pf-00299804 and Glioma

Intratumoural heterogeneity underlies tumour escape from molecularly targeted therapy in glioblastoma. A cell-based model preserving the evolving molecular profiles of a tumour during treatment is key to understanding the recurrence mechanisms and development of strategies to overcome resistance. In this study, we established a matched pair of glioblastoma stem-like cell (GSC) cultures from patient glioblastoma samples before and after epidermal growth factor receptor (EGFR)-targeted therapy. A patient with recurrent glioblastoma (MGG70R) harboring focal, high-level EGFR amplification received the irreversible EGFR tyrosine kinase inhibitor dacomitinib. The tumour that subsequently recurred (MGG70RR) showed diploid EGFR, suggesting inhibitor-mediated elimination of EGFR-amplified tumour cells and propagation of EGFR non-amplified cell subpopulations. The MGG70R-GSC line established from MGG70R formed xenografts retaining EGFR amplification and EGFR overexpression, while MGG70RR-GSC established from MGG70RR generated tumours that lacked EGFR amplification and EGFR overexpression. MGG70R-GSC-derived intracranial xenografts were more proliferative than MGG70RR-GSC xenografts, which had upregulated mesenchymal markers, mirroring the pathological observation in the corresponding patient tumours. In vitro MGG70R-GSC was more sensitive to EGFR inhibitors than MGG70RR-GSC. Thus, these molecularly distinct GSC lines recapitulated the subpopulation alteration that occurred during glioblastoma evasion of targeted therapy, and offer a valuable model facilitating therapeutic development for recurrent glioblastoma.

Topics: Animals; Brain Neoplasms; ErbB Receptors; Glioblastoma; Glioma; Heterografts; Humans; Mice; Molecular Targeted Therapy; Neoplasm Recurrence, Local; Quinazolinones; Tumor Cells, Cultured

Glioma is one of the most epidemic and obstinate types of cancer in the central nervous system (CNS) with poor survival rate. Dacomitinib inhibited cell viability and proliferation of epidermal growth factor receptor (. A C6 rat glioma model was evaluated using proton magnetic resonance spectroscopy (. The obtained data indicated that metabolite ratios were significantly decreased (all. The results demonstrated that the Dacomitinib could suppress glioma cell necrosis and proliferation.

Topics: Animals; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Glioma; Humans; Ki-67 Antigen; Male; Necrosis; Proton Magnetic Resonance Spectroscopy; Quinazolinones; Rats, Sprague-Dawley

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.

Topics: Animals; Cell Line, Tumor; Cysteine; ErbB Receptors; Female; Glioma; Humans; Ligands; Mice; Mice, Inbred BALB C; Mutation; Phosphorylation; Protein Multimerization; Protein Structure, Tertiary; Quinazolinones

Cancer cells emit extracellular vesicles (EVs) containing unique molecular signatures. Here, we report that the oncogenic EGF receptor (EGFR) and its inhibitors reprogram phosphoproteomes and cargo of tumor cell-derived EVs. Thus, phosphorylated EGFR (P-EGFR) and several other receptor tyrosine kinases can be detected in EVs purified from plasma of tumor-bearing mice and from conditioned media of cultured cancer cells. Treatment of EGFR-driven tumor cells with second generation EGFR kinase inhibitors (EKIs), including CI-1033 and PF-00299804 but not with anti-EGFR antibody (Cetuximab) or etoposide, triggers a burst in emission of exosome-like EVs containing EGFR, P-EGFR, and genomic DNA (exo-gDNA). The EV release can be attenuated by treatment with inhibitors of exosome biogenesis (GW4869) and caspase pathways (ZVAD). The content of P-EGFR isoforms (Tyr-845, Tyr-1068, and Tyr-1173), ERK, and AKT varies between cells and their corresponding EVs and as a function of EKI treatment. Immunocapture experiments reveal the presence of EGFR and exo-gDNA within the same EV population following EKI treatment. These findings suggest that targeted agents may induce cancer cells to change the EV emission profiles reflective of drug-related therapeutic stress. We suggest that EV-based assays may serve as companion diagnostics for targeted anticancer agents.

Topics: Animals; Antineoplastic Agents; Biomarkers; Brain Neoplasms; Cell Line, Tumor; Cetuximab; Culture Media, Conditioned; DNA; ErbB Receptors; Etoposide; Exosomes; Extracellular Vesicles; Glioma; Humans; Mice; Mice, SCID; Morpholines; Neoplasm Transplantation; Neoplasms; Phosphoproteins; Phosphorylation; Proteomics; Quinazolinones; Transfection