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.

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 which bind to the inactive EGFR conformation, on the other hand, 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 two cancer types.

Many proteins contain ubiquitin-binding domains or motifs (UBDs), such as the UIM (ubiquitin-interacting motif) and are referred to as ubiquitin receptors. Ubiquitin receptors themselves are frequently monoubiquitinated by a process that requires the presence of a UBD and is referred to as coupled monoubiquitination. Using a UIM-containing protein, eps15, as a model, we show here that coupled monoubiquitination strictly depends on the ability of the UIM to bind to monoubiquitin (mUb). We found that the underlying molecular mechanism is based on interaction between the UIM and a ubiquitin ligase (E3), which has itself been modified by ubiquitination. Furthermore, we demonstrate that the in vivo ubiquitination of members of the Nedd4 family of E3 ligases correlates with their ability to monoubiquitinate eps15. Thus, our results clarify the mechanism of coupled monoubiquitination and identify the ubiquitination of E3 ligases as a critical determinant in this process.

The phosphatase and tensin homolog (PTEN) is a tumor suppressor that is inactivated in many human cancers. PTEN loss has been associated with resistance to inhibitors of the epidermal growth factor receptor (EGFR), but the molecular basis of this resistance is unclear. It is believed that unopposed phosphatidylinositol-3-kinase (PI3K) activation through multiple receptor tyrosine kinases (RTKs) can relieve PTEN-deficient cancers from their "dependence" on EGFR or any other single RTK for survival. Here we report a distinct resistance mechanism whereby PTEN inactivation specifically raises EGFR activity by impairing the ligand-induced ubiquitylation and degradation of the activated receptor through destabilization of newly formed ubiquitin ligase Cbl complexes. PTEN-associated resistance to EGFR kinase inhibitors is phenocopied by expression of dominant negative Cbl and can be overcome by more complete EGFR kinase inhibition. PTEN inactivation does not confer resistance to inhibitors of the MET or PDGFRA kinase. Our study identifies a critical role for PTEN in EGFR signal termination and suggests that more potent EGFR inhibition should overcome resistance caused by PI3K pathway activation.T he phosphatidylinositol-3-kinase (PI3K) pathway has emerged as the most frequently deregulated signaling pathway in human cancer. Activation of this pathway in cancer cells can occur through a variety of mechanisms, including mutations in genes encoding the catalytic (PIK3CA) or regulatory (PIK3R1) subunit of class IA PI3Ks, the phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Akt family members, Ras family members, neurofibromin 1, and/or various growth factor receptors. Inactivation of PTEN through missense mutations, deletions, and epigenetic mechanisms represents the most common cause of PI3K pathway activation in human cancer (1).The PTEN protein exhibits dual protein and lipid phosphatase activity. Most of PTEN's tumor suppressor functions have been attributed to its lipid phosphatase activity which hydrolyzes phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] at the D3 position of the inositol ring and directly antagonizes the function of phosphatidylinositol-3-kinase. PTEN loss leads to accumulation of PtdIns(3,4,5)P 3 which recruits proteins containing pleckstrin homology domains to cellular membranes, including the serine/threonine kinase Akt. In addition to its role in tumor suppression, PTEN has emerged as a determinant of tumor cell response to ATP-site competitive inhibitors of the epidermal growth factor receptor (EGFR) in EGFR amplified cancer cell lines (2) and in glioblastoma (GBM) patients whose tumors expressed the oncogenic EGFR variant III (EGFRvIII) mutant receptor (3). How PTEN's functions as tumor suppressor and drug response modifier relate to each other is currently unclear. One possibility is that PTEN inactivation relieves EGFR mutant cancer cells from their dependence on EGFR for survival by allowing sufficient PtdIns(3,4,5)P 3 accumulation and Akt activation through oth...

The serine–threonine kinase AKT regulates proliferation and survival by phosphorylating a network of protein substrates. In this study, we describe a kinase-independent function of AKT. In cancer cells harboring gain-of-function alterations in MET, HER2, or Phosphatidyl-Inositol-3-Kinase (PI3K), catalytically inactive AKT (K179M) protected from drug induced cell death in a PH-domain dependent manner. An AKT kinase domain mutant found in human melanoma (G161V) lacked enzymatic activity in vitro and in AKT1/AKT2 double knockout cells, but promoted growth factor independent survival of primary human melanocytes. ATP-competitive AKT inhibitors failed to block the kinase-independent function of AKT, a liability that limits their effectiveness compared to allosteric AKT inhibitors. Our results broaden the current view of AKT function and have important implications for the development of AKT inhibitors for cancer.DOI: http://dx.doi.org/10.7554/eLife.03751.001

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ.

In this work, the authors report the first proteome-wide analysis of EGF-regulated ubiquitination, revealing surprisingly pervasive growth factor-induced ubiquitination across a broad range of cellular systems and signaling pathways.

21Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is 22 now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade 23 gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct 24 repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT 25 promoter hypermethylation is currently the only known biomarker for TMZ response in 26 glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic 27 rearrangements that lead to MGMT overexpression, independently from changes in its promoter 28 methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT 29 rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements 30 contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can 31 be detected in tumor-derived exosomes and could potentially represent an early detection marker 32 of tumor recurrence in a subset of patients treated with TMZ. Introduction 34The therapeutic benefits of TMZ depend on its ability to methylate DNA, which takes place at 35 the N-7 and O-6 positions of guanine and N-3 position of adenine. Although the minor product 36 O6-Methylguanine (O6-meG) accounts for less than 10% of total alkylation, it exerts the greatest 37 potential for apoptosis induction 1 . O6-meG pairs with thymine as opposed to cytosine during 38 DNA replication. The O6-meG:thymine mismatch can be recognized by the post-replication 39Mismatch Repair (MMR) system and, according to the futile repair hypothesis, ultimately 40 induces DNA double-strand breaks, cell cycle arrest and cell death 2 . The O-6-methylguanine-41 DNA methyltransferase (MGMT) is responsible for the direct repair of O6-meG lesion by 42 transferring the alkyl group from guanine to a cysteine residue. Epigenetic silencing, due to 43 promoter methylation, of the MGMT gene prevents the synthesis of this enzyme, and as a 44 consequence increases the tumours sensitivity to the cytotoxic effects induced by TMZ and other 45 alkylating compounds 3,4 . As today, MGMT promoter hypermethylation is the only known 46 biomarker for TMZ response 4 . However, the discordance between promoter methylation and 47 Nevertheless, the desired genomic rearrangements were further validated using a break-apart 132 fluorescence in situ hybridization (FISH) assay ( Supplementary Fig. 4). 133 134