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The molecular signature that defines tumor microvasculature will likely provide clues as to how vascular-dependent tumor proliferation is regulated. Using purified endothelial cells, we generated a database of gene expression changes accompanying vascular proliferation in invasive breast cancer. In contrast to normal mammary vasculature, invasive breast cancer vasculature expresses extracellular matrix and surface proteins characteristic of proliferating and migrating endothelial cells. We define and validate the up-regulated expression of VE-cadherin and osteonectin in breast tumor vasculature. In contrast to other tumor types, invasive breast cancer vasculature induced a high expression level of specific transcription factors, including SNAIL1 and HEYL, that may drive gene expression changes necessary for breast tumor neovascularization. We demonstrate the expression of HEYL in tumor endothelial cells and additionally establish the ability of HEYL to both induce proliferation and attenuate programmed cell death of primary endothelial cells in vitro. We also establish that an additional intracellular protein and previously defined metastasis-associated gene, PRL3, appears to be expressed predominately in the vasculature of invasive breast cancers and is able to enhance the migration of endothelial cells in vitro. Together, our results provide unique insights into vascular regulation in breast tumors and suggest specific roles for genes in driving tumor angiogenesis.

Patients with non–small cell lung carcinoma (NSCLC) with activating mutations in epidermal growth factor receptor (EGFR) initially respond well to the EGFR inhibitors erlotinib and gefitinib. However, all patients relapse because of the emergence of drug-resistant mutations, with T790M mutations accounting for approximately 60% of all resistance. Second-generation irreversible EGFR inhibitors are effective against T790M mutations in vitro, but retain affinity for wild-type EGFR (EGFRWT). These inhibitors have not provided compelling clinical benefit in T790M-positive patients, apparently because of dose-limiting toxicities associated with inhibition of EGFRWT. Thus, there is an urgent clinical need for therapeutics that overcome T790M drug resistance while sparing EGFRWT. Here, we describe a lead optimization program that led to the discovery of four potent irreversible 2,4-diaminopyrimidine compounds that are EGFR mutant (EGFRmut) selective and have been designed to have low affinity for EGFRWT. Pharmacokinetic and pharmacodynamic studies in H1975 tumor–bearing mice showed that exposure was dose proportional resulting in dose-dependent EGFR modulation. Importantly, evaluation of normal lung tissue from the same animals showed no inhibition of EGFRWT. Of all the compounds tested, compound 3 displayed the best efficacy in EGFRL858R/T790M-driven tumors. Compound 3, now renamed CO-1686, is currently in a phase I/II clinical trial in patients with EGFRmut-advanced NSCLC that have received prior EGFR-directed therapy. Mol Cancer Ther; 13(6); 1468–79. ©2014 AACR.

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 EGFRT790M 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 first 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. Cancer Discov; 3(12); 1404–15. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 1317

Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45−/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow–derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC. [Mol Cancer Ther 2008;7(8):2536–46]

Protein tyrosine phosphatase PRL-3 mRNA was found highly expressed in colon cancer endothelium and metastases. We sought to associate a function with PRL-3 expression in both endothelial cells and malignant cells using in vitro models. PRL-3 mRNA levels were determined in several normal human endothelial cells exposed or unexposed to the phorbol ester phorbol 12-myristate 13-acetate (PMA) and in 27 human tumor cell lines. In endothelial cells, PRL-3 mRNA expression was increased in human umbilical vascular endothelial cells and human microvascular endothelial cells (HMVEC) exposed to PMA. An oligonucleotide microarray analysis revealed that PRL-3 was among the 10 genes with the largest increase in expression on PMA stimulation. Phenotypically, PMA-treated HMVEC showed increased invasion, tube formation, and growth factor–stimulated proliferation. A flow cytometric analysis of cell surface markers showed that PMA-treated HMVEC retained endothelial characteristics. Infection of HMVEC with an adenovirus expressing PRL-3 resulted in increased tube formation. In tumor cells, PRL-3 mRNA levels varied markedly with high expression in SKNAS neuroblastoma, MCF-7 and BT474 breast carcinoma, Hep3B hepatocellular carcinoma, and HCT116 colon carcinoma. Western blotting analysis of a subset of cell line lysates showed a positive correlation between PRL-3 mRNA and protein levels. PRL-3 was stably transfected into DLD-1 colon cancer cells. PRL-3-overexpressing DLD-1 subclones were assessed for doubling time and invasion. Although doubling time was similar among parental, empty vector, and PRL-3 subclones, invasion was increased in PRL-3-expressing subclones. In models of endogenous expression, we observed that the MCF-7 cell line, which expresses high levels of PRL-3, was more invasive than the SKBR3 cell line, which expresses low levels of PRL-3. However, the MDA-MB-231 cell line was highly invasive with low levels of PRL-3, suggesting that in some models invasion is PRL-3 independent. Transfection of a PRL-3 small interfering RNA into MCF-7 cells inhibited PRL-3 expression and cell invasion. These results indicate that PRL-3 is functional in both endothelial cells and malignant cells and further validate PRL-3 as a potentially important molecular target for anticancer therapy. [Mol Cancer Ther 2006;5(2):219–29]

Introduction: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations initially respond well to the EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib. However, clinical efficacy is limited by the development of resistance. The most common mechanism of resistance is a second site mutation within exon 20 of EGFR (T790M), observed in ∼50% of cases. CO-1686 is an irreversible kinase inhibitor that targets the mutant forms of EGFR by inhibiting the common activating mutations (L858R, delE746-A750) and the gatekeeper mutation (T790M) but not the wild-type receptor. Therefore, CO-1686 has the potential to effectively treat first- and second-line NSCLC patients with EGFR mutations without causing the dose limiting toxicities associated with approved EGFR kinase inhibitors or those in clinical development. Experimental procedures: Using structure-based drug design, CO-1686, a covalent, irreversible small molecule, which selectively inhibits mutant EGFR, was identified. CO-1686 potency was assessed against four common EGFR mutations (L858R, delE746-A750, L858R/T790M and delE746-A750/T790M) using in vitro biochemical and cell-based assays. Antitumor activity of CO-1686 as a single agent was assessed in NSCLC xenograft models harboring EGFR mutations: H1975 (L858R/T790M) and HCC827 (delE746-A750). Pharmacodynamic studies were performed to evaluate effects on cell survival and EGFR signaling. Results: In vitro and in vivo pharmacology studies focused on evaluating CO-1686 potency in four EGFR mutations common in NSCLC patients: L858R, delE746-A750, L858R/T790M and delE746-A750/T790M. CO-1686 was shown to be active against all four EGFR mutants. Effects of CO-1686 on cell proliferation and EGFR signaling were evaluated in HCC827 cells (delE746-A750) and its erlotinib-resistant clone, HCC827-EPR harboring the second site mutation T790M (delE746-A750/T790M). CO-1686 inhibited cell proliferation in both cell lines equally (GI50 values of 12 nM in the parental line and 19 nM in the T790M-positive clone). In mouse xenograft studies, oral dosing of CO-1686 in H1975 double mutant (L858R/T790M) and in HCC827 single mutant (delE746-A750) models caused tumor shrinkage as a single agent in a dose-dependent manner. Different dosing schedules were explored. Conclusions: Our results establish CO-1686 as a potent, mutant-selective EGFR inhibitor with excellent in vivo activity in mice bearing tumors with activating EGFR mutations as well as the resistance mutation T790M. These data suggest that treatment with CO-1686 as a single agent can overcome T790M-mediated drug resistance in NSCLC. Initially, clinical development will focus on NSCLC patients with mutant EGFR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1791. doi:1538-7445.AM2012-1791

Introduction: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations initially respond well to EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by the development of resistance. The most common mechanism of resistance is a second site mutation within exon 20 of EGFR (T790M), observed in ∼50% of cases. Our goal was to develop a mutant-selective EGFR inhibitor that potently inhibits activating EGFR mutations as well as the T790M resistance mutation while sparing wild-type EGFR for the treatment of NSCLC patients. Such a drug has the potential to effectively treat first- and second-line NSCLC patients with EGFR mutations without causing the dose limiting toxicities associated with EGFR kinase inhibitors currently in clinical development. Experimental procedures: Using structure-based drug design, we identified CO-1686, a covalent, irreversible small molecule, which selectively inhibits mutant EGFR. We assessed antitumor activity of CO-1686 both in vitro and in vivo in two NSCLC cell lines harboring EGFR mutations: H1975 (EGFR L858R/T790M) and HCC827 (EGFR delE746-A750). We evaluated inhibition of EGFR phosphorylation and downstream signaling by immunoblot analysis in cells and tissue samples. IHC staining on skin samples was performed to address effects on wild-type EGFR. Results: CO-1686 is a potent inhibitor of cell proliferation and EGFR signaling in NSCLC cells harboring the single activating mutation EGFR delE746-A750 as well as the double mutation EGFR L858R/T790M. When administered orally, CO-1686 (3 − 100 mg/kg) significantly suppresses tumor growth of H1975 cells (L858R/T790M) in a dose-dependent manner causing tumor regressions at the highest dose (100 mg/kg) without affecting body weight. Erlotinib at the same dose exhibits no effect against H1975 xenografts. In HCC827 (delE746-A750) xenografts, both agents cause tumor shrinkage. In both NSCLC mouse models, inhibition of EGFR phosphorylation in tumors correlate with the observed anti-tumor activity, while no effect on EGFR signaling is observed in normal lung or skin tissues with CO-1686 treatment, confirming that CO-1686 does not inhibt wild-type EGFR. Conclusions: Our results establish CO-1686 as a mutant-selective, wild-type sparing EGFR inhibitor with in vivo efficacy against tumors with activating EGFR mutations as well as the resistance mutation T790M. These data suggest that treatment with CO-1686 as a single agent can overcome T790M-mediated drug resistance in NSCLC. This hypothesis will be tested clinically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C189.