NF-κB drives acquired resistance to a novel mutant-selective EGFR inhibitor.

The clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) harbouring activating EGFR mutations is limited by the emergence of acquired resistance, mostly ascribed to the secondary EGFR-T790M mutation. Selective EGFR-T790M inhibitors have been proposed as a new, extremely relevant therapeutic approach. Here, we demonstrate that the novel irreversible EGFR-TKI CNX-2006, a structural analog of CO-1686, currently tested in a phase-1/2 trial, is active against in vitro and in vivo NSCLC models expressing mutant EGFR, with minimal effect on the wild-type receptor. By integration of genetic and functional analyses in isogenic cell pairs we provide evidence of the crucial role played by NF-κB1 in driving CNX-2006 acquired resistance and show that NF-κB activation may replace the oncogenic EGFR signaling in NSCLC when effective and persistent inhibition of the target is achieved in the presence of the T790M mutation. In this context, we demonstrate that the sole, either genetic or pharmacologic, inhibition of NF-κB is sufficient to reduce the viability of cells that adapted to EGFR-TKIs. Overall, our findings support the rational inhibition of members of the NF-κB pathway as a promising therapeutic option for patients who progress after treatment with novel mutant-selective EGFR-TKIs.

Discovery of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC.

UNLABELLED: Patients with non-small cell lung cancer (NSCLC) with activating EGF receptor (EGFR) mutations initially respond to first-generation reversible EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by acquired resistance, frequently driven by the EGFR(T790M) mutation. CO-1686 is a novel, irreversible, and orally delivered kinase inhibitor that specifically targets the mutant forms of EGFR, including T790M, while exhibiting minimal activity toward the wild-type (WT) receptor. Oral administration of CO-1686 as single agent induces tumor regression in EGFR-mutated NSCLC tumor xenograft and transgenic models. Minimal activity of CO-1686 against the WT EGFR receptor was observed. In NSCLC cells with acquired resistance to CO-1686 in vitro, there was no evidence of additional mutations or amplification of the EGFR gene, but resistant cells exhibited signs of epithelial-mesenchymal transition and demonstrated increased sensitivity to AKT inhibitors. These results suggest that CO-1686 may offer a novel therapeutic option for patients with mutant EGFR NSCLC. SIGNIFICANCE: We report the preclinical development of a novel covalent inhibitor, CO-1686, that irreversibly and selectively inhibits mutant EGFR, in particular the T790M drug-resistance mutation, in NSCLC models. CO-1686 is the fi rst drug of its class in clinical development for the treatment of T790M-positive NSCLC, potentially offering potent inhibition of mutant EGFR while avoiding the on-target toxicity observed with inhibition of the WT EGFR.

Adventitial endothelial implants reduce matrix metalloproteinase-2 expression and increase luminal diameter in porcine arteriovenous grafts.

OBJECTIVE: Vascular access dysfunction is a major problem in hemodialysis patients. Only 50% of arteriovenous grafts (AVGs) will remain patent 1 year after surgery. AVGs frequently develop stenoses and occlusions at the venous anastomoses in the venous outflow tract. Lumen diameter is not only determined by intimal thickening but is also influenced by remodeling of the vessel wall. Vascular remodeling requires degradation and reorganization of the extracellular matrix by the degradation enzymes, matrix metalloproteinases (MMPs). In this study, we aimed to provide further insight into the mechanism of endothelial regulation of vascular remodeling and luminal narrowing in AVGs. METHODS: End-to-side carotid artery-jugular vein polytetrafluoroethylene grafts were created in 20 domestic swine. The anastomoses and outflow vein were treated with Gelfoam matrices (Pfizer, New York, NY) containing allogeneic porcine aortic endothelial (PAE, n = 10) cells or control matrices without cells (n = 10), and the biologic responses to PAE implants were investigated 3 and 28 days postoperatively. Angiograms before euthanasia were compared with baseline angiograms. Tissue sections were stained with hematoxylin and eosin, Verhoeff elastin, and antibodies specific to MMP-9 and MMP-2 and underwent histopathologic, morphometric and immunohistochemical analysis. RESULTS: Veins treated with PAE cell implants had a 2.8-fold increase in venous lumen diameter compared with baseline (P < .05), a 2.3-fold increase in lumen diameter compared with control, and an 81% decrease in stenosis (P < .05) compared with control at 28 days. The increase in lumen diameter by angiographic analysis correlated with morphometric analysis of tissue sections. PAE implants increased the venous lumen area 2.3-fold (P < .05), decreased venous luminal occlusion 66%, and increased positive venous remodeling 1.9-fold (P < .05) compared with control at 28 days. PAE cell implants reduced MMP-2 expression and neovascularization at 3 and 28 days and adventitial fibrosis at 28 days, suggesting a role of the implants in controlling the affects of medial and adventitial cells in the response to vascular injury. CONCLUSIONS: These results demonstrate that the adventitial application of endothelial implants significantly reduced MMP-2 expression within the venous wall, and increased venous lumen diameter and positive remodeling in a porcine arteriovenous graft model. Adventitial endothelial implants may be useful in decreasing luminal narrowing in a clinical setting.

Laminin modulates morphogenic properties of the collagen XVIII endostatin domain.

We have shown previously that the oligomeric endostatin domain of collagen XVIII (NC1) functioned as a motility-inducing factor regulating the extracellular matrix-dependent morphogenesis of endothelial cells. This motogenic activity gave rise to structures resembling filipodia and lamellipodia and was dependent on Rac, Cdc42, and mitogen-activated protein kinase. Here, we demonstrate that these properties of endostatin are primarily mediated by laminin in the basement membrane and heparan sulfates on the cell surface. The sites of interaction between laminin and oligomeric endostain include the N-terminal regions of all three laminin chains (amino acids 204-1243 of the alpha chain, 932-1161 of the beta chain, and 150-965 of the gamma chain). A monoclonal antibody that blocks the interactions between endostatin and laminin was utilized to inhibit the motogenic activity of endostatin. In parallel, we have engineered selective point mutations and produced recombinant forms that lack binding to heparan sulfates on the cell surface. Our data are consistent with a model of endostatin with two binding sites: one mainly to laminin in the basement membrane and the other to heparan sulfates on the cell surface. The two binding domains on endostatin appear to be separate with the possibility of some overlap between the two sites.