Nimotuzumab enhances temozolomide-induced growth suppression of glioma cells expressing mutant EGFR in vivo.
ABSTRACT: A mutant form of epidermal growth factor receptor (EGFR), EGFRvIII, is common in glioblastoma (GBM) and confers enhanced tumorigenic activity and drug resistance. Nimotuzumab, an anti-EGFR antibody, has shown preclinical and clinical activity to GBM, but its specific activity against EGFRvIII has not been fully investigated. Human glioma U87MG or LNZ308 cells overexpressing either wild-type (wt) EGFR or EGFRvIII were treated with nimotuzumab, temozolomide, or both. Expression and phosphorylation status of molecules were determined by Western blot analysis. Methylation status of promoter region of O(6) -methylguanine-DNA methyltransferase (MGMT) was detected by methylation-specific PCR. Antitumor activity was tested using nude mice bearing either subcutaneous or intracerebral xenografts along with analyses of EGFR phosphorylation status, proliferation, apoptosis, and vessel density. Nimotuzumab treatment resulted in reduction of EGFRvIII tyrosine phosphorylation with a decrease in Akt phosphorylation that was greater than that of wtEGFR. Correspondingly, antitumor effects, growth suppression and survival elongation, were more significant in mice bearing either subcutaneous or intracerebral tumor expressing EGFRvIII than in those expressing wtEGFR. These effects were markedly increased when temozolomide was combined with nimotuzumab. The post-treatment recurrent brain tumors exhibited a decrease in expression of the mismatch repair (MMR) proteins, MSH6 and MLH1, but their methylated MGMT status did not changed. Nimotuzumab has in vivo antitumor activity against GBM, especially those expressing EGFRvIII, when combined with temozolomide. This could provide a basis for preselection of patients with GBM by EGFR status who might benefit from the nimotuzumab and temozolomide combination therapy.
Project description:Glioblastoma multiforme (GBM) is a malignant primary brain tumor with a mean survival of 15 months with the current standard of care. Genetic profiling efforts have identified the amplification, overexpression, and mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) in ? 50% of GBM patients. The genetic aberration of wtEGFR is frequently accompanied by the overexpression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2-7EGFR, ?EGFR), which is expressed in 30% of GBM tumors. The molecular mechanisms of tumorigenesis driven by EGFRvIII overexpression in human tumors have not been fully elucidated. To identify specific therapeutic targets for EGFRvIII driven tumors, it is important to gather a broad understanding of EGFRvIII specific signaling. Here, we have characterized signaling through the quantitative analysis of protein expression and tyrosine phosphorylation across a panel of glioblastoma tumor xenografts established from patient surgical specimens expressing wtEGFR or overexpressing wtEGFR (wtEGFR+) or EGFRvIII (EGFRvIII+). S100A10 (p11), major vault protein, guanylate-binding protein 1(GBP1), and carbonic anhydrase III (CAIII) were identified to have significantly increased expression in EGFRvIII expressing xenograft tumors relative to wtEGFR xenograft tumors. Increased expression of these four individual proteins was found to be correlated with poor survival in patients with GBM; the combination of these four proteins represents a prognostic signature for poor survival in gliomas. Integration of protein expression and phosphorylation data has uncovered significant heterogeneity among the various tumors and has highlighted several novel pathways, related to EGFR trafficking, activated in glioblastoma. The pathways and proteins identified in these tumor xenografts represent potential therapeutic targets for this disease.
Project description:Background:RNAs within extracellular vesicles (EVs) have potential as diagnostic biomarkers for patients with cancer and are identified in a variety of biofluids. Glioblastomas (GBMs) release EVs containing RNA into cerebrospinal fluid (CSF). Here we describe a multi-institutional study of RNA extracted from CSF-derived EVs of GBM patients to detect the presence of tumor-associated amplifications and mutations in epidermal growth factor receptor (EGFR). Methods:CSF and matching tumor tissue were obtained from patients undergoing resection of GBMs. We determined wild-type (wt)EGFR DNA copy number amplification, as well as wtEGFR and EGFR variant (v)III RNA expression in tumor samples. We also characterized wtEGFR and EGFRvIII RNA expression in CSF-derived EVs. Results:EGFRvIII-positive tumors had significantly greater wtEGFR DNA amplification (P = 0.02) and RNA expression (P = 0.03), and EGFRvIII-positive CSF-derived EVs had significantly more wtEGFR RNA expression (P = 0.004). EGFRvIII was detected in CSF-derived EVs for 14 of the 23 EGFRvIII tissue-positive GBM patients. Conversely, only one of the 48 EGFRvIII tissue-negative patients had the EGFRvIII mutation detected in their CSF-derived EVs. These results yield a sensitivity of 61% and a specificity of 98% for the utility of CSF-derived EVs to detect an EGFRvIII-positive GBM. Conclusion:Our results demonstrate CSF-derived EVs contain RNA signatures reflective of the underlying molecular genetic status of GBMs in terms of wtEGFR expression and EGFRvIII status. The high specificity of the CSF-derived EV diagnostic test gives us an accurate determination of positive EGFRvIII tumor status and is essentially a less invasive "liquid biopsy" that might direct mutation-specific therapies for GBMs.
Project description:Background: Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) antibody that has shown preclinical and clinical anticancer activity in cerebral glioblastoma multiforme (GBM). We conducted a phase II, single-arm, multicenter clinical trial to evaluate the benefit of adding nimotuzumab to current standard chemo-radiotherapy for patients with GBM with positive EGFR expression. Methods: Newly diagnosed patients with histologically proven single supratentorial GBM and epidermal growth factor receptor (EGFR) positive expressions were recruited. All patients were treated with nimotuzumab, administered once a week intravenously for 6 weeks in addition to radiotherapy with concomitant and adjuvant temozolomide after surgery. The primary endpoints were overall survival (OS) and progression-free survival (PFS). Secondary objectives included objective response rate (ORR) and toxicity. Results: A total of 39 patients were enrolled and 36 patients were evaluated for efficacy. The ORR at the end of RT was 72.2%. Median OS and PFS were 24.5 and 11.9 months. The 1-year OS and PFS rates were 83.3% and 49.3%. The 2-year OS and PFS rates were 51.1% and 29.0%. O (6)-methylquanine DNA methyl-tranferase (MGMT) expression is known to affect the efficacy of chemotherapy and status of its expression is examined. No significant correlation between treatment outcomes and MGMT status was found. Most frequent treatment-related toxicities were mild to moderate and included constipation, anorexia, fatigue, nausea, vomiting, and leucopenia. Conclusions: Our study show that nimotuzumab in addition to standard treatment is well tolerable and has increased survival in newly diagnosed GBM patients with EGFR positive expression.
Project description:The epidermal growth factor receptor variant III (EGFRvIII) is associated with increased proliferation of glioma cells. However, the impact of EGFRvIII on survival of patients with glioblastoma (GBM) has not been definitively established. In the present study, we prospectively evaluated 73 patients with primary GBM treated with surgical resection and standard radio/chemotherapy. The EGFRvIII was assessed by reverse transcription-polymerase chain reaction (PCR), O(6)-methylguanine methyltransferase (MGMT) promoter methylation was assessed by methylation-specific PCR, and phosphatase and tension homolog (PTEN) expression was assessed by immunohistochemistry. In 14 patients of this series, who presented with tumor recurrence, EGFRvIII was determined by real-time PCR. Sensitivity to temozolomide (TMZ) was assessed in vitro on GBM neurosphere cell cultures with different patterns of EGFRvIII expression. Age 60 years or younger, preoperative Karnofsky Performance Status score of 70 or higher, recursive partitioning analysis score III and IV, methylated MGMT, and Ki67 index of 20% or less were significantly associated with longer overall survival (OS; P = .0069, P = .0035, P = .0007, P = .0437, and P = .0286, respectively). EGFRvIII identified patients with significantly longer OS (P = .0023) and the association of EGFRvIII/Ki67 of 20% or less, EGFRvIII/normal PTEN, EGFRvIII/methylated MGMT, and EGFRvIII/normal PTEN/methylated MGMT identified subgroups of GBM patients with better prognosis. In recurred GBMs, EGFRvIII expression was approximately two-fold lower than in primary tumors. In vitro, the EGFRvIII-negative GBM neurosphere cells were more resistant to TMZ than the positive ones. In conclusion, in contrast with previous studies, we found that EGFRvIII is associated with prolonged survival of GBM patients treated with surgery and radio/chemotherapy. Depletion of EGFRvIII in recurrent GBMs as well as differential sensitivity to TMZ in vitro indicates that the EGFRvIII-negative cell fraction is involved in resistance to radio/chemotherapy and tumor repopulation.
Project description:A constitutively active epidermal growth factor receptor (EGFR) mutant, EGFR variant III (EGFRvIII), has been detected at high frequencies in certain human cancers. This study evaluated transactivation and trafficking of erbB family members as a result of constitutive EGFR activity in a cancer cell line. Expression of EGFRvIII modulated erbB family members through different mechanisms; the erbB3 mRNA level was reduced, whereas wild-type EGFR (wtEGFR) and erbB2 protein levels were diminished, with no change in their mRNA levels, and there was no change in the erbB4 expression level. Both EGFR and erbB2 were internalized as a result of EGFRvIII's activity and redistributed to the cell surface upon addition of AG1478, an inhibitor of wtEGFR/EGFRvIII catalytic activity. Acute activation of EGFRvIII by removing AG1478 from cells increased phosphorylation of both wtEGFR and erbB2 and caused differential trafficking of EGFRvIII's activation partners; wtEGFR was directed primarily to lysosomal compartments and partially to recycling compartments, whereas erbB2 was directed primarily to recycling compartments and partially to lysosomal compartments. Our data demonstrate that the constitutive activity of EGFRvIII is sufficient to trigger endocytosis and trafficking of wtEGFR and erbB2, which may play a role in activating signaling pathways that are triggered during receptor endocytosis.
Project description:Glioblastoma (GB) is the most frequent primary brain tumor in adults with a dismal prognosis despite aggressive treatment including surgical resection, radiotherapy and chemotherapy with the alkylating agent temozolomide. Thus far, the successful implementation of the concept of targeted therapy where a drug targets a selective alteration in cancer cells was mainly limited to model diseases with identified genetic drivers. One of the most commonly altered oncogenic drivers of GB and therefore plausible therapeutic target is the epidermal growth factor receptor (EGFR). Trials targeting this signaling cascade, however, have been negative, including the phase III OSAG 101-BSA-05 trial. This highlights the need for further patient selection to identify subgroups of GB with true EGFR-dependency. In this retrospective analysis of treatment-naïve samples of the OSAG 101-BSA-05 trial cohort, we identify the EGFR signaling activity markers phosphorylated PRAS40 and phosphorylated ribosomal protein S6 as predictive markers for treatment efficacy of the EGFR-blocking antibody nimotuzumab in MGMT promoter unmethylated GBs. Considering the total trial population irrespective of MGMT status, a clear trend towards a survival benefit from nimotuzumab was already detectable when tumors had above median levels of phosphorylated ribosomal protein S6. These results could constitute a basis for further investigations of nimotuzumab or other EGFR- and downstream signaling inhibitors in selected patient cohorts using the reported criteria as candidate predictive biomarkers.
Project description:BACKGROUND:Glioblastoma (GBM) is an aggressive brain tumor with universal recurrence and poor prognosis. The recurrence is largely driven by chemoradiation resistant cancer stem cells (CSCs). Epidermal growth factor receptor (EGFR) and its mutant EGFRvIII are amplified in ~ 60% and ~ 30% of GBM patients, respectively; however, therapies targeting EGFR have failed to improve disease outcome. EGFRvIII-mediated cross-activation of tyrosine kinase receptor, cMET, regulates GBM CSC maintenance and promote tumor recurrence. Here, we evaluated the efficacy of pan-EGFR inhibitor afatinib and Temozolomide (TMZ) combination on GBM in vitro and in vivo. METHODS:We analyzed the effect of afatinib and temozolomide (TMZ) combination on GBM cells U87MG and U251 engineered to express wild type (WT) EGFR, EGFRvIII or EGFRvIII dead kinase, CSCs isolated from U87 and U87EGFRvIII in vitro. The therapeutic utility of the drug combination was investigated on tumor growth and progression using intracranially injected U87EGFRvIII GBM xenografts. RESULTS:Afatinib and TMZ combination synergistically inhibited the proliferation, clonogenic survival, motility, invasion and induced senescence of GBM cells compared to monotherapy. Mechanistically, afatinib decreased U87EGFRvIII GBM cell proliferation and motility/invasion by inhibiting EGFRvIII/AKT, EGFRvIII/JAK2/STAT3, and focal adhesion kinase (FAK) signaling pathways respectively. Interestingly, afatinib specifically inhibited EGFRvIII-cMET crosstalk in CSCs, resulting in decreased expression of Nanog and Oct3/4, and in combination with TMZ significantly decreased their self-renewal property in vitro. More interestingly, afatinib and TMZ combination significantly decreased the xenograft growth and progression compared to single drug alone. CONCLUSION:Our study demonstrated significant inhibition of GBM tumorigenicity, CSC maintenance in vitro, and delayed tumor growth and progression in vivo by combination of afatinib and TMZ. Our results warrant evaluation of this drug combination in EGFR and EGFRvIII amplified GBM patients.
Project description:The efficacy of novel targeted therapies is often tested at the time of tumor recurrence. However, for glioblastoma (GBM) patients, surgical resections at recurrence are performed only in a minority of patients; therefore, molecular data are predominantly derived from the initial tumor. Molecular data of the initial tumor for patient selection into personalized medicine trials can therefore be used only when the specific genetic change is retained in the recurrent tumor.In this study we determined whether EGFR amplification and expression of the most common mutation in GBMs (EGFRvIII) is retained at tumor recurrence. Because retention of genetic changes may be dependent on the initial treatment, we only used a cohort of GBM samples that were uniformly treated according to the current standard of care (ie, chemo-irradiation with temozolomide).Our data show that, in spite of some quantitative differences, the EGFR amplification status remains stable in the majority (84%) of tumors evaluated. EGFRvIII expression remained similar in 79% of GBMs. However, within the tumors expressing EGFRvIII at initial diagnosis, approximately one-half lose their EGFRvIII expression at tumor recurrence.The relative stability of EGFR amplification indicates that molecular data obtained in the primary tumor can be used to predict the EGFR status of the recurrent tumor, but care should be taken in extrapolating EGFRvIII expression from the primary tumor, particularly when expressed at first diagnosis.
Project description:The EGF-receptor (EGFR) is amplified and mutated in glioblastoma (GBM) where its common mutation, (∆EGFR, also called EGFRvIII) has a variety of activities that promote growth and inhibit death, thereby conferring a strong tumor-enhancing effect. This range of activities suggested to us that ∆EGFR might exert its influence through pleiotropic effectors and we hypothesized that microRNAs (miRs) might serve such a function. To test this, we determined the miR profiles of GBM cells with activated wild type EGFR (wtEGFR) and mutant EGFR (∆EGFR) to cells with non-activated EGFR or kinase dead ∆EGFR. To identify miRs regulated by EGFR, RNA from 2 different glioma cell lines (U87 and U373) were hybridized to miR expression arrays and analyzed. Each cell type was engineered to express wild type EGFR (wtEGFR), dead kinase ∆EGFR (DK) or ∆EGFR at elevated levels similar to those observed in primary glioblastomas displaying EGFR overexpression. Parental cells expressing endogenous EGFR and wtEGFR cells stimulated with EGF for 1hr were also included in the analyses.
Project description:Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-? production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB.