Discovery of N-aryl-N′-pyrimidin-4-yl ureas as irreversible L858R/T790M mutant selective epidermal growth factor receptor inhibitors

A novel series of N-aryl-N′-pyrimidin-4-yl ureas has been optimized to afford potent and selective inhibitors of the EGFR L858R/T790M. The most representative compound 28 showed high activity against EGFR L858R/T790M kinase (IC₅₀?=?4?nM) and 22-fold selectivity against wild type EGFR. Moreover, compound 28 potently inhibited EGFR L858R/T790M phosphorylation (IC₅₀?=?41?nM) and cellular proliferation (IC₅₀?=?37?nM) in the H1975 cell line, while being significantly less toxic to A431 cells. Further, compound 28 exhibited a great selectivity in a mini-panel of kinases.     

Gefitinib induces epidermal growth factor receptor dimers which alters the interaction characteristics with ¹²⁵I-EGF

The tyrosine kinase inhibitor gefitinib inhibits growth in some tumor types by targeting the epidermal growth factor receptor (EGFR). Previous studies show that the affinity of the EGF-EGFR interaction varies between hosting cell line, and that gefitinib increases the affinity for some cell lines. In this paper, we investigate possible mechanisms behind these observations. Real-time interaction analysis in LigandTracer® Grey revealed that the HER2 dimerization preventing antibody pertuzumab clearly modified the binding of ¹²⁵I-EGF to EGFR on HER2 overexpressing SKOV3 cells in the presence of gefitinib. Pertuzumab did not affect the binding on A431 cells, which express low levels of HER2. Cross-linking measurements showed that gefitinib increased the amount of EGFR dimers 3.0–3.8 times in A431 cells in the absence of EGF. In EGF stimulated SKOV3 cells the amount of EGFR dimers increased 1.8–2.2 times by gefitinib, but this effect was cancelled by pertuzumab. Gefitinib treatment did not alter the number of EGFR or HER2 expressed in tumor cell lines A431, U343, SKOV3 and SKBR3. Real-time binding traces were further analyzed in a novel tool, Interaction Map, which deciphered the different components of the measured interaction and supports EGF binding to multiple binding sites. EGFR and HER2 expression affect the levels of EGFR monomers, homodimers and heterodimers and EGF binds to the various monomeric/dimeric forms of EGFR with unique binding properties. Taken together, we conclude that dimerization explains the varying affinity of EGF – EGFR in different cells, and we propose that gefitinib induces EGFR dimmers, which alters the interaction characteristics with ¹²⁵I-EGF.     

Epidermal growth factor, transforming growth factor-α, and epidermal growth factor receptor expression and localization in the canine endometrium during the estrous cycle and in bitches with pyometra

Gene expression and immunohistochemical localization of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and epidermal growth factor receptor (EGF-R) were compared between the endometrium of bitches (Canis familiaris) with pyometra accompanied by cystic endometrial hyperplasia (CEH) and that of healthy bitches at similar stages of the estrous cycle. In normal bitches, endometrial TGF-α mRNA levels were highest at proestrus and gradually decreased as the cycle progressed to anestrus. Epidermal growth factor receptor mRNA levels were not significantly affected by the stage of the estrous cycle. Epidermal growth factor mRNA levels were higher at Day 35 of diestrus than at other stages of the estrous cycle (P < 0.05). In bitches with pyometra, endometrial TGF-α and EGF-R mRNA levels did not differ significantly from those at diestrus in normal bitches, but EGF mRNA levels were lower than those at Day 35 of diestrus in normal bitches (P < 0.05). In normal bitches, positive immunohistochemical staining for TGF-α, EGF, and EGF-R was mainly present in the glandular and luminal epithelial cells of the endometrium. In contrast, in bitches with pyometra, immunoreactivity for EGF was clearly present in endometrial stromal cells. Inflammatory cells that had infiltrated the endometrial stroma stained strongly for TGF-α and EGF-R. Luminal and glandular epithelial cells also stained positive for EGF-R. In conclusion, expression of TGF-α by inflammatory cells and a low level of expression and differential localization of EGF may be involved in aberrant growth of endometrial glands and development of CEH.     

Production of macrophage-activated killer cells for targeting of glioblastoma cells with bispecific antibody to FcγRI and the epidermal growth factor receptor

Objective: The aim was to determine the ability of macrophage-activated killer cells (MAK cells) obtained from peripheral blood of normal volunteers to kill glioblastoma multiforme (GBM) cell lines. Another goal was to investigate whether a bispecific antibody (bsAb) MDX-447, recognizing the high-affinity Fc receptor for IgG (FcγRI) and epidermal growth factor receptor (EGFR), would enhance MAK cell tumoricidal activity. Methods: Monocytes, from leukapheresis product, were isolated by countercurrent elutriation and differentiated into MAK cells by culture with granulocyte/macrophage-colony-stimulating factor, vitamin D₃ and interferon γ. Cells were checked for sterility, endotoxin and phenotypic markers. MAK cell functional activity was measured by a flow-cytometric phagocytosis assay. Target cells, a carcinoma cell line and two glioma cell lines expressing EGFR, were stained with PKH-26. MAK cells were labeled with fluorescein-conjugated anti-CD14. Combined effectors, targets and bsAb were incubated and the percentage of MAK cells with phagocytosed targets was determined by flow cytometry. Conclusion: We demonstrate that a large number of highly purified monocytes, isolated from peripheral blood, can be differentiated into MAK cells for use as an adjuvant for cancer treatment. After culture these cells are sterile, endotoxin-free and comprise more than 95% MAK cells. Increased amounts of CD14, CD64 and HLA-DR, which are characteristics of macrophage activation, were expressed. MAK cells were extremely phagocytic in comparison to monocytes, even in the absence of bsAb. Moreover, bsAb enhanced the tumoricidal activity of elutriated MAK cells targeted against GBM cell lines. Therefore, intracavity MAK cells armed with MDX-447 could be an effective adoptive immunotherapy for EGFR-positive GBM. Received: 5 April 2000 / Accepted: 12 July 2000     

CD19 chimeric antigen receptor–redirected T cells combined with epidermal growth factor receptor pathway substrate 8 peptide–derived dendritic cell vaccine in leukemia

BackgroundChimeric antigen receptor (CAR)–T cell therapy opens a new era for cancer treatment. However, in prolonged follow-up, relapse has emerged as one of the major obstacles. Dendritic cell (DC) vaccination is a promising treatment to eradicate tumor cells and prevent relapse. The epidermal growth factor receptor (EGFR) pathway substrate 8 (Eps8) gene is involved in regulating cancer progression and is considered an attractive target for specific cancer immunotherapy. The purpose of this study was to explore a combinatorial therapy using CAR-T cells and a DC vaccine such as Eps8-DCs to increase leukemia treatment efficacy.MethodsWe pulsed DCs with Eps8-derived peptides to generate Eps8-DCs, engineered T cells to express a second-generation CAR specific for CD19, and analyzed the effects of the Eps8-DCs on the in vitro expansion, phenotype and effector functions of the CD19 CAR-T cells.ResultsThe Eps8-DCs significantly reduced the activation-induced cell death and enhanced the proliferative potential of CAR-T cells during in vitro expansion. In addition, the expanded T cells co-cultured with the Eps8-DCs exhibited an increased percentage of central memory T cells (Tcms) and a decreased percentage of effector memory T cells (Tems). The Eps8-DCs enhanced CD19 CAR-T cell immune functions, including cytokine production, CD107a degranulation activity and cytotoxicity.DiscussionThis study demonstrates that Eps8-DCs exert synergistic effect on CD19 targeting CAR-T cells and paves the way for clinical trials using the combination of DC vaccination and engineered T cells in relapsed leukemia.     

CD8+T cells from a patient with colon carcinoma,specific for a mutant p21-Ras-derived peptide (GLY13→ASP), are cytotoxic towards a carcinoma cell line harbouring the same mutation

Several T lymphocyte clones (TLC), specific for a p21-Ras-derived peptide expressing a Gly¹³Asp mutation and of the CD8⁺ subtype, were generated from peripheral blood of a colon carcinoma patient. The TLC exerted cytotoxicity against an interferon- (IFN)-pretreated colon carcinoma cell line, HCT116, which harbours the Gly¹³Asp mutation and shares both HLA-A2 and HLA-B12(44) with the patient. This cytotoxic effect could be blocked by a monoclonal antibody (mAb) against CD8 molecules, as well as with a mAb against HLA class I molecules and a polyclonal antiserum against HLA-B12, identifying B12(44) as the antigen-presenting molecule. In growth-inhibition experiments, the growth of both IFN-pretreated and untreated target cells were strongly inhibited by the presence of the CD8⁺TLC. Together these data indicate that human cancer cells harbouring a spontaneousras mutation can process aberrant p21 Ras and express peptide/HLA-class-I complexes on their surface in sufficient density to be recognized by Ras-specific cytotoxic T lymphocytes.     

Thyroid hormone is a MAPK-dependent growth factor for human myeloma cells acting via αvβ3 integrin

Experimental and clinical observations suggest that thyroid hormone [l-thyroxine (T(4)) and 3,5,3'-triiodo-l-thyronine (T(3))] can support cancer cell proliferation. T(3) and T(4) promote both tumor cell division and angiogenesis by activating mitogen-activated protein kinase (MAPK) via binding to a hormone receptor on the αvβ3 integrin, overexpressed on many cancer cells. We have studied the responsiveness of several MM cell lines to T(3) and T(4) and characterized hormonal effects on cell survival, proliferation, and MAPK activation. Overnight T(3) (1-100 nmol/L) and T(4) (100 nmol/L) incubation enhanced, up to 50% (P < 0.002), MM cell viability (WST-1 assay) and increased cell proliferation by 30% to 60% (P < 0.01). Short exposure (10 minutes) to T(3) and T(4) increased MAPK activity by 2.5- to 3.5-fold (P < 0.03). Pharmacologic MAPK inhibition blocked the proliferative action of T(3) and T(4). Antibodies to the integrin αvβ3 dimer and αv and β3 monomers (but not β1) inhibited MAPK activation and subsequent cell proliferation in response to thyroid hormone, indicating dependence upon this integrin. Moreover, tetraiodothyroacetic acid (tetrac), a non-agonist T(4) analogue previously shown to selectively block T(3)/T(4) binding to αvβ3 receptor site, blocked induction of MAPK by the hormones in a dose-dependent manner. This demonstration of the role of thyroid hormones as growth factors for MM cells may offer novel therapeutic approaches.     

Estrogen receptor alpha is essential for the proliferation of prostatic smooth muscle cells stimulated by 17β-estradiol and insulin-like growth factor 1

Estradiol (E₂) and its receptor estrogen receptor alpha (ERα) are implicated in the pathology of stromal‐predominant benign prostatic hyperplasia (BPH). Insulin‐like growth factor 1(IGF1) is produced primarily by stromal cells in the prostate. Recent study showed that E₂‐mediated regulation of IGF1 in mouse uterus requires the DNA binding ability of ERα. However, the crosstalk between ERα and IGF1 in the prostate has not been addressed yet. Therefore, in this study we employed mouse prostatic smooth muscle cells (PSMCs) as a model to demonstrate that E₂ stimulated the proliferation of PSMCs and up‐regulated the expression of IGF1 and its receptor IGF1R as well as cyclin D1 in PSMCs, all of which could be inhibited by shRNA‐mediated knockdown of ERα. Furthermore, we found that exogenous IGF1 could not promote cell proliferation and cyclin D1 expression in PSMCs subjected to shRNA‐mediated knockdown of ERα. Interestingly, blockage of IGF1R by antibody could inhibit E₂‐stimulated PSMCs proliferation. Altogether our present study provides the first line of evidence that there is crosstalk between ERα and IGF1 signaling in PSMCs proliferation in which ERα up‐regulates the expression of IGF1 and IGF1R, and IGF1 signaling is indispensable to mediate downstream effects of E₂ and ERα. Copyright © 2011 John Wiley & Sons, Ltd.     

Therapeutic potential of the epidermal growth factor receptor transactivation in hypertension: a convergent signaling pathway of vascular tone, oxidative stress, and hypertrophic growth downstream of vasoactive G-protein-coupled receptors?

The concurrence of enhanced vascular tone, oxidative stress, and hypertrophic growth is a hallmark of hypertension, the condition characterized by sustained elevated blood pressure. However, it is unclear how and why such apparently distinct processes coincide in hypertension. Elevated levels of certain vasoactive G-protein-coupled receptor agonists (such as catecholamines, endothelin-1, and angiotensin II) can explain, at least in part, the development and progression of many hypertensive disorders. Here, we review findings made by other investigators and ourselves suggesting that enhanced vascular tone, oxidative stress, and hypertrophic growth characteristically induced by these agonists involve the transactivation of growth factor receptors. The first step in this transactivation mechanism is agonist-induced activation of metalloproteinase-dependent shedding of growth factors. Shed growth factors then trigger intracellular signaling cascades necessary for growth, production of reactive oxygen species, and maintenance of vascular tone. If this hypothesis is proven generally correct, then transactivation blockers have general therapeutic potential in hypertension regardless of the causative agonist.     

Neural cell adhesion molecule potentiates the growth of murine melanoma via β-catenin signaling by association with fibroblast growth factor receptor and glycogen synthase kinase-3β

The neural cell adhesion molecule (NCAM) was recently shown to be involved in the progression of various tumors with diverse effects. We previously demonstrated that NCAM potentiates the cellular invasion and metastasis of melanoma. Here we further report that the growth of melanoma is obviously retarded when the expression of NCAM is silenced. We found that the proliferation of murine B16F0 melanoma cells, their colony formation on soft agar, and growth of transplanted melanoma in vivo are clearly inhibited by the introduction of NCAM siRNA. Interestingly, change of NCAM expression level is shown to regulate the activity of Wnt signaling molecule, β-catenin, markedly. This novel machinery requires the function of FGF receptor and glycogen synthase kinase-3β but is independent of the Wnt receptors, MAPK-Erk and PI3K/Akt pathways. In addition, NCAM is found to form a functional complex with β-catenin, FGF receptor, and glycogen synthase kinase-3β. Moreover, up-regulation of NCAM140 and NCAM180 appears more potent than NCAM120 in activation of β-catenin, suggesting that the intracellular domain of NCAM is required for facilitating the β-catenin signaling. Furthermore, the melanoma cells also exhibit distinct differentiation phenotypes with the NCAM silencing. Our findings reveal a novel regulatory role of NCAM in the progression of melanoma that might serve as a new therapeutic target for the treatment of melanoma.     

The SH3 domain, but not the catalytic domain, is required for phospholipase C-γ1 to mediate epidermal growth factor-induced mitogenesis

Phospholipase C-γ1 (PLC-γ1) is a multiple-domain protein and plays an important role in epidermal growth factor (EGF)-induced cell mitogenesis, but the underlying mechanism is unclear. We have previously demonstrated that PLC-γ1 is required for EGF-induced mitogenesis of squamous cell carcinoma (SCC) cells, but the mitogenic function of PLC-γ1 is independent of its lipase activity. Earlier studies suggest that the Src homology 3 (SH3) domain of PLC-γ1 possesses mitogenic activity. In the present study, we sought to determine the role of the SH3 domain of PLC-γ1 in EGF-induced SCC cell mitogenesis. We examined the effect of overexpression of PLC-γ1, a catalytically active PLC-γ1 mutant lacking the SH3 domain or a catalytically inactive PLC-γ1 mutant lacking the X domain on EGF-induced SCC4 (tongue squamous cell carcinoma) cell mitogenesis. We found that overexpression of PLC-γ1 enhanced EGF-induced SCC4 cell mitogenesis. This enhancement was abolished by deletion of the SH3 domain but not by deletion of the X catalytic domain. These data suggest that the SH3 domain, but not the catalytic domain, is required for PLC-γ1 to mediate EGF-induced SCC4 cell mitogenesis.     

Identification of a novel mutation of the gene for gap junction protein α3 (GJA3) in a Chinese family with congenital cataract

Cataract, defined as any opacity of the crystallin lens, can be divided into early onset (congenital or infantile) and age-related. It is the leading cause of visual disability in children, and mutations in many genes have currently been linked with this disorder. In the present study, we identified a genetic defect in a Chinese family with congenital cataract. Genomic DNA was extracted from the venous blood of the family and 100 normal controls. To screen for the disease-causing mutation, we sequenced eight candidate genes, and to predict the functional consequences of the mutation, a structural model of the protein was developed using the Protein Data Bank and PyMOL 1.1r1. We found a novel variant (c.163 A > G transition) in the gene for gap junction protein α3, or the connexin46 gene. This mutation resulted in the substitution of a highly conserved asparagine at codon 55 by aspartic acid (p.N55D). There were no nucleotide polymorphisms in the other candidate genes sequenced.     

Complete paternal uniparental isodisomy for chromosome 1 revealed by mutation analyses of the<Emphasis Type="Italic"> TRKA (NTRK1)</Emphasis> gene encoding a receptor tyrosine kinase for nerve growth factor in a patient with congenital insensitivity to pain with anhidrosis

Uniparental disomy (UPD) is defined as the presence of a chromosome pair that derives from only one parent in a diploid individual. The human TRKA gene on chromosome 1q21-q22 encodes a receptor tyrosine kinase for nerve growth factor and is responsible for an autosomal recessive genetic disorder: congenital insensitivity to pain with anhidrosis (CIPA). We report here the second case of paternal UPD for chromosome 1 in a male patient with CIPA who developed normally at term and did not show overt dysmorphisms or malformations. He had only the usual features of CIPA with a homozygous mutation at the TRKA locus and a normal karyotype with no visible deletions or evidence of monosomy 1. Haplotype analysis of the TRKA locus and allelotype analyses of whole chromosome 1 revealed that the chromosome pair was exclusively derived from his father. Non-maternity was excluded by analyses of autosomes other than chromosome 1. Thus, we have identified a complete paternal isodisomy for chromosome 1 as the cause of reduction to homozygosity of the TRKA gene mutation, leading to CIPA. Our findings further support the idea that there are no paternally imprinted genes on chromosome 1 with a major effect on phenotype. UPD must be considered as a rare but possible cause of autosomal recessive disorders when conducting genetic testing.