Disrupting NOTCH Slows Diffuse Intrinsic Pontine Glioma Growth, Enhances Radiation Sensitivity, and Shows Combinatorial Efficacy With Bromodomain Inhibition

Abstract

NOTCH regulates stem cells during normal development and stem-like cells in cancer but the roles of NOTCH in the lethal pediatric brain tumor diffuse intrinsic pontine glioma (DIPG) remain unknown. Because DIPGs express stem cell factors such as SOX2 and MYCN, we hypothesized that NOTCH activity would be critical for DIPG growth. We determined that primary DIPGs expressed high levels of NOTCH receptors, ligands, and downstream effectors. Treatment of the DIPG cell lines JHH-DIPG1 and SF7761 with the γ-secretase inhibitor MRK003 suppressed the level of the NOTCH effectors HES1, HES4, HES5, inhibited DIPG growth by 75%, and caused a 3-fold induction of apoptosis. Short hairpin RNAs targeting the canonical NOTCH pathway caused similar effects. Pre-treatment of DIPG cells with MRK003 suppressed clonogenic growth by more than 90% and enhanced the efficacy of radiation therapy. The high level of MYCN in DIPG led us to test sequential therapy with the bromodomain inhibitor JQ1 and MRK003, and we found that JQ1 and MRK003 inhibited DIPG growth and induced apoptosis. Together, these results suggest that dual targeting of NOTCH and MYCN in DIPG may be an effective therapeutic strategy in DIPG and that adding a γ-secretase inhibitor during radiation therapy may be efficacious initially or during re-irradiation.

Transcriptional Dependencies in Diffuse Intrinsic Pontine Glioma

“…This may represent a key list for developing targeted therapy in DIPG. Consistent with this notion, we observed enrichment for a number of pathways previously described as dysregulated and important to DIPG biology, including NOTCH (Taylor et al 2015), Hedgehog (Monje et al 2011), and PDGF (Paugh et al 2013). These signaling pathways enriched for common downstream kinases ( MAPK1 , MAPK3K1 , MAP3K2 , MAPK8 , MAPK11 , MAPAPK2 , PRKCA , PRKCD , PRKCE , PRKCZ ).…”

“…As previously shown, transcriptional inhibition mediated by JQ1 bromodomain inhibition impedes DIPG cell viability in vitro (Taylor et al 2015). We expand upon this finding by examining a range of bromodomain inhibitors and by demonstrating BRD4 as the key target using shRNA-mediated BRD4 knockdown.…”

“…SU-pcGBM2 is histone-3 WT and exhibits a TP53 mutation and EGFR amplification (Table S1; Venkatesh et al 2015). To confirm and extend the observation that BET inhibition reduces DIPG cell viability (Taylor et al 2015), we treated these patient-derived cell cultures with a range of concentrations of JQ1 and observed a dose-dependent reduction in DIPG cell viability across all cell cultures, particularly at later time points (72-hour IC 50 >1 mM in most cultures, 6-day IC 50 median: 0.35 μM, range: 0.076 – 2.06 mM; Figure 1A and S1B). Interestingly, while the H3WT culture VUMC-DIPG-10 responded to JQ1 treatment similarly to other DIPG cultures, the H3WT pediatric glioblastoma cell culture SU-pcGBM2 showed minimal vulnerability to JQ1 treatment (Figure 1A).…”

The dual mTOR kinase inhibitor TAK228 inhibits tumorigenicity and enhances radiosensitization in diffuse intrinsic pontine glioma

“…JHH-DIPG1, SF7761, and SU-DIPG-XIII DIPG neurosphere lines were maintained in DMEM/F12 medium supplemented with 20 ng/ml epidermal growth factors (EGF) and 20 ng/ml fibroblast growth factors (FGF) (EF media) [26]. To avoid differentiation of SU-DIPG-XIII cells, EF media without retinoic acid was used [17].…”

“…To avoid differentiation of SU-DIPG-XIII cells, EF media without retinoic acid was used [17]. The JHH-DIPG1 line was derived from a rapid autopsy specimen and established in our laboratory as previously described [26]. The SF7761 line is a kind gift from Rintaro Hashizume and Nalin Gupta (University of California, San Francisco, CA) [27], and the SU-DIPG-XIII line is a kind gift of Michelle Monje (Stanford University School of Medicine, Stanford, CA) [28].…”