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Heterozygous isocitrate dehydrogenase (IDH) R132H mutation (IDH1R132H/WT) is an early event during gliomagenesis. Clinically, patients with glioma carrying mutant IDH1 respond better to antitumor therapies. However, the mechanism by which IDH1 mutations contribute to gliomagenesis and therapeutic response remains elusive. Here we report that senescence is involved in the improved therapeutic responses of mutant IDH1 glioma cells. Knocking-in IDH1R132H/WT in glioma cells significantly enhanced gliomas cell senescence in response to temozolomide and radiation via a DNA-damage mediated mechanism. We further asked if senescence plays a role in IDH1R132H/WT-induced gliomagenesis. Together with ATRX knockout and p53/RB loss, IDH1R132H/WT transformed nonneoplastic human astroglial cells to form tumors in mouse brains. In-depth characterization revealed that a subset of these precancerous cells underwent senescence-like phenotypic changes, including flat and enlarged-cell morphology, increased senescence marker expression, decreased cell proliferation, and cell-cycle arrest at the G2–M phase. Mechanistic studies indicated that the combination of glioma driver genes (p53/RB/IDH1/ATRX) dramatically increased DNA damage and activated DNAdamage response (DDR) pathways ATR/ATR and Chk1/Chk2 in senescent cells. To determine how senescent cells drive tumor formation, we investigated non–cell-autonomous mechanisms such as senescence-associated secretory phenotype (SASP), a panel of proinflammatory and tissue-remodeling factors implicated in a tumor-permissive microenvironment. We found that astroglial cells carrying p53/RB/ATRX loss and IDH1R132H/WT upregulated key factors in SASP via an epigenetic-mediated mechanism. Our work suggests that drugs that specifically eliminate senescent cells could help kill precancerous cells and senescent tumor cells following antitumor therapies.Implications:The mechanisms by which IDH1 mutations contribute to gliomagenesis and therapeutic responses remain incompletely characterized; this work reveals senescence as a novel mechanism of IDH-mutant–mediated biological impact and describes new therapeutic opportunities concerning IDH1-mutant gliomas.

Supplementary Table 1. Primers used for qRT-PCR Supplementary Fig. 1. Heterozygous IDH1R132H/WT enhanced therapy-induced senescence in glioma cells. Supplementary Fig. 2. a. Schematic graph of genome editing strategy with two guide RNAs to knock out human ATRX. b. The ATRX gene sequence of two clones (C4, C10) after genome editing with CRISPR/Cas9 and two ARTX guide RNAs. c. Transwell assays of cell migration showing ATRX knockout in mIDH1 WT cells decreased cell migration. Supplementary Fig. 3. ATRX knockout in SVG IDH1 WT cells did not affect cell growth and migration. Supplementary Fig. 4. a, b. BrdU incorporation assays. c. Cell cycle analysis with PI and flow cytometry. Cells were synchronized by serum withdrawing for 24 h, followed by serum replenishing for 18 h. d. Western blot analyses of Y-H2AX and DNA damage response pathways in cells. Quantification of protein level changes was from the average of three independent experiments. Supplementary Fig. 5. IDH1R132H/WT and ATRX knockout did not induce ALT and alter chromosome numbers in SVG astroglial cells. Supplementary Fig. 6. Effect of AGI-5198 (a) and 5-Aza (b) on SASP expression. Supplementary Fig. 7. M0 human primary macrophages responses to LPS and IL4. Supplementary Fig. 8. ABT-263 decreased SA-beta-gal staining.

High-grade astrocytoma with piloid features (HGAP) is a recently recognized glioma type whose classification is dependent on its global epigenetic signature. HGAP is characterized by alterations in the mitogen-activated protein kinase (MAPK) pathway, often co-occurring with CDKN2A/B homozygous deletion and/or ATRX mutation. Experience with HGAP is limited and to better understand this tumor type, we evaluated an expanded cohort of patients (n = 144) with these tumors, as defined by DNA methylation array testing, with a subset additionally evaluated by next-generation sequencing (NGS). Among evaluable cases, we confirmed the high prevalence CDKN2A/B homozygous deletion, and/or ATRX mutations/loss in this tumor type, along with a subset showing NF1 alterations. Five of 93 (5.4%) cases sequenced harbored TP53 mutations and RNA fusion analysis identified a single tumor containing an NTRK2 gene fusion, neither of which have been previously reported in HGAP. Clustering analysis revealed the presence of three distinct HGAP subtypes (or groups = g) based on whole-genome DNA methylation patterns, which we provisionally designated as gNF1 (n = 18), g1 (n = 72), and g2 (n = 54) (median ages 43.5 years, 47 years, and 32 years, respectively). Subtype gNF1 is notable for enrichment with patients with Neurofibromatosis Type 1 (33.3%, p = 0.0008), confinement to the posterior fossa, hypermethylation in the NF1 enhancer region, a trend towards decreased progression-free survival (p = 0.0579), RNA processing pathway dysregulation, and elevated non-neoplastic glia and neuron cell content (p < 0.0001 and p < 0.0001, respectively). Overall, our expanded cohort broadens the genetic, epigenetic, and clinical phenotype of HGAP and provides evidence for distinct epigenetic subtypes in this tumor type.

The purpose of this study was to present a case of diffuse noncaseating granulomas involving the corneal stroma in a patient with ocular and pulmonary sarcoidosis.This was a single case report.A 31-year-old female patient presented with a 6-year history of panuveitis of the right eye along with a history of pulmonary sarcoidosis and a conjunctival biopsy of the right eye that was reported as positive for sarcoidosis. At presentation to our clinic, the patient had band keratopathy, vascularization of the inferonasal cornea, and active anterior uveitis of the right eye. When the patient returned for a follow-up of 15 months after the initial presentation, the cornea of the right eye exhibited widespread stromal scarring and vascularization. Because of the corneal scarring, the patient underwent an implantation of a Boston type 1 keratoprosthesis in the right eye. Histopathological examination of the host corneal tissue removed at the time of the keratoprosthesis procedure revealed extensive noncaseating granulomas in the deep corneal stroma. The patient underwent penetrating keratoplasty 8 months later, and histopathological examination again demonstrated noncaseating granulomas, this time at the edges of the donor corneal graft used during the keratoprosthesis implantation.We present the histopathological evidence of sarcoidosis involving the corneal stroma. Interestingly, the stromal keratitis also subsequently involved the donor cornea tissue after the patient underwent a keratoprosthesis implantation. It seems that sarcoidosis is a rare cause of stromal keratitis.

We have previously identified high activation of both mTORC1 and mTORC2 in the aggressive infantile brain tumor, atypical teratoid/rhabdoid tumor (AT/RT). Paxalisib is a highly brain penetrant PI3k inhibitor which acts upstream of mTOR to simultaneously inhibit mTORC1/2. Paxalisib is well-tolerated in ongoing pediatric clinical trials. In murine orthotopic xenograft models of AT/RT, Paxalisib significantly extended survival (CHLA-06: 40 to 54 days, p=0.001; BT12: 21 to 35 days, p=0.02). However, to improve the durability of this single agent therapy, we evaluated the efficacy of rational therapeutic partners. Analysis of RNAseq following mTOR inhibition (mTORi) of 4 cell lines representative of AT/RT showed an upregulation of genes associated with the integrated stress response (ISR), (ATF4: CHLA-06, t-test p Citation Format: Tyler Findlay, Kristen Malebranche, Anupa Geethadevi, Eric Raabe, Charles Eberhart, Jeffrey Rubens. Combination of the PI3k inhibitor Paxalisib with the nucleoside analog Gemcitabine over-activates the integrated stress response and induces atypical teratoid/rhabdoid tumor cell death. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5490.

Atypical teratoid/rhabdoid tumors (AT/RT) are deadly infantile brain tumors. Their aggressive phenotype results from a single recurring biallelic loss-of-function mutation in genes encoding components of the SWI/SNF chromatin-remodeling complex - SMARCB1 in the majority of tumors and SMARCA4 in remaining tumors. Residual SWI/SNF activity continues to inhibit EZH2 methyltransferase except at gene promoter regions where EZH2 co-localizes with the REST complex. At these sites, EZH2 increases repressive H3K27me3 marks. EZH2 co-localizes with REST on neuronal differentiation and tumor suppressor genes. As a result, this epigenetic abnormality helps maintain AT/RT in a stem cell-like state, driving its aggressive growth and therapy resistance. We study the consequences of disrupting SWI/SNF inhibition at sites of EZH2/REST co-localization to target AT/RT precisely. While there are no known interactions between the REST complex and SWI/SNF, there are interactions between REST’s primary co-factor CoREST and SWI/SNF. The CoREST complex consists of LSD1, HDAC1, and the scaffolding protein RCOR. We disrupted the CoREST complex with lentiviral knockdown of RCOR2 (kdRCOR2). KdRCOR2 reversed AT/RT’s stem cell-like state, driving neuronal differentiation and slowing AT/RT growth. Interestingly, kdRCOR1 had no impact on cell differentiation (microscopy and qRT-PCR) and had a modest impact on cell growth (MUSE cell viability assay). We next used Corin to pharmacologically target the CoREST complex in 3 AT/RT cell lines (CHLA05, CHLA06, and BT37). Corin is a bi-functional inhibitor of LSD1 and HDAC1/2, the component enzymes of the CoREST complex. Similar to kdRCOR2, Corin induced neuronal differentiation in AT/RT with increased adherence to plastic and new axonal development (microscopy). Cells also increased expression of neuronal differentiation markers identified by immunofluorescence (MAP2) and qRT-PCR (CEND1, and NEUROD2, t-test p Citation Format: Anupa Geethadevi, Marianne Collard, Nikhil Vaidya, Tyler Findlay, Kristen Malebranche, Charles Eberhart, Eric Raabe, Philip Cole, Rhoda Alani, Jeffrey Rubens. The CoREST complex as a novel target to disrupt AT/RT’s abnormal epigenetic landscape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1141.

Diffuse midline glioma (DMG) is an aggressive pediatric glioma with no curative treatment options. Mutation in MAPK pathway is common in many DMG tumors, however, has not been largely explored before. E6201 is a dual inhibitor of ACVR1 and MEK 1/2. It is currently in a phase 1 clinical trial for BRAF/MEK mutant melanoma. E6201 achieves higher levels in brain compared to other MEK inhibitors, suggesting it might be a promising therapeutic option for DMG. Previously, E6201 was effective in DMG cell lines with ACVR1 mutation, such as HSJD007. We have evaluated the effect of E6201 in two MAP kinase pathway activated, novel DMG cell lines, DIPG2JA and JHH-DIPG17. DIPG2JA has a KRAS mutation, leading to upregulation of MAPK pathway, and JHH-DIPG17 has two NF1 alterations predicted to disrupt this tumor suppressor, resulting in activation of MAPK pathway. Treatment of DIPG2JA with E6201 identified an IC50 of 44 nM, which is comparable to an IC50 of 71nM in the ACVR1 mutant DMG HSJD007. E6201 suppressed phospho—ERK and induced apoptosis as measured by cPARP western blot in DMG cells. Evaluation of cell viability by CellTiterBlue viability assay demonstrated that E6201 suppressed DMG growth between 0.05uM to 1uM concentrations. (DIPG2JA p=0.0004, JHH-DIPG17 p Citation Format: Hyuk Jean Kwon, Abigail Tindall, Charles Eberhart, Jeffrey Rubens, Eric Raabe. Dual inhibitor of ACVR1 and MEK 1/2 E6201 and PI3K/mTOR inhibitor paxalisib synergistically inhibit cell growth in DMG. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4990.

H3K27-altered diffuse midline glioma (DMG) and atypical teratoid/rhabdoid tumor (AT/RT) are aggressive pediatric brain tumors with dismal outcomes. DMG and AT/RT have increased baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that enables cells to tolerate various forms of stress. This activation is manifested by elevated levels of ATF4 and NRF2 in DMG and AT/RT, respectively. Transient or low-level expression of these transcription factors protect cells from stress, while sustained, high-level activation leads to cell death. Ixazomib is an orally bioavailable proteasome inhibitor that causes endoplasmic reticulum stress, which is a major upstream activator of the ISR. We hypothesized that the high baseline level of ATF4 in DMG and NRF2 in AT/RT would make these tumors susceptible to ISR activators such as ixazomib. After determining the IC50 of ixazomib, DMG and AT/RT cell lines (JHH-DIPG1, JHH-DIPG16A, and CHLA06) were treated with increasing concentrations of the drug. Cleaved caspase 3 immunofluorescence showed a significant increase of apoptosis in all cell lines (JHH-DIPG16A and CHLA06 p Citation Format: Orlandi Valencia Novak, Tyler Findlay, Jeffrey Rubens, Charles Eberhart, Eric H. Raabe. Using proteasome inhibition to hyperactivate the integrated stress response in aggressive pediatric brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6720.

Medulloblastoma (MB) is an aggressive brain tumor that predominantly affects children. Recent high-throughput sequencing studies suggest that the non-coding RNA genome, in particular long non-coding RNAs (lncRNAs), contributes to MB formation and tumor progression. Here we report the identification of a novel lncRNA, lnc-HLX-2-7, as a potential therapeutic target in group 3 MBs. In this study, we report that lnc-HLX-2-7 RNA specifically accumulates in the HLX (host gene of lnc-HLX-2-7) promoter region and activates HLX expression by recruiting multiple factors including enhancer elements. RNA sequencing and chromatin immunoprecipitation revealed that HLX directly binds to the promoters of several tumor-promoting genes, including MYC, and activates their expression. Furthermore, intravenous treatment with antisense oligonucleotides targeting lnc-HLX-2-7 coated with cerium-oxide nanoparticle (CNP-lnc-HLX-2-7) reduced tumor growth (40-50%) in intracranial MB xenograft mouse model (n = 10, p < 0.01, t-test). We found that the combinatorial therapy of CNP-lnc-HLX-2-7 and cisplatin further inhibits tumor growth and significantly prolongs mouse survival compared to CNP-lnc-HLX-2-7 monotherapy (n = 10, p < 0.01, t-test) only. We report here the importance of the lnc-HLX-2-7-HLX-MYC axis in regulating group 3 MB progression and provide a strong rationale for using lnc-HLX-2-7 as a specific and potent therapeutic target for the group 3 MBs in children.

Medulloblastoma is a central nervous system tumor that primarily affects children and requires aggressive therapy. Patients often suffer from treatment-related side effects, and treatment-resistant recurrences are common, with high mortality rates. There are four major molecular MB subgroups (Wnt- and Shh-activated MBs G3 and G4 MBs). G3 MB is the most aggressive subtype, and the diagnosis and management remain challenging. Medulloblastomas develop through various genetic, epigenetic, and noncoding (nc) RNA-related mechanisms. However, the roles played by ncRNAs, (microRNAs, long noncoding RNAs, circular RNAs, etc.) in MB development remain poorly defined. Here we address this knowledge gap with an exemplar microRNA, microRNA 211 (miR-211) implicated in G3 MB development and progression. Our preliminary results support that miR-211 is an attractive therapeutic agent to treat this aggressive MB subtype. miR-211 is significantly downregulated in medulloblastoma cell lines compared to normal cerebellum, underscoring its important role as a therapeutic agent and a biomarker. miR-211 ectopic expression in G3 MB cells significantly reduced cell proliferation and 3D colony formation and induced apoptosis. In vivo, miR-211force-expression in G3 MB cells injected into mouse cerebellum produce smaller tumors than those derived from parental cells. We identified that Long-chain-fatty-acid—CoA ligase 4 (ACSL4), and the oncogene Ras-related protein Rab22A are miR-211 targets genes for G3 medulloblastomas. The preliminary results of this study are encouraging and will provide a pre-clinical foundation for further therapeutic testing.

Diffuse Intrinsic Pontine Glioma (DIPG) is a pediatric brain tumor characterized by epigenetic dysregulation with median survival of 9-11 months. Currently, radiation is the only therapy option, and therefore calls for a new, effective treatment method. RG2833 is a brain penetrant, selective HDAC 1/3 inhibitor. Western blot confirmed that RG2833 treatment alone downregulates the NFkB pathway in multiple DIPG cell lines. We showed that RG2833 treatment decreases expression of NFkB regulated anti-apoptotic genes XIAP, BCL-2, and BCL-XL in vitro. In vivo results in DIPG flank tumors confirmed that BCL-2 and BCL-XL expression are decreased with RG2833 treatment. We have also confirmed through Western blot that RG2833 treatment induces p16, p21, and p27 expression, which negatively regulate cell cycle progression, in multiple DIPG cell lines. RNAseq data showed that PI3K/mTOR pathway is upregulated in DIPG after RG2833 treatment. Paxalisib is a brain penetrant PI3K/mTOR inhibitor currently in clinical trials for DIPG. We hypothesized that RG2833 and paxalisib combination treatment would yield synergistic effects. RG2833 synergizes with paxalisib to suppress growth as measured by CellTiterBlue. ZIP synergy scores calculated by SynergyFinder were 10.5 for SUDIPG06 and 9.2 for HSJD007. Western blot demonstrated that combination treatment induced c-PARP and FOXO1 in the SUDIPG06 cell line, and decreased p-RB expression in HSJD007 cell line. Induction of c-PARP indicates increased apoptosis, and decreased p-RB expression indicates decreased cell proliferation. In vivo experiments evaluating the efficacy of combination treatment are in progress. Our data suggest the combination of RG2833 and paxalisib may be a promising treatment option for DIPG.

How dysregulation of neurodevelopment relates to medulloblastoma (MB), the most common pediatric brain tumor, remains elusive. Here, we uncovered a neurodevelopmental epigenomic program being hijacked to induce MB metastatic dissemination. Unsupervised analyses by integrating publicly available datasets with our newly generated data revealed that SMARCD3/BAF60C regulates DAB1-mediated Reelin signaling in Purkinje cell migration and MB metastasis by orchestrating cis-regulatory elements (CREs) at the DAB1 locus. We further identified that a core set of transcription factors, enhancer of zeste homolog 2 (EZH2) and nuclear factor I X (NFIX), coordinates with the CREs at the SMARCD3 locus to form a chromatin hub for controlling SMARCD3 expression in the developing cerebellum and metastatic MB. Elevated SMARCD3 activates Reelin/DAB1-mediated Src kinase signaling, resulting in MB response to Src inhibition. These data deepen our understanding of how neurodevelopmental programming influences disease progression and provide a potential therapeutic option for MB patients.

Sezen Karakus,1 James Foster,1 Xi Dai,1,2 Anthony Gonzales,1 Xi Zhu,1,3 Charles Eberhart,1 William Hsu1,4 1Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2Johns Hopkins University School of Medicine, Baltimore, MD, USA; 3Department of Biology, Public Health Studies, Krieger School of Arts and Sciences, Johns Hopkins University, Baltimore, MD, USA; 4Department of Molecular and Cell Biology, Krieger School of Arts and Sciences, Johns Hopkins University, Baltimore, MD, USACorrespondence: Sezen KarakusWilmer Eye Institute, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Maumenee 741, Baltimore, MD, 21287, USATel +1 410 955 0128Fax +1 410 614 1670Email skarakus@jhmi.eduPurpose: To investigate the prevalence of SARS-CoV-2-associated conjunctivitis among patients presenting with conjunctivitis to ophthalmology clinics in the absence of respiratory symptoms suggesting COVID-19 during the first year of the pandemic.Patients and Methods: This prospective, observational, cross-sectional study enrolled patients aged 18 years and older who presented with acute conjunctivitis between May 2020 and May 2021. After reviewing demographics, ocular and systemic symptoms, a slit lamp examination was performed. Five samples were collected (conjunctival swab from each eye, nasal swab from each nostril, and a nasopharyngeal swab) in separate tubes for a reverse transcription-polymerase chain reaction test for SARS-CoV-2. Patients were called at 1 and 2 weeks to follow up on their ocular and systemic symptoms and collect information about conjunctival adenovirus test results and COVID-19 test results if performed outside of the study.Results: A total of 36 patients were enrolled. The most common ocular symptom was redness (35/36, 97%), and 14 patients (39%) had symptoms in both eyes at the time of the presentation. SARS-CoV-2 RNA was not detected in any samples collected during the study visit (95% confidence interval [CI] from 0 to 0.08). None of the study participants were diagnosed with COVID-19 following conjunctivitis during the 2-week follow-up period. We found that 25 patients had a conjunctival adenovirus test done on the day of their study visit, 9 of which reported positive results.Conclusion: Our data suggest that it may not be valuable to obtain routine testing of SARS-CoV-2 in patients presenting with conjunctivitis in the absence of COVID-19.Keywords: adenovirus, conjunctivitis, coronavirus, COVID-19, SARS-CoV-2

Low-grade gliomas (LGG) are slow-growing tumors that affect neurologic function by pressing on the surrounding parts of the brain. Pediatric low-grade glioma (pLGG) is the most common childhood brain tumors. Patients with pLGG often have a significant life-long disabilities. The most common mutations in pLGG are in the mitogen-activated protein kinase (MAPK) pathway including tumor suppressor neurofibromin (NF1), and the oncogene BRAF. BRAF activates MEK1/2, which activates of ERK1/2, leading to tumorigenesis. BRAFV600E mutation results in the activation segment of the kinase “monomeric form of BRAF”, leading to constitutive activation of MAP kinase and mTOR pathways Combination treatment of TAK228 (mTOR inhibitor) and trametinib (MEK1/2 inhibitor), suppressed both mTOR and MAP kinase pathways and inhibited BRAFV600E glioma tumor growth. To further investigate and understand the molecular mechanism of TAK228 and trametinib combinatorial efficacy, we performed comprehensive proteomic analysis of BRAFV600E glioma tumors treated with TAK228 and trametinib. We found increased levels of many proteins in the glycolysis and TCA cycle. Specifically we found increases at the protein level in citrate synthase, aconitase and malate dehydrogenase in the TCA cycle. In glycolysis, we observed increased glucose transporter expression (SLC2A1), increased hexokinase 2, and increased GAPDH. Upregualtion of these proteins may represent attempts to bypass the effects of mTOR and MAP kinase blockade on the activity of these pathways. We are currently performing flux metabolic profiling using uniformly labeled glucose to assess the activity of the pathways after combined mTOR and MAP kinase blockade. Our results may increase our understanding of how glioma cells use glucose and how mTOR and MAP kinase pathway inhibition affects basic metabolic pathways in glioma cells.

Medulloblastoma is a central nervous system tumor that develops through various genetic, epigenetic, and non-coding (nc) RNA-related mechanisms, but the roles played by ncRNAs, particularly circular RNAs (circRNAs), remain poorly defined. CircRNAs are increasingly recognized as stable noncoding RNA therapeutic targets in many cancers, but little is known about their function, subtype specificity, and therapeutic potential in medulloblastomas. To determine medulloblastoma subgroup-specific circRNAs, we subjected RNA-seq data from 175 clinical medulloblastoma samples in four subgroups (SSH, WNT, G3, and G4) to a statistical and machine learning (random forest) pipeline and identified a group of medulloblastoma specific circular RNAs. CircRNA, circ_63706 was identified as sonic hedgehog (SHH) group specific and confirmed its expression by RNA-FISH analysis in clinical tissue samples (tissue microarrays). To identify the molecular function of circ_63706, we depleted circ_63706 in DAOY and ONS76 cells and subjected them to global RNA sequencing and lipid profiling. Circ_63706 resides in the coding gene Pericentrin (PCNT), which is known to be involved in congenital disorders. When Circ_63706 gets depleted by shRNA, it shows a significant decrease in cell proliferation and invasion in SSH cells, and mice implanted with circ_63706-deleted cells showed reduced tumor growth and extended survival compared to parental cells implant. At the molecular level, we identified circ_63706-deleted cells elevated total ceramide and oxidized lipids and reduced total triglyceride (TG). Our study implicates an identification of a novel oncogenic circular RNA in the medulloblastoma subgroup SSH and establishes its potential as a future therapeutic target.

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.

While loss of RB function is the most common genetic alteration in the pediatric embryonal eye tumor retinoblastoma, mutations leading to BCL-6 corepressor (BCOR) loss of function are the second most common alteration. BCOR mutations are found in more aggressive tumors, and are associated with metastasis and poor prognosis. We investigated the effects of BCOR loss or overexpression in retinoblastoma cells in order to better understand its functional role, and to determine if it represents a new therapeutic target in these tumors. Six established retinoblastoma cell lines were examined, and BCOR protein was most highly expressed in WERI-RB1, but absent in Y79 and two other lines. BCOR knock down in WERI-RB1 cells using shRNA caused markedly increased migration and proliferation. BCOR was also knocked out in these cells using CRSPR, resulting in more than three-fold increased size of intraocular murine xenografts and extensive extraocular extension of tumors. In terms of mechanism, BCOR loss of function induces IGF2 transcription, resulting in an activation of IGF1R and the ERK signaling pathway. In contrast, overexpressing BCOR in Y79 retinoblastoma cells with no baseline protein had opposite effects on IGF2. Targeting IGF2/IGF1R signaling pharmacologically using Lincitinib inhibited proliferation and migration in BCOR-negative retinoblastoma cells. Increased IGF2 transcription was regulated at least in part by hyper methylation of CCCTC-binding factor (CTCF)-binding regions in the IGF2/H19 imprinting control region (ICR), suggesting that BCOR knock down promotes increased methylation at this site. In summary, our studies revealed that BCOR loss promotes the growth and invasion of retinoblastoma cells in vitro and in vivo, at least in part via modulating IGF2 transcription, suggesting new potential therapeutic targets for these aggressive childhood tumors of the eye.

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.

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brain tumor. We have previously shown that the brain penetrant class 1 HDAC inhibitor RG2833 (RGFP109) inhibits cell growth (MTS assay for HSJD007 p=0.0004, JHH-DIPG1 p=0.001, SF-7761 p=0.04, SU-DIPG13 p=0.01 by t-test), reduces proliferation (phospho-Rb Western blot, BrdU immunofluorescence HSJD007 p=0.008, JHH-DIPG1 p=0.0002 by t-test), and induces apoptosis (cPARP Western blot, cleaved caspase 3 immunofluorescence HSJD007 p20). Evaluation of combination treatment for apoptotic effects in vitro and in vivo are ongoing. These findings identify RG2833 and Paxalisib as a promising combination therapy for DIPG. Citation Format: Katherine Barnett, Hyuk Jean Kwon, Orlandi Novak, Charles Eberhart, Eric Raabe. Brain penetrant HDAC and PI3K/mTOR inhibitors synergize to induce DIPG cell death [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 3987.

Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive pediatric brain tumors and the most common malignant brain tumors of infancy. The four-year event-free survival rate is only 37%. For patients with relapsed AT/RT, there are limited treatment options, but novel precision therapies may help improve survival for patients with this deadly disease. We have previously identified strong activation of the PI3K-AKT-mTOR (mTOR) signaling pathways in AT/RT. Paxalisib is a highly brain-penetrant PI3K inhibitor acting upstream of mTOR. We find that Paxalisib slows tumor growth in orthotopic xenograft models of AT/RT and extends median survival from 40 to 54 days (p=0.001, log-rank test). RNASeq after mTOR pathway inhibition identifies reflexive activation of the MAPK pathway as a possible mechanism of therapy resistance (KEGG pathway analysis). TAK580 (DAY 101) is a highly brain penetrant, pan-RAF kinase inhibitor, currently in clinical trials in pediatric low grade gliomas (NCT03429803, NCT04775485). Paxalisib combines synergistically with TAK580 to reduce AT/RT growth and viability (Bliss synergy score 24.22). A pilot study suggests combination therapy is well tolerated in mice bearing AT/RT orthotopic xenografts and slows tumor growth compared to Paxalisib alone and vehicle controls. This novel combination of precision therapies can translate into a clinical trial aimed at improving survival in this deadly disease. Citation Format: Kristen Malebranche, Tyler Findlay, Charles Eberhart, Eric Raabe, Jeffrey Rubens. The PI3K inhibitor Paxalisib combines synergistically with the pan-Raf inhibitor TAK580 (DAY 101) to extend survival in orthotopic xenograft models of atypical teratoid/rhabdoid tumors [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 5226.

Atypical teratoid/rhabdoid tumors (AT/RT) are the most common malignant brain tumors of infancy with a four-year event-free survival of 37%. Previously, we have identified high activation of both mTORC1 and mTORC2 through immunohistochemistry of 18 primary human AT/RT. The PI3K-Akt-mTOR signaling pathway drives tumorigenicity by promoting cancer cell growth and survival. Paxalisib is a highly brain-penetrant PI3K inhibitor acting upstream of mTORC1/2 to fully inhibit the activation of both complexes. Ongoing pediatric clinical trials have reported Paxalisib as safe and well-tolerated, with minimal dose limiting toxicities. Paxalisib treatment in mice bearing orthotopic xenografts of AT/RT slowed tumor growth and significantly extended survival from 40 to 54 days (bioluminescent imaging, log rank test p=0.0011). However, due to limited durability of single agent therapy, we conducted pilot studies to identify rational combination therapies to enhance Paxalisib survival benefits in AT/RT. RG2833 is a novel, highly brain penetrant, histone deacetylase 1/3 (HDAC1/3) inhibitor. Our preliminary data has shown that Paxalisib and RG2833 combine synergistically to decrease AT/RT cell growth (SynergyFinder ZIP score 11.1; CellTiter-Blue Cell Viability Assay, p Citation Format: Tyler Findlay, Kristen Malebranche, Charles Eberhart, Eric Raabe, Jeffrey Rubens. The PI3K inhibitor Paxalisib combines with the novel HDAC1/3 inhibitor RG2833 to improve survival in mice bearing orthotopic xenografts of atypical teratoid/rhabdoid tumors [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 5225.

We previously identified high activation of mTORC1 and mTORC2 in AT/RT by immunohistochemistry of 18 primary tumors expanding over each molecular subgroup of AT/RT. Paxalisib is a highly brain-penetrant PI3k inhibitor acting upstream of mTORC1/2 to fully inhibit the activation of both complexes. Ongoing pediatric clinical trials have reported Paxalisib as safe and well-tolerated. We find that Paxalisib leads to minimal toxicities in mice bearing AT/RT orthotopic xenografts, slows tumor growth (as determined by bioluminescent imaging), and significantly extends survival (CHLA-06: 40 to 54 days, p=0.0011; BT12: 21 to 35 days, p=0.02). However, due to limited durability of single agent therapy, we conducted pilot studies to identify rational combination therapies to further enhance these survival benefits. RG2833 is a novel, highly brain penetrant, histone deacetylase 1/3 (HDAC1/3) inhibitor. HDAC inhibitors have previously been identified as synergistic partners with PI3k/mTOR inhibitors through complementary activation of FOXO signaling pathways. We hypothesized that RG2833 would synergize with Paxalisib, minimize toxicities compared with pan-HDAC inhibitors, and maximize therapeutic benefits due to superior CNS penetration. We demonstrate that Paxalisib and RG2833 combine synergistically to decrease AT/RT cell growth (SynergyFinder ZIP score 11.1; CellTiter-Blue Cell Viability Assay, p

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.

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.

To determine whether p16, a molecular marker of cellular senescence, and CD68, a microglial marker, are detectible in optic nerve glioma tissue stored for decades, thus providing potential targets for pharmacologic intervention.Cases were retrieved from the Armed Forces Institute of Pathology Registry of Ophthalmic Pathology. Clinical information was tabulated. In specimens with sufficient tissue, a tissue microarray was constructed to conduct molecular studies.Ninety-two cases were included: gender distribution was in a ratio of one male to 1.6 females, and age range was 2 months to 50 years (average age, 10.8 years). Neurofibromatosis type 1 was identified in 10 cases (10.8%). The majority presented with decreased vision and exophthalmos. Forty-eight cases were studied by a tissue microarray construction. Glial fibrillary acidic protein, a control for immunoreactivity, was positive in 46 cases (96%). Immunoreactivity for p16 protein was seen in 36 cases (75%) and CD68-positive cells in 34 (71%). Limitations include referral bias, limited clinical information, limited amount of tissue, and extended period of tissue preservation.Optic nerve glioma is a tumor of the visual axis in young individuals, which is generally indolent but with a variable clinical course. Traditional histopathologic techniques have not been reliably predictive of clinical course. This microarray contains tumors with representative demographic, clinical, and histologic characteristics for optic nerve glioma. Immunoreactivity for p16 protein and CD68 is positive in the majority. These findings suggest a possible explanation for the variable clinical course and identify therapeutic targets in the cell senescence and microglial pathways.

Ink4 proteins inhibit the enzymatic activities of cyclin D-dependent kinases, thereby governing transcriptional programs that depend on the activities of the retinoblastoma protein and other retinoblastoma family members (p107 and p130). Mice lacking Ink4c and p53 spontaneously develop a broad spectrum of neoplasms, usually presenting with multiple tumors of different histological types and dying of cancer by 6 months of age. Whereas thymic lymphomas or pituitary tumors predominate in mice lacking p53 or Ink4c, respectively, animals lacking both genes develop many vascular tumors and also present with medulloblastomas not observed in the parental strains. Unlike p53, loss of the Arf tumor suppressor did not contribute to the appearance of vascular or cerebellar tumors. Vascular tumors ranged in severity from angiomas to hemangiosarcomas, some of which could be transplanted into immunocompromised mice. Intriguingly, loss of Ink4c but maintenance of at least one Ink4d allele was required for formation of medulloblastomas in p53-null mice. In situ hybridization revealed that, in newborn mice, Ink4c is detected in the pia mater and in an adjacent layer of rapidly dividing cells within the cerebellar external granule layer (EGL), whereas Ink4d is primarily expressed in Purkinje neurons. Because the pia mater and Purkinje cells sandwich the cerebellar EGL from which medulloblastomas are presumed to arise, Ink4 proteins might function in a cell-autonomous manner in governing neuronal cell cycle exit as well as in a non-cell-autonomous manner in controlling the production of diffusible mitogens and chemokines that influence postnatal development of the cerebellar EGL.