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Deregulation of the phosphoinositide 3-kinase (PI3K) signaling pathway such as by PTEN loss or PIK3CA mutation occurs frequently in human cancer and contributes to resistance to antitumor therapies. Inhibition of key signaling proteins in the pathway therefore represents a valuable targeting strategy for diverse cancers. PF-04691502 is an ATP-competitive PI3K/mTOR dual inhibitor, which potently inhibited recombinant class I PI3K and mTOR in biochemical assays and suppressed transformation of avian fibroblasts mediated by wild-type PI3K γ, δ, or mutant PI3Kα. In PIK3CA-mutant and PTEN-deleted cancer cell lines, PF-04691502 reduced phosphorylation of AKT T308 and AKT S473 (IC50 of 7.5–47 nmol/L and 3.8–20 nmol/L, respectively) and inhibited cell proliferation (IC50 of 179–313 nmol/L). PF-04691502 inhibited mTORC1 activity in cells as measured by PI3K-independent nutrient stimulated assay, with an IC50 of 32 nmol/L and inhibited the activation of PI3K and mTOR downstream effectors including AKT, FKHRL1, PRAS40, p70S6K, 4EBP1, and S6RP. Short-term exposure to PF-04691502 predominantly inhibited PI3K, whereas mTOR inhibition persisted for 24 to 48 hours. PF-04691502 induced cell cycle G1 arrest, concomitant with upregulation of p27 Kip1 and reduction of Rb. Antitumor activity was observed in U87 (PTEN null), SKOV3 (PIK3CA mutation), and gefitinib- and erlotinib-resistant non–small cell lung carcinoma xenografts. In summary, PF-04691502 is a potent dual PI3K/mTOR inhibitor with broad antitumor activity. PF-04691502 has entered phase I clinical trials. Mol Cancer Ther; 10(11); 2189–99. ©2011 AACR.

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Deregulation of the phosphoinositide 3-kinase (PI3K) signaling pathway such as by PTEN loss or PIK3CA mutation occurs frequently in human cancer and contributes to resistance to antitumor therapies. Inhibition of key signaling proteins in the pathway therefore represents a valuable targeting strategy for diverse cancers. PF-04691502 is an ATP-competitive PI3K/mTOR dual inhibitor, which potently inhibited recombinant class I PI3K and mTOR in biochemical assays and suppressed transformation of avian fibroblasts mediated by wild-type PI3K γ, δ, or mutant PI3Kα. In PIK3CA-mutant and PTEN-deleted cancer cell lines, PF-04691502 reduced phosphorylation of AKT T308 and AKT S473 (IC50 of 7.5–47 nmol/L and 3.8–20 nmol/L, respectively) and inhibited cell proliferation (IC50 of 179–313 nmol/L). PF-04691502 inhibited mTORC1 activity in cells as measured by PI3K-independent nutrient stimulated assay, with an IC50 of 32 nmol/L and inhibited the activation of PI3K and mTOR downstream effectors including AKT, FKHRL1, PRAS40, p70S6K, 4EBP1, and S6RP. Short-term exposure to PF-04691502 predominantly inhibited PI3K, whereas mTOR inhibition persisted for 24 to 48 hours. PF-04691502 induced cell cycle G1 arrest, concomitant with upregulation of p27 Kip1 and reduction of Rb. Antitumor activity was observed in U87 (PTEN null), SKOV3 (PIK3CA mutation), and gefitinib- and erlotinib-resistant non–small cell lung carcinoma xenografts. In summary, PF-04691502 is a potent dual PI3K/mTOR inhibitor with broad antitumor activity. PF-04691502 has entered phase I clinical trials. Mol Cancer Ther; 10(11); 2189–99. ©2011 AACR.

A 10 yr old female cat presented for an acute onset of back arching, regurgitation, and open mouth breathing. Radiographs indicated the presence of a large intrathoracic mass. Computed tomography confirmed the presence of a large mass of fatty density in the dorso-caudal mediastinum. The mass was removed via right intercostal thoracotomy, and histopathology confirmed the mass as a lipoma. The cat was continuing to recover well as of 21 mo after surgery. This is the first reported case of an intrathoracic lipoma in a cat.

Aberrant apoptosis-mediated cell death is believed to result in a number of different human diseases. For example, excessive apoptosis in the liver can result in fulminant and autoimmune forms of hepatitis. We have explored the possibility that inhibition of Fas expression in mice would reduce the severity of fulminant hepatitis. To do this, we have developed a chemically modified 2'-O-(2-methoxy)ethyl antisense oligonucleotide (ISIS 22023) inhibitor of mouse Fas expression. In tissue culture, this oligonucleotide induced a reduction in Fas mRNA expression that was both concentration- and sequence-specific. In Balb/c mice, dosing with ISIS 22023 reduced Fas mRNA and protein expressions in liver by 90%. The ID50 for this response was 8-10 mg kg-1 daily dosing, and the reduction was highly dependent on oligonucleotide sequence, oligonucleotide concentration in liver, and treatment time. Pretreatment with ISIS 22023 completely protected mice from fulminant hepatitis induced by agonistic Fas antibody, by a mechanism entirely consistent with an oligonucleotide antisense mechanism of action. In addition, oligonucleotide-mediated suppression of Fas expression reduced the severity of acetaminophen-mediated fulminant hepatitis, but was without effect on concanavalin A-mediated hepatitis. Our results demonstrate that 2'-O-(2-methoxy)ethyl containing antisense oligonucleotides targeting Fas can exert in vivo pharmacological activity in liver, and suggest that oligonucleotide inhibitors of Fas may be useful in the treatment of human liver disease.

Matrix metalloproteases (MMPs) secreted by both tumor and endothelial cells proteolytically degrade collagen during tumor growth and neo-vascularization. This exposes cryptic binding sites on collagen with functional relevance for angiogenesis. In this report, we characterized a novel humanized monoclonal IgG1 antibody, D93. After humanization, the antibody retained the binding specificity of the parental murine IgM antibody for denatured (dn) collagen. D93 recognized dn-collagen but not native (nat) collagen of different species, including mouse, chicken, and human, indicating that its cryptic binding site(s) is conserved across species. In immunohistochemistry (IHC) studies, D93 stained the basement membrane of blood vessels in several xenograft human tumors or in surgically removed tumor tissues from patients with different types of malignancies. D93 staining was rarely or not present in normal blood vessels of healthy tissues. In in vivo experiments, D93 significantly inhibited basic fibroblast growth factor (bFGF)-induced angiogenesis in chick embryo chorioallantoic membrane (CAM) and the tumor growth of pre-established orthotopic human breast (MDA-MB-435) tumors in mice. D93 i.v. administered in mice was subsequently detected in the subendothelial basement membrane of tumor blood vessels but not blood vessels of normal tissues. Inhibition of growth of pre-established orthotopic human breast MDA-MB-435 tumors was more effective when D93 was combined with Taxol, than either treatment alone. In addition, tumors from animals treated with D93 and/or Taxol showed significantly reduced levels of the endothelial cell-marker CD31. Our data suggest that blockade of cryptic epitopes exposed on collagen IV during angiogenesis and tumor growth by a monoclonal antibody may provide a novel therapeutic modality for treatment of cancer and pathogenic neo-vascularization.

EML4-ALK fusion was recently characterized as an “Addicted Oncogene” in a subset of human lung adenocarcinoma, and it plays an essential role in regulation of the tumor cell survival, growth and metastasis. Crizotinib is a potent and selective ATP competitive small molecule inhibitor of ALK and c-Met. It is currently in clinical trials for advanced non-small cell lung cancers positive for ALK fusion. This report summarizes the pre-clinical pharmacology studies for crizotinib in the EML4-Alk positive tumor models to assess the pharmacodynamic inhibition of EML4-ALK, antitumor efficacy, PKPD relationships and antitumor mechanism of action in vitro and in vivo. Crizotinib potently inhibited the catalytic activity of ALK kinase (Ki = 0.5 nM) and the autophosphorylation of cellular EML4-ALK V1, V2, V3a and V3b with IC50 values ranging from 26–74 nM. Crizotinib also inhibited cell proliferation and induced apoptosis in NCI-H3122 human NSCLC cells harboring EML4-ALK V1 fusion with IC50s of 63 nM and 110 nM respectively. In the EML4-ALK V3a/b positive NCI-H2228 human NSCLC cells, crizotinib completely inhibited ALK phosphorylation (IC50 = 74 nM) but only partial inhibited cell proliferation and survival. This observation is consistent with the notion that only a portion of H2228 cells are EML4-ALK positive by Exon Array analysis. Crizotinib demonstrated marked tumor growth inhibition and regression in H3122 xenograft model at well tolerated dose levels. The antitumor efficacy of crizotinib was dose dependent and demonstrated a strong correlation to pharmacodynamic inhibition of ALK phosphorylation in vivo. PKPD modeling was conducted to understand the relationships between crizotinib plasma concentration to ALK target inhibition (EC50 = 19 nM) and antitumor efficacy (EC50 = 23 nM). Collectively, the results from H3122 model indicated that significant inhibition of EML4-ALK during the entire treatment period was necessary to achieve robust antitumor efficacy. Additional in vivo studies with crizotinib demonstrated dose dependent inhibition of EML4-ALK mediated signal transduction (STAT3, AKT, Erk, PLCγ, c-Myc), tumor cell proliferation (Ki67) and induction of apoptosis (caspase-3). Crizotinib also dose dependently inhibited total EML4-ALK levels in H3122 tumors indicating an additional antitumor mechanism of action by crizotinib in these tumors. Furthermore, a dose dependent increase in phospho-EGFR levels was observed after 4 days of drug treatment in H3122 model, indicating a compensation mechanism of “oncogene switching” in tumor cell signaling, and a potential resistance mechanism that may compromise “patient” responses to crizotinib treatment in these tumors. In conclusion, crizotinib was shown to be a potent inhibitor of EML4-ALK. It demonstrated marked antitumor efficacy in EML4-ALK dependent human NSCLC tumors in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-390. doi:10.1158/1538-7445.AM2011-LB-390