Qi Xie

Mitochondrial Control by DRP1 in Brain Tumor Initiating Cells

Brain tumor initiating cells (BTICs) co-opt the neuronal high affinity glucose transporter, GLUT3, to withstand metabolic stress. We investigated another mechanism critical to brain metabolism, mitochondrial morphology, in BTICs. BTIC mitochondria were fragmented relative to non-BTIC tumor cell mitochondria, suggesting that BTICs increase mitochondrial fission. The essential mediator of mitochondrial fission, dynamin-related protein 1 (DRP1), showed activating phosphorylation in BTICs and inhibitory phosphorylation in non-BTIC tumor cells. Targeting DRP1 using RNA interference or pharmacologic inhibition induced BTIC apoptosis and inhibited tumor growth. Downstream, DRP1 activity regulated the essential metabolic stress sensor, AMP-activated protein kinase (AMPK), and targeting AMPK rescued the effects of DRP1 disruption. Cyclin-dependent kinase 5 (CDK5) phosphorylated DRP1 to increase its activity in BTICs, whereas Ca2+-calmodulin-dependent protein kinase 2 (CAMK2) inhibited DRP1 in non-BTIC tumor cells, suggesting that tumor cell differentiation induces a regulatory switch in mitochondrial morphology. DRP1 activation correlated with poor prognosis in glioblastoma, suggesting that mitochondrial dynamics may represent a therapeutic target for BTICs.

Transcription elongation factors represent in vivo cancer dependencies in glioblastoma

Glioblastoma is a universally lethal cancer with a median survival time of approximately 15 months. Despite substantial efforts to define druggable targets, there are no therapeutic options that notably extend the lifespan of patients with glioblastoma. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology for use in orthotopic patient-derived xenograft models, creating a high-throughput negative-selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators needed for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause–release and elongation factors as one set of in vivo-specific cancer dependencies, and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause–release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, suggesting that targeting transcription elongation machinery may be an effective therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of ‘cancer dependencies’ not identified by previous in vitro approaches, and could supply new opportunities for therapeutic intervention.

Arsenic trioxide disrupts glioma stem cells via promoting PML degradation to inhibit tumor growth.

Glioblastoma multiforme (GBM) is the most lethal brain tumor. Tumor relapse in GBM is inevitable despite maximal therapeutic interventions. Glioma stem cells (GSCs) have been found to be critical players in therapeutic resistance and tumor recurrence. Therapeutic drugs targeting GSCs may significantly improve GBM treatment. In this study, we demonstrated that arsenic trioxide (As2O3) effectively disrupted GSCs and inhibited tumor growth in the GSC-derived orthotopic xenografts by targeting the promyelocytic leukaemia (PML). As2O3 treatment induced rapid degradation of PML protein along with severe apoptosis in GSCs. Disruption of the endogenous PML recapitulated the inhibitory effects of As2O3 treatment on GSCs both in vitro and in orthotopic tumors. Importantly, As2O3 treatment dramatically reduced GSC population in the intracranial GBM xenografts and increased the survival of mice bearing the tumors. In addition, As2O3 treatment preferentially inhibited cell growth of GSCs but not matched non-stem tumor cells (NSTCs). Furthermore, As2O3 treatment or PML disruption potently diminished c-Myc protein levels through increased poly-ubiquitination and proteasome degradation of c-Myc. Our study indicated a potential implication of As2O3 in GBM treatment and highlighted the important role of PML/c-Myc axis in the maintenance of GSCs.

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

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.

Deubiquitinase USP13 maintains glioblastoma stem cells by antagonizing FBXL14-mediated Myc ubiquitination

Glioblastoma is the most lethal brain tumor and harbors glioma stem cells (GSCs) with potent tumorigenic capacity. The function of GSCs in tumor propagation is maintained by several core transcriptional regulators including c-Myc. c-Myc protein is tightly regulated by posttranslational modification. However, the posttranslational regulatory mechanisms for c-Myc in GSCs have not been defined. In this study, we demonstrate that the deubiquitinase USP13 stabilizes c-Myc by antagonizing FBXL14-mediated ubiquitination to maintain GSC self-renewal and tumorigenic potential. USP13 was preferentially expressed in GSCs, and its depletion potently inhibited GSC proliferation and tumor growth by promoting c-Myc ubiquitination and degradation. In contrast, overexpression of the ubiquitin E3 ligase FBXL14 induced c-Myc degradation, promoted GSC differentiation, and inhibited tumor growth. Ectopic expression of the ubiquitin-insensitive mutant T58A–c-Myc rescued the effects caused by FBXL14 overexpression or USP13 disruption. These data suggest that USP13 and FBXL14 play opposing roles in the regulation of GSCs through reversible ubiquitination of c-Myc.

Purine synthesis promotes maintenance of brain tumor initiating cells in glioma

Brain tumor initiating cells (BTICs), also known as cancer stem cells, hijack high-affinity glucose uptake active normally in neurons to maintain energy demands. Here we link metabolic dysregulation in human BTICs to a nexus between MYC and de novo purine synthesis, mediating glucose-sustained anabolic metabolism. Inhibiting purine synthesis abrogated BTIC growth, self-renewal and in vivo tumor formation by depleting intracellular pools of purine nucleotides, supporting purine synthesis as a potential therapeutic point of fragility. In contrast, differentiated glioma cells were unaffected by the targeting of purine biosynthetic enzymes, suggesting selective dependence of BTICs. MYC coordinated the control of purine synthetic enzymes, supporting its role in metabolic reprogramming. Elevated expression of purine synthetic enzymes correlated with poor prognosis in glioblastoma patients. Collectively, our results suggest that stem-like glioma cells reprogram their metabolism to self-renew and fuel the tumor hierarchy, revealing potential BTIC cancer dependencies amenable to targeted therapy.

The Tailless Root of Glioma: Cancer Stem Cells

In this issue of Cell Stem Cell, Zhu et al. (2014) demonstrate that a genetically engineered glioma model displays a functional cellular hierarchy defined by expression of the nuclear orphan receptor Tlx. Targeting cancer stem cells through genetic deletion of TLX promotes cancer stem cell death and differentiation and extends survival.

MYC-regulated mevalonate metabolism maintains brain tumor–initiating cells

Metabolic dysregulation drives tumor initiation in a subset of glioblastomas harboring isocitrate dehydrogenase (IDH) mutations, but metabolic alterations in glioblastomas with wild-type IDH are poorly understood. MYC promotes metabolic reprogramming in cancer, but targeting MYC has proven notoriously challenging. Here, we link metabolic dysregulation in patient-derived brain tumor-initiating cells (BTIC) to a nexus between MYC and mevalonate signaling, which can be inhibited by statin or 6-fluoromevalonate treatment. BTICs preferentially express mevalonate pathway enzymes, which we find regulated by novel MYC-binding sites, validating an additional transcriptional activation role of MYC in cancer metabolism. Targeting mevalonate activity attenuated RAS-ERK-dependent BTIC growth and self-renewal. In turn, mevalonate created a positive feed-forward loop to activate MYC signaling via induction of miR-33b. Collectively, our results argue that MYC mediates its oncogenic effects in part by altering mevalonate metabolism in glioma cells, suggesting a therapeutic strategy in this setting. Cancer Res; 77(18); 4947-60. ©2017 AACR.