A common mutation of the epidermal growth factor receptor (EGFR) in glioblastoma multiforme (GBM) is an extracellular truncation known as the de2-7 EGFR (or EGFRvIII). Hepatocyte growth factor (HGF) is the ligand for the receptor tyrosine kinase (RTK) c-Met, and this signaling axis is often active in GBM. The expression of the HGF/c-Met axis or de2-7 EGFR independently enhances GBM growth and invasiveness, particularly through the phosphatidylinositol-3 kinase/pAkt pathway. Using RTK arrays, we show that expression of de2-7 EGFR in U87MG GBM cells leads to the coactivation of several RTKs, including platelet-derived growth factor receptor beta and c-Met. A neutralizing antibody to HGF (AMG102) did not inhibit de2-7 EGFR-mediated activation of c-Met, demonstrating that it is ligand-independent. Therapy for parental U87MG xenografts with AMG 102 resulted in significant inhibition of tumor growth, whereas U87MG.Delta 2-7 xenografts were profoundly resistant. Treatment of U87MG.Delta 2-7 xenografts with panitumumab, an anti-EGFR antibody, only partially inhibited tumor growth as xenografts rapidly reverted to the HGF/c-Met signaling pathway. Cotreatment with panitumumab and AMG 102 prevented this escape leading to significant tumor inhibition through an apoptotic mechanism, consistent with the induction of oncogenic shock. This observation provides a rationale for using panitumumab and AMG 102 in combination for the treatment of GBM patients. These results illustrate that GBM cells can rapidly change the RTK driving their oncogene addiction if the alternate RTK signals through the same downstream pathway. Consequently, inhibition of a dominant oncogene by targeted therapy can alter the hierarchy of RTKs resulting in rapid therapeutic resistance.

Background: Aberrant processing of the pro-IGF-II transcript produces pro-and big-IGF-II, which are secreted in a range of cancers. Results: These induce potent receptor activation and cell proliferation and retard ternary complex formation with ALS and IGFBP-3 and -5. Conclusion: They elicit unique biological responses that can be completely different from IGF-II. Significance: Understanding the effects induced by these individual isoforms is crucial to elucidate their role in tumorigenesis.

High-grade glioma (HGG) is an incurable brain cancer. The transcriptomes of cells within HGG tumors are highly heterogeneous. This renders the tumors unresponsive or able to adapt to therapeutics targeted at single pathways, thereby causing treatment failure. To overcome this, we focused on cyclin-dependent kinase 7 (CDK7), a ubiquitously expressed molecule involved in two major drivers of HGG pathogenesis: cell cycle progression and RNA polymerase-II-based transcription. We tested the activity of THZ1, an irreversible CDK7 inhibitor, on patient-derived primary HGG cell lines and ex vivo HGG patient tissue slices, using proliferation assays, microarray analysis, high-resolution respirometry, cell cycle analysis and in vivo tumor orthografts. The cellular processes affected by CDK7 inhibition were analyzed by reverse transcriptase–quantitative PCR, western blot, flow cytometry and immunofluorescence. THZ1 perturbed the transcriptome and disabled CDK activation, leading to cell cycle arrest at G2 and DNA damage. THZ1 halted transcription of the nuclear-encoded mitochondrial ribosomal genes, reducing mitochondrial translation and oxidative respiration. It also inhibited the expression of receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFR-α), reducing signaling flux through the AKT, extracellular-signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3) downstream pathways. Finally, THZ1 disrupted nucleolar, Cajal body and nuclear speckle formation, resulting in reduced cytosolic translation and malfunction of the spliceosome and thus leading to aberrant mRNA processing. These findings indicate that CDK7 is crucial for gliomagenesis, validate CDK7 as a therapeutic target and provide new insight into the cellular processes that are affected by THZ1 and induce antitumor activity.

A truncation mutant of the epidermal growth factor receptor, EGFRvIII, is commonly expressed in glioma, an incurable brain cancer. EGFRvIII is tumorigenic, in part, through its transactivation of other receptor tyrosine kinases (RTKs). Preventing the effects of this transactivation could form part of an effective therapy for glioma; however, the mechanism by which the transactivation occurs is unknown. Focusing on the RTK MET, we show that MET transactivation in U87MG human glioma cells in vitro is proportional to EGFRvIII activity and involves MET heterodimerization associated with a focal adhesion kinase (FAK) scaffold. The transactivation of certain other RTKs was, however, independent of FAK. Simultaneously targeting EGFRvIII (with panitumumab) and the transactivated RTKs themselves (with motesanib) in an intracranial mouse model of glioma resulted in significantly greater survival than with either agent alone, indicating that cotargeting these RTKs has potent antitumor efficacy and providing a strategy for treating EGFRvIII-expressing gliomas, which are usually refractory to treatment.

The epidermal growth factor receptor (EGFR) is overexpressed or mutated in glioma. Recently, a series of missense mutations in the extracellular domain (ECD) of EGFR were reported in glioma patients. Some of these mutations clustered within a cysteine-rich region of the EGFR targeted by the therapeutic antibody mAb806. This region is only exposed when EGFR activates and appears to locally misfold during activation. We expressed two of these mutations (R324L and E330K) in NR6 mouse fibroblasts, as they do not express any EGFR-related receptors. Both mutants were autophosphorylated in the absence of ligand and enhanced cell survival and anchorage-independent and xenograft growth. The ECD truncation that produces the de2-7EGFR (or EGFRvIII), the most common EGFR mutation in glioma, generates a free cysteine in this same region. Using a technique optimized for detecting disulfide-bonded dimers, we definitively demonstrated that the de2-7EGFR is robustly dimerized and that ablation of the free cysteine prevents dimerization and activation. Modeling of the R324L mutation suggests it may cause transient breaking of disulfide bonds, leading to similar disulfide-bonded dimers as seen for the de2-7EGFR. These ECD mutations confirm that the cysteine-rich region of EGFR around the mAb806 epitope has a significant role in receptor activation.

A feature of many gliomas is the amplification of the epidermal growth factor receptor (EGFR), resulting in its overexpression. Missense mutations or deletions within the extracellular domain are associated with this amplification and can lead to constitutive activation of the receptor, with the Domain I/II deletion, EGFRvIII, being the most common. These changes have also been associated with increased sensitivity to EGFR inhibition using small molecule inhibitors. We have expressed, in human glioma cells, EGFR containing four glioma-specific EGFR missense mutations within Domain IV (C620Y, C624F, C628Y and C636Y) to analyze their biological properties and sensitivity to EGFR inhibition. One of these mutants, C620Y, exhibited an enhanced basal phosphorylation, which was partially dependent on an EGFR-ligand autocrine loop. All Domain IV mutants responded equally as well as wildtype EGFR (wtEGFR) to ligand stimulation. Biochemical analysis revealed that a pre-formed, disulfide-bonded dimer associated with these mutations was underglycosylated, inactive and cytoplasmically retained. Ligand stimulation resulted in the formation of a tyrosine-phosphorylated, disulfide-bonded dimer for all Domain IV mutants but not for wtEGFR. Following treatment with the next-generation, irreversible pan-ErbB inhibitor dacomitinib, the C620Y, C624F and EGFRvIII mutants were inactivated, covalently dimerized and were retained in the cytoplasm, resulting in cell-surface receptor loss and, for C620Y and C624F, decreased binding of EGF. Dacomitinib treatment significantly reduced the in vivo growth of human glioma xenografts bearing C620Y, but not wtEGFR. Collectively, these data indicate that the unique biochemical traits of Domain IV EGFR cysteine mutants can be exploited for enhanced sensitivity to EGFR small molecule inhibitors, with potential clinical applications.

Combining gefitinib with methotrexate potently inhibits placental cell growth in vitro and in mouse models. The combination may have potential in treating ectopic pregnancies.

The c-MET receptor has a function in many human cancers and is a proven therapeutic target. Generating antagonistic or therapeutic monoclonal antibodies (mAbs) targeting c-MET has been difficult because bivalent, intact anti-Met antibodies frequently display agonistic activity, necessitating the use of monovalent antibody fragments for therapy. By using a novel strategy that included immunizing with cells expressing c-MET, we obtained a range of mAbs. These c-MET mAbs were tested for binding specificity and anti-tumor activity using a range of cell-based techniques and in silico modeling. The LMH 80 antibody bound an epitope, contained in the small cysteine-rich domain of c-MET (amino acids 519–561), that was preferentially exposed on the c-MET precursor. Since the c-MET precursor is only expressed on the surface of cancer cells and not normal cells, this antibody is potentially tumor specific. An interesting subset of our antibodies displayed profound activities on c-MET internalization and degradation. LMH 87, an antibody binding the loop connecting strands 3d and 4a of the 7-bladed β-propeller domain of c-MET, displayed no intrinsic agonistic activity but promoted receptor internalization and degradation. LMH 87 inhibited HGF/SF-induced migration of SK-OV-3 ovarian carcinoma cells, the proliferation of A549 lung cancer cells and the growth of human U87MG glioma cells in a mouse xenograft model. These results indicate that c-MET antibodies targeting epitopes controlling receptor internalization and degradation provide new ways of controlling c-MET expression and activity and may enable the therapeutic targeting of c-MET by intact, bivalent antibodies.