[Show abstract][Hide abstract] ABSTRACT: Intratumoral heterogeneity contributes to cancer drug resistance, but the underlying mechanisms are not understood. Single-cell
analyses of patient-derived models and clinical samples from glioblastoma patients treated with epidermal growth factor receptor
(EGFR) tyrosine kinase inhibitors (TKIs) demonstrate that tumor cells reversibly up-regulate or suppress mutant EGFR expression,
conferring distinct cellular phenotypes to reach an optimal equilibrium for growth. Resistance to EGFR TKIs is shown to occur
by elimination of mutant EGFR from extrachromosomal DNA. After drug withdrawal, reemergence of clonal EGFR mutations on extrachromosomal DNA follows. These results indicate a highly specific, dynamic, and adaptive route by which
cancers can evade therapies that target oncogenes maintained on extrachromosomal DNA.
[Show abstract][Hide abstract] ABSTRACT: Aerobic glycolysis (the Warburg effect) is a core hallmark of cancer, but the molecular mechanisms underlying it remain unclear. Here, we identify an unexpected central role for mTORC2 in cancer metabolic reprogramming where it controls glycolytic metabolism by ultimately regulating the cellular level of c-Myc. We show that mTORC2 promotes inactivating phosphorylation of class IIa histone deacetylases, which leads to the acetylation of FoxO1 and FoxO3, and this in turn releases c-Myc from a suppressive miR-34c-dependent network. These central features of activated mTORC2 signaling, acetylated FoxO, and c-Myc levels are highly intercorrelated in clinical samples and with shorter survival of GBM patients. These results identify a specific, Akt-independent role for mTORC2 in regulating glycolytic metabolism in cancer.
[Show abstract][Hide abstract] ABSTRACT: mTOR pathway hyperactivation occurs in nearly 90% of glioblastomas, but the allosteric mTOR inhibitor rapamycin has failed in the clinic. Here we examine the efficacy of the newly discovered ATP-competitive mTOR kinase inhibitors CC214-1 and CC214-2 in glioblastoma, identifying molecular determinants of response and mechanisms of resistance, and develop a pharmacological strategy to overcome it.
We performed in vitro and in vivo studies in glioblastoma cell lines and an intracranial model to: determine the potential efficacy of the recently reported mTOR kinase inhibitors CC214-1 (in vitro use) and CC214-2 (in vivo use) at inhibiting rapamycin resistant signaling and blocking GBM growth and a novel single cell technology, DNA Encoded Antibody Libraries, was used to identify mechanisms of resistance.
Here we demonstrate that CC214-1 and CC214-2 suppress rapamycin-resistant mTORC1 signaling; block mTORC2 signaling and significantly inhibit the growth of glioblastomas in vitro and in vivo. EGFRvIII expression and PTEN loss enhance sensitivity to CC214 compounds, consistent with enhanced efficacy in strongly mTOR-activated tumors. Importantly, CC214 compounds potently induce autophagy, preventing tumor cell death. Genetic or pharmacologic inhibition of autophagy greatly sensitizes GBM cells and orthotopic xenografts to CC214-1 and CC214-2 induced cell death.
These results identify CC214-1 and CC214-2 as potentially efficacious mTOR kinase inhibitors in GBM and suggest a strategy for identifying patients most likely to benefit from mTOR inhibition. This study also demonstrates a central role for autophagy in preventing mTOR-kinase inhibitor-mediated tumor cell death, and suggests a pharmacological strategy for overcoming it.
Full-text · Article · Sep 2013 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Alternative splicing contributes to diverse aspects of cancer pathogenesis including altered cellular metabolism, but the specificity of the process or its consequences are not well understood. We characterized genome-wide alternative splicing induced by the activating EGFRvIII mutation in glioblastoma (GBM). EGFRvIII upregulates the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 splicing factor, promoting glycolytic gene expression and conferring significantly shorter survival in patients. HnRNPA1 promotes splicing of a transcript encoding the Myc-interacting partner Max, generating Delta Max, an enhancer of Myc-dependent transformation. Delta Max, but not full-length Max, rescues Myc-dependent glycolytic gene expression upon induced EGFRvIII loss, and correlates with hnRNPA1 expression and downstream Myc-dependent gene transcription in patients. Finally, Delta Max is shown to promote glioma cell proliferation in vitro and augment EGFRvIII expressing GBM growth in vivo. These results demonstrate an important role for alternative splicing in GBM and identify Delta Max as a mediator of Myc-dependent tumor cell metabolism.
[Show abstract][Hide abstract] ABSTRACT: Despite their nearly universal activation of mammalian target of rapamycin (mTOR) signaling, glioblastomas (GBMs) are strikingly resistant to mTOR-targeted therapy. We analyzed GBM cell lines, patient-derived tumor cell cultures, and clinical samples from patients in phase 1 clinical trials, and find that the promyelocytic leukemia (PML) gene mediates resistance to mTOR-targeted therapies. Direct mTOR inhibitors and EGF receptor (EGFR) inhibitors that block downstream mTOR signaling promote nuclear PML expression in GBMs, and genetic overexpression and knockdown approaches demonstrate that PML prevents mTOR and EGFR inhibitor-dependent cell death. Low doses of the PML inhibitor, arsenic trioxide, abrogate PML expression and reverse mTOR kinase inhibitor resistance in vivo, thus markedly inhibiting tumor growth and promoting tumor cell death in mice. These results identify a unique role for PML in mTOR and EGFR inhibitor resistance and provide a strong rationale for a combination therapeutic strategy to overcome it.
Full-text · Article · Feb 2013 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Activation of the epidermal growth factor receptor (EGFR) in glioblastoma (GBM) occurs through mutations or deletions in the extracellular (EC) domain. Unlike lung cancers with EGFR kinase domain (KD) mutations, GBMs respond poorly to the EGFR inhibitor erlotinib. Using RNAi, we show that GBM cells carrying EGFR EC mutations display EGFR addiction. In contrast to KD mutants found in lung cancer, glioma-specific EGFR EC mutants are poorly inhibited by EGFR inhibitors that target the active kinase conformation (e.g., erlotinib). Inhibitors that bind to the inactive EGFR conformation, however, potently inhibit EGFR EC mutants and induce cell death in EGFR-mutant GBM cells. Our results provide first evidence for single kinase addiction in GBM and suggest that the disappointing clinical activity of first-generation EGFR inhibitors in GBM versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these 2 cancer types. SIGNIFICANCE: Approximately 40% of human glioblastomas harbor oncogenic EGFR alterations, but attempts to therapeutically target EGFR with first-generation EGFR kinase inhibitors have failed. Here, we demonstrate selective sensitivity of glioma-specific EGFR mutants to ATP-site competitive EGFR kinase inhibitors that target the inactive conformation of the catalytic domain.
[Show abstract][Hide abstract] ABSTRACT: Although it is known that mTOR complex 2 (mTORC2) functions upstream of Akt, the role of this protein kinase complex in cancer is not well understood. Through an integrated analysis of cell lines, in vivo models, and clinical samples, we demonstrate that mTORC2 is frequently activated in glioblastoma (GBM), the most common malignant primary brain tumor of adults. We show that the common activating epidermal growth factor receptor (EGFR) mutation (EGFRvIII) stimulates mTORC2 kinase activity, which is partially suppressed by PTEN. mTORC2 signaling promotes GBM growth and survival and activates NF-κB. Importantly, this mTORC2-NF-κB pathway renders GBM cells and tumors resistant to chemotherapy in a manner independent of Akt. These results highlight the critical role of mTORC2 in the pathogenesis of GBM, including through the activation of NF-κB downstream of mutant EGFR, leading to a previously unrecognized function in cancer chemotherapy resistance. These findings suggest that therapeutic strategies targeting mTORC2, alone or in combination with chemotherapy, will be effective in the treatment of cancer. SIGNIFICANCE: This study demonstrates that EGFRvIII-activated mTORC2 signaling promotes GBM proliferation, survival, and chemotherapy resistance through Akt-independent activation of NF-κB. These results highlight the role of mTORC2 as an integrator of two canonical signaling networks that are commonly altered in cancer, EGFR/phosphoinositide-3 kinase (PI3K) and NF-κB. These results also validate the importance of mTORC2 as a cancer target and provide new insights into its role in mediating chemotherapy resistance, suggesting new treatment strategies.
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma (GBM) is the most common malignant primary brain tumor of adults and one of the most lethal of all cancers. Epidermal growth factor receptor (EGFR) mutations (EGFRvIII) and phosphoinositide 3-kinase (PI3K) hyperactivation are common in GBM, promoting tumor growth and survival, including through sterol regulatory element-binding protein 1 (SREBP-1)-dependent lipogenesis. The role of cholesterol metabolism in GBM pathogenesis, its association with EGFR/PI3K signaling, and its potential therapeutic targetability are unknown. In our investigation, studies of GBM cell lines, xenograft models, and GBM clinical samples, including those from patients treated with the EGFR tyrosine kinase inhibitor lapatinib, uncovered an EGFRvIII-activated, PI3K/SREBP-1-dependent tumor survival pathway through the low-density lipoprotein receptor (LDLR). Targeting LDLR with the liver X receptor (LXR) agonist GW3965 caused inducible degrader of LDLR (IDOL)-mediated LDLR degradation and increased expression of the ABCA1 cholesterol efflux transporter, potently promoting tumor cell death in an in vivo GBM model. These results show that EGFRvIII can promote tumor survival through PI3K/SREBP-1-dependent upregulation of LDLR and suggest a role for LXR agonists in the treatment of GBM patients.
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma, the most common malignant brain tumor, is among the most lethal and difficult cancers to treat. Although epidermal growth factor receptor (EGFR) mutations are frequent in glioblastoma, their clinical relevance is poorly understood. Studies of tumors from patients treated with the EGFR inhibitor lapatinib revealed that EGFR induces the cleavage and nuclear translocation of the master transcriptional regulator of fatty acid synthesis, sterol regulatory element-binding protein 1 (SREBP-1). This response was mediated by Akt; however, clinical data from rapamycin-treated patients showed that SREBP-1 activation was independent of the mammalian target of rapamycin complex 1, possibly explaining rapamycin's poor efficacy in the treatment of such tumors. Glioblastomas without constitutively active EGFR signaling were resistant to inhibition of fatty acid synthesis, whereas introduction of a constitutively active mutant form of EGFR, EGFRvIII, sensitized tumor xenografts in mice to cell death, which was augmented by the hydroxymethylglutaryl coenzyme A reductase inhibitor atorvastatin. These results identify a previously undescribed EGFR-mediated prosurvival metabolic pathway and suggest new therapeutic approaches to treating EGFR-activated glioblastomas.
Full-text · Article · Dec 2009 · Science Signaling
[Show abstract][Hide abstract] ABSTRACT: Activating epidermal growth factor receptor (EGFR) mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We show that the Src family kinases (SFK) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. Expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR- and EGFRvIII-dependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression, and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples showed frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies.
[Show abstract][Hide abstract] ABSTRACT: Epidermal growth factor receptor variant III (EGFRvIII) is an oncogenic, constitutively active mutant form of the EGFR that is commonly expressed in glioblastoma and is also detected in a number of epithelial cancers. EGFRvIII presents a unique antigenic target for anti-EGFRvIII vaccines and it has been shown to modulate response to EGFR kinase inhibitor therapy. Thus, detection in clinical samples may be warranted. Existing patents preclude the use of anti-EGFRvIII antibodies for clinical detection. Further, frozen tissue is not routinely available, particularly for patients treated in the community. Thus, detection of EGFRvIII in formalin-fixed paraffin-embedded (FFPE) clinical samples is a major challenge.
We developed a real-time reverse transcription-PCR (RT-PCR) assay for detecting EGFRvIII in FFPE samples and analyzed 59 FFPE glioblastoma clinical samples with paired frozen tissue from the same surgical resection. We assessed EGFRvIII protein expression by immunohistochemistry using two distinct specific anti-EGFRvIII antibodies and examined EGFR gene amplification by fluorescence in situ hybridization.
The FFPE RT-PCR assay detected EGFRvIII in 16 of 59 (27%) samples, exclusively in cases with EGFR amplification, consistent with the expected frequency of this alteration. The FFPE RT-PCR assay was more sensitive and specific for detecting EGFRvIII than either of the two antibodies alone, or in combination, with a sensitivity of 93% (95% confidence interval, 0.78-1.00) and a specificity of 98% (95% confidence interval, 0.93-1.00).
This assay will facilitate accurate assessment of EGFRvIII in clinical samples and may aid in the development of strategies for stratifying patients for EGFRvIII-directed therapies.
Preview · Article · Feb 2008 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: There is much discussion in the cancer drug development community about how to incorporate molecular tools into early-stage clinical trials to assess target modulation, measure anti-tumor activity, and enrich the clinical trial population for patients who are more likely to benefit. Small, molecularly focused clinical studies offer the promise of the early definition of optimal biologic dose and patient population.
Based on preclinical evidence that phosphatase and tensin homolog deleted on Chromosome 10 (PTEN) loss sensitizes tumors to the inhibition of mammalian target of rapamycin (mTOR), we conducted a proof-of-concept Phase I neoadjuvant trial of rapamycin in patients with recurrent glioblastoma, whose tumors lacked expression of the tumor suppressor PTEN. We aimed to assess the safety profile of daily rapamycin in patients with glioma, define the dose of rapamycin required for mTOR inhibition in tumor tissue, and evaluate the antiproliferative activity of rapamycin in PTEN-deficient glioblastoma. Although intratumoral rapamycin concentrations that were sufficient to inhibit mTOR in vitro were achieved in all patients, the magnitude of mTOR inhibition in tumor cells (measured by reduced ribosomal S6 protein phosphorylation) varied substantially. Tumor cell proliferation (measured by Ki-67 staining) was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test) but not the intratumoral rapamycin concentration. Tumor cells harvested from the Ki-67 nonresponders retained sensitivity to rapamycin ex vivo, indicating that clinical resistance to biochemical mTOR inhibition was not cell-intrinsic. Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test).
Rapamycin has anticancer activity in PTEN-deficient glioblastoma and warrants further clinical study alone or in combination with PI3K pathway inhibitors. The short-term treatment endpoints used in this neoadjuvant trial design identified the importance of monitoring target inhibition and negative feedback to guide future clinical development.
[Show abstract][Hide abstract] ABSTRACT: The epidermal growth factor receptor (EGFR) is commonly amplified, overexpressed, and mutated in glioblastoma, making it a compelling molecular target for therapy. We have recently shown that coexpression of EGFRvIII and PTEN protein by glioblastoma cells is strongly associated with clinical response to EGFR kinase inhibitor therapy. PTEN loss, by dissociating inhibition of the EGFR from downstream phosphatidylinositol 3-kinase (PI3K) pathway inhibition, seems to act as a resistance factor. Because 40% to 50% of glioblastomas are PTEN deficient, a critical challenge is to identify strategies that promote responsiveness to EGFR kinase inhibitors in patients whose tumors lack PTEN. Here, we show that the mammalian target of rapamycin (mTOR) inhibitor rapamycin enhances the sensitivity of PTEN-deficient tumor cells to the EGFR kinase inhibitor erlotinib. In two isogenic model systems (U87MG glioblastoma cells expressing EGFR, EGFRvIII, and PTEN in relevant combinations, and SF295 glioblastoma cells in which PTEN protein expression has been stably restored), we show that combined EGFR/mTOR kinase inhibition inhibits tumor cell growth and has an additive effect on inhibiting downstream PI3K pathway signaling. We also show that combination therapy provides added benefit in promoting cell death in PTEN-deficient tumor cells. These studies provide strong rationale for combined mTOR/EGFR kinase inhibitor therapy in glioblastoma patients, particularly those with PTEN-deficient tumors.
[Show abstract][Hide abstract] ABSTRACT: The epidermal growth factor receptor (EGFR) is frequently amplified, overexpressed, or mutated in glioblastomas, but only 10 to 20 percent of patients have a response to EGFR kinase inhibitors. The mechanism of responsiveness of glioblastomas to these inhibitors is unknown.
We sequenced kinase domains in the EGFR and human EGFR type 2 (Her2/neu) genes and analyzed the expression of EGFR, EGFR deletion mutant variant III (EGFRvIII), and the tumor-suppressor protein PTEN in recurrent malignant gliomas from patients who had received EGFR kinase inhibitors. We determined the molecular correlates of clinical response, validated them in an independent data set, and identified effects of the molecular abnormalities in vitro.
Of 49 patients with recurrent malignant glioma who were treated with EGFR kinase inhibitors, 9 had tumor shrinkage of at least 25 percent. Pretreatment tissue was available for molecular analysis from 26 patients, 7 of whom had had a response and 19 of whom had rapid progression during therapy. No mutations in EGFR or Her2/neu kinase domains were detected in the tumors. Coexpression of EGFRvIII and PTEN was significantly associated with a clinical response (P<0.001; odds ratio, 51; 95 percent confidence interval, 4 to 669). These findings were validated in 33 patients who received similar treatment for glioblastoma at a different institution (P=0.001; odds ratio, 40; 95 percent confidence interval, 3 to 468). In vitro, coexpression of EGFRvIII and PTEN sensitized glioblastoma cells to erlotinib.
Coexpression of EGFRvIII and PTEN by glioblastoma cells is associated with responsiveness to EGFR kinase inhibitors.
Full-text · Article · Dec 2005 · New England Journal of Medicine
[Show abstract][Hide abstract] ABSTRACT: A critical challenge in cancer research is to identify genetic lesions that sensitize patients to chemotherapy. p53, which is mutated in nearly one-third to half of glioblastomas, may be such a lesion. In this paper, we demonstrate that p53 disruption dramatically sensitizes glioblastoma cells to DNA topoisomerase I inhibitor-mediated apoptosis. Using 19 glioblastoma cell lines, including 15 low-passage ex vivo cell lines derived from patients, as well as isogenic glioblastoma cells varying in p53 status, we show that clinically relevant levels of SN-38 potently induce cell cycle arrest and temporary senescence in glioblastoma cells with wild-type p53 while causing massive apoptosis in p53-deficient cells (P<0.0002). We demonstrate that glioblastoma cells with wild-type p53 proliferate when recultured in drug-free medium, whereas p53-deficient cells do not. We also show that p16 protein expression is neither necessary nor sufficient for initiation and/or maintenance of SN-38-induced arrest/senescence. These results indicate that p53 disruption has a dramatic effect on how glioblastoma cells process topoisomerase I inhibitor-mediated DNA damage.