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

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.

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Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

10.1101/2021.05.10.443452 ◽

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.

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Diffuse intrinsic pontine glioma: current insights and future directions

Chinese Neurosurgical Journal ◽

10.1186/s41016-020-00218-w ◽

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.

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H3.3K27M Mutation Promotes The Migration and Invasion of Glioma Cells By Activating β-Catenin/USP1 Signaling

10.21203/rs.3.rs-1215261/v1 ◽

2022 ◽

Author(s):

Zhiyuan Sun ◽

Yufu Zhu ◽

Xia Feng ◽

Xiaoyun Liu ◽

Kunlin Zhou ◽

...

Keyword(s):

Glioma Cells ◽

High Grade Glioma ◽

Diffuse Intrinsic Pontine Glioma ◽

Migration And Invasion ◽

Mrna Levels ◽

High Grade ◽

Protein Levels ◽

Adult Glioma

Abstract H3.3K27M is a newly identified molecular pathology marker in glioma and is especially correlated with the malignancy of diffuse intrinsic pontine glioma (DIPG). In recent years, accumulating research has revealed that other types of glioma also contain the H3.3K27M mutation. However, the role of H3.3K27M in high-grade adult glioma, which is the most malignant glioma, has not been investigated. In this study, we focused on exploring the expression and function of H3.3K27M in high-grade adult glioma patients. We found that H3.3K27M is partly highly expressed in high-grade glioma tissues. Then, we introduced H3.3K27M into H3.3 wild-type glioma cells, U87 cells and LN229 cells. We found that H3.3K27M did not regulate the growth of glioma in vitro and in vivo; however, the survival of mice with transplanted tumors was significantly reduced. Further investigation revealed that H3.3K27M expression mainly promoted the migration and invasion of glioma cells. Moreover, we certified that H3.3K27M overexpression enhanced the protein levels of ꞵ-catenin and p-ꞵ-catenin, the protein and mRNA levels of ubiquitin-specific protease 1 (USP1), and the protein level of enhancer of zeste homolog 2 (EZH2). Importantly, the ꞵ-catenin inhibitor XAV-939 significantly attenuated the upregulation of the aforementioned proteins. Overall, the H3.3K27M mutation is present in a certain proportion of high-grade glioma patients and facilitates a poor prognosis by promoting the metastasis of glioma by regulating the ꞵ-catenin/USP1/EZH2 pathway.

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

Neuro-Oncology ◽

10.1093/neuonc/noab196.861 ◽

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.

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In Vitro and In Vivo Anti-Myeloma Activity of PRLX, an Orally-Bioavailable Agent Against Cells with Constitutive Activation of Ras or Its Downstream Pathway.

Blood ◽

10.1182/blood.v110.11.540.540 ◽

2007 ◽

Vol 110 (11) ◽

pp. 540-540

Author(s):

Steffen Klippel ◽

Jana Jakubikova ◽

Lauren Jenkins ◽

Joseph M. Negri ◽

Yevgenia Tesmenitsky ◽

...

Keyword(s):

Cell Death ◽

Cell Lines ◽

Target Cells ◽

Pharmacokinetic Studies ◽

In Vivo Models ◽

Ras Mutations ◽

Chemical Screening ◽

Orally Bioavailable

Abstract Ras mutations occur in 40–60% of multiple myeloma (MM) patients and are implicated in progression to advanced MM (including plasma cell leukemia/extramedullary lesions). The small molecule PRLX (Prolexys Pharmaceuticals) was identified in the context of “synthetic lethal” chemical screening for genotype-selective cytotoxicity against cells transformed with forced expression of mutant Ras (but not against isogenic normal cell counterparts) and was tested for anti-MM activity. In MTT survival assays, 34 of 46 human MM cell lines (74%) responded to 48hr treatment with sub-μM PRLX concentrations (achievable in preclinical pharmacokinetic studies). (24 MM lines had IC50 values <300nM). PRLX anti-MM activity compared favorably with its in vitro activity against cell lines from leukemias, lymphomas, and solid tumors. PRLX activity was not restricted to cells with known Ras mutations: it was also observed in cells with FGF-R3 overexpression and/or activating mutation, suggesting that PRLX may target cells with hyper-active signaling through either upstream receptors, Ras or its downstream effectors, rather than targeting Ras biochemical activity itself. PRLX was active against MM cells resistant to conventional (Dex, alkylators, anthracyclines) and/or novel (lenalidomide, CC-4047, bortezomib, multitargeted kinase inhibitors) anti-MM agents. No antagonism was observed when PRLX was combined in vitro with other anti-MM agents, including Dex, bortezomib or CC-4047. Co-culture with BMSCs did not protect MM cells against PRLX (at doses non-toxic to BMSCs). Cell death commitment assays revealed that a pharmacologically relevant 5hr pulse with 300nM PRLX is sufficient to commit OPM-2 MM cells to cell death. Gene expression profiles (with Affymetrix U133 2.0plus oligonucleotide microarrays) showed early (<2hr) PRLX-induced modulation of broad spectrum of genes involved in regulation of cellular bioenergetics. FACS analyses of PRLX-treated cells revealed decreased mitochondrial membrane potential followed by apoptotic and then necrotic features of cell death. Reducing agents, such as tocopherol, can protect MM cells against PRLX, suggesting that release of reactive oxidative species may be an ultimate downstream mediator of the mechanism of action of this agent. The in vivo anti-MM activity of PRLX was evaluated in 2 separate in vivo models of diffuse MM lesions in SCID-beige mice, generated by i.v.injections of OPM-2 and MM.1S cells, respectively. In both models, mice were sublethally irradiated, injected i.v. with 1x106 MM cells and then randomly assigned to receive by oral gavage either PRLX 100 mg/kg (n=14) or vehicle only (n=14) on cyclical schedule of 5 days-on/2 days-off treatment. In both models, >90% of PRLX-treated mice were alive at 140 and 64 days from treatment onset, in the OPM-2 and MM.1S model respectively. In contrast, median overall survival was 35 days (95% CI: 23–47) and 35 days (95%C.I.: 31–39) in the respective control groups (Kaplan-Meier analyses, p<0.0001 in both models, by log-rank test). PRLX represents a promising novel orally bioavailable agent that merits further evaluation for possible clinical trials in advanced MM.

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HGG-12. HUMAN IPSC-DERIVED H3.3K27M NEUROSPHERES: A NOVEL MODEL FOR INVESTIGATING DIPG PATHOGENESIS AND DRUG RESPONSE

Neuro-Oncology ◽

10.1093/neuonc/noab090.078 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i19-i20

Author(s):

Kasey Skinner ◽

Tomoyuki Koga ◽

Shunichiro Miki ◽

Robert F Gruener ◽

R Stephanie Huang ◽

...

Keyword(s):

Principal Component ◽

High Grade Glioma ◽

Diffuse Intrinsic Pontine Glioma ◽

Genetically Engineered ◽

High Grade ◽

Primary Tumors ◽

Secondary Tumor ◽

Immunodeficient Mice

Abstract Diffuse intrinsic pontine glioma (DIPG) is a subset of high-grade glioma that occurs predominantly in children and has no cure. Up to 80% of DIPG harbor a heterozygous point mutation that results in a lysine 27 to methionine substitution in histone variant H3.3 (H3.3K27M). Existing DIPG models have provided insight into the role of H3.3K27M but have limitations: genetically engineered murine models often rely on overexpression of the mutant histone to form tumors; patient-derived xenografts (PDX) are more genetically faithful but preclude examination of the effect of individual mutations on pathogenesis. To address these shortcomings and better recapitulate the genetics of human tumors, we designed a novel DIPG model based on human induced pluripotent stem cells (iPSC) edited via CRISPR to express heterozygous H3.3K27M. Edited iPSC were chemically differentiated into neural progenitor cells, which upon implantation into the brainstems of immunodeficient mice formed diffusely invasive tumors that were histologically consistent with high-grade glioma. Further, neurospheres cultured from primary tumors formed secondary tumors upon reimplantation with more diffuse invasion, suggesting in vivo evolution. To validate this model’s relevance to DIPG transcriptionally, we performed RNA-sequencing on a cohort of primary and secondary tumor neurospheres (termed primary and secondary iDIPG) and compared them to published RNA-seq data from pediatric PDX and patient tumor samples. Hierarchical clustering and principal component analysis on differentially expressed genes (P<0.05) showed that H3.3K27M iDIPG cluster with H3.3K27M PDX and patient tumors. Further, ssGSEA showed that H3.3K27M iDIPG are enriched for astrocytic and mesenchymal signature genes, a defining feature of H3.3K27M DIPG. Finally, we found that primary H3.3K27M iDIPG neurospheres are sensitive to panobinostat, an HDAC inhibitor shown to be effective against H3.3K27M DIPG cells in vitro. Overall, these data suggest that H3.3K27M iDIPG are a promising tool for investigating DIPG biology and new therapeutic strategies.

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The preventive effect of Mangifera foetida L. leaf extract administered simultaneously to excess iron on markers of iron overload in Spraque-Dawley rats

Medical Journal of Indonesia ◽

10.13181/mji.v26i4.1829 ◽

2018 ◽

Vol 26 (4) ◽

pp. 246-52

Author(s):

Purnama Fajri ◽

Ari Estuningtyas ◽

Melva Louisa ◽

Hans-Joachim Freisleben

Keyword(s):

Iron Overload ◽

Leaf Extract ◽

Blood Levels ◽

Sprague Dawley ◽

Leaf Extracts ◽

In Vivo Models ◽

Excess Iron ◽

Before And After

Background: Recently, there is no agent available for the prevention of iron overload (IO) in thalassemia patients. Previous studies showed that Mangifera foetida L. leaf extracts reduced the levels of iron in IO in vitro and in vivo models. The present study aimed to determine the efficacy of Mangifera foetida L. leaf extract in the prevention of IO induced in rats.Methods: Thirty male Sprague-Dawley rats were divided into 5 groups: control (untreated), IO, 3 treatment groups with leaf extract equivalent to 50, 100, and 200 mg of mangiferin per kg BW. Fe-dextran (15 mg) was administered intraperitoneally twice a week for 4 weeks to all groups except control (IO, DSM50, DSM100, and DSM200). Urine and blood samples were taken before and after treatments. After 4 weeks of treatment, rats were terminated, and samples of spleen, liver, and heart were taken. Ferritin and mangiferin levels and SOD activities were determined in plasma. Iron levels were measured in plasma, urine, and spleen.Results: Experimental IO increased plasma Fe content 4.23 times and plasma ferritin levels 6.9 times vs normal. Mangifera foetida L. leaf extract DSM50 resulted in the highest blood levels of 212 ng mangiferin per mL and moderately, although not significant, prevented increased plasma ferritin levels and IO in organs and protected against oxidative stress.Conclusion: Aqueous Mangifera foetida L. leaf extract may be useful to prevent IO and oxidative stress in thalassemia patients.

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

Neuro-Oncology ◽

10.1093/neuonc/noaa222.060 ◽

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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Everolimus improves the efficacy of dasatinib in PDGFRα-driven glioma

10.1101/2019.12.28.19015974 ◽

2020 ◽

Author(s):

Zachary Miklja ◽

Viveka Nand Yadav ◽

Rodrigo T. Cartaxo ◽

Ruby Siada ◽

Chase C. Thomas ◽

...

Keyword(s):

Prolonged Exposure ◽

Pediatric Brain Tumor ◽

High Grade Glioma ◽

Pharmacokinetic Studies ◽

Exploratory Research ◽

Research Award ◽

Tumor Accumulation ◽

Dasatinib Treatment

AbstractBackgroundPediatric and adult high-grade glioma (HGG) frequently harbor PDGFRA alterations. We hypothesized that co-treatment with everolimus may improve the efficacy of dasatinib in PDGFRα-driven glioma through combinatorial synergism and increased tumor accumulation of dasatinib.MethodsDose response, synergism studies, P-gp inhibition and pharmacokinetic studies were performed on in vitro and in vivo human and mouse models of HGG. Six patients with recurrent PDGFRα-driven glioma were treated with dasatinib and everolimus.ResultsDasatinib effectively inhibited the proliferation of mouse and human primary HGG cells with a variety of PDGFRA alterations. Dasatinib exhibited synergy with everolimus in the treatment of HGG cells at low nanomolar concentrations of both agents, with reduction in mTOR signaling that persists after dasatinib treatment alone. Prolonged exposure to everolimus significantly improved the CNS retention of dasatinib and extended survival of PPK tumor bearing mice. Pediatric patients (n=6) with glioma tolerated this combination without significant adverse events. Recurrent patients (n=4) demonstrated median overall survival of 8.5 months.ConclusionEfficacy of dasatinib treatment of PDGFRα-driven HGG is improved with everolimus and suggests a promising route for improving targeted therapy for this patient population.Trial RegistrationClinicalTrials.gov NCT03352427FundingThe authors thank the patients and their families for participation in this study. CK is supported by NIH/NINDS K08-NS099427-01, the University of Michigan Chad Carr Pediatric Brain Tumor Center, the Chad Tough Foundation, Hyundai Hope on Wheels, Catching up With Jack, Prayers from Maria Foundation, U CAN-CER VIVE FOUNDATION, Morgan Behen Golf Classic, and the DIPG Collaborative. The PEDS-MIONCOSEQ study was supported by grant 1UM1HG006508 from the National Institutes of Health Clinical Sequencing Exploratory Research Award (PI: Arul Chinnaiyan).

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