Characterization of a full-length infectious cDNA clone and a GFP reporter derivative of the oncolytic picornavirus SVV-001.

Seneca Valley virus (SVV-001) is an oncolytic picornavirus with selective tropism for a subset of human cancers with neuroendocrine differentiation. To characterize further the specificity of SVV-001 and its patterns and kinetics of intratumoral spread, bacterial plasmids encoding a cDNA clone of the full-length wild-type virus and a derivative virus expressing GFP were generated. The full-length cDNA of the SVV-001 RNA genome was cloned into a bacterial plasmid under the control of the T7 core promoter sequence to create an infectious cDNA clone, pNTX-09. A GFP reporter virus cDNA clone, pNTX-11, was then generated by cloning a fusion protein of GFP and the 2A protein from foot-and-mouth disease virus immediately following the native SVV-001 2A sequence. Recombinant GFP-expressing reporter virus, SVV-GFP, was rescued from cells transfected with in vitro RNA transcripts from pNTX-11 and propagated in cell culture. The proliferation kinetics of SVV-001 and SVV-GFP were indistinguishable. The SVV-GFP reporter virus was used to determine that a subpopulation of permissive cells is present in small-cell lung cancer cell lines previously thought to lack permissivity to SVV-001. Finally, it was shown that SVV-GFP administered to tumour-bearing animals homes in to and infects tumours whilst having no detectable tropism for normal mouse tissues at 1×10(11) viral particles kg(-1), a dose equivalent to that administered in ongoing clinical trials. These infectious clones will be of substantial value in further characterizing the biology of this virus and as a backbone for the generation of additional oncolytic derivatives.

Therapeutic efficacy of CEP-33779, a novel selective JAK2 inhibitor, in a mouse model of colitis-induced colorectal cancer.

Constitutively activated STAT3 and STAT5 are expressed in a wide variety of human malignancies including solid and hematopoietic cancers and often correlate with a poor prognosis and resistance to multiple therapies. Given the well established role of STAT3 in tumorigenesis, inhibition of Janus-activated kinase 2 (JAK2) activity might represent an attractive therapeutic approach. Using a mouse model of colitis-induced colorectal cancer, we show that a novel, orally active, selective JAK2 inhibitor, CEP-33779, induced regression of established colorectal tumors, reduced angiogenesis, and reduced proliferation of tumor cells. Histopathologic analysis confirmed reduced incidence of histologic-grade neoplasia by CEP-33779. Tumor regression correlated with inhibition of STAT3 and NF-κB (RelA/p65) activation in a CEP-33779 dose-dependent manner. In addition, the expression of proinflammatory, tumor-promoting cytokines interleukin (IL)-6 and IL-1ß was strongly reduced upon JAK2 inhibition. The ability of CEP-33779 to suppress growth of colorectal tumors by inhibiting the IL-6/JAK2/STAT3 signaling suggests a potential therapeutic utility of JAK2 inhibitors in multiple tumors types, particularly those with a strong inflammatory component.

Novel, orally active, proteasome inhibitor, delanzomib (CEP-18770), ameliorates disease symptoms and glomerulonephritis in two preclinical mouse models of SLE.

Current therapies for late-stage systemic lupus erythematosus (SLE) are limited to cytotoxic agents. Delanzomib (CEP-18770) is an orally active, reversible P2 threonine boronic acid inhibitor of the 26S mammalian proteasome. Delanzomib was tested in a head-to-head comparison against bortezomib to protect and treat mice with fatal lupus nephritis (LN). Age matched MRL/lpr or NZBWF1 mice with established SLE or LN, respectively, were treated with delanzomib either 3 mg/kg once or twice weekly intravenously or orally at 10 mg/kg. Mice were also treated with reference agent bortezomib at 0.5 mg/kg, intraperitoneally, once a week or 0.3 mg/kg once or twice a week. Reductions in the frequencies of specific anti-chromatin, smith and dsDNA antibody secreting cells and levels of the corresponding circulating antinuclear antibodies, were observed following delanzomib treatment. Reductions in several serum pro-inflammatory cytokines were observed in delanzomib-treated animals. Delanzomib treatment suppressed the development and progression of renal tissue damage and extended the survival of ill mice. Proteinuria was significantly decreased and severity of various renal histopathologies reduced relative to vehicle-treated nephritic mice. Treatment of lupus in these models demonstrated that delanzomib treatment lead to greater tolerability and rate of response resulting in improved stabilization of disease.

Animal models of systemic lupus erythematosus (SLE) and ex vivo assay design for drug discovery.

Systemic Lupus Erythematosus (SLE) is a debilitating and often fatal autoimmune disease that involves multiple organ systems. It can develop for years before being diagnosed. Current treatments for SLE usually involve the use of cytotoxic or immunosuppressive agents that can lead to infection or cancer. The design of appropriate models and assays will determine the efficiency and speed with which an investigator can test a new chemical entity (NCE) or expect results to move a drug discovery program forward. This unit describes a series of preclinical assays for the identification of new agents for the treatment of SLE. Most importantly, this unit will guide the reader through a step-by-step process to select appropriate models, validation drugs, and readouts, depending on the objective of the study. The reader will acquire a working knowledge of what models are available and the potential advantages and disadvantages of each, including ex vivo assays relevant to the discovery of new SLE therapeutics.

Depletion of autoreactive plasma cells and treatment of lupus nephritis in mice using CEP-33779, a novel, orally active, selective inhibitor of JAK2.

Accumulating evidence suggests that autoreactive plasma cells play an important role in systemic lupus erythematosus (SLE). In addition, several proinflammatory cytokines promote autoreactive B cell maturation and autoantibody production. Hence, therapeutic targeting of such cytokine pathways using a selective JAK2 inhibitor, CEP-33779 (JAK2 enzyme IC(50) = 1.3 nM; JAK3 enzyme IC(50)/JAK2 enzyme IC(50) = 65-fold), was tested in two mouse models of SLE. Age-matched, MRL/lpr or BWF1 mice with established SLE or lupus nephritis, respectively, were treated orally with CEP-33779 at 30 mg/kg (MRL/lpr), 55 mg/kg or 100 mg/kg (MRL/lpr and BWF1). Studies included reference standard, dexamethasone (1.5 mg/kg; MRL/lpr), and cyclophosphamide (50 mg/kg; MRL/lpr and BWF1). Treatment with CEP-33779 extended survival and reduced splenomegaly/lymphomegaly. Several serum cytokines were significantly decreased upon treatment including IL-12, IL-17A, IFN-α, IL-1ß, and TNF-α. Anti-nuclear Abs and frequencies of autoantigen-specific, Ab-secreting cells declined upon CEP-33779 treatment. Increased serum complement levels were associated with reduced renal JAK2 activity, histopathology, and spleen CD138(+) plasma cells. The selective JAK2 inhibitor CEP-33779 was able to mitigate several immune parameters associated with SLE advancement, including the protection and treatment of mice with lupus nephritis. These data support the possibility of using potent, orally active, small-molecule inhibitors of JAK2 to treat the debilitative disease SLE.

A highly selective, orally active inhibitor of Janus kinase 2, CEP-33779, ablates disease in two mouse models of rheumatoid arthritis.

INTRODUCTION: Janus kinase 2 (JAK2) is involved in the downstream activation of signal transducer and activator of transcription 3 (STAT3) and STAT5 and is responsible for transducing signals for several proinflammatory cytokines involved in the pathogenesis of rheumatoid arthritis (RA), including interleukin (IL)-6, interferon γ (IFNγ) and IL-12. In this paper, we describe the efficacy profile of CEP-33779, a highly selective, orally active, small-molecule inhibitor of JAK2 evaluated in two mouse models of RA. METHODS: Collagen antibody-induced arthritis (CAIA) and collagen type II (CII)-induced arthritis (CIA) were established before the oral administration of a small-molecule JAK2 inhibitor, CEP-33779, twice daily at 10 mg/kg, 30 mg/kg, 55 mg/kg or 100 mg/kg over a period of 4 to 8 weeks. RESULTS: Pharmacodynamic inhibition of JAK2 reduced mean paw edema and clinical scores in both CIA and CAIA models of arthritis. Reduction in paw cytokines (IL-12, IFNγ and tumor necrosis factor α) and serum cytokines (IL-12 and IL-2) correlated with reduced spleen CII-specific T helper 1 cell frequencies as measured by ex vivo IFNγ enzyme-linked immunosorbent spot assay. Both models demonstrated histological evidence of disease amelioration upon treatment (for example, reduced matrix erosion, subchondral osteolysis, pannus formation and synovial inflammation) and reduced paw phosphorylated STAT3 levels. No changes in body weight or serum anti-CII autoantibody titers were observed in either RA model. CONCLUSIONS: This study demonstrates the utility of using a potent and highly selective, orally bioavailable JAK2 inhibitor for the treatment of RA. Using a selective inhibitor of JAK2 rather than pan-JAK inhibitors avoids the potential complication of immunosuppression while targeting critical signaling pathways involved in autoimmune disease progression.

Novel method of monitoring trace cytokines and activated STAT molecules in the paws of arthritic mice using multiplex bead technology.

BACKGROUND: The use of mouse models to study human disease provides useful data that can provide support for research projects or an existing drug discovery program. How well a model recapitulates the human condition and the ease and reproducibility of data collected will determine how much confidence a scientist can place on results obtained. Designing new treatments for rheumatic diseases, such as rheumatoid arthritis (RA), requires complex immunocompetent models that depend on intricate cytokine networks. Using local cytokines, signal transduction and transcription factor molecules as potential biomarkers to monitor disease and treatment efficacy is the best method to follow the progression of tissue damage and repair when testing an unknown compound or biologic. Described here in this report, a novel method for the non-enzymatic extraction and measurement of cytokines and signal transducers and activators of transcription (STAT) molecules using Luminex® bead array technology in two different mouse models for human RA--collagen antibody-dependent arthritis (CAIA) and collagen-induced arthritis (CIA). RESULTS: Dynamic expression of several pro-inflammatory cytokines responsible for promoting disease augmentation overtime were monitored, such as IL-1ß, TNFα, IL-6 and IL-12, locally in the paws of affected animals directly ex vivo. Local cytokine responses could be matched with serum cytokine levels and joint pathology results. In addition, STAT1, 3, and 5a/b activation status could be monitored with confidence using specifically formulated extraction buffer that protected the phosphorylation site. STAT3 activation followed paw swelling and cytokine levels in both models and correlates of disease could be ablated upon treatment with dexamethasone. Here reported a novel method of extracting joint fluid from the paws of inflamed mice coupled with powerful multiplex bead technology allowing us to measure cytokine responses, pharmacodynamic markers such as STATs and pharmacokinetic analysis of dosed agent all from the same sample directly ex vivo. CONCLUSIONS: This method is powerful in that it is applicable to multiple autoimmunity model types, streamlines ex vivo readouts in a high-throughput manner, and allows multiplexing providing the investigator with an array of options and possible analytes when developing preclinical animal models to support drug discovery efforts in the search for new treatments for rheumatic diseases.

Activated TNF-alpha/NF-kappaB signaling via down-regulation of Fas-associated factor 1 in asbestos-induced mesotheliomas from Arf knockout mice.

The human CDKN2A locus encodes 2 distinct proteins, p16(INK4A) and p14(ARF) [mouse p19(Arf)], designated INK4A (inhibitor of cyclin dependent kinase 4) and ARF (alternative reading frame) here, that are translated from alternatively spliced mRNAs. Human ARF is implicated as a tumor suppressor gene, mainly in association with the simultaneous deletion of INK4A. However, questions remain as to whether loss of ARF alone is sufficient to drive tumorigenesis. Here, we report that mice deficient for Arf are susceptible to accelerated asbestos-induced malignant mesothelioma (MM). MMs arising in Arf (+/-) mice consistently exhibit biallelic inactivation of Arf, but, unexpectedly, do not acquire additional recurrent genetic alterations that we previously identified in asbestos-induced MMs arising in Nf2 (+/-) mice. Array CGH analysis was used to detect a recurrent deletion at chromosome 4C6 in MMs from Arf (+/-) mice. A candidate gene in this region, Faf1 (FAS-associated factor 1), was further explored, because it encodes a protein implicated in tumor cell survival and in the pathogenesis of some human tumor types. We confirmed hemizygous loss of Faf1 and down-regulation of Faf1 protein in a series of MMs from Arf (+/-) mice, and we then showed that Faf1 regulates TNF-alpha-mediated NF-kappaB signaling, a pathway previously implicated in asbestos-induced oncogenesis of human mesothelial cells. Collectively, these data indicate that Arf inactivation has a significant role in driving MM pathogenesis, and implicate Faf1 as a key component in the TNF-alpha/NF-kappaB signaling node that has now been independently implicated in asbestos-induced oncogenesis.