Anthony Polverino

Multiple ras functions can contribute to mammalian cell transformation

We have developed a generalized approach, using two hybrid interactions, to isolate Ha-Ras effector loop mutations that separate the ability of Ha-Ras to interact with different downstream effectors. These mutations attenuate or eliminate Ha-ras(G12V) transformation of mammalian cells, but retain complementary activity, as demonstrated by synergistic induction of foci of growth-transformed cells, and by the ability to activate different downstream components. The transformation defect of Ha-ras(G12V, E37G) is rescued by a mutant, raf1, that restores interaction. These results indicate that multiple cellular components, including Raf1, are activated by Ha-Ras and contribute to Ha-Ras-induced mammalian cell transformation.

Osteoprotegerin Ligand Modulates Murine Osteoclast Survival in Vitro and in Vivo

Osteoprotegerin ligand (OPGL) targets osteoclast precursors and osteoclasts to enhance differentiation and activation, however, little is known about OPGL effects on osteoclast survival. In vitro, the combination of OPGL + colony-stimulating factor-1 (CSF-1) is required for optimal osteoclast survival. Ultrastructurally, apoptotic changes were observed in detached cells and culture lysates exhibited elevated caspase 3 activity, particularly in cultures lacking CSF-1. DEVD-FMK (caspase 3 inhibitor) partially protected cells when combined with OPGL, but not when used alone or in combination with CSF-1. CSF-1 maintained NF-kappaB activation and increased the expression of bcl-2 and bcl-X(L) mRNA, but had no effect on JNK activation. In contrast, OPGL enhanced both NF-kappaB and JNK kinase activation and increased the expression of c-src, but not bcl-2 and bcl-X(L) mRNA. These data suggest that aspects of both OPGLs and CSF-1s signaling/survival pathways are required for optimal osteoclast survival. In mice, a single dose of OPG, the OPGL decoy receptor, led to a >90% loss of osteoclasts because of apoptosis within 48 hours of exposure without impacting osteoclast precursor cells. Therefore, OPGL is essential, but not sufficient, for osteoclast survival and endogenous CSF-1 levels are insufficient to maintain osteoclast viability in the absence of OPGL.

AMG 706, an Oral, Multikinase Inhibitor that Selectively Targets Vascular Endothelial Growth Factor, Platelet-Derived Growth Factor, and Kit Receptors, Potently Inhibits Angiogenesis and Induces Regression in Tumor Xenografts

The growth of solid tumors is dependent on the continued stimulation of endothelial cell proliferation and migration resulting in angiogenesis. The angiogenic process is controlled by a variety of factors of which the vascular endothelial growth factor (VEGF) pathway and its receptors play a pivotal role. Small-molecule inhibitors of VEGF receptors (VEGFR) have been shown to inhibit angiogenesis and tumor growth in preclinical models and in clinical trials. A novel nicotinamide, AMG 706, was identified as a potent, orally bioavailable inhibitor of the VEGFR1/Flt1, VEGFR2/kinase domain receptor/Flk-1, VEGFR3/Flt4, platelet-derived growth factor receptor, and Kit receptors in preclinical models. AMG 706 inhibited human endothelial cell proliferation induced by VEGF, but not by basic fibroblast growth factor in vitro, as well as vascular permeability induced by VEGF in mice. Oral administration of AMG 706 potently inhibited VEGF-induced angiogenesis in the rat corneal model and induced regression of established A431 xenografts. AMG 706 was well tolerated and had no significant effects on body weight or on the general health of the animals. Histologic analysis of tumor xenografts from AMG 706-treated animals revealed an increase in endothelial apoptosis and a reduction in blood vessel area that preceded an increase in tumor cell apoptosis. In summary, AMG 706 is an orally bioavailable, well-tolerated multikinase inhibitor that is presently under clinical investigation for the treatment of human malignancies.

Selective Activation of Caspases During Apoptotic Induction in HL-60 Cells EFFECTS OF A TETRAPEPTIDE INHIBITOR

Apoptosis is a highly regulated biochemical process that results in the selective death of cells. Members of the caspase family of cysteine proteases play a pivotal role in the effector phase of apoptosis. We show that, in HL-60 cells, the addition of either anisomycin, a protein synthesis inhibitor, or geranylgeraniol, an intermediate in the cholesterol biosynthetic pathway, results in a rapid and en masse induction of apoptosis. The levels of actin, p42 and p44 MAPK, JNK1, JNK2, p38, and PCNA were not substantially altered during this process. Although these treatments appear to function by diverse pathways, they both result in the processing and activation of caspase-3 (CPP32β/Yama/Apopain). In contrast, no activation of caspase-1 (interleukin-1β converting enzyme (ICE)) was observed. Furthermore, we obtained ambiguous results regarding the activation of caspase-2 (Ich-1) depending on the antibody used. Pretreatment of the cells with benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD.fmk), a tetrapeptide inhibitor of caspases, prevented the induction of apoptosis for 24 h. Even after 72 h of treatment, some cells were still alive and progressing through the cell cycle, suggesting that blockage of caspase activity is able to protect cells. These results suggest that selective activation of some caspases is necessary to induce apoptosis in HL-60 cells.

ACTIVATION OF MITOGEN-ACTIVATED PROTEIN KINASE CASCADES BY P21-ACTIVATED PROTEIN KINASES IN CELL-FREE EXTRACTS OF XENOPUS OOCYTES

In the evolutionarily distant yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, genetic evidence suggests that activation of pheromone-induced mitogen-activated protein kinase (MAPK) cascades involves the function of the p21-activated protein kinases (PAKs) Ste20 and Shk1, respectively. In this report, we show that purified Ste20 and Shk1 were each capable of inducing p42 activation in cell-free extracts of Xenopus laevis oocytes, while a mammalian Ste20/Shk1-related protein kinase, p65 (Pak1), did not induce activation of p42. In contrast to p42, activation of JNK/SAPK in Xenopus oocyte extracts was induced by both the yeast Ste20 and Shk1 kinases, as well as by mammalian Pak1. Our results demonstrate that MAPK cascades that are responsive to PAKs are conserved in higher eukaryotes and suggest that distinct PAKs may regulate distinct MAPK modules.

Naphthamides as novel and potent vascular endothelial growth factor receptor tyrosine kinase inhibitors: design, synthesis, and evaluation.

A series of naphthyl-based compounds were synthesized as potential inhibitors of vascular endothelial growth factor (VEGF) receptors. Investigations of structure-activity relationships led to the identification of a series of naphthamides that are potent inhibitors of the VEGF receptor tyrosine kinase family. Numerous analogues demonstrated low nanomolar inhibition of VEGF-dependent human umbilical vein endothelial cell (HUVEC) proliferation, and of these several compounds possessed favorable pharmacokinetic (PK) profiles. In particular, compound 48 demonstrated significant antitumor efficacy against established HT29 human colon adenocarcinoma xenografts implanted in athymic mice. A full account of the preparation, structure-activity relationships, pharmacokinetic properties, and pharmacology of analogues within this series is presented.

Evaluation of a Series of Naphthamides as Potent, Orally Active Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitors¶

We have previously shown N-arylnaphthamides can be potent inhibitors of vascular endothelial growth factor receptors (VEGFRs). N-Alkyl and N-unsubstituted naphthamides were prepared and found to yield nanomolar inhibitors of VEGFR-2 (KDR) with an improved selectivity profile against a panel of tyrosine and serine/threonine kinases. The inhibitory activity of this series was retained at the cellular level. Naphthamides 3, 20, and 22 exhibited good pharmacokinetics following oral dosing and showed potent inhibition of VEGF-induced angiogenesis in the rat corneal model. Once-daily oral administration of 22 for 14 days led to 85% inhibition of established HT29 colon cancer and Calu-6 lung cancer xenografts at doses of 10 and 20 mg/kg, respectively.

Novel 2,3-dihydro-1,4-benzoxazines as potent and orally bioavailable inhibitors of tumor-driven angiogenesis.

Angiogenesis is vital for solid tumor growth, and its prevention is a proven strategy for the treatment of disease states such as cancer. The vascular endothelial growth factor (VEGF) pathway provides several opportunities by which small molecules can act as inhibitors of endothelial proliferation and migration. Critical to these processes is signaling through VEGFR-2 or the kinase insert domain receptor (KDR) upon stimulation by its ligand VEGF. Herein, we report the discovery of 2,3-dihydro-1,4-benzoxazines as inhibitors of intrinsic KDR activity (IC 50 < 0.1 microM) and human umbilical vein endothelial cell (HUVEC) proliferation with IC 50 < 0.1 microM. More specifically, compound 16 was identified as a potent (KDR: < 1 nM and HUVEC: 4 nM) and selective inhibitor that exhibited efficacy in angiogenic in vivo models. In addition, this series of molecules is typically well-absorbed orally, further demonstrating the 2,3-dihydro-1,4-benzoxazine moiety as a promising platform for generating kinase-based antiangiogenic therapeutic agents.

On the complexities of ceramide changes in cells undergoing apoptosis: lack of evidence for a second messenger function in apoptotic induction

The generation of cellular ceramides as a second messenger has been implicated as a regulatory and required step for the induction of apoptosis. In this study, we have applied a recently developed mass spectrometric technique to the determination of changes in physiological ceramide levels during apoptosis induced by tumor necrosis factor plus cycloheximide in U937 cells and the chemical agents anisomycin or geranylgeraniol in HL-60 cells. The mass spectrometric method has significant advantages over traditional methods for ceramide quantitation in that it determines the relative abundance of all ceramide species present in complex biological lipid mixtures individually and simultaneously. We quantitiated ceramides ranging from C14 to C26, finding that their basal levels and relative distribution varied significantly, both within and between different cell types. However, we were not able to detect any significant changes in either total ceramide content or species distribution until 1 h or more post-stimulation with any of these treatments, by which time the cells were in an advanced stage of apoptosis. Differences were also seen between all three treatments in the ceramide species distribution observed in these late stages of apoptosis. These data indicate that in vivo ceramide generation occurs as a consequence of apoptosis rather than as an essential second messenger involved in its induction. They also pose new questions about the potential roles that certain ceramide species may play in the late stages of apoptosis, and demonstrate a clear need to utilize the resolving power of mass spectrometry-based assays in any future investigations into the biological function of ceramides.

Broad Antitumor Activity in Breast Cancer Xenografts by Motesanib, a Highly Selective, Oral Inhibitor of Vascular Endothelial Growth Factor, Platelet-Derived Growth Factor, and Kit Receptors

Purpose: Angiogenesis plays a critical role in breast cancer development and progression. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that regulates endothelial cell proliferation and survival. We investigated the effects of motesanib, a novel, oral inhibitor of VEGF receptors 1, 2, and 3; platelet-derived growth factor receptor; and Kit receptor, on the growth of xenografts representing various human breast cancer subtypes. Experimental Design: Athymic nude mice were implanted with MCF-7 (luminal) or MDA-MB-231 (mesenchymal) tumor fragments or Cal-51 (mixed/progenitor) tumor cells. Once tumors were established, animals were randomized to receive increasing doses of motesanib alone or motesanib plus cytotoxic chemotherapy (docetaxel, doxorubicin, or tamoxifen). Results: Across all three xenograft models, motesanib treatment resulted in significant dose-dependent reductions in tumor growth, compared with vehicle-treated controls, and in marked reductions in viable tumor fraction and blood vessel density. No significant effect on body weight was observed with compound treatment compared with control-treated animals. Motesanib did not affect the proliferation of tumor cells in vitro. There was a significantly greater reduction in xenograft tumor growth when motesanib was combined with docetaxel (MDA-MB-231 tumors) or with the estrogen receptor modulator tamoxifen (MCF-7 tumors), compared with either treatment alone, but not when combined with doxorubicin (Cal-51 tumors). Conclusions: Treatment with motesanib alone or in combination with chemotherapy inhibits tumor growth in vivo in various models of human breast cancer. These data suggest that motesanib may have broad utility in the treatment of human breast cancer.