TAMI-79. THERAPEUTIC REVERSAL OF PRENATAL PONTINE ID1 SIGNALING IN DIPG

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi215-vi215
Author(s):  
Viveka Nand Yadav ◽  
Micah K Harris ◽  
Dana Messinger ◽  
Chase Thomas ◽  
Jessica R Cummings ◽  
...  
Keyword(s):  
Tumor Model ◽  
Pediatric Brain Tumor ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Normal Brain ◽  
Sequencing Analysis ◽  
In Vivo Models ◽  
Chip Sequencing ◽  
Tumor Expression

Abstract Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor with rare survival beyond two years. This poor prognosis is largely due to the tumor's highly infiltrative and invasive nature. Nearly 80% of DMGs harbor K27M mutation in the genes encoding histone H3.1 (H3F3A) or H3.3 (HISTIH3B), often with concurrent ACVR1 mutation. Inhibitor of DNA-binding (ID) proteins are key transcriptional regulators of genes involved in lineage commitment and are associated with invasiveness and poor clinical outcomes in multiple human cancers. Introduction of H3K27M and ACVR1 mutations increase ID1 expression in cultured astrocytes, but this has not been confirmed in human tumors or targeted therapeutically. We developed an in-utero electroporation (IUE) murine H3K27M-driven tumor model, which demonstrates increased ID1 expression in H3K27M- and ACVR1-mutated tumor cells. Exome and transcriptome sequencing analysis of multi-focal DMG tumors (n=52) and normal brain tissue revealed that increased ID1 expression is associated with H3K27M/ACVR1-mutation and brainstem location, and correlates with poor survival in patients. ChIP-sequencing for H3K27ac and H3K27me3 in multiple DMG tumors (n=5) revealed that the ID1 gene is epigenetically active, which matches the epigenetic state of murine prenatal hindbrain cells. Higher ID1-expressing astrocyte-like DIPG cells share a similar transcriptional program with ID1+/SPARCL1+ positive oligo/astrocyte-precursor (OAPC) cells from the developing human brain and demonstrate upregulation of gene sets involved in regulation of cell migration. Both genetic and pharmacologic [cannabidiol (CBD)] suppression of ID1 result in decreased DIPG cell invasion/migration in vitro and invasion/tumor growth in multiple in vivo models. Mechanistically, CBD reduces proliferation through production of reactive oxygen species. Further, DIPG patients treated off-trial with CBD (n=15) displayed reduced ID1 tumor expression and improved overall survival. In summary, ID1 is upregulated in DIPG through K27M-mediated epigenetic reactivation of a developmental OAPC-like transcriptional state, and ID1-driven invasiveness of DIPG is therapeutically targetable with CBD.

scholarly journals Diffuse intrinsic pontine glioma: current insights and future directions

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Dilakshan Srikanthan ◽  
Michael S. Taccone ◽  
Randy Van Ommeren ◽  
Joji Ishida ◽  
Stacey L. Krumholtz ◽  
...  
Keyword(s):  
Brain Tumor ◽  
Gene Mutations ◽  
Pediatric Brain Tumor ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Pontine Glioma ◽  
Current Standard ◽  
In Vivo Models ◽  
Landscape Models

AbstractDiffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor and the leading cause of brain tumor–related death in children. As several clinical trials over the past few decades have led to no significant improvements in outcome, the current standard of care remains fractionated focal radiation. Due to the recent increase in stereotactic biopsies, tumor tissue availabilities have enabled our advancement of the genomic and molecular characterization of this lethal cancer. Several groups have identified key histone gene mutations, genetic drivers, and methylation changes in DIPG, providing us with new insights into DIPG tumorigenesis. Subsequently, there has been increased development of in vitro and in vivo models of DIPG which have the capacity to unveil novel therapies and strategies for drug delivery. This review outlines the clinical characteristics, genetic landscape, models, and current treatments and hopes to shed light on novel therapeutic avenues and challenges that remain.

scholarly journals Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

2021 ◽  
Author(s):  
Viveka Nand Yadav ◽  
Micah K. Harris ◽  
Dana Messinger ◽  
Chase Thomas ◽  
Jessica R. Cummings ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Tumor Model ◽  
High Grade Glioma ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Human Tumors ◽  
In Vivo Models ◽  
Gene Sets ◽  
Transcriptional State

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive brain tumor with rare survival beyond two years. This poor prognosis is largely due to the tumor's highly infiltrative and invasive nature. Previous reports demonstrate upregulation of the transcription factor ID1 with H3K27M and ACVR1 mutations, but this has not been confirmed in human tumors or therapeutically targeted. We developed an in utero electroporation (IUE) murine H3K27M-driven tumor model, which demonstrates increased ID1 expression in H3K27M- and ACVR1-mutated tumor cells. In human tumors, elevated ID1 expression is associated with H3K27M/ACVR1-mutation, brainstem location, and reduced survival. The ID1 promoter demonstrates a similar active epigenetic state in H3K27M tumor cells and murine prenatal hindbrain cells. In the developing human brain, ID1 is expressed highest in oligo/astrocyte-precursor cells (OAPCs). These ID1+/SPARCL1+ cells share a transcriptional program with astrocyte-like (AC-like) DIPG cells, and demonstrate upregulation of gene sets involved with regulation of cell migration. Both genetic and pharmacologic [cannabidiol (CBD)] suppression of ID1 results in decreased DIPG cell invasion/migration in vitro and invasion/tumor growth in multiple in vivo models. CBD reduces proliferation through reactive oxygen species (ROS) production at low micromolar concentrations, which we found to be achievable in the murine brainstem. Further, pediatric high-grade glioma patients treated off-trial with CBD (n=15) demonstrate tumor ID1 reduction and improved overall survival compared to historical controls. Our study identifies that ID1 is upregulated in DIPG through reactivation of a developmental OAPC transcriptional state, and ID1-driven invasiveness of DIPG is therapeutically targetable with CBD.

scholarly journals PDTM-25. STUDY OF ONC201 IN PRE-CLINICAL MODELS OF DIPG

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi192-vi192
Author(s):  
Ajay Sharma ◽  
Yanlai Lai ◽  
Bridget Kennis ◽  
Sreepradha Sridharan ◽  
Tara Dobson ◽  
...  
Keyword(s):  
Intraperitoneal Administration ◽  
Pediatric Brain Tumor ◽  
Standard Of Care ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Tumor Burden ◽  
Phosphorylated Akt ◽  
Ic50 Values ◽  
Different Response

Abstract Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable pediatric brain tumor that occur in the pons and brainstem and have a peak onset of age between 6–9 years of age. Radiation is currently used as standard of care. Chemotherapy has shown no improvements in survival. Here, we report our study of ONC201, a first-in-class anticancer small molecule developed by Oncoceutics, Inc., against DIPG cells in vitro and in mouse orthotopic models. ONC201 was discovered in a screen as a p53-independent inducer of the pro-apoptotic cytokine TRAIL. It is known to directly and selectively inhibit dopamine receptor D2 (DRD2), a member of the G protein-coupled receptor (GPCR) family. MTT assays to determine the sensitivity of DIPG cells to ONC201 revealed a slight but not significantly different response to the drug based on their expression of wild type (WT) histone H3 or histone H3K27M mutant protein, with IC50 values in the range of 3-8mM. Decrease in cell growth was associated with a decrease in AKT and ERK phosphorylation and an increase in TRAIL expression. In vivo, intraperitoneal administration of ONC201 to mice bearing pontine DIPG tumors, once every week for 6 weeks, caused a significant reduction in tumor burden relative to untreated controls as measured by bioluminescence assays. However, stoppage of treatment resulted in tumor regrowth within 6 weeks, suggesting the existence of a population that were not eliminated by the current schedule of ONC210. Single cell proteomic analyses-based comparison of untreated and ONC201-treated DIPG cells showed an expected global reduction in pro-survival signals such as phosphorylated AKT and ERK. Molecules with potential to predict susceptibility of cells to ONC201 were also revealed, and are being confirmed by transcriptome analyses. Results of a chemical screen to target ONC201-refractory tumor cells will be discussed.

scholarly journals Breast Tumor Cells Highly Resistant to Drugs Are Controlled Only by the Immune Response Induced in an Immunocompetent Mouse Model

2019 ◽  
Vol 18 ◽  
pp. 153473541984804 ◽  
Author(s):  
Paola Lasso ◽  
Mónica Llano Murcia ◽  
Tito Alejandro Sandoval ◽  
Claudia Urueña ◽  
Alfonso Barreto ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Immune Response ◽  
Tumor Cells ◽  
High Capacity ◽  
Cancer Cell Line ◽  
Tumor Model ◽  
Main Element ◽  
In Vivo Models

Background: The tumor cells responsible for metastasis are highly resistant to chemotherapy and have characteristics of stem cells, with a high capacity for self-regeneration and the use of detoxifying mechanisms that participate in drug resistance. In vivo models of highly resistant cells allow us to evaluate the real impact of the immune response in the control of cancer. Materials and Methods: A tumor population derived from the 4T1 breast cancer cell line that was stable in vitro and highly aggressive in vivo was obtained, characterized, and determined to exhibit cancer stem cell (CSC) phenotypes (CD44+, CD24+, ALDH+, Oct4+, Nanog+, Sox2+, and high self-renewal capacity). Orthotopic transplantation of these cells allowed us to evaluate their in vivo susceptibility to chemo and immune responses induced after vaccination. Results: The immune response induced after vaccination with tumor cells treated with doxorubicin decreased the formation of tumors and macrometastasis in this model, which allowed us to confirm the immune response relevance in the control of highly chemotherapy-resistant ALDH+ CSCs in an aggressive tumor model in immunocompetent animals. Conclusions: The antitumor immune response was the main element capable of controlling tumor progression as well as metastasis in a highly chemotherapy-resistant aggressive breast cancer model.

The effect of everolimus on CNS penetration and efficacy of dasatinib in the treatment of PDGFRA-driven glioma.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e13508-e13508
Author(s):  
Zachary Miklja ◽  
Brendan Mullan ◽  
Ruby Siada ◽  
Stefanie Stallard ◽  
Viveka Nand Yadav ◽  
...  
Keyword(s):  
High Grade Glioma ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Pharmacokinetic Studies ◽  
Glycoprotein P ◽  
In Vivo Models ◽  
Phase 2 Trial ◽  
Before And After ◽  
Cns Penetration

e13508 Background: Pediatric and adult high-grade glioma (HGG) frequently harbor PDGFRA alterations. The CNS penetration of PDGFRA inhibitors, such as dasatinib, is limited by the tumor-efflux protein P-glycoprotein (P-gp). We hypothesized that co-treatment with everolimus, which has been shown to block P-gp, will increase CNS penetration and efficacy of dasatinib in in vitro and in vivo models as well as in human PDGFRA-driven glioma. Methods: Tumors were generated in mice using an intra-uterine electroporation (IUE) model [introduction of TP53, PDGFRA and H3K27M mutations in pre-natal cortex]. Dose response, synergism studies, P-GP inhibition and pharmacodynamics/pharmacokinetic studies were then performed on in vitro and in vivo models employing this IUE system. A phase 2 trial employing dasatinib and everolimus was established for children with HGG and diffuse intrinsic pontine glioma (DIPG) that contain PDGFRA alterations (NCT03352427). Paired CSF/plasma samples (before and after addition of everolimus) were collected from enrolled patients. Results: Dasatinib effectively treated mouse HGG cells with an IC50 of 100 nM. Dose-dependent reduction in PDGFRA and pPDGFRA was found. P-gp inhibitor assay confirmed that everolimus strongly blocks P-gp activity at 1 uM (p = 0.0028 vs untreated). Mice treated with dasatinib and everolimus had extended survival as compared to control. Two-hour exposure to everolimus resulted in sub-IC50 dasatinib concentration in cortex (23 nM) and tumor (65 nM). 24-hour exposure to everolimus resulted in greater cortex (235 nM) and tumor (509 nM) concentrations. Two trial patients, recurrent HGG ( PDGFRA-amplified) and recurrent DIPG ( PDGFRA D842V) respectively, survived 6 months and 9 months (ongoing) after progression, which compares very favorably to historical controls. A paired CSF sample from the PDGFRA-amplified patient showed a 50% increase in CSF dasatinib level after addition of everolimus. Conclusions: Dasatinib treatment of PDGFRA-driven HGG is improved with everolimus blockade of P-gp and represents a novel route for improving CNS penetration and efficacy of therapies for HGG. Clinical trial information: NCT03352427.

scholarly journals DIPG-22. DISSECTING THE ONCOGENIC ROLE OF FOXR2 IN DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii291-iii291
Author(s):  
Jessica W Tsai ◽  
Smruti K Patel ◽  
Heather Bear ◽  
Frank Dubois ◽  
Prasidda Khadka ◽  
...  
Keyword(s):  
Cell Lines ◽  
Therapeutic Target ◽  
Transcriptional Profiling ◽  
Genomic Analysis ◽  
In Utero ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Sequencing Analysis ◽  
Integrative Genomics

Abstract BACKGROUND Diffuse intrinsic pontine gliomas (DIPGs) pose particular challenges for treatment. We recently completed a genomic analysis of close to 200 DIPGs and high-grade gliomas. We identified that nearly 10% of all DIPGs have increased expression of the fork head domain transcription factor FOXR2. We hypothesize that FOXR2 accelerates gliomagenesis in histone mutant DIPGs and represents a previously unexplored therapeutic target. METHODS To determine whether FOXR2 is sufficient to mediate gliomagenesis, we applied an integrative genomics approach using both in vitro and in vivo DIPG models: mouse neural stem cell models expressing FOXR2, in vivo mouse models using in utero brainstem electroporation, patient-derived DIPG cell lines, and RNA sequencing analysis of human and mouse tumors expressing FOXR2. RESULTS Our data shows that FOXR2 indeed is an oncogene that rapidly accelerates gliomagenesis using an in vivo brainstem in utero electroporation model of DIPG. In human tumors, increased FOXR2 expression is mutually exclusive with MYC amplification suggesting functional redundancy. In vivo, FOXR2 results in large brainstem gliomas and rapid neurologic decline of animals. Transcriptional profiling of these tumors demonstrates activation of MYC signaling pathways. In vitro, we have further identified patient-derived cell lines with increased expression of FOXR2. CONCLUSION FOXR2 is sufficient to enhance gliomagenesis and represents a previously understudied therapeutic target for patients with the devastating disease DIPG.

DDRE-07. TARGETING TUMOR HYPOXIA AND MITOCHONDRIAL METABOLISM WITH ANTI-PARASITIC DRUGS AS AN APPROACH TO IMPROVE THE RADIOSENSITIVITY OF DIFFUSE INTRINSIC PONTINE GLIOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi75-vi75
Author(s):  
Faiqa Mudassar ◽  
Cecilia Chang ◽  
Prunella Ing ◽  
Kristina Cook ◽  
Geraldine O'Neill ◽  
...  
Keyword(s):  
Hypoxia Inducible Factor ◽  
Tumor Hypoxia ◽  
Pediatric Brain Tumor ◽  
Diffuse Intrinsic Pontine Glioma ◽  
In Vivo Studies ◽  
Pontine Glioma ◽  
Antiproliferative Effects ◽  
Extracellular Flux

Abstract Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brain tumor with a median survival of 12 months. Current management is limited to radiotherapy; however, the tumor recurs secondary to radioresistance. Tumor hypoxia appears to be one of the major contributors to radioresistance of DIPG, as oxygenation is critical to successful radiotherapy treatment. Therefore, strategies to alleviate hypoxia could enhance the effectiveness of radiotherapy and result in improved survival outcomes of patients with DIPG. Recent approaches to target tumor hypoxia are predicated on inhibiting mitochondrial respiration of the tumors to decrease oxygen consumption rate (OCR) and increase oxygenation. Here, we aimed to identify a safe but potent mitochondrial inhibitor that could decrease OCR and hypoxia, and improve the radiosensitivity of DIPG. A subset of anti-parasitic drugs (atovaquone, ivermectin, quinacrine, mefloquine and proguanil) which are known mitochondrial inhibitors were studied against a panel of patient-derived DIPG cell lines. We assessed their antiproliferative effects, OCR inhibition and radiosensitising efficacy using cell proliferation, extracellular flux and colony formation assays. Among the five tested drug candidates, atovaquone was found to be the most potent OCR inhibitor with minimal antiproliferative effects on DIPG cultures. It also decreased hypoxia in 3-dimensional DIPG neurospheres, reduced the expression of hypoxia-inducible factor-1α and improved the radiosensitivity of neurospheres of DIPG. Its anti-mitochondrial role was further confirmed by inhibition of various mitochondrial parameters and increase in reactive oxygen species. Overall, these results provide promising in vitro evidence of atovaquone as a hypoxia modifier and radiosensitiser in DIPG and pave a way for rapid translation to in vivo studies.

scholarly journals Modeling Traumatic Brain Injury in Human Cerebral Organoids

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2683
Author(s):  
Santiago Ramirez ◽  
Abhisek Mukherjee ◽  
Sofia Sepulveda ◽  
Andrea Becerra-Calixto ◽  
Nicolas Bravo-Vasquez ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Brain Injury ◽  
Metabolic Response ◽  
Neuronal Damage ◽  
Neuron Specific Enolase ◽  
Brain Regions ◽  
Normal Brain ◽  
In Vivo Models

Traumatic brain injury (TBI) is a head injury that disrupts the normal brain structure and function. TBI has been extensively studied using various in vitro and in vivo models. Most of the studies have been done with rodent models, which may respond differently to TBI than human nerve cells. Taking advantage of the recent development of cerebral organoids (COs) derived from human induced pluripotent stem cells (iPSCs), which resemble the architecture of specific human brain regions, here, we adapted the controlled cortical impact (CCI) model to induce TBI in human COs as a novel in vitro platform. To adapt the CCI procedure into COs, we have developed a phantom brain matrix, matching the mechanical characteristics of the brain, altogether with an empty mouse skull as a platform to allow the use of the stereotactic CCI equipment on COs. After the CCI procedure, COs were histologically prepared to evaluate neurons and astrocyte populations using the microtubule-associated protein 2 (MAP2) and the glial fibrillary acidic protein (GFAP). Moreover, a marker of metabolic response, the neuron-specific enolase (NSE), and cellular death using cleaved caspase 3 were also analyzed. Our results show that human COs recapitulate the primary pathological changes of TBI, including metabolic alterations related to neuronal damage, neuronal loss, and astrogliosis. This novel approach using human COs to model TBI in vitro holds great potential and opens new alternatives for understanding brain abnormalities produced by TBI, and for the development and testing of new therapeutic approaches.

Maize transcription factor ZmBES1/BZR1-5 positively regulates kernel size

2020 ◽  
Author(s):  
Fuai Sun ◽  
Lei Ding ◽  
Wenqi Feng ◽  
Yang Cao ◽  
Fengzhong Lu ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Kernel Weight ◽  
Sequencing Analysis ◽  
Kernel Size ◽  
Kernel Development ◽  
Chip Sequencing ◽  
Transposon Insertion ◽  
Expression Of Genes

Abstract The BES1/BZR1 transcription factors regulate the expression of genes responsive to brassinosteroids and play pivotal roles in plant development, but their role in regulating kernel development in maize remains unclear. In this study, we found that ZmBES1/BZR1-5 positively regulates kernel size. Association analysis of candidate genes in 513 diverse maize inbred lines indicated that three SNPs related to ZmBES1/BZR1-5 were significantly associated with kernel width and whilst four SNPs were related to 100-kernel weight. Overexpression of ZmBES1/BZR1-5 in Arabidopsis and rice both significantly increased seed size and weight, and smaller kernels were produced in maize Mu transposon insertion and EMS mutants. The ZmBES1/BZR1-5 protein locates in the nucleus, contains bHLH and BAM domains, and shows no transcriptional activity as a monomer but forms a homodimer through the BAM domain. ChIP-sequencing analysis, and yeast one-hybrid and dual-luciferase assays demonstrated that the protein binds to the promoters of AP2/EREBP genes (Zm00001d010676 and Zm00001d032077) and inhibits their transcription. cDNA library screening showed that ZmBES1/BZR1-5 interacts with casein kinase II subunit β4 (ZmCKIIβ4) and ferredoxin 2 (ZmFdx2) in vitro and in vivo, respectively. Taken together, our study suggests that ZmBES1/BZR1-5 positively regulates kernel size, and provides new insights into understanding the mechanisms of kernel development in maize.

scholarly journals DIPG-10. OPTIMAL HDAC INHIBITION IN DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii288-iii289
Author(s):  
Nicholas Vitanza ◽  
Matt Biery ◽  
Carrie Myers ◽  
Eric Ferguson ◽  
Giulia Park ◽  
...  
Keyword(s):  
Cell Viability ◽  
Hdac Inhibitors ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Model Systems ◽  
Hdac Inhibition ◽  
Pontine Glioma ◽  
In Vivo Models ◽  
Drug Concentrations

Abstract As the majority of diffuse intrinsic pontine glioma (DIPG) have H3K27M mutations, epigenetic-targeting agents have been studied, though evaluations have been limited by their model systems, untranslatable drug concentrations, and/or evasive mechanisms of action. To develop a more translational model, we used biopsy samples from newly diagnosed DIPG patients to create treatment-naïve in vitro and in vivo models (molecular aberrations in parentheses), including PBT-09FH (H3FA3, PI3KCA), PBT-22FH (H3F3A, TP53), PBT-24FH (PMS2), and PBT-27FH (HIST1H3B, TP53, NTRK2). Models demonstrated radiation-resistance similar to the patient from whom the culture was generated, supporting the models’ relevance (e.g. cell viability after 8 Gy was 36%, 81%, 71%, and 61% in PBT-09FH, -22FH, -24FH, and -27FH, respectively, compared to 7% in the medulloblastoma model MED-411FH). We evaluated cell viability and apoptosis following treatment with a panel of HDAC inhibitors, identifying the low nanomolar IC50 of quisinostat (~50 nM) and romidepsin (~5 nM). While RNA expression changes induced by 100 nM panobinostat and quisinostat included shared overexpression of the top 20/25 genes (e.g. FSTL5, ITIH5) and shared downregulation of the top 22/25 (e.g. GPR37L1, HEPACAM), only 9/25 were downregulated by panobinostat, quisinostat, and romidepsin (e.g. C21orf62, IFIT2), identifying these as potential vulnerabilities or biomarkers of lethal HDAC inhibition. Mass-spectrometry (LC-MS) demonstrated panobinostat as the greatest acetylator of cortactin, potentially related to thrombocytopenia. While PBT-09 flank models demonstrated quisinostat’s on-target acetylation and efficacy, orthotopic xenograft models did not, supporting our model’s intact blood-brain barrier and emphasizing the need for CNS penetrant versions of potentially efficacious agents.

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