Ingo K. Mellinghoff

IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype

Both genome-wide genetic and epigenetic alterations are fundamentally important for the development of cancers, but the interdependence of these aberrations is poorly understood. Glioblastomas and other cancers with the CpG island methylator phenotype (CIMP) constitute a subset of tumours with extensive epigenomic aberrations and a distinct biology. Glioma CIMP (G-CIMP) is a powerful determinant of tumour pathogenicity, but the molecular basis of G-CIMP remains unresolved. Here we show that mutation of a single gene, isocitrate dehydrogenase 1 (IDH1), establishes G-CIMP by remodelling the methylome. This remodelling results in reorganization of the methylome and transcriptome. Examination of the epigenome of a large set of intermediate-grade gliomas demonstrates a distinct G-CIMP phenotype that is highly dependent on the presence of IDH mutation. Introduction of mutant IDH1 into primary human astrocytes alters specific histone marks, induces extensive DNA hypermethylation, and reshapes the methylome in a fashion that mirrors the changes observed in G-CIMP-positive lower-grade gliomas. Furthermore, the epigenomic alterations resulting from mutant IDH1 activate key gene expression programs, characterize G-CIMP-positive proneural glioblastomas but not other glioblastomas, and are predictive of improved survival. Our findings demonstrate that IDH mutation is the molecular basis of CIMP in gliomas, provide a framework for understanding oncogenesis in these gliomas, and highlight the interplay between genomic and epigenomic changes in human cancers.

Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR.

Recent evidence places the FRAP/mTOR kinase downstream of the phosphatidyl inositol 3-kinase/Akt-signaling pathway, which is up-regulated in multiple cancers because of loss of the PTEN tumor suppressor gene. We performed biological and biochemical studies to determine whether PTEN-deficient cancer cells are sensitive to pharmacologic inhibition of FRAP/mTOR by using the rapamycin derivative CCI-779. In vitro and in vivo studies of isogenic PTEN+/+ and PTEN−/− mouse cells as well as human cancer cells with defined PTEN status showed that the growth of PTEN null cells was blocked preferentially by pharmacologic FRAP/mTOR inhibition. Enhanced tumor growth caused by constitutive activation of Akt in PTEN+/+ cells also was reversed by CCI-779 treatment, indicating that FRAP/mTOR functions downstream of Akt in tumorigenesis. Loss of PTEN correlated with increased S6 kinase activity and phosphorylation of ribosomal S6 protein, providing evidence for activation of the FRAP/mTOR pathway in these cells. Differential sensitivity to CCI-779 was not explained by differences in biochemical blockade of the FRAP/mTOR pathway, because S6 phosphorylation was inhibited in sensitive and resistant cell lines. These results provide rationale for testing FRAP/mTOR inhibitors in PTEN null human cancers.

IDH mutation impairs histone demethylation and results in a block to cell differentiation

Recurrent mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 have been identified in gliomas, acute myeloid leukaemias (AML) and chondrosarcomas, and share a novel enzymatic property of producing 2-hydroxyglutarate (2HG) from α-ketoglutarate. Here we report that 2HG-producing IDH mutants can prevent the histone demethylation that is required for lineage-specific progenitor cells to differentiate into terminally differentiated cells. In tumour samples from glioma patients, IDH mutations were associated with a distinct gene expression profile enriched for genes expressed in neural progenitor cells, and this was associated with increased histone methylation. To test whether the ability of IDH mutants to promote histone methylation contributes to a block in cell differentiation in non-transformed cells, we tested the effect of neomorphic IDH mutants on adipocyte differentiation in vitro. Introduction of either mutant IDH or cell-permeable 2HG was associated with repression of the inducible expression of lineage-specific differentiation genes and a block to differentiation. This correlated with a significant increase in repressive histone methylation marks without observable changes in promoter DNA methylation. Gliomas were found to have elevated levels of similar histone repressive marks. Stable transfection of a 2HG-producing mutant IDH into immortalized astrocytes resulted in progressive accumulation of histone methylation. Of the marks examined, increased H3K9 methylation reproducibly preceded a rise in DNA methylation as cells were passaged in culture. Furthermore, we found that the 2HG-inhibitable H3K9 demethylase KDM4C was induced during adipocyte differentiation, and that RNA-interference suppression of KDM4C was sufficient to block differentiation. Together these data demonstrate that 2HG can inhibit histone demethylation and that inhibition of histone demethylation can be sufficient to block the differentiation of non-transformed cells.

Assessing the significance of chromosomal aberrations in cancer: Methodology and application to glioma

Comprehensive knowledge of the genomic alterations that underlie cancer is a critical foundation for diagnostics, prognostics, and targeted therapeutics. Systematic efforts to analyze cancer genomes are underway, but the analysis is hampered by the lack of a statistical framework to distinguish meaningful events from random background aberrations. Here we describe a systematic method, called Genomic Identification of Significant Targets in Cancer (GISTIC), designed for analyzing chromosomal aberrations in cancer. We use it to study chromosomal aberrations in 141 gliomas and compare the results with two prior studies. Traditional methods highlight hundreds of altered regions with little concordance between studies. The new approach reveals a highly concordant picture involving ≈35 significant events, including 16–18 broad events near chromosome-arm size and 16–21 focal events. Approximately half of these events correspond to known cancer-related genes, only some of which have been previously tied to glioma. We also show that superimposed broad and focal events may have different biological consequences. Specifically, gliomas with broad amplification of chromosome 7 have properties different from those with overlapping focalEGFR amplification: the broad events act in part through effects on MET and its ligand HGF and correlate with MET dependence in vitro. Our results support the feasibility and utility of systematic characterization of the cancer genome.

An Inhibitor of Mutant IDH1 Delays Growth and Promotes Differentiation of Glioma Cells

IDHology Among the most exciting drug targets to emerge from cancer genome sequencing projects are two related metabolic enzymes, isocitrate dehydrogenases 1 and 2 (IDH1, IDH2). Mutations in the IDH1 and IDH2 genes are common in certain types of human cancer. Whether inhibition of mutant IDH activity might offer therapeutic benefits is unclear (see the Perspective by Kim and DeBerardinis). F. Wang et al. (p. 622, published online 4 April) isolated a small molecule that selectively inhibits mutant IDH2, describe the structural details of its binding to the mutant enzyme, and show that this compound suppresses the growth of patient-derived leukemia cells harboring the IDH2 mutation. Rohle et al. (p. 626, published online 4 April) show that a small molecule inhibitor of IDH1 selectively slows the growth of patient-derived brain tumor cells with the IDH1 mutation. A small molecule that inhibits a mutant enzyme in tumors slows malignant growth by inducing cancer cell differentiation. [Also see Perspective by Kim and DeBerardinis] The recent discovery of mutations in metabolic enzymes has rekindled interest in harnessing the altered metabolism of cancer cells for cancer therapy. One potential drug target is isocitrate dehydrogenase 1 (IDH1), which is mutated in multiple human cancers. Here, we examine the role of mutant IDH1 in fully transformed cells with endogenous IDH1 mutations. A selective R132H-IDH1 inhibitor (AGI-5198) identified through a high-throughput screen blocked, in a dose-dependent manner, the ability of the mutant enzyme (mIDH1) to produce R-2-hydroxyglutarate (R-2HG). Under conditions of near-complete R-2HG inhibition, the mIDH1 inhibitor induced demethylation of histone H3K9me3 and expression of genes associated with gliogenic differentiation. Blockade of mIDH1 impaired the growth of IDH1-mutant—but not IDH1–wild-type—glioma cells without appreciable changes in genome-wide DNA methylation. These data suggest that mIDH1 may promote glioma growth through mechanisms beyond its well-characterized epigenetic effects.

Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer

Inhibitors of the kinase mammalian target of rapamycin (mTOR) have shown sporadic activity in cancer trials, leading to confusion about the appropriate clinical setting for their use. Here we show that loss of the Von Hippel-Lindau tumor suppressor gene (VHL) sensitizes kidney cancer cells to the mTOR inhibitor CCI-779 in vitro and in mouse models. Growth arrest caused by CCI-779 correlates with a block in translation of mRNA encoding hypoxia-inducible factor (HIF1A), and is rescued by expression of a VHL-resistant HIF1A cDNA lacking the 5′ untranslated region. VHL-deficient tumors show increased uptake of the positron emission tomography (PET) tracer fluorodeoxyglucose (FDG) in an mTOR-dependent manner. Our findings provide preclinical rationale for prospective, biomarker-driven clinical studies of mTOR inhibitors in kidney cancer and suggest that FDG-PET scans may have use as a pharmacodynamic marker in this setting.

Antitumor activity of rapamycin in a phase I trial for patients with recurrent PTEN-deficient glioblastoma

Background 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. Methods and Findings 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). Conclusions 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. Trial registration: http://www.ClinicalTrials.gov (#NCT00047073).

Dual targeting of EGFR can overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer

EGFR is a major anticancer drug target in human epithelial tumors. One effective class of agents is the tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. These drugs induce dramatic responses in individuals with lung adenocarcinomas characterized by mutations in exons encoding the EGFR tyrosine kinase domain, but disease progression invariably occurs. A major reason for such acquired resistance is the outgrowth of tumor cells with additional TKI-resistant EGFR mutations. Here we used relevant transgenic mouse lung tumor models to evaluate strategies to overcome the most common EGFR TKI resistance mutation, T790M. We treated mice bearing tumors harboring EGFR mutations with a variety of anticancer agents, including a new irreversible EGFR TKI that is under development (BIBW-2992) and the EGFR-specific antibody cetuximab. Surprisingly, we found that only the combination of both agents together induced dramatic shrinkage of erlotinib-resistant tumors harboring the T790M mutation, because together they efficiently depleted both phosphorylated and total EGFR. We suggest that these studies have immediate therapeutic implications for lung cancer patients, as dual targeting with cetuximab and a second-generation EGFR TKI may be an effective strategy to overcome T790M-mediated drug resistance. Moreover, this approach could serve as an important model for targeting other receptor tyrosine kinases activated in human cancers.

AKT Activity Determines Sensitivity to Mammalian Target of Rapamycin (mTOR) Inhibitors by Regulating Cyclin D1 and c-myc Expression

Prior work demonstrates that AKT activity regulates sensitivity of cells to G1 arrest induced by mammalian target of rapamycin (mTOR) inhibitors such as rapamycin and CCI-779. To investigate this, a novel high-throughput microarray polysome analysis was performed to identify genes whose mRNA translational efficiency was differentially affected following mTOR inhibition. The analysis also allowed the assessment of steady-state transcript levels. We identified two transcripts, cyclin D1 and c-myc, which exhibited differential expression in an AKT-dependent manner: High levels of activated AKT resulted in rapamycin-induced down-regulation of expression, whereas low levels resulted in up-regulation of expression. To ectopically express these proteins we exploited the finding that the p27kip1 mRNA was efficiently translated in the face of mTOR inhibition irrespective of AKT activity. Thus, the p27kip1 5′-untranslated region was fused to the cyclin D1 and c-myc coding regions and these constructs were expressed in cells. In transfected cells, expression of cyclin D1 or c-myc was not decreased by rapamycin. Most importantly, this completely converted sensitive cells to a phenotype resistant to G1 arrest. Furthermore, the AKT-dependent differential expression patterns of these two genes was also observed in a mouse xenograft model following in vivo treatment with CCI-779. These results identify two critical downstream molecular targets whose expression is regulated by AKT activity and whose down-regulation is required for rapamycin/CCI-779 sensitivity.

Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain

Background Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. Methods and Findings Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. Conclusions Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.