In this study we examined the activation of the non-canonical NF kappa B signalling pathway in endothelial cells. In HUVECs, LIGHT stimulated a delayed induction of serine 866/870 p100 phosphorylation linked to p52 NF kappa B formation. Surprisingly, the canonical ligand, IL-1 beta, stimulated a rapid phosphorylation or p100 which was not associated with p52 formation. Inhibition of IKK alpha activity, using DN-IKK alpha adenovirus, IKK alpha siRNA or a novel first-in-class selective IKK alpha inhibitor, SU1261, revealed IL-1 beta induced p100 phosphorylation to be dependent on IKK alpha. In contrast, IKK beta inhibition was found to be without effect. The NIK inhibitor, CW15337, did not affect IL1 beta induced p100 phosphorylation however, both p100 and pIKK alpha/ beta phosphorylation was substantially reduced by inhibition of the upstream kinase TAK-1, suggesting phosphorylation of p100 is mediated by IKK alpha from within the canonical NEMO/IKK beta /IKK alpha complex. IL-1 beta also stimulated a rapid increase in nuclear translocation of p52, which was not affected by NIK inhibition, suggesting a source of p52 independent of p100 processing. Inhibition of TAK-1 abolished p52 and p65 nuclear translocation in response to IL-1 beta. SiRNA deletion or inhibition with dominant-negative virus of IKK alpha activity partially reduced p52 translocation, however pharmacological inhibition of IKK alpha was without effect. Inhibition of IKK beta abolished both p52 and p65 translocation. Taken together these results show that IL-1 beta stimulates a novel IKK alpha -dependent axis within the non-canonical NF kappa B pathway in endothelial cells which is NIK-independent and regulated by TAK-1. However, this pathway is not primarily responsible for the early nuclear translocation of p52, which is dependent on IKK beta. Elucidation of both these new pathways may be significant for NF kappa B biology within the endothelium.

The inhibitory-kappaB kinases (IKKs) IKKα and IKKβ play central roles in regulating the non-canonical and canonical NF-κB signalling pathways. Whilst the proteins that transduce the signals of each pathway have been extensively characterised, the clear dissection of the functional roles of IKKα-mediated non-canonical NF-κB signalling versus IKKβ-driven canonical signalling remains to be fully elucidated. Progress has relied upon complementary molecular and pharmacological tools; however, the lack of highly potent and selective IKKα inhibitors has limited advances. Herein, we report the development of an aminoindazole-pyrrolo[2,3-b]pyridine scaffold into a novel series of IKKα inhibitors. We demonstrate high potency and selectivity against IKKα over IKKβ in vitro and explain the structure–activity relationships using structure-based molecular modelling. We show selective target engagement with IKKα in the non-canonical NF-κB pathway for both U2OS osteosarcoma and PC-3M prostate cancer cells by employing isoform-related pharmacodynamic markers from both pathways. Two compounds (SU1261 [IKKα Ki = 10 nM; IKKβ Ki = 680 nM] and SU1349 [IKKα Ki = 16 nM; IKKβ Ki = 3352 nM]) represent the first selective and potent pharmacological tools that can be used to interrogate the different signalling functions of IKKα and IKKβ in cells. Our understanding of the regulatory role of IKKα in various inflammatory-based conditions will be advanced using these pharmacological agents.

The pathogenesis and drug resistance of symptomatic CLL patients involves genetic changes associated with the CLL clone as well as changes within the microenvironment. To further understand these processes, we compared early stage CLL to symptomatic late stage using gene expression and serum cytokine profiling to gain insight of the genetic and microenvironment changes associated with the most severe form of the disease. Patients were classified into low stage (Rai stage 0/I/II) and high stage (Rai stage III/IV). Gene expression profiles were obtained on pretreatment samples using the HG-U133A 2.0 Affymetrix platform. A comparison of low versus high stage CLL revealed a set of 21 genes differentially expressed genes. 15 genes were up regulated in the high stage compared to low stage while 6 genes were down regulated. Analysis of GO molecular function revealed 9 of 21 genes were involved in transcription factor activity. Serum cytokine profiles showed six cytokines to be significantly different in high stage patients. Two chemokines, SDF-1/CXCL12 and uPAR known to be involved in stem cell mobilization and homing were increased in serum of high stage patients. This study has identified therapeutic targets for symptomatic CLL patients.

Prostate cancer is a common disease in men and at present there is no effective therapy available due to its recurrence despite androgen deprivation therapy. The epidermal growth factor receptor family (EGFR/HER1, HER2/neu and HER3)/PI3K/Akt signaling axis has been implicated in prostate cancer development and progression. However, Erlotinib, an EGFR tyrosine kinase inhibitor, has less effect on proliferation and apoptosis in prostate cancer cell lines. In this study, we evaluate whether MP470, a novel receptor tyrosine kinase inhibitor alone or in combination with Erlotinib has inhibitory effect on prostate cancer in vitro and in vivo.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease, with surgery being the only curative modality for localized disease, and gemcitabine with or without erlotinib remains the standard of therapy for unresectable or metastatic disease. CEACAM6 is overexpressed in human PDA independent of stage or grade and causes anoikis resistance when dysregulated. Because murine monoclonal antibody 13-1 possesses target-specific cytotoxicity in human PDA cell lines, we designed a humanized anti-CEACAM6 single-chain variable fragment (scFv) based on monoclonal antibody 13-1. PEGylation of the glycine-serine linker was used to enhance plasma half-life. These scFvs bound CEACAM6 with high affinity, exhibited cytotoxic activity, and induced dose-dependent poly(ADP-ribose) polymerase cleavage. Murine PDA xenograft models treated with humanized scFv alone elicited tumor growth inhibition, which was enhanced in combination with gemcitabine. Immunohistochemistry showed significant apoptosis, with inhibition of angiogenesis and proliferation, and preservation of the target. Collectively, our results have important implications for the development of novel antibody-based therapies against CEACAM6 in PDA.

Gene expression profiling (GEP) of 8 stage 0/I untreated Chronic Lymphocytic Leukemia (CLL) patients showed over-expression of Frizzled 3 (FZD3)/ROR-1 receptor tyrosine kinase (RTK), FLT-3 RTK and CXCR3 G-protein coupled receptor (GPCR). RT-PCR of 24 genes in 21 patients of the WNT pathway corroborated the GEP. Transforming growth factorβ, fibromodulin, TGFβRIII and SMAD2 are also over-expressed by GEP. Serum cytokine profiling of 26 low stage patients showed elevation of IFNγ, CSF3, Flt-3L and insulin-like growth factor binding protein 4. In order to ascertain why CLL cells grow poorly in culture, a GEP of 4 CLL patients cells at 0 hr and 24 hr in culture demonstrated over expression of CXCL5, CCL2 and CXCL3, that may recruit immune cells for survival. Treatment with thalidomide, an immunomodulatory agent, showed elevation of CCL5 by GEP but was not cytotoxic to CLL cells. Our data suggest an interplay of several oncogenic pathways, cytokines and immune cells that promote a survival program in CLL.

This paper examines the current legal treatment of software innovation. It argues that recent judicial standards for the regulation of software innovation do not adequately protect innovation. It presents an original standard for the regulation of software innovation, one intended to guide judicial decisions in contributory copyright liability, in interpretations of the Digital Millennium Copyright Act, and in every courtroom where a developer is on trial for the mere creation and distribution of software. The standard presented in this paper separates the questions of liability and remedy in order to produce an optimal dynamic balance of interests.

3042 Background: Novel Therapies for pancreatic cancer (PC) based on biological insight are urgently needed. CEACAM6, a member of the carcinoembryonic antigen (CEA) family, is a cell surface oncogene on PC cells. It is a glycoprotein composed of 3 Ig-like domains (Mr ∼35.2kD) and is linked to the plasma membrane via a glycophospholipid linked anchor. It is over-expressed on ≥95% PC patients’ found irrespective of stage of disease and represents a target for antibody therapy. Methods: Murine anti-CEACAM6 monoclonal antibody (Mab) 13–1 was humanized by a structure-based approach and single chain variable fragments (scFv) designed consisting of a Gly/Ser-linker which includes a cysteine residue for PEGylation. Murine scFv and 4 humanized scFv fragments (Version.1, 2, 7, 8) were bacterially expressed, purified, PEGylated and evaluated for activity alone or in combination with gemcitabine in human PC cell lines and mouse xenograft tumors. Cell viability, apoptosis and in-cell Westerns were done for efficacy and binding. Mouse xenograft tumors were treated with scFv or PEGylated scFv or combination with gemcitabine. Tumors (treated Vs untreated) were analyzed by immunohistochemistry for Ki-67, CD31, CEACAM6 and Caspase 3 Results: Mab 13–1, murine and 4 humanized scFvs’ were cytotoxic to CEACAM6 expressing PC cells (BxPC-3, HPAF-2) and not to PC cells that lack CEACAM6 (CaPan-2) with increased PARP-cleavage that was dose dependent (IC50=10μg/mL). Western blotting of culture media and serum from xenograft mice showed that CEACAM6 is not shed. In-cell Western confirmed humanized V.7 to be the best binder (KD=1–10μg/mL) confirming protein-protein interaction studies performed in silico with homology models of murine and humanized scFvs’ and CEACAM6. The murine scFv alone or the humanized scFV (V.8) in combination with gemcitabine delayed tumor growth by >50%. This tumor reduction correlated well with the immunohistochemical markers of response. Conclusions: We have demonstrated that CEACAM6 is a clinically relevant target in PC and that a PEGylated humanized scFv is a novel effective therapy alone and/or in combination with gemcitabine. Humanized scFv V.7 is undergoing mouse xenograft evaluation and expect will be the most effective agent to take forward into clinical development. No significant financial relationships to disclose.

Background: Chronic lymphocytic leukemia (CLL) is a common leukemia that is incurable. Survival curves for Rai stage 0/I and III/IV are distinct and a specific gene expression signature may be associated with stage progression (SP). However, distinct oncogenic pathways that differentiate stage progression are not well defined.

Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer and death in North America. In the year 2002, adenocarcinoma of the exocrine pancreas accounted for ≈30,300 new ases and 29,700 deaths. The current treatment strategy for localized pancreatic cancer is multimodality therapy combining pancreaticoduodenectomy with postoperative adjuvant chemotherapy and external beam radiation therapy, which results in a median overall survival of 12 to 24 months. For locally advanced and metastatic pancreatic cancer, the median survival is 6 to 12 months with currently available palliative chemotherapy. CEACAM6 (carcinoembryonic antigen cell adhesion molecule 6 [CD66c]) is an oncogene expressed on the cell surface as a GPI-linked glycoprotein comprised of 3 Ig-like domains capable of homophilic and heterophilic (CEACAM 5 and 8) interactions. It is highly expressed in > 90% of patients with pancreatic ductal adenocarcinoma. Deregulated expression of CEACAM6 inhibits differentiation and apoptosis of cells deprived of their anchorage to the extracellular matrix. We have previously demonstrated that CEACAM6-expressing pancreatic cancer cell lines treated with a murine monoclonal antibody (13-1) display reduced cellular viability by MTS assay, an indication of cytotoxicity. A murine scFv based on mAb (13-1) as well as a humanized form of the murine scFv also showed in vitro activity in CEACAM6-expressing cancer cell lines. In vivo studies using an SCID mouse model xenograft with BXPC-3 cells indicated a significant decrease in tumor burden as a result of treatment with a PEGylated form of the previously mentioned humanized scFv. The humanized scFv drug reduced the tumor burden as a stand-alone agent and in combination with gemcitabine compared with the control animal groups. Immunohistochemical analysis of tumors excised from animals treated with the humanized scFv and untreated animals indicated a marked increase in apoptosis (H&E) as well as a marked decrease in proliferation (Ki67) in the treated group relative to the untreated group. This observed in vivo effect on proliferation and apoptosis is currently being confirmed in vitro using CEACAM6-expressing cancer cell lines.

ABSTRACT Seven family-level metrics were determined from benthic macroinvertebrate samples collected monthly across four different landuse sites in the Cazenovia Creek watershed in western New York. We evaluated which metrics were most appropriate for, and effective in, discerning differences among the sites. Dramatic and consistent seasonal differences in metric scores were observed across all sites. All metric scores indicated markedly better biological health during winter months than during summer months. The biological impairment designation was highest in the summer at the suburban and agricultural sites. Coefficients of variation were considerably greater at the lower stream order sites. The metrics that were most appropriate and effective in assessing benthic assemblage health in this study were: richness, percent model affinity, family-level biotic index, and Ephemeroptera-Plecoptera-Trichoptera index. These indices correlated with a low amount of redundancy in a Pearson matrix, had significant discriminatory power in assessing biological impairments across sites, and had low variation within sites and seasons. This suggests these metrics should be selected for bioassessments in similar Great Lakes watersheds.

KIT or α-platelet-derived growth factor receptor (α-PDGFR) activating mutations are the pathogenic mechanisms that characterize gastrointestinal stromal tumors (GIST). Despite excellent responses to imatinib mesylate (IM), patients are relapsing. We developed an IM-resistant GIST cell line (GIST-R) from the IM-sensitive GIST882 cell line (GIST-S) by growing these cells in IM. Gene expression profiling (GEP) of GIST-S, GIST-R cells and two IM resistant GIST patients demonstrated that KIT is downregulated implying a major role in IM resistance. Instead, GIST-R cells have acquired IM resistance by overexpressing the oncogenic receptor tyrosine kinase – AXL – in a ‘kinase switch’. Further, the two IM resistant GIST patients express AXL and not c-Kit, seen by immunohistochemistry (IHC). Real time reverse transcriptase–polymerase chain reaction and Western blotting of the GIST-S and GIST-R cells confirmed the switch from Kit to AXL. In GIST-R, AXL is tyrosine phosphorylated and its ligand growth-arrest-specific gene 6 is overexpressed implying autocrine activation. The kinase switch is associated with a morphological change from spindle to epithelioid. Molecular modeling of the kinase domain of mutant c-Kit (V654A) and AXL showed no binding to IM but efficient binding to MP470, a novel c-Kit/AXL kinase inhibitor. MP470 synergizes with docetaxel (taxotere) and is cytotoxic to GIST cells.

The scattering characteristics of an infinite cylinder are strongly influenced by the incidence angle relative to its axis. If the incident wave propagates in the plane normal to the axis of the cylinder, the polarization of the scattered wave remains unchanged and the scattered wave propagates in the same plan as the incident wave. At off-axis incidence such that the incident direction makes an oblique angle with the cylinder axis, the scattered wave is depolarized, and its spatial distribution becomes three-dimensional. This paper presents the scattering solution for oblique incidence on multiple parallel cylinders located near a planar interface by an evanescent wave that is generated by total internal reflection of the source wave propagating in the higher refractive index substrate. Hertz potentials are utilized to formulate the interaction of inhomogeneous waves with the cylinders, scattering at the substrate interface, and near field scattering between the cylinders. Analytic formulas are derived for the electromagnetic fields and Poynting vector of scattered radiation in the near-field and their asymptotic forms in the far-field. Numerical examples are shown to illustrate scattering of evanescent wave by multiple cylinders at off-axis incidence.

Background: B-cell chronic lymphocytic leukemia (B-CLL) the most common leukemia in the Western world accounts for 25% of all newly diagnosed leukemias. Despite new therapeutic advances, B-CLL is currently not curable. Potential oncogenic signaling pathways in B-CLL require elucidation. We identified ROR-1 receptor tyrosine kinase (RTK) in the WNT/Planar Cell Polarity non-canonical pathway as a possible mechanism of oncogenesis by gene expression profiling (GEP). Methods: Mononuclear cells from 8 low stage CLL patients were obtained through an IRB approved protocol and analyzed utilizing the HG-U133A 2.0 Affymetrix array (~18,400 transcripts, 22,000 probe sets) after isolating and purifying total RNA (Qiagen, RNAeasy). The control RNA samples were isolated from 8 normal volunteer peripheral blood (PB) B-cells (AllCell, CA) and a normal reactive lymph node. Tumor lineage was confirmed by immunohistochemistry (IHC). Quantitative real time RT-PCR was performed on 20 selected genes to validate the microarray GEP. Twenty one B-CLL patients (includes the 8 patients from the GEP) were evaluated by RT-PCR for several key components of oncogenic signaling pathways based on the GEP with gene specific probes. A serum cytokine profile (120 cytokines) on 12 CLL patients (8 for which GEP is available and 4 additional patients) and 4 normal volunteers were performed for identification of a signature for diagnostic and prognostic value. Results: Data are represented as “robust” increases or decreases of relative gene expression common to 8 patients. ROR-2, a close member of ROR-1, has been shown to bind Wnt-5A via the cysteine rich domain (CRD) and to activate the JNK signaling pathway. Our findings identify over-expression of members of the non-canonical WNT/PCP- ROR-1- signaling pathway genes that were validated by RT-PCR. A serum cytokine profile of 12 patients provides a signature that may be useful in CLL diagnosis and prognosis. Conclusions: GEP identified WNT/PCP-ROR-1 as key components of an autocrine pathway that helps B-CLL avoid apoptosis. Several serum cytokines are elevated and require validation as potential diagnostic and prognostic markers. GEP of B-CLL in combination with quantitative real time RT-PCR has identified several novel targets for therapy. The identification of ROR-1 RTK has led to the development of a molecular target for future therapeutic application. Several lead compounds have been identified and are being evaluated as potential therapies in B-CLL.

A Native American‐Indian female presenting with anemia and thrombocytosis was diagnosed with myelodysplastic syndrome (MDS, refractory anemia). Over the course of 5 years she developed cytopenias and periods of leukocytosis with normal bone marrow (BM) blast counts, features of an unclassifiable MDS/MPS syndrome. The patient ultimately progressed to acute myelogenous leukemia (AML, FAB M2) and had a normal karyotype throughout her course. The episodes of leukocytosis were associated with infectious complications. Transformation to AML was characterized by a BM blast percentage of 49%. Peripheral blood and BM samples were obtained for serum protein analysis and gene expression profiling (GEP) to elucidate her disease process. An ELISA assay of the serum analyzed ∼80 cytokines, which demonstrated that hepatocyte growth factor/scatter factor and insulin‐like growth factor binding protein 1 were markedly elevated compared to normal. GEP demonstrated a unique “tumor molecular profile,” which included overexpression of oncogenes (HOXA9, N‐MYC, KOC1), proliferative genes (PAWR, DLG5, AKR1C3), invasion/metastatic genes (FN1, N‐CAM‐1, ITGB5), pro‐angiogenesis genes (c‐Kit), and down regulation of tumor suppressor genes (SUI1, BARD1) and anti‐apoptotic genes (PGLYRP, SERPINB2, MPO). Hence, a biomics approach has provided insight into elucidating disease mechanisms, molecular prognostic factors, and discovery of novel targets for therapeutic intervention. Am. J. Hematol., 2006. © 2006 Wiley‐Liss, Inc.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with a poor prognosis where incidence mirrors mortality. Gemcitabine and gemcitabine plus erlotinib (epidermal growth factor receptor tyrosine kinase inhibitor) are the only FDA approved therapies for unresectable or metastatic PDA and are at best palliative. Hence, considerable efforts have been initiated to identify novel targets for monoclonal antibody (Mab) therapies that may safely and effectively be combined with gemcitabine. Mabs to cell surface receptors and/or their ligands have shown efficacy in pre-clinical and clinical studies in both solid and hematological malignancies and can safely be given with chemotherapy. A number of clinical trials have evaluated the safety and efficacy of Mabs targeting the tumor and/or tumor micro-environment and in combination with chemotherapy for PDA with very little success. Here we review the rationale for Mab therapies, targeted clinical trials, rational basis for target selection, pre-clinical models and promising novel cell surface targets and/or growth factor ligands that are amenable to ongoing and future Mab therapies that hold promise and hope for patients and their families with this devastating disease.

BACKGROUND AND AIMS:Barrett's esophagus (BE) is a premalignant lesion of the distal esophagus in which squamous epithelial cells are replaced by metaplastic intestinal-like columnar epithelium that contains goblet cells. The factors that contribute to the progression from normal squamous mucosa to BE, Barrett's dysplasia, and adenocarcinoma are not well understood at the molecular level. Since reflux of bile acids is associated with BE development, we speculate that cells with an apoptosis-resistant phenotype are selected after long-term repeated exposure to pulses of bile acids. This will result in the survival of cells with unrepaired DNA damage, and a consequent increase in genomic instability leading to cancer progression. The major goal of this study is to compare sensitivity to apoptosis induced by the bile acid, deoxycholate (DOC), a known inducer of apoptosis, in normal esophageal squamous epithelium, normal colon epithelium, and BE. METHODS:Thirteen patients with a confirmed diagnosis of BE and four patients who had undergone clinically indicated colectomy were included in the present study. Freshly obtained biopsies were incubated with control medium or medium supplemented with 1 mM DOC for 3 h and then evaluated for apoptotic changes using transmission electron microscopy and immunohistochemical staining for two apoptotic markers, cleaved caspase 3 and cleaved cytokeratin 18. RESULTS:Our results indicate that BE is resistant to apoptosis induced by DOC compared to esophageal squamous epithelium and normal colon epithelium. In addition, electron micrographs revealed mitochondrial swelling in squamous epithelial cells treated ex vivo with DOC, which was absent in epithelial cells of BE. Formation of swollen mitochondria is an early marker of apoptotic cell death. Altogether, the data indicate that reduced apoptosis capability in BE tissue may contribute to progression to esophageal adenocarcinoma.