA4-Ig and anti-CD154 antibodies to modulate T-cell activation in the context of allogeneic glial-restricted progenitor transplantation. We found that co-stimulation blockade successfully prevented rejection of allogeneic glial-restricted progenitors from immunocompetent mouse brains. The long-term engrafted glial-restricted progenitors myelinated dysmyelinated adult mouse brains within one month. Furthermore, we identified a set of plasma miRNAs whose levels specifically correlated to the dynamic changes of immunoreactivity and as such could serve as biomarkers for graft rejection or tolerance. We put forward a successful strategy to induce alloantigen-specific hyporesponsiveness towards stem cells in the CNS, which will foster effective therapeutic application of allogeneic stem cells.

Pediatric low-grade glioma (PLGG) is the most common brain tumor of childhood. We and others have identified mTORC and MEK-activation in PLGG. The dual mTORC1/2-inhibitor, TAK228, and the FDA approved MEK-inhibitor, trametinib, are promising candidates for targeted PLGG therapy. We treated five different patient-derived PLGG cell models with TAK228 and/or trametinib: JHH_NF1_PA1 (NF1 mutation), BT66_SV40/hTERT (BRAF:KIAA1549 fusion), BT40 (BRAFV600E) Res186 (PTEN deletion) and Res259 (PDGFRα amplification and CDKN2A deletion). In vitro, treatment with TAK228 or trametinib reduces cell proliferation in a dose and time depended manner investigated via MTS-assay. Both drugs exert a synergistic effect at 5-20nM in JHH_NF1_PA1, Res186, and Res259 cells. BT66_SV40/hTERT cells have a 70% reduction in cell growth with 10nM TAK228 (p

Die Kalte. Eine Isolation als vierter Teil der autobiographischen Prosa ist laut Typoskript rund zwei Jahre nach Erscheinen des dritten Teils, Der Atem, wahrend eines Aufenthalts gemeinsam mit Hedwig Stavianicek in Lovran, im ehemaligen Jugoslawien und heutigen Kroatien, am 6. Mai 1980 fertiggestellt worden. Der Text erscheint Anfang des Jahres 1981 im Salzburger Residenz Verlag mit einer Auflage von 7.000 Exemplaren. Zuvor hatte der Suhrkamp-Verleger Siegfried Unseld mehrfach vergeblich versucht, die autobiographische Prosa fur seinen Verlag zu sichern. Er erfahrt im Juli 1980 von der Fertigstellung der Kalte. Am 22. Februar 1981 schreibt Bernhard: »Die ›Kalte‹ ist ein unerfreuliches Buch, das aber notwendig ist, wenn ich weiterkommen will und ich habe ganz einfach die Hemmungen des Stiefvaters was dieses Buch betrifft und schicke es nicht, sondern bringe es einmal mit« (Bernhard/Unseld 2009, 623 f.). Unseld halt fest, dass zu dieser Zeit drei weitere Bande autobiographischer Prosa in Planung gewesen seien (vgl. ebd., 596–598).

The therapeutic effect of glial progenitor transplantation in diseases of dysmyelination is currently attributed to the formation of new myelin. Using magnetic resonance imaging (MRI), we show that the therapeutic outcome in dysmyelinated shiverer mice is dependent on the extent of cell migration but not the presence of mature and compact myelin. Human or mouse glial restricted progenitors (GRPs) were transplanted into rag2-/- shiverer mouse neonates and followed for over one year. Mouse GRPs produced mature myelin as detected with multi-parametric MRI, but showed limited migration without extended animal lifespan. In sharp contrast, human GRPs migrated extensively and significantly increased animal survival, but production of mature myelin did not occur until 46weeks post-grafting. We conclude that human GRPs can extend the survival of transplanted shiverer mice prior to production of mature myelin, while mouse GRPs fail to extend animal survival despite the early presence of mature myelin. This paradox suggests that transplanted GRPs provide therapeutic benefits through biological processes other than the formation of mature myelin capable to foster rapid nerve conduction, challenging the current dogma of the primary role of myelination in regaining function of the central nervous system.

Artikelbeginn: Als Peter Härtling am 7.11.2014 mit dem Hessischen Kulturpreis ausgezeichnet wird, spielt er auf die reformpädagogischen »fromme[n] Wünsche« an, die mit dem von Ellen Key 1902 ausgerufenen »Jahrhundert des Kindes« für das 20. Jahrhundert postuliert worden waren, und die, wie er selbst als Kind während und nach dem Zweiten Weltkrieg erfahren musste, »unerfüllbar« gewesen seien. Sein Rückblick lässt ihn allerdings zu dem Schluss kommen, dass das 21. Jahrhundert noch »schlimmer« sei, denn es handele sich um das »Jahrhundert des Flüchtlingskindes«. Es liegt folglich nahe, angesichts seines im Herbst 2016 erschienenen »Romans für Kinder«, Djadi, Flüchtlingsjunge, auf den erinnernden Autor zu blicken, der mit 13 Jahren kurz nach Ende des Zweiten Weltkriegs zum Waisen wurde.

Pediatric low-grade glioma (pLGG) is the most common brain tumor in children. We and others have recently identified constitutive activation of the mTOR and MEK-pathway in pLGG. This led us to investigate the antitumor activity of TAK228, a second generation mTORC1/2-inhibitor, and the FDA approved MEK-inhibitor, trametinib, in mono- and combination therapy in pLGG. We examined antitumor activity in five different patient-derived pLGG cell models treated with TAK228, trametinib, and combination: JHH_NF1_PA1NF1mut, BT66_SV40BRAF:KIAA1549, BT40BRAFV600E, Res186PTEN-/- and Res259CDKN2A-/-. In all cell lines, as measured by western blot (WB), trametinib treatment led to suppression of phospho-ERK at low levels (1-5nM). Similarly, TAK228 treatment led to inhibition of mTORC1 and mTORC2 in the low nanomolar range. Treatment with TAK228 or trametinib reduced cell proliferation in a dose and time dependent manner (MTS-assay). The combination treatment exerted a synergistic effect at 5-20nM in JHH_NF1_PA1, Res186, and Res259 cells (by Chou-Talay method). The BT66 cell line had a 70% reduction in cell growth with 10nM TAK228 (p Citation Format: Antje Arnold, Lauren Harris, Ming Yuan, Fausto Rodriguez, Charles Eberhart, Eric Raabe. Inhibition of the mTOR and MAPK pathways suppresses growth, induces apoptosis, and decreases vascularity in pediatric low grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3108.

Pediatric low-grade glioma (pLGG) is the most common brain tumor in children. We and others have identified constitutive activation of the mTOR and MEK-pathway in pLGG. This led us to investigate TAK228, a mTORC1/2-inhibitor, and the FDA approved MEK-inhibitor, trametinib, in mono- and combination therapy in pLGG. We examined antitumor activity in five patient-derived pLGG cell models treated with TAK228, trametinib, and combination: JHH_NF1_PA1(NF1mut), BT66_SV40(BRAF:KIAA1549), BT40(BRAFV600E), Res186(PTEN-/-) and Res259(CDKN2A-/-). In all cell lines, trametinib treatment led to suppression of phospho-ERK at low levels (1-5nM; Western Blot). TAK228 treatment led to inhibition of mTORC1/2 in low nanomolar range. Treatment with TAK228 or trametinib reduced cell proliferation in a dose and time dependent manner (MTS-assay). The combination treatment exerted a synergistic effect at 5-20nM in JHH_NF1_PA1, Res186, and Res259 cells (Chou-Talay method). We tested trametinib and TAK228 against the mutant BT40(BRAFV600E) patient-derived xenograft cells in immunodeficient mice. Combination of TAK228 with trametinib showed greater antitumor activity than that of either mono-treatment in vivo. BT40 tumor growth was significantly decreased in combination compared to vehicle or either agent alone (p Our study provides evidence that pLGG-derived cell lines in vitro and in vivo are sensitive to mTORC1/2 kinase inhibition and MEK inhibition. Combination treatment with TAK228 and trametinib had a significant anti-tumor activity in vivo shown in survival rate, decreased tumor size, and reduced vascularity. These results provide a strong rationale for combination therapy with TAK228 and trametinib for clinical consideration in pLGG.

10–15% of all pediatric gliomas harbor a point mutation in the BRAF gene, BRAF(V600E), and it is especially common in certain low grade subtypes. Targeting this mutation with BRAF inhibitors has generated impressive responses in pediatric brain tumor patients with multiple clinical trials ongoing. In melanoma, where BRAF(V600E) mutation is common, responses to BRAF inhibition have been robust but not durable. However, long term remission has been achieved with immune checkpoint inhibitors, including those of the programmed death (PD) pathway. Response to PD inhibition has been linked to the expression of PD-L1 on the surface of tumor cells. To evaluate a potential relationship between BRAF(V600E) mutation and PD-L1 expression, we examined the expression of PD-L1 in pediatric high and low grade glioma cell lines as well as a cohort of pediatric low grade glioma patient samples of diverse and rare histologic subtypes. Transient transfection of BRAF wild-type cell lines with a plasmid expressing mutant BRAF(V600E) increased baseline PD-L1 expression in 2 of 3 cell lines. Moreover, a BRAF(V600E) mutant cell line also had high baseline PD-L1 expression. All tumors in our cohort expressed PD-L1, and some cases had high levels, >50%. There was a trend toward increased PD-L1 in the BRAF mutant cases but some wild-type tumors also had robust expression. These findings suggest that the programmed death pathway may be active in subsets of pediatric low grade glioma as a mechanism of immune evasion regardless of BRAF mutational status. Although low grade gliomas often have excellent overall survival when they can be removed surgically, unresectable tumors are associated with significant morbidity and often present a treatment challenge for the neuro-oncologist. Clinical trials evaluating the effect of PD pathway inhibitors in pediatric low grade glioma patients will be critical in determining whether PD-L1 expression indicates a likely therapeutic benefit.

The derivation of induced pluripotent stem cells (iPS) from human cell sources using transduction based on viral vectors has been reported by several laboratories. Viral vector-induced integration is a potential cause of genetic modification. We have derived iPS cells from human foreskin, adult Huntington fibroblasts, and adult skin fibroblasts of healthy donors using a nonviral and nonintegrating procedure based on mRNA transfer. In vitro transcribed mRNA for 5 factors, oct-4, nanog, klf-4, c-myc, sox-2 as well as for one new factor, hTERT, was used to induce pluripotency. Reprogramming was analyzed by qPCR analysis of pluripotency gene expression, differentiation, gene expression array, and teratoma assays. iPS cells were shown to express pluripotency markers and were able to differentiate towards ecto-, endo-, and mesodermal lineages. This method may represent a safer technology for reprogramming and derivation of iPS cells. Cells produced by this method can more easily be transferred into the clinical setting.

Pediatric low-grade glioma (PLGG) is one of the most common childhood tumors. If the tumor is located in a brain region that is not accessible for surgical resection, additional therapies are needed. Recent studies highlighted the important role of mTORC and MEK-activation in PLGG. The dual mTORC1/2-inhibitor, TAK-228, and MEK-inhibitor, trametinib, are promising candidates for targeted therapy. We hypothesized that TAK-228 and trametinib would show synergistic effects in vitro and in vivo in PLGG models. We treated the PLGG-derived cell lines Res186 and Res259 and our human MAP-kinase-driven glioma model with TAK-228 and trametinib. Cell growth was investigated using MTT-assay, DNA replication with bromodeoxyuridine (BrdU) assay, and apoptosis through cleaved-caspase-3 (CC-3) staining. Activation of MAPK pathway was detected via Western Blot by pERK and mTOR pathway by pAKT, pS6, and p4E-BP1 to total protein amount, and β-actin. Synergy was calculated with the Chou-Talalay method. Treatment of Res186 and Res259 with TAK-228 or trametinib reduced cell growth and proliferation in a dose- and time-depended manner. The combination of TAK-228 (20nM) and trametinib (100nM) resulted in a synergistic effect in Res259. No synergy was detected for Res186. Staining for CC-3 showed a significant increase for apoptosis in Res259 after treatment with TAK-228 or trametinib (**p

Pediatric low grade glioma (PLGG) is the most common brain tumor of childhood. If the tumor is located in a region of the brain that is not accessible for surgical resection, additional therapies are needed. We and others have identified mTORC and MEK-activation in PLGG. The dual mTORC1/2-inhibitor, TAK228, and the FDA approved MEK-inhibitor, trametinib, are promising candidates for targeted PLGG therapy. We hypothesized that TAK228 and trametinib would show synergistic effects in vitro and in vivo PLGG models. We treated in vitro four different patient-derived PLGG cell lines with TAK228, trametinib, or vehicle control: JHH_NF1_PA1 (NF1 mutation), BT66_SV40/hTERT (BRAFfusion), Res186 and Res259 (both cell lines with mTOR and MAPK activation). In vivo, BT40 (BRAFV600E) tumor cells were investigated with both agents in immunodeficient mice. Cell growth was investigated using MTS-assay compared to vehicle control. Activation of MAPK pathway was detected via Western Blot by phosphorylated ERK compared to total ERK, and β-actin. mTOR pathway was investigated with pAKT, pS6, and p4E-BP1 compared to the total protein amount and β-actin. In all of the cell lines, treatment with TAK228 or trametinib reduces cell growth and proliferation in a dose and time depended manner. We have found a robust synergistic (via Chou-Talalay method) effect for JHH_NF1_PA1, Res186, and Res259 cells in clinically relevant doses of both drugs (5nM, 10nM, 20nM). BT66_SV40/hTERT cells have a significant reduction in cell growth under TAK228 treatment after 4 days by up to 70% (p Citation Format: Antje Arnold, Ming Yuan, Fausto J. Rodriguez, Charles G. Eberhart, Eric H. Raabe. Synergistic growth inhibitor effect on a patient derived NF1 pilocytic astrocytoma cell line with the dual mTORC1/2 inhibitor TAK228 and MEK inhibitor trametinib [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 4629.

The mTOR pathway contributes to the tumorigenicity of aggressive pediatric low grade glioma (pLGG) and TORC1 inhibitors have demonstrated efficacy in treating pLGG cell lines. The FDA-approved TORC1 inhibitor, everolimus, is currently in clinical trials for patients with recurrent/refractory pLGG. We hypothesized that metabolic alterations downstream of TORC1 inhibition could be targeted to synergize with everolimus and reduce pLGG tumorigenicity. We identified through unbiased metabolic screens and stable isotope tracing that glutathione was downregulated (p

Isolation and propagation of neural stem cells derived from human brain tissue uniquely enables the study of human neurogenesis in vitro. In addition, ex vivo-expanded human neural stem/precursor cells (NPCs) may offer novel therapeutic strategies. We investigated the effects of extracellular nucleotides on the proliferation and differentiation of human mesencephalic neural stem/precursor cells (hmNPCs). When combined with the mitogens epidermal growth factor and fibroblast growth factor 2, UTP (1 microm) boosted proliferation of hmNPCs as shown by increased expression of the proliferation marker proliferating cell nuclear antigen (330%). UTP-induced proliferation was abrogated by the preferential P2Y receptor blocker pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). UTP also stimulated dopaminergic differentiation. Treatment with UTP (100 microm) increased the number of tyrosine hydroxylase (TH)-positive cells and TH protein by 267 and 319% respectively. UTP-stimulated dopaminergic differentiation of hmNPCs was blocked by the P2 receptor antagonists suramin (10 microm) and PPADS (100 microm). In addition, UDP (1 microm) enhanced TH protein expression by 194%. During differentiation, treatment with UTP stimulated the extracellular signal-regulated kinase (ERK) pathway. Both ERK1/2 phosphorylation and dopaminergic differentiation were inhibited by U0126, a selective ERK kinase inhibitor, as well as by suramin. When other P2 receptor agonists (ATP, ADP and adenosine 5'-O-(2-thiophosphate) (ADPbetaS); all 100 microm) were applied, both proliferation and dopaminergic differentiation of NPCs were compromised. We conclude that uracil nucleotides exert specific P2 receptor-mediated effects on midbrain-derived human NPCs, and may be used to enhance both proliferation and dopaminergic differentiation.

Most members of the large family of rhodopsin-like G-protein-coupled receptors possess an evolutionarily conserved Asp-Arg-Tyr (DRY) motif in the C-terminal region of the third transmembrane domain. Mutations of residues within this motif usually abolish receptor function and, when they occur naturally, can even cause human diseases. By analyzing over 100 mammalian orthologs of the chemoattractant receptor GPR33 we identified several polymorphic and fixed sequence variations within the DRY motif. Unexpectedly, the naturally occurring mutation of Arg(3.50) to His in mouse GPR33 showed no difference from the wild-type receptor in several functional tests. Sequence analysis of GPR33 from Asian house mice revealed the polymorphic existence of Arg(3.50) and His(3.50) alleles in wild-trapped populations, further supporting the functional equivalence of both allelic variants. In contrast, the Arg(3.50) to Gly mutation found in hamster GPR33 inactivates the receptor and may have contributed to pseudogenization of this gene in this species. Functional data with GPR33 variants indicate different receptor- and context-specific consequences of DRY mutations. Our study also reveals GPR33 as a new example illustrating missense mutations as a first step in the pseudogenization process.

Pediatric low grade glioma (PLGG) is one of the most common childhood tumors. If the tumor is located in a region of the brain that is not accessible for surgical resection or if the tumor recurs after surgery, additional therapies are needed. Recent studies highlighted the important role of mTORC and MEK-activation in PLGG. The dual mTORC1/2-inhibitor, TAK-228, and the FDA approved MEK-inhibitor, Trametinib, have good brain penetration and are promising candidates for targeted therapy PLGG. We hypothesized that TAK-228 and Trametinib would show synergistic effects both in vitro and in vivo in PLGG models. We treated the PLGG derived cell lines Res186 and Res259 with TAK-228, Trametinib, and vehicle. Cell growth was investigated using MTT-assay over different days and compared to the treatment with the vehicle. DNA replication was measured through bromodeoxyuridine (BrdU) incorporation assay and apoptosis was evaluated through cleaved caspase 3 (CC-3) staining. Cells were analyzed and counted with ImageJ. Activation of MAPK pathway was detected via Western Blot by phosphorylated pERK compared to total pERK, and β-actin. Treatment of Res186 and Res259 with TAK-288 or Trametinib reduces cell growth and proliferation in a dose and time depended manner. Res186 have a significant reduction in cell growth after 4 days treatment (TAK-288: 20nM, **p Citation Format: Antje Arnold, Fausto Rodriguez, Charles George Eberhart, Eric Hutton Raabe. Combinatory treatment for pediatric low grade glioma with the dual mTORC1/2 inhibitor TAK228 and MEK inhibitor Trametinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 703. doi:10.1158/1538-7445.AM2017-703

The discovery that somatic cells can be induced into a pluripotent state by the expression of reprogramming factors has enormous potential for therapeutics and human disease modeling. With regard to aging and rejuvenation, the reprogramming process resets an aged, somatic cell to a more youthful state, elongating telomeres, rearranging the mitochondrial network, reducing oxidative stress, restoring pluripotency, and making numerous other alterations. The extent to which induced pluripotent stem cell (iPSC)s mime embryonic stem cells is controversial, however, as iPSCs have been shown to harbor an epigenetic memory characteristic of their tissue of origin which may impact their differentiation potential. Furthermore, there are contentious data regarding the extent to which telomeres are elongated, telomerase activity is reconstituted, and mitochondria are reorganized in iPSCs. Although several groups have reported that reprogramming efficiency declines with age and is inhibited by genes upregulated with age, others have successfully generated iPSCs from senescent and centenarian cells. Mixed findings have also been published regarding whether somatic cells generated from iPSCs are subject to premature senescence. Defects such as these would hinder the clinical application of iPSCs, and as such, more comprehensive testing of iPSCs and their potential aging signature should be conducted.