Role of Interleukin 12 and Costimulators in T Cell Anergy In Vivo

The induction of T cell anergy in vivo is thought to result from antigen recognition in the absence of co-stimulation and inflammation, and is associated with a block in T cell proliferation and Th1 differentiation. Here we have examined the role of interleukin (IL)-12, a potent inducer of Th1 responses, in regulating this process. T cell tolerance was induced by the administration of protein antigen without adjuvant in normal mice, and in recipients of adoptively transferred T cells from T cell receptor transgenic mice. The administration of IL-12 at the time of tolerance induction stimulates Th1 differentiation, but does not promote antigen-specific T cell proliferation. Conversely, inhibiting CTLA-4 engagement during anergy induction reverses the block in T cell proliferation, but does not promote full Th1 differentiation. T cells exposed to tolerogenic antigen in the presence of both IL-12 and anti–CTLA-4 antibody are not anergized, and behave identically to T cells which have encountered immunogenic antigen. These results suggest that two processes contribute to the induction of anergy in vivo; CTLA-4 engagement, which leads to a block in the ability of T cells to proliferate to antigen, and the absence of a prototypic inflammatory cytokine, IL-12, which prevents the differentiation of T cells into Th1 effector cells. The combination of IL-12 and anti–CTLA-4 antibody is sufficient to convert a normally tolerogenic stimulus to an immunogenic one

Genome-wide analyses implicate 33 loci in heritable dog osteosarcoma, including regulatory variants near CDKN2A/B

Background: Canine osteosarcoma is clinically nearly identical to the human disease, but is common and highly heritable, making genetic dissection feasible. Results: Through genome-wide association analyses in three breeds (greyhounds, Rottweilers, and Irish wolfhounds), we identify 33 inherited risk loci explaining 55% to 85% of phenotype variance in each breed. The greyhound locus exhibiting the strongest association, located 150 kilobases upstream of the genes CDKN2A/B, is also the most rearranged locus in canine osteosarcoma tumors. The top germline candidate variant is found at a >90% frequency in Rottweilers and Irish wolfhounds, and alters an evolutionarily constrained element that we show has strong enhancer activity in human osteosarcoma cells. In all three breeds, osteosarcoma-associated loci and regions of reduced heterozygosity are enriched for genes in pathways connected to bone differentiation and growth. Several pathways, including one of genes regulated by miR124, are also enriched for somatic copy-number changes in tumors. Conclusions: Mapping a complex cancer in multiple dog breeds reveals a polygenic spectrum of germline risk factors pointing to specific pathways as drivers of disease

Phase I Evaluation of STA-1474, a Prodrug of the Novel HSP90 Inhibitor Ganetespib, in Dogs with Spontaneous Cancer

The novel water soluble compound STA-1474 is metabolized to ganetespib (formerly STA-9090), a potent HSP90 inhibitor previously shown to kill canine tumor cell lines in vitro and inhibit tumor growth in the setting of murine xenografts. The purpose of the following study was to extend these observations and investigate the safety and efficacy of STA-1474 in dogs with spontaneous tumors.This was a Phase 1 trial in which dogs with spontaneous tumors received STA-1474 under one of three different dosing schemes. Pharmacokinetics, toxicities, biomarker changes, and tumor responses were assessed. Twenty-five dogs with a variety of cancers were enrolled. Toxicities were primarily gastrointestinal in nature consisting of diarrhea, vomiting, inappetence and lethargy. Upregulation of HSP70 protein expression was noted in both tumor specimens and PBMCs within 7 hours following drug administration. Measurable objective responses were observed in dogs with malignant mast cell disease (n = 3), osteosarcoma (n = 1), melanoma (n = 1) and thyroid carcinoma (n = 1), for a response rate of 24% (6/25). Stable disease (>10 weeks) was seen in 3 dogs, for a resultant overall biological activity of 36% (9/25).This study provides evidence that STA-1474 exhibits biologic activity in a relevant large animal model of cancer. Given the similarities of canine and human cancers with respect to tumor biology and HSP90 activation, it is likely that STA-1474 and ganetespib will demonstrate comparable anti-cancer activity in human patients

Anti-Angiogenic Activity of a Small Molecule STAT3 Inhibitor LLL12

Background: Recent data indicate the Signal Transducer and Activator of Transcription 3 (STAT3) pathway is required for VEGF production and angiogenesis in various types of cancers. STAT3 inhibitors have been shown to reduce tumor microvessel density in tumors but a direct anti-angiogenic activity has not been described. Methodology/Principal Findings: We investigated the direct action of a small molecule inhibitor of STAT3 (LLL12) in human umbilical cord vascular endothelial cells (HUVECs) in vitro, in a Matrigel model for angiogenesis in vivo, and its antitumor activity in a xenograft model of osteosarcoma. LLL12 (100 nM) significantly inhibited VEGF-stimulated STAT3 phosphorylation in HUVECs, reduced their proliferation/migration and inhibited VEGF-induced tube formation. Morphologic analysis of LLL12 treated HUVECs demonstrated marked changes in actin/tubulin distribution and bundling. In scid mice, LLL12 reduced microvessel invasion into VEGF-infused Matrigel plugs by,90 % at a dose of 5 mg/kg daily. Following a period of tumor progression (2 weeks), LLL12 completely suppressed further growth of established OS-1 osteosarcoma xenografts. Pharmacodynamic studies showed robust phosphorylated STAT3 in control tumors, whereas phospho-STAT3 was not detected in LLL12-treated OS-1 tumors. Treated tumors demonstrated decreased proliferation (Ki67 staining), and decreased microvessel density (CD34 staining), but no significant increase in apoptosis (TUNEL staining), relative to controls. Assay of angiogenic factors, using an antibody array, showed VEGF, MMP-9, Angiopoietin1/2, Tissue Factor and FGF-

Cathepsin W, T-cell receptor-associated transmembrane adapter 1, lymphotactin and killer cell lectin like receptor K1 are sensitive and specific RNA biomarkers of canine epitheliotropic lymphoma

Cutaneous T-cell lymphoma (CTCL) is an uncommon type of lymphoma involving malignant skin-resident or skin-homing T cells. Canine epitheliotropic lymphoma (EL) is the most common form of CTCL in dogs, and it also spontaneously arises from T lymphocytes in the mucosa and skin. Clinically, it can be difficult to distinguish early-stage CTCLs apart from other forms of benign interface dermatitis (ID) in both dogs and people. Our objective was to identify novel biomarkers that can distinguish EL from other forms of ID, and perform comparative transcriptomics of human CTCL and canine EL. Here, we present a retrospective gene expression study that employed archival tissue from biorepositories. We analyzed a discovery cohort of 6 canines and a validation cohort of 8 canines with EL which occurred spontaneously in client-owned companion dogs. We performed comparative targeted transcriptomics studies using NanoString to assess 160 genes from lesional skin biopsies from the discovery cohort and 800 genes from the validation cohort to identify any significant differences that may reflect oncogenesis and immunopathogenesis. We further sought to determine if gene expression in EL and CTCL are conserved across humans and canines by comparing our data to previously published human datasets. Similar chemokine profiles were observed in dog EL and human CTCL, and analyses were performed to validate potential biomarkers and drivers of disease. In dogs, we found enrichment of T cell gene signatures, with upregulation of IFNG, TNF, PRF1, IL15, CD244, CXCL10, and CCL5 in EL in dogs compared to healthy controls. Importantly, CTSW, TRAT1 and KLRK1 distinguished EL from all other forms of interface dermatitis we studied, providing much-needed biomarkers for the veterinary field. XCL1/XCL2 were also highly specific of EL in our validation cohort. Future studies exploring the oncogenesis of spontaneous lymphomas in companion animals will expand our understanding of these disorders. Biomarkers may be useful for predicting disease prognosis and treatment responses. We plan to use our data to inform future development of targeted therapies, as well as for repurposing drugs for both veterinary and human medicine

Preclinical Evaluation of the Novel, Orally Bioavailable Selective Inhibitor of Nuclear Export (SINE) KPT-335 in Spontaneous Canine Cancer: Results of a Phase I Study

The purpose of this study was to evaluate the activity of Selective Inhibitors of Nuclear Export (SINE) compounds that inhibit the function of the nuclear export protein Exportin 1 (XPO1/CRM1) against canine tumor cell lines and perform a Phase I clinical trial of KPT-335 in dogs with spontaneous cancer to provide a preliminary assessment of biologic activity and tolerability.Canine tumor cell lines derived from non-Hodgkin lymphoma (NHL), mast cell tumor, melanoma and osteosarcoma exhibited growth inhibition and apoptosis in response to nanomolar concentrations of SINE compounds; NHL cells were particularly sensitive with IC50 concentrations ranging from 2-42 nM. A Phase I clinical trial of KPT-335 was performed in 17 dogs with NHL (naive or relapsed), mast cell tumor or osteosarcoma. The maximum tolerated dose was 1.75 mg/kg given orally twice/week (Monday/Thursday) although biologic activity was observed at 1 mg/kg. Clinical benefit (CB) including partial response to therapy (PR, n = 2) and stable disease (SD, n = 7) was observed in 9/14 dogs with NHL with a median time to progression (TTP) for responders of 66 days (range 35-256 days). A dose expansion study was performed in 6 dogs with NHL given 1.5 mg/kg KPT-335 Monday/Wednesday/Friday; CB was observed in 4/6 dogs with a median TTP for responders of 83 days (range 35-354 days). Toxicities were primarily gastrointestinal consisting of anorexia, weight loss, vomiting and diarrhea and were manageable with supportive care, dose modulation and administration of low dose prednisone; hepatotoxicity, anorexia and weight loss were the dose limiting toxicities.This study provides evidence that the novel orally bioavailable XPO1 inhibitor KPT-335 is safe and exhibits activity in a relevant, spontaneous large animal model of cancer. Data from this study provides critical new information that lays the groundwork for evaluation of SINE compounds in human cancer

Oncostatin M promotes STAT3 activation, VEGF production, and invasion in osteosarcoma cell lines

<p>Abstract</p> <p>Background</p> <p>We have previously demonstrated that both canine and human OSA cell lines, as well as 8 fresh canine OSA tumor samples, exhibit constitutive phosphorylation of STAT3, and that this correlates with enhanced expression of matrix metalloproteinase-2 (MMP2). While multiple signal transduction pathways can result in phosphorylation of STAT3, stimulation of the cytokine receptor gp130 through either IL-6 or Oncostatin M (OSM) is the most common mechanism through which STAT3 is activated. The purpose of this study was to evaluate the role of IL-6 and OSM stimulation on both canine and human OSA cell lines to begin to determine the role of these cytokines in the biology of OSA.</p> <p>Methods</p> <p>RT-PCR and Western blotting were used to interrogate the consequences of OSM and IL-6 stimulation of OSA cell lines. OSA cells were stimulated with OSM and/or hepatocyte growth factor (HGF) and the effects on MMP2 activity (gel zymography), proliferation (CyQUANT), invasion (Matrigel transwell assay), and VEGF production (Western blotting, ELISA) were assessed. The small molecule STAT3 inhibitor LLL3 was used to investigate the impact of STAT3 inhibition following OSM stimulation of OSA cells.</p> <p>Results</p> <p>Our data demonstrate that the OSM receptor (OSMR), but not IL-6 or its receptor, is expressed by all human and canine OSA cell lines and canine OSA tumor samples; additionally, OSM expression was noted in all tumor samples. Treatment of OSA cell lines with OSM induced phosphorylation of STAT3, Src, and JAK2. OSM stimulation also resulted in a dose dependent increase in MMP2 activity and VEGF expression that was markedly reduced following treatment with the small molecule STAT3 inhibitor LLL3. Lastly, OSM stimulation of OSA cell lines enhanced invasion through Matrigel, particularly in the presence of rhHGF. In contrast, both OSM and HGF stimulation of OSA cell lines did not alter their proliferative capacity.</p> <p>Conclusions</p> <p>These data indicate OSM stimulation of human and canine OSA cells induces STAT3 activation, thereby enhancing the expression/activation of MMP2 and VEGF, ultimately promoting invasive behavior and tumor angiogenesis. As such, OSM and its receptor may represent a novel target for therapeutic intervention in OSA.</p