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Abstract Details

Cloning and overexpression of multi-domain enhancer protein p300, to study its involvement in childhood-diffuse intrinsic pontine glioma (DIPG) cell proliferation.
Neuro-oncology
P13 - Poster Session 13 (8:00 AM-9:00 AM)
4-003

DIPG accounts for 10% of all childhood central nervous system tumors, are highly aggressive and difficult to treat brain tumors at brainstem. ~ 80% of DIPG patients harbor mutations on lysine 27 of histone 3 which colocalizes with bromodomain proteins to sites of active transcription. p300 is a multi-domain enhancer protein which includes bromo domain (BrD) and PHD domain, is expressed in 80% of gliomas. Based on our findings, bromodomain inhibitors (BrDi) have been suggested as a potential therapeutic target for DIPG and have shown antiproliferative activity in vitro. We therefore hypothesize that p300 -BrD inhibition is a potential therapeutic strategy that must be urgently explored in DIPG.

Characterization of p300 (or EP300) and studying its role in childhood DIPG cell proliferation.
The patient-derived DIPG cell lines, SU-DIPG-IV (H3.1 mutation) and SF-8628 (H3.3 mutation) were cultured in regular media. The proliferation rate, viability and apoptosis were measured by SYTOX cell event assay. Western blot analysis was done using dose-dependent p300-BrDi to understand the BrDi-mediated mechanism of action. To perform the binding experiments with p300-domains, we have generated different domain-specific constructs.
Treatment of DIPG with dose-dependent p300-BrDi (PF-CBP1, CPI-637) demonstrated growth inhibition in patient-derived cell lines. We confirmed the decrease of p300 acetylation and upregulation of H3K27me3 (an oncogene silencing marker) by western blot in cultured DIPG-IV cells with PF-CBP1 treatment. For binding experiments with specific domains of p300, we have generated Flag-tagged full length p300, BrD-domain deletion construct and PHD- domain deletion construct for establishment of stable DIPG cell lines.
p300-BrDi decrease DIPG proliferation in a dose dependent manner. It also decrease p300 acetylation followed by H3K27me3 upregulation which signifies its therapeutic property. To further validate our hypothesis, molecular characterization and binding studies of p300-domains will be performed which will help to develop a novel therapeutic strategy against this deadly disease. 
Authors/Disclosures
Steven Summers
PRESENTER
Mr. Summers has nothing to disclose.
No disclosure on file