OLIG2 maintenance is not essential for diffuse intrinsic pontine glioma cell line growth but regulates tumor phenotypes

Abstract Background Diffuse intrinsic pontine glioma (DIPG) is a pediatric lethal high-grade brainstem glioma with no effective therapies. OLIG2 was reported to be critical for the growth of a DIPG cell line CCHMC-DIPG-1. Surprisingly, we found that the CCHMC-DIPG-1 cells express little OLIG2 and exhibit a mesenchymal phenotype, which raised a question regarding the role of OLIG2 in the growth of DIPG cells. Methods We evaluated the function of OLIG2 in different DIPG cell lines through molecular and genetic approaches and performed transcriptomic and genomic landscape profiling including whole-genome bisulfite-sequencing, RNA-seq, ATAC-seq, and ChIP-seq. shRNA-mediated knockdown and CRISPR-Cas9-mediated knockout approaches were utilized to assess OLIG2 functions in DIPG cell growth. Results We found that DIPG cells are phenotypically heterogeneous and exhibit the characteristics of distinct malignant gliomas including proneural, classical, and mesenchymal subtypes. OLIG2 knockdown did not impact the growth of CCHMC-DIPG-1 cells, wherein OLIG2 is epigenetically silenced. Moreover, OLIG2 deletion did not substantially impair OLIG2-expressing proneural-like DIPG growth but led to an upregulation of HIPPO-YAP1 and EGFR signaling and a tumor phenotype shift. Targeting HIPPO-YAP1 and EGFR signaling in OLIG2-deficient DIPG cells inhibited tumor cell growth. Conclusions Our data indicate that OLIG2 is dispensable for DIPG growth but regulates the phenotypic switch of DIPG tumor cells. OLIG2 downregulation leads to deregulation of adaptive YAP1 and EGFR signaling. Targeting YAP1 and EGFR pathways inhibits the growth of OLIG2-deficient DIPG cells, pointing to a therapeutic potential by targeting adaptive signaling to treat DIPG tumors with nominal OLIG2 expression.

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Characterization of a diffuse intrinsic pontine glioma cell line: implications for future investigations and treatment

Journal of Neuro-Oncology ◽

10.1007/s11060-012-0973-6 ◽

2012 ◽

Vol 110 (3) ◽

pp. 305-313 ◽

Cited By ~ 43

Author(s):

Rintaro Hashizume ◽

Ivan Smirnov ◽

Sharon Liu ◽

Joanna J. Phillips ◽

Jeanette Hyer ◽

...

Keyword(s):

Cell Line ◽

Glioma Cell ◽

Diffuse Intrinsic Pontine Glioma ◽

Glioma Cell Line ◽

Pontine Glioma

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Immunobiology of primary intracranial tumors

Journal of Neurosurgery ◽

10.3171/jns.1983.59.2.0208 ◽

1983 ◽

Vol 59 (2) ◽

pp. 208-216 ◽

Cited By ~ 19

Author(s):

M. Stephen Mahaley ◽

G. Yancey Gillespie ◽

Ritchie P. Gillespie ◽

Pamela J. Watkins ◽

Darell D. Bigner ◽

...

Keyword(s):

Tissue Culture ◽

Cell Line ◽

Glioma Cell ◽

Malignant Gliomas ◽

Osteogenic Sarcoma ◽

Culture Cell ◽

Glioma Cell Line ◽

Tissue Culture Cell ◽

Serological Studies ◽

Line Following

✓ Serial serological studies were carried out on 19 of 20 patients with malignant gliomas who were actively immunized with one of two human glioma tissue culture cell lines (D-54MG or U-251MG). Most patients mounted a significant serum reaction to histocompatibility antigens (HLA's), as well as an antibody response to fetal bovine serum (FBS) which was added to the glioma-cell inoculum. These two sources of antibody accounted for greater than 90% of the antibody induced by these inoculations. Two patients continued to have significant amounts of binding antibody to the original immunizing cell line following exhaustive absorptions of FBS and HLA antibodies. One of these two had all remaining significant antibody removed by further absorption of the serum against the 2-T osteogenic sarcoma tissue culture cell line known to possess antigens cross-reactive with human gliomas. One single patient continued to show significant antibody binding to the original glioma cell line following absorption against FBS, human platelets, and the 2-T cell line, and therefore seems to have produced glioma-distinctive antibodies in response to immunization. The antibody preparation from this patient was also cytotoxic against the original glioma cell line, as well as another recently cultured human glioblastoma cell line. The significance of these serological studies is discussed as it relates to immunological responses patients with gliomas may make to active immunization.

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DIPG-50. CHROMATIN IMMUNOPRECIPITATION OF DIFFUSE INTRINSIC PONTINE GLIOMA TUMOR TISSUE IS FEASIBLE AND SHOW DIFFERENT ENRICHMENT COMPARED TO PRIMARY CELL LINE

Neuro-Oncology ◽

10.1093/neuonc/noy059.143 ◽

2018 ◽

Vol 20 (suppl_2) ◽

pp. i59-i59

Author(s):

Yong Yean Kim ◽

Jaclyn Andricovich ◽

Sridevi Yadavilli ◽

Madhuri Kambhampati ◽

Alexandros Tzatsos ◽

...

Keyword(s):

Cell Line ◽

Chromatin Immunoprecipitation ◽

Tumor Tissue ◽

Diffuse Intrinsic Pontine Glioma ◽

Primary Cell ◽

Pontine Glioma ◽

Primary Cell Line ◽

Glioma Tumor

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Therapeutic targeting of transcriptional elongation in diffuse intrinsic pontine glioma

Neuro-Oncology ◽

10.1093/neuonc/noab009 ◽

2021 ◽

Author(s):

Hiroaki Katagi ◽

Nozomu Takata ◽

Yuki Aoi ◽

Yongzhan Zhang ◽

Emily J Rendleman ◽

...

Keyword(s):

Cell Growth ◽

Rna Polymerase Ii ◽

Therapeutic Strategy ◽

Diffuse Intrinsic Pontine Glioma ◽

Transcriptional Elongation ◽

Pontine Glioma ◽

Elongation Complex ◽

Pol Ii ◽

Chip Sequencing ◽

Animal Survival

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

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Molecular Mechanism of EGFR Signaling Pathway Mediating Proliferation and Migration of U251 Glioma Cell Line

International Journal of Sciences ◽

10.18483/ijsci.1148 ◽

2016 ◽

Vol 2 (11) ◽

pp. 61-66

Author(s):

Linlin YUE ◽

Ling LI ◽

Bin WANG ◽

Xiangmin YU ◽

Lingling DING ◽

...

Keyword(s):

Cell Line ◽

Signaling Pathway ◽

Molecular Mechanism ◽

Glioma Cell ◽

Glioma Cell Line ◽

Egfr Signaling ◽

Egfr Signaling Pathway ◽

And Migration ◽

Proliferation And Migration

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Preclinical Validation of Ubiquitin Receptor PSMD4 As Therapeutic Target in Multiple Myeloma

Blood ◽

10.1182/blood-2019-122952 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4403-4403

Author(s):

Yan Song ◽

Ting DU ◽

Arghya Ray ◽

Dharminder Chauhan ◽

Kenneth C. Anderson

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Cell Line ◽

Therapeutic Potential ◽

Statistical Significance ◽

Annexin V ◽

Endoplasmic Reticulum Stress Response ◽

In Vivo Models

Background and Rationale Our preclinical studies have focused on Identification and validation of components within the ubiquitin proteasome system which can be targeted with novel therapies to overcome proteasome-inhibitor (PI)-resistance in multiple myeloma (MM). Our siRNA screening studies identified ubiquitin Receptor (UbR) PSMD4/Rpn10 as a mediator of MM cell growth and survival. Rpn10 is localized on the 19S regulatory lid of the proteasome, and plays a key role in chaperoning ubiquitinated substrate proteins for downstream 20S proteasomal degradation. Here, we show that inhibition of Rpn10 triggers potent anti-MM activity using both in in vitro and in vivo models of MM, including against PI-resistant MM cells. Materials and Methods Rpn10 knockout 293 cell line was generated using CRISPR/Cas9. Rpn10-inducible knockdown (KD) MM.1S cell line was generated using shRNA. Drug sensitivity, cell viability, and apoptosis assays were performed using WST/CellTiter-Glo assay, and Annexin V staining, respectively. Cell signaling and caspase activity were determined by western blotting. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2012, 22:345-358). A xenograft human MM model was used to characterize the role of Rpn10 on tumor progression. Statistical significance was assessed with Student's t test. Results 1) Analysis of MM patient gene expression profiling database showed that Rpn10 expression inversely correlates with overall survival (n=175; p= 0.00064). 2)Real-time PCR, immunoblotting, and immunohistochemistry of MM patient bone marrow showed higher Rpn10 levels in patient MM cells and MM cell lines versus normal cells. 3)Transient transfection of MM cells with Rpn10-siRNA decreased their viability; conversely, transfection with Rpn10-WT specifically rescued cells from growth-inhibitory activity of Rpn10-siRNA. Immunoblot analysis confirmed significant knockdown of Rpn10 by Rpn10-siRNA versus scrambled (scr)-siRNA, and restoration of Rpn10 levels in cells transfected with Rpn10-WT versus Rpn10-siRNA. 4)Genetic blockade of Rpn10 decreased viability in bortezomib-resistant MM cells. 5)CRISPR/Cas9-mediated knockout (KO) of Rpn10 decreased cell growth. 5)Both Rpn10-KO and inducible Rpn10-KD cells showed elevated levels of polyubiquitylated proteins. 6)Inhibition of Rpn10 induced apoptosis, cell-cycle arrest, activation of caspases, and endoplasmic reticulum stress response signaling. 7)Reduced tumor growth was noted in mice xenografted with Rpn10-KD MM.1S cells versus mice engrafted with WT-MM.1S cells. Conclusion Our data demonstrate the therapeutic potential of targeting ubiquitin receptor Rpn10/PSMD4, and provide the preclinical basis for development of Rpn10 inhibitors to overcome PI-resistance in MM. Disclosures Chauhan: C4 Therapeutics.: Equity Ownership; Stemline Therapeutics: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

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