Cellular and functional actions of tofacitinib related to the pathophysiology of hibernoma development.

Tofacitinib is an oral JAK inhibitor for the treatment of rheumatoid arthritis. In the 2-year carcinogenicity study with tofacitinib, increased incidence of hibernoma (a neoplasm of brown adipose tissue [BAT]) was noted in female rats at ≥30 mg/kg/day (≥41x human exposure multiples). Thus, signaling pathways within BAT were investigated by measuring BAT: weight, cell proliferation biomarkers, content of basal and prolactin-induced phosphorylated Signal Transducer and Activator of Transcription (STAT), and uncoupling protein 1 (UCP-1). The relationship between cardiovascular hemodynamics and plasma norepinephrine (NE) levels was also investigated. Tofacitinib administered to female rats at doses of 10, 30, or 75 mg/kg/day for 14 days increased BAT weight at 75 mg/kg/day and cell proliferation at ≥30 mg/kg/day. JAK inhibition, observed as lower pSTAT3 and pSTAT5 in BAT, was noted at ≥10 mg/kg/day, while lower activity of BAT was observed as lower UCP-1 protein at ≥30 mg/kg/day. In cultured brown adipocytes, prolactin-induced increase in pSTAT5 and pSTAT3 were inhibited by tofacitinib in a concentration-dependent manner. Tofacitinib lowered blood pressure, increased heart rate, and resulted in dose-dependent increases in circulating NE. Thus, JAK/STAT inhibition in BAT and sympathetic stimulation are two factors which might contribute to the genesis of hibernomas by tofacitinib in rats.

Regulatory Forum Commentary* Counterpoint: Dose Selection for rasH2 Mouse Carcinogenicity Studies.

Dose selection for the 6-month rasH2 mouse carcinogenicity studies depends heavily on the maximum tolerated dose (MTD) obtained from 1-month range-finding studies. A retrospective evaluation of range-finding studies and pivotal 6 month rasH2 mouse studies for 11 compounds demonstrated that the MTD based on at least a 10% decrease in body weight gain, mortality, and target organ toxicity in range-finding studies appropriately identified high doses for pivotal studies for 8 of 11 compounds. Two of the selected high doses were based on decreased body weight gain alone, while 7 were based on mortality at higher doses in shorter duration range-finding studies. High-dose selection was based on the maximum feasible dose for one study. The Center for Drug Evaluation and Research, U.S. Food and Drug Administration Executive Carcinogenicity Assessment Committee often suggested different doses than those proposed by the sponsor. High mortality was observed in only one pivotal study and the high dose was lowered during the course of that study.

Two-year carcinogenicity study in rats with a nonnucleoside reverse transcriptase inhibitor.

Administration of lersivirine, a nonnucleotide reverse transcriptase inhibitor, daily by oral gavage to Sprague-Dawley rats for up to 2 yr was associated with decreased survival, decreased body weights, and an increase in neoplasms and related proliferative lesions in the liver, thyroid, kidney, and urinary bladder. Thyroid follicular adenoma and carcinoma, the associated thyroid follicular hypertrophy/hyperplasia, hepatocellular adenoma/adenocarcinoma, altered cell foci, and hepatocellular hypertrophy were consistent with lersivirine-related induction of hepatic microsomal enzymes. Renal tubular adenoma and renal tubular hyperplasia were attributed to the lersivirine-related exacerbation of chronic progressive nephropathy (CPN), while urinary bladder hyperplasia and transitional cell carcinoma in the renal pelvis and urinary bladder were attributed to urinary calculi. Renal tubular neoplasms associated with increased incidence and severity of CPN, neoplasms of transitional epithelium attributed to crystalluria, and thyroid follicular and hepatocellular neoplasms related to hepatic enzyme induction have low relevance for human risk assessment.

Toxicities associated with 1-month treatment with propylthiouracil (PTU) and methimazole (MMI) in male rats.

Thionamides such as propylthiouracil (PTU) and methimazole (MMI) have been used for more than 50 years to treat the more common causes of thyrotoxicosis/hyperthyroidism such as Graves' disease. Serious adverse effects associated with thionamides in humans include idiosyncratic liver damage, agranulocytosis, aplastic anemia, and vasculitis. Both prospective and retrospective clinical studies with these drugs have failed to identify predictive biomarker for these adverse effects. To assess whether rat is a good model for predicting drug-related adverse events in the liver and in the bone marrow, we conducted a comprehensive study in male rats with multiple doses of PTU and MMI. As expected, euthyroid animals became hypothyroid along with several secondary changes associated with hypothyroidism. There were slight reductions in red blood cell parameters along with some marginal effects on the bone marrow elements. However, there was no evidence of significant neutropenia and liver injury in both PTU-treated and MMI-treated cohorts. MMI-related effects were noted in the seminiferous tubules of the testes. Overall, 1-month daily treatment of euthyroid rats with PTU or MMI resulted in hypothyroidism, minor bone marrow effects, and several secondary effects associated with hypothyroidism, but without any evidence of adverse effects reported in humans including liver injury and agranulocytosis.

Regulatory Forum commentary: alternative mouse models for future cancer risk assessment.

International regulatory and pharmaceutical industry scientists are discussing revision of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S1 guidance on rodent carcinogenicity assessment of small molecule pharmaceuticals. A weight-of-evidence approach is proposed to determine the need for rodent carcinogenicity studies. For compounds with high human cancer risk, the product may be labeled appropriately without conducting rodent carcinogenicity studies. For compounds with minimal cancer risk, only a 6-month transgenic mouse study (rasH2 mouse or p53+/- mouse) or a 2-year mouse study would be needed. If rodent carcinogenicity testing may add significant value to cancer risk assessment, a 2-year rat study and either a 6-month transgenic mouse or a 2-year mouse study is appropriate. In many cases, therefore, one rodent carcinogenicity study could be sufficient. The rasH2 model predicts neoplastic findings relevant to human cancer risk assessment as well as 2-year rodent models, produces fewer irrelevant neoplastic outcomes, and often will be preferable to a 2-year rodent study. Before revising ICH S1 guidance, a prospective evaluation will be conducted to test the proposed weight-of-evidence approach. This evaluation offers an opportunity for a secondary analysis comparing the value of alternative mouse models and 2-year rodent studies in the proposed ICH S1 weight-of-evidence approach for human cancer risk assessment.

The rasH2 mouse model for assessing carcinogenic potential of pharmaceuticals.

A factor limiting widespread use of the transgenic rasH2 mouse model for carcinogenicity testing of pharmaceuticals is the paucity of published data on actual drug candidates in rasH2 mice. This report addresses this gap by highlighting rasH2 mouse study data for 10 pharmaceutical candidates. These results were compared with findings in the 2-year studies in Sprague-Dawley rats for the same 10 compounds. In the 6-month rasH2 studies, only 2 of the 10 compounds tested positive for carcinogenicity and these correlated with positive findings in the companion 2-year rat studies. One compound, sunitinib, produced gastroduodenal carcinoma in both sexes and increased hemangiosarcoma in spleen and uterus in female rasH2 mice; in rats it produced gastroduodenal carcinoma and increased pheochromocytoma (males only). The second compound, bazedoxifene, produced ovarian granulosa cell neoplasms in rasH2 mice and rats, and renal tubular neoplasms associated with increased chronic progressive nephropathy only in rats. The higher percentage of carcinogenicity positive rat bioassays could be attributed to rat-specific phenomena with little or low relevance to man. Thus, this article confirms previous reports that rasH2 mice develop rodent-specific neoplasms less frequently than rats and positive findings, when present, are accompanied by similar positive results in the rat.

A protein kinase C α and ß inhibitor blunts hyperphagia to halt renal function decline and reduces adiposity in a rat model of obesity-driven type 2 diabetes.

Type 2 diabetes (T2D) and its complications can have debilitating, sometimes fatal consequences for afflicted individuals. The disease can be difficult to control, and therapeutic strategies to prevent T2D-induced tissue and organ damage are needed. Here we describe the results of administering a potent and selective inhibitor of Protein Kinase C (PKC) family members PKCα and PKCß, Cmpd 1, in the ZSF1 obese rat model of hyperphagia-induced, obesity-driven T2D. Although our initial intent was to evaluate the effect of PKCα/ß inhibition on renal damage in this model setting, Cmpd 1 unexpectedly caused a marked reduction in the hyperphagic response of ZSF1 obese animals. This halted renal function decline but did so indirectly and indistinguishably from a pair feeding comparator group. However, above and beyond this food intake effect, Cmpd 1 lowered overall animal body weights, reduced liver vacuolation, and reduced inguinal adipose tissue (iWAT) mass, inflammation, and adipocyte size. Taken together, Cmpd 1 had strong effects on multiple disease parameters in this obesity-driven rodent model of T2D. Further evaluation for potential translation of PKCα/ß inhibition to T2D and obesity in humans is warranted.

Spontaneous tumor incidence in rasH2 mice: review of internal data and published literature.

Alternate transgenic mouse models are accepted as replacements for the standard carcinogenicity mouse bioassay by regulatory agencies with a companion 2-year rat bioassay. The slower rate of industry acceptance of these shorter transgenic mouse cancer bioassays has been due to lack of historical data and diagnostic criteria, and the use of nonstandardized terminologies in published data. To address these issues, especially that of generating a large historical database, a retrospective analysis of the spontaneous tumor incidences in rasH2 mice from internally sponsored 6-month carcinogenicity studies was compared to the published literature. Incidences of common spontaneous tumors (incidences > 1%) observed in these studies were lung bronchiolo-alveolar adenomas (mean 3.9-9.9%; range 0-18%), lung bronchiolo-alveolar adenocarcinomas (mean 1.4-2.4%; range 0-5%), splenic hemangiosarcomas (mean 3.0-3.9%; range 0-17%), cutaneous squamous cell papillomas (mean 1.1-1.2%; range 0-4%), Harderian gland adenoma (mean 0.8-1.2%; range 0-4%), and hepatocellular adenomas (mean 1.8%; 0-9% in males only). The remarkable similarity in the tumor incidences in multiple rasH2 studies over a decade and the observed stability of the inserted human gene are important indicators of the minimal drift in this model. Overall, the historical control data for spontaneous neoplasms should assist in the interpretation of future rasH2 mouse studies.

Development of a CD8 co-receptor independent T-cell receptor specific for tumor-associated antigen MAGE-A4 for next generation T-cell-based immunotherapy.

BACKGROUND: The cancer-testis antigen MAGE-A4 is an attractive target for T-cell-based immunotherapy, especially for indications with unmet clinical need like non-small cell lung or triple-negative breast cancer. METHODS: An unbiased CD137-based sorting approach was first used to identify an immunogenic MAGE-A4-derived epitope (GVYDGREHTV) that was properly processed and presented on human leukocyte antigen (HLA)-A2 molecules encoded by the HLA-A*02:01 allele. To isolate high-avidity T cells via subsequent multimer sorting, an in vitro priming approach using HLA-A2-negative donors was conducted to bypass central tolerance to this self-antigen. Pre-clinical parameters of safety and activity were assessed in a comprehensive set of in vitro and in vivo studies. RESULTS: A MAGE-A4-reactive, HLA-A2-restricted T-cell receptor (TCR) was isolated from primed T cells of an HLA-A2-negative donor. The respective TCR-T-cell (TCR-T) product bbT485 was demonstrated pre-clinically to have a favorable safety profile and superior in vivo potency compared with TCR-Ts expressing a TCR derived from a tolerized T-cell repertoire to self-antigens. This natural high-avidity TCR was found to be CD8 co-receptor independent, allowing effector functions to be elicited in transgenic CD4+ T helper cells. These CD4+ TCR-Ts supported an anti-tumor response by direct killing of MAGE-A4-positive tumor cells and upregulated hallmarks associated with helper function, such as CD154 expression and release of key cytokines on tumor-specific stimulation. CONCLUSION: The extensive pre-clinical assessment of safety and in vivo potency of bbT485 provide the basis for its use in TCR-T immunotherapy studies. The ability of this non-mutated high-avidity, co-receptor-independent TCR to activate CD8+ and CD4+ T cells could potentially provide enhanced cellular responses in the clinical setting through the induction of functionally diverse T-cell subsets that goes beyond what is currently tested in the clinic.

An "Omics" based survey of human colon cancer.

Despite an increased understanding of the molecular pathogenesis of colorectal cancer (CRC) during the past two decades, reliable and robust biomarkers to enable screening, surveillance, and primary prevention of this disease are lacking. CRC diagnosis and therapy remain dependent upon descriptive classification and staging systems, based primarily on morphology and histology. The traditional approach of understanding complex biological systems by studying smaller, discrete units of the whole system has been less fruitful for understanding complex diseases. The implicit assumption of traditional methods, that a single or even only a few factors, play a dominant role in a complex disease might be inadequate when studying multifactorial diseases such as cancer. The burgeoning field of systems biology adopts a holistic approach, wherein the integration of individual parts of the system is sought. The cornerstone of a systems biology approach has been the development of a variety of high-throughput "omics" sciences, including genomics, transcriptomics, proteomics, and metabolomics. This review will focus on the "omics" literature in the field of sporadic human CRC and present examples of how a systems approach has been extremely useful in understanding concepts that would have been difficult to develop using traditional methods.

Agammaglobulinemia and Staphylococcus aureus botryomycosis in a cohort of related sentinel Swiss Webster mice.

Sentinel mouse seroconversion to infectious agents is critical for laboratory animal facility disease monitoring. We report spontaneous emergence of non-sex-linked agammaglobulinemia with B-cell deficiency and cutaneous Staphylococcus aureus granulomatosis (botryomycosis) in a cohort of related Swiss Webster sentinel mice. Our experience reinforces the importance of immunocompetency validation in surveillance programs.

Proinflammatory CD4+ CD45RB(hi) lymphocytes promote mammary and intestinal carcinogenesis in Apc(Min/+) mice.

Cancers of breast and bowel are increasingly frequent in humans. Chronic inflammation is known to be a risk factor for these malignancies, yet cellular and molecular mechanisms linking inflammation and carcinogenesis remain poorly understood. Here, we apply a widely used T-cell transfer paradigm, involving adoptive transfer of proinflammatory CD4+ CD45RB(hi) (T(E)) cells to induce inflammatory bowel disease (IBD) in mice, to investigate roles of inflammation on carcinogenesis in the Apc(Min/+) mouse model of intestinal polyposis. We find that transfer of T(E) cells significantly increases adenoma multiplicity and features of malignancy in recipient Apc(Min/+) mice. Surprisingly, we find that female Apc(Min/+) recipients of T(E) cells also rapidly develop mammary tumors. Both intestinal polyposis and mammary adenocarcinoma are abolished by cotransfer of anti-inflammatory CD4+ CD45RB(lo) regulatory lymphocytes or by neutralization of key proinflammatory cytokine tumor necrosis factor-alpha. Lastly, down-regulation of cyclooxygenase-2 and c-Myc expression is observed coincident with tumor regression. These findings define a novel mouse model of inflammation-driven mammary carcinoma and suggest that epithelial carcinogenesis can be mitigated by anti-inflammatory cells and cytokines known to regulate IBD in humans and mice.

Innate immune inflammatory response against enteric bacteria Helicobacter hepaticus induces mammary adenocarcinoma in mice.

Inflammation associated with bacterial infections is a risk factor for cancers in humans, yet its role in breast cancer remains poorly understood. We have previously shown that innate immune inflammatory response against intestinal bacteria is sufficient to induce colon cancer. Here we report that infecting Rag2-deficient C57BL/6 Apc(Min/+) mice with an intestinal bacterial pathogen, Helicobacter hepaticus, significantly promotes mammary carcinoma in females and enhances intestinal adenoma multiplicity by a tumor necrosis factor alpha (TNFalpha)-dependent mechanism. The mammary and intestinal tumor development as well as the increase in proinflammatory mediators is suppressed by adoptive transfer of interleukin 10-competent CD4+CD45RB(lo)CD25+ regulatory (T(R)) cells. Furthermore, prior exposure of donor mice to H. hepaticus significantly enhances antitumor potency of their T(R) cells. Interestingly, these microbially experienced T(R) cells suppress tumorigenesis more effectively in recipient mice irrespective of their tumor etiology. These data suggest that infections with enteric pathogens enhance T(R)-cell potency and protect against epithelial cancers later in life, potentially explaining paradoxical increases in cancer risk in developed countries having more stringent hygiene practices. The possibility that dysregulated gut microbial infections in humans may lead to cancer in anatomically distant organs, such as breast, highlights the need for novel immune-based strategies in cancer prevention and treatment.

Rapid reversal of interleukin-6-dependent epithelial invasion in a mouse model of microbially induced colon carcinoma.

Chronic inflammation of mucosal surfaces renders them increasingly susceptible to epithelial cancers both in humans and mice. We have previously shown that anti-inflammatory CD4(+)CD45RB(lo)CD25(+) regulatory (Treg or T(R)) lymphocytes down-regulate inflammation and block development of bacteria-triggered colitis and colorectal cancer (CRC) in 129/SvEv Rag2-/- mice. Interestingly, T(R) cells collected from Interleukin (IL)-10-deficient cell donors not only failed to suppress carcinogenesis but instead promoted invasive mucinous colonic carcinoma with a strong gender bias expressing in male mice. We found we show that peritoneal invasion in this model is dependent on pleiotropic cytokine IL-6. Mucinous carcinoma arose rapidly and consistently after treatment with IL10-/- T(R) cells, which were found to express Foxp3+ and localize throughout tumor tissue. Carcinogenesis was rapidly reversible with transfer of wild type IL10-competent T(R) cells. Likewise, treatment with IL10-Ig fusion protein was sufficient to revert the lesions histologically, and restore inflammatory cytokine and oncogene expression to base line levels. These studies indicate an essential role for IL 6 in this CRC phenotype. Furthermore, immune-competent T(R) cells were important not only for preventing pathology but also for constructive remodeling of bowel following tumorigenic microbial insults. These data provide insights into etiopathogenesis of inflammation-associated epithelial invasion and maintenance of epithelial homeostasis.

CD4+CD25+ regulatory lymphocytes induce regression of intestinal tumors in ApcMin/+ mice.

Colorectal cancer in humans results from sequential genetic changes in intestinal epithelia commencing with inactivation of the APC tumor suppressor gene. Roles for host immunity in epithelial tumorigenesis are poorly understood. It has been previously shown that CD4+CD25+ lymphocytes inhibit colitis-associated epithelial tumors in Rag-deficient mice. Here we show that addition of CD4+CD25+ lymphocytes in ApcMin/+ mice reduces multiplicity of epithelial adenomas. Interleukin-10 was required in regulatory cells for therapeutic effect. Recipients of regulatory cells showed increased apoptosis and down-regulation of cyclooxygenase-2 within tumors coincident with tumor regression. These data suggest a role for regulatory lymphocytes in epithelial homeostasis in the ApcMin/+ mouse model of intestinal polyposis. Similarities with cancer of the breast, prostate, lung, and other sites raise the possibility of broader roles for regulatory lymphocytes in prevention and treatment of epithelial cancers in humans.

Circumvention and reactivation of the p53 oncogene checkpoint in mouse colon tumors.

The p53 tumor suppressor protein is sequence-normal in azoxymethane (AOM)-induced mouse colon tumors, making them a good model for human colon cancers that retain a wild type p53 gene. Cellular localization and co-immunoprecipitation experiments using a cell line derived from an AOM-induced colon tumor (AJ02-NM(0) cells) pointed to constitutively expressed Mdm2 as being an important negative regulator of p53 in these cells. Although the Mdm2 inhibitory protein p19/ARF was expressed in AJ02-NM(0) cells, its level of expression was not sufficient for p53 activation. We tested the response of AJ02-NM(0) cells to the recently developed Mdm2 inhibitor, Nutlin-3. Nutlin-3 was found to activate p53 DNA binding in AJ02-NM(0) cells, to a level comparable to doxorubicin and 5-fluorouracil (5-FU). In addition, Nutlin-3 increased expression of the p53 target genes Bax and PERP to a greater extent than doxorubicin or 5-FU, and triggered a G2/M phase arrest in these cells, compared to a G1 arrest triggered by doxorubicin and 5-FU. The differences in the cellular response may be related to differences in the kinetics of p53 activation and/or its post-translational modification status. In an ex vivo experiment, Nutlin-3 was found to activate p53 target gene expression and apoptosis in AOM-induced tumor tissue, but not in normal adjacent mucosa. Our data indicate that Mdm2 inhibitors may be an effective means of selectively targeting colon cancers that retain a sequence-normal p53 gene while sparing normal tissue and that the AOM model is an appropriate model for the preclinical development of these drugs.

Enterohepatic Helicobacter species isolated from the ileum, liver and colon of a baboon with pancreatic islet amyloidosis.

Microaerobic bacteria were isolated from a baboon with pancreatic islet amyloidosis and hepatitis. Phenotypic and molecular analyses identified two distinct helicobacters. Analyses of 16S rRNA demonstrated "Helicobacter macacae" in the ileum and liver, and Helicobacter cinaedi in the colon. To the best of the authors' knowledge, this is the first report describing the isolation of enterohepatic Helicobacter species from a baboon.

Immunomodulatory effects of in vitro exposure to organochlorines on T-cell proliferation in marine mammals and mice.

Marine mammals bioaccumulate various environmental contaminants such as organochlorines (OCs), which biomagnify via the food web. While the immunomodulatory effects of individual OCs have been studied, the effects of mixtures are not well understood. The immunomodulatory effects of polychlorinated biphenyl (PCB) 138, 153, 169, and 180 as well as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and all possible mixtures were examined in marine mammals and mice. Lymphocyte proliferation was significantly modulated by OCs in all species tested, mostly by non-coplanar PCBs, as shown using regression analyses. Correlation analyses showed significant correlations (interpreted as additive effects) between OCs in mice, killer whales, and Steller sea lions. Nonadditive synergistic and antagonistic interactions between OCs were detected in most of the species tested. Toxic equivalency (TEQ) values used for OC toxicity assessment failed to predict the immunomodulatory effects measured in mice and marine mammals. The commonly used mouse model failed to predict immunomodulatory effects in other species. Clustering data suggested that phylogeny does not predict toxicity of OCs. Overall, our data suggest the presence of species-specific sensitivities to different mixtures, in which OCs interactions may be complex and that may exert their effects through dioxinlike or dioxin-independent pathways. Lastly, lymphocyte proliferation, an important part of adaptive immunity, was significantly modulated in mice and marine mammals, suggesting the possibility of increased susceptibility to diseases. These findings will be useful to better characterize the risk associated with OC exposure and possibly lead to new conservation and management strategies.

Role of the alternating reading frame (P19)-p53 pathway in an in vivo murine colon tumor model.

Considering the importance of the oncogene checkpoint function of the alternating reading frame(ARF)-p53 pathway, studies were undertaken to evaluate the status of this pathway in azoxymethane (AOM)-induced mouse colon tumors. A PCR-based analysis of ARF and p53 cDNAs in normal colon tissues and AOM-induced colon tumors failed to detect mutations in either of these two critical tumor suppressor genes. In addition, laser capture microdissection of tumors followed by PCR-based sequencing of exons 5-7 of genomic p53 showed that even the most pleomorphic cancer cells were p53 normal. A marked increase in ARF mRNA and protein levels was observed in colon tumors, indicating activation of the ARF-p53 pathway in these tumors. High levels of ARF protein stabilized p53 protein in the tumors, but the p53 protein showed little biochemical activity. Compared with a mouse colonocyte cell line that expresses high levels of wild-type p53 (YAMC), the p53 protein in tumors had no detectable DNA binding activity nor did it activate p21 expression. In fact, p21 levels were lower in tumor tissue relative to normal mucosa, even though p53 levels were approximately 30-fold higher in tumors relative to control. Within the A/J tumors, we also used a cDNA microarray approach to screen a panel of genes that are transcriptionally up- or down-regulated by functional p53. The expression patterns of these p53-regulated genes were consistent with a lack of functional p53. This work demonstrates that the ARF-p53 oncogene checkpoint can be overcome without p53 mutations and that the mechanism used to overcome this checkpoint involves the suppression of p53 transcriptional activating activity. The AOM colon cancer model may be well suited for studying tumor promotion events that precede p53 disruption.