RBPJ maintains brain tumor–initiating cells through CDK9-mediated transcriptional elongation
Qi Xie, … , Shideng Bao, Jeremy N. Rich
Qi Xie, … , Shideng Bao, Jeremy N. Rich
Published July 1, 2016; First published June 20, 2016
Citation Information: J Clin Invest. 2016;126(7):2757-2772. https://doi.org/10.1172/JCI86114.
View: Text | PDF
Categories: Research Article Oncology Stem cells

RBPJ maintains brain tumor–initiating cells through CDK9-mediated transcriptional elongation

Abstract

Glioblastomas co-opt stem cell regulatory pathways to maintain brain tumor–initiating cells (BTICs), also known as cancer stem cells. NOTCH signaling has been a molecular target in BTICs, but NOTCH antagonists have demonstrated limited efficacy in clinical trials. Recombining binding protein suppressor of hairless (RBPJ) is considered a central transcriptional mediator of NOTCH activity. Here, we report that pharmacologic NOTCH inhibitors were less effective than targeting RBPJ in suppressing tumor growth. While NOTCH inhibitors decreased canonical NOTCH gene expression, RBPJ regulated a distinct profile of genes critical to BTIC stemness and cell cycle progression. RBPJ was preferentially expressed by BTICs and required for BTIC self-renewal and tumor growth. MYC, a key BTIC regulator, bound the RBPJ promoter and treatment with a bromodomain and extraterminal domain (BET) family bromodomain inhibitor decreased MYC and RBPJ expression. Proteomic studies demonstrated that RBPJ binds CDK9, a component of positive transcription elongation factor b (P-TEFb), to target gene promoters, enhancing transcriptional elongation. Collectively, RBPJ links MYC and transcriptional control through CDK9, providing potential nodes of fragility for therapeutic intervention, potentially distinct from NOTCH.

Authors

Qi Xie, Qiulian Wu, Leo Kim, Tyler E. Miller, Brian B. Liau, Stephen C. Mack, Kailin Yang, Daniel C. Factor, Xiaoguang Fang, Zhi Huang, Wenchao Zhou, Kareem Alazem, Xiuxing Wang, Bradley E. Bernstein, Shideng Bao, Jeremy N. Rich

×

Figure 1

NOTCH inhibition does not attenuate cell autonomous BTIC growth.

Options: View larger image (or click on image) Download as PowerPoint
NOTCH inhibition does not attenuate cell autonomous BTIC growth. (A) Lef...
(A) Left: Matched sets of BTICs and non-BTICs (387, 3691, and 4121) were transfected with a 4× RBPJ luciferase reporter together with a tk-renilla reporter. Data are displayed as mean ± SEM for the ratio of firefly-to-renilla luciferase (t test, **P < 0.01, n = 3). Right: 3691 and 4121 BTICs were transfected with a 4× RBPJ luciferase reporter together with a tk-renilla reporter, and then treated with the NOTCH antagonist, DAPT (5 μM), or vehicle control (DMSO). Data are displayed as mean ± SEM for the ratio of firefly-to-renilla luciferase (Student’s t test, **P < 0.01, n = 3). (B) Effects of DAPT or vehicle control (DMSO) on cell proliferation were tested in two BTIC models (3691 and 4121). Data are displayed as the mean values for each time point. (C) Cleaved NOTCH1 (NOTCH intracellular domain [NICD]) levels were analyzed by immunoblot after treatment with vehicle control or DAPT in two BTIC models (3691 and 4121). (D) Effects of the stapled peptide NOTCH inhibitor, SAHM1, or vehicle control (DMSO) on cell proliferation were tested in two BTIC models (3691 and 4121). Data are displayed as the mean values for each time point. (E) Effects of SAHM1 treatment on downstream NOTCH target gene expression (HES1 and HES5) were tested in two BTIC models (3691 and 4121). Cells were treated with SAHM1 for two days. Total RNA was isolated and cDNA was synthesized by reverse transcription. The mRNA levels of indicated genes were detected by qPCR (t test, *P < 0.05, n = 3).