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John Christoforidis1, Robert Ricketts1, Cedric Pratt1, Jordan Pierce1, Scott Bean1, Michael Wells1, Xiaoli Zhang2, Krista La Perle31College of Medicine, 2Center for Biostatistics, 3College of Veterinary Medicine, The Ohio State University, Columbus, OH, USAPurpose: To investigate the effect of intravitreal pegaptanib, bevacizumab, and ranibizumab on blood-vessel formation during cutaneous wound healing in a rabbit model and to compare this effect to placebo controls.Methods: Forty New Zealand albino rabbits underwent full thickness cutaneous wounds using 6-mm dermatologic punch biopsies. The rabbits were assigned to four groups of ten, each receiving intravitreal injections of pegaptanib, bevacizumab, ranibizumab, or no injection (untreated controls). Five rabbits from each group underwent wound harvesting on day 7 and five from each group on day 14. The skin samples were stained with hematoxylin and eosin (HE), Masson's trichrome (MT), and CD34 for vascular endothelial cells. Semiquantitative evaluation of HE- and MT-stained slides was performed by one pathologist. Quantitative assessment of mean neovascularization (MNV) scores was obtained from five contiguous biopsy margin 400× fields of CD34-stained sections by four independent observers.Results: Week 1 MNV scores in CD-34 stained sections were: untreated controls: 11.51 ± 4.36; bevacizumab: 7.41 ± 2.82 (P = 0.013); ranibizumab: 8.71 ± 4.08 (P = 0.071); and pegaptanib: 10.15 ± 5.59 (P = 0.378). Week 2 MNV data were: untreated controls: 6.14 ± 2.25; bevacizumab: 7.25 ± 2.75 (P = 0.471); ranibizumab: 4.53 ± 3.12 (P = 0.297); and, pegaptanib: 6.35 ± 3.09 (P = 0.892). Interobserver variability using intraclass correlation coefficient was 0.961.Conclusions: At week 1, all three anti-VEGF agents had suppressed MNV scores compared to controls. Although not statistically significant, there was an inhibitory trend, particularly with bevacizumab and ranibizumab. These effects were diminished at 2 weeks, reflecting a transition between the proliferative and remodeling phases of wound healing.Keywords: anti-VEGF, wound healing, intraocular, pegaptanib, bevacizumab, ranibizumab

Information on a forum sponsored by the American College of Veterinary Pathologists (ACVP) on veterinary students interested in pathology residencies is presented. Topics include the challenges that the veterinary pathologists face who provide research pathology support in medical schools or research institution. The symposium proposes a basic curriculum that serves as guideline for an institution that wants to establish a fellowship that allows participants to be exposed to mouse pathobiology.

Human respiratory syncytial virus (RSV) is the leading viral cause of lower respiratory tract disease in infants and children worldwide. Currently, there are no FDA-approved vaccines to combat this virus. The large (L) polymerase protein of RSV replicates the viral genome and transcribes viral mRNAs. The L protein is organized as a core ring-like domain containing the RNA-dependent RNA polymerase and an appendage of globular domains containing an mRNA capping region and a cap methyltransferase region, which are linked by a flexible hinge region. Here, we found that the flexible hinge region of RSV L protein is tolerant to amino acid deletion or insertion. Recombinant RSVs carrying a single or double deletion or a single alanine insertion were genetically stable, highly attenuated in immortalized cells, had defects in replication and spread, and had a delay in innate immune cytokine responses in primary, well-differentiated, human bronchial epithelial (HBE) cultures. The replication of these recombinant viruses was highly attenuated in the upper and lower respiratory tracts of cotton rats. Importantly, these recombinant viruses elicited high levels of neutralizing antibody and provided complete protection against RSV replication. Taken together, amino acid deletions or insertions in the hinge region of the L protein can serve as a novel approach to rationally design genetically stable, highly attenuated, and immunogenic live virus vaccine candidates for RSV. IMPORTANCE Despite tremendous efforts, there are no FDA-approved vaccines for human respiratory syncytial virus (RSV). A live attenuated RSV vaccine is one of the most promising vaccine strategies for RSV. However, it has been a challenge to identify an RSV vaccine strain that has an optimal balance between attenuation and immunogenicity. In this study, we generated a panel of recombinant RSVs carrying a single and double deletion or a single alanine insertion in the large (L) polymerase protein that are genetically stable, sufficiently attenuated, and grow to high titer in cultured cells, while retaining high immunogenicity. Thus, these recombinant viruses may be promising vaccine candidates for RSV., (Copyright © 2020 American Society for Microbiology.)

The Akt family is comprised of three unique homologous proteins with isoform-specific effects, but isoform-specific in vivo data are limited in follicular thyroid cancer (FTC), a PI3 kinase-driven tumor. Prior studies demonstrated that PI3K/Akt signaling is important in thyroid hormone receptor β PV/PV knock-in (PV) mice that develop metastatic thyroid cancer that most closely resembles FTC. To determine the roles of Akt isoforms in this model we crossed Akt1 -/- , Akt2 -/- , and Akt3 -/- mice with PV mice. Over 12 months, thyroid size was reduced for the Akt null crosses (p < 0.001). Thyroid cancer development and local invasion were delayed in only the PVPV-Akt1 knock out (KO) mice in association with increased apoptosis with no change in proliferation. Primary-cultured PVPV-Akt1KO thyrocytes uniquely displayed a reduced cell motility. In contrast, loss of any Akt isoform reduced lung metastasis while vascular invasion was reduced with Akt1 or 3 loss. Microarray of thyroid RNA displayed incomplete overlap between the Akt KO models. The most upregulated gene was the dendritic cell (DC) marker CD209a only in PVPV-Akt1KO thyroids. Immunohistochemistry demonstrated an increase in CD209a-expressing cells in the PVPV-Akt1KO thyroids. In summary, Akt isoforms exhibit common and differential functions that regulate local and metastatic progression in this model of thyroid cancer.

Typhoid fever, caused primarily by Salmonella enterica serovar Typhi ( S Typhi), is a life-threatening systemic disease responsible for significant morbidity and mortality worldwide. Three to 5% of individuals infected with S Typhi become chronic carriers due to bacterial persistence in the gallbladder. We have demonstrated that Salmonella forms biofilms on gallstones to establish gallbladder carriage. However, an in-depth molecular understanding of chronic carriage in the gallbladder, from the perspective of both the pathogen and host, is poorly defined. To examine the dynamics of the gallbladder in response to Salmonella infection, we performed transcriptional profiling in the mouse gallbladder at early (7 days) and chronic (21 days) time points. Transcriptome sequencing (RNA-Seq) revealed a shift from a Th1 proinflammatory response at 7 days postinfection (dpi) toward an anti-inflammatory Th2 response by 21 dpi, characterized by increased levels of immunoglobulins and the Th2 master transcriptional regulator, GATA3. Additionally, bioinformatic analysis predicted the upstream regulation of characteristic Th2 markers, including interleukin-4 (IL-4) and Stat6. Immunohistochemistry and fluorescence-activated cell sorter (FACS) analysis confirmed a significant increase in lymphocytes, including T and B cells, at 21 dpi in mice with gallstones. Interestingly, the levels of Salmonella -specific CD4 T cells were 10-fold higher in the gallbladder of mice with gallstones at 21 dpi. We speculate that the biofilm state allows Salmonella to resist the initial onslaught of the Th1 inflammatory response, while yet undefined events influence a switch in the host immunity toward a more permissive type 2 response, enabling the establishment of chronic infection. IMPORTANCE The existence of chronic typhoid carriers has been in the public eye for over 100 years in part because of the publicity around Typhoid Mary. Additionally, it has been known for decades that the gallbladder is the main site of persistence and recently that gallstones play a key role. Despite this, very little is known about the physiological conditions that allow Salmonella enterica serovar Typhi to persist in the gallbladder. In this study, we analyze the transcriptional profile of the gallbladder in a mouse model of chronic carriage. We found a shift from an early proinflammatory immune response toward a later anti-inflammatory response, which could explain the stalemate that allows Salmonella persistence. Interestingly, we found a 10-fold increase in the number of Salmonella -specific T cells in mice with gallstones. This work moves us closer to understanding the mechanistic basis of chronic carriage, with a goal toward eradication of the disease., (Copyright © 2019 González et al.)

Purpose: Naturally occurring primary canine lung cancers share clinicopathologic features with human lung cancers in never-smokers, but the genetic underpinnings of canine lung cancer are unknown. We have charted the genomic landscape of canine lung cancer and performed functional characterization of novel, recurrent HER2 (ERBB2) mutations occurring in canine pulmonary adenocarcinoma (cPAC)., Experimental Design: We performed multiplatform genomic sequencing of 88 primary canine lung tumors or cell lines. Additionally, in cPAC cell lines, we performed functional characterization of HER2 signaling and evaluated mutation-dependent HER2 inhibitor drug dose-response., Results: We discovered somatic, coding HER2 point mutations in 38% of cPACs (28/74), but none in adenosquamous (cPASC, 0/11) or squamous cell (cPSCC, 0/3) carcinomas. The majority (93%) of HER2 mutations were hotspot V659E transmembrane domain (TMD) mutations comparable to activating mutations at this same site in human cancer. Other HER2 mutations were located in the extracellular domain and TMD. HER2 V659E was detected in the plasma of 33% (2/6) of dogs with localized HER2 V659E tumors. HER2 V659E cPAC cell lines displayed constitutive phosphorylation of AKT and significantly higher sensitivity to the HER2 inhibitors lapatinib and neratinib relative to HER2 -wild-type cell lines (IC 50 < 200 nmol/L in HER2 V659E vs. IC 50 > 2,500 nmol/L in HER2 WT )., Conclusions: This study creates a foundation for molecular understanding of and drug development for canine lung cancer. These data also establish molecular contexts for comparative studies in dogs and humans of low mutation burden, never-smoker lung cancer, and mutant HER2 function and inhibition., (©2019 American Association for Cancer Research.)

Background: Evidence suggests that antimicrobial peptides, components of the innate immune response, protect the kidneys and bladder from bacterial challenge. We previously identified ribonuclease 7 (RNase 7) as a human antimicrobial peptide that has bactericidal activity against uropathogenic Escherichia coli (UPEC). Functional studies assessing RNase 7's contributions to urinary tract defense are limited., Methods: To investigate RNase 7's role in preventing urinary tract infection (UTI), we quantified urinary RNase 7 concentrations in 29 girls and adolescents with a UTI history and 29 healthy female human controls. To assess RNase 7's antimicrobial activity in vitro in human urothelial cells, we used siRNA to silence urothelial RNase 7 production and retroviral constructs to stably overexpress RNase 7; we then evaluated UPEC's ability to bind and invade these cells. For RNase 7 in vivo studies, we developed humanized RNase 7 transgenic mice, subjected them to experimental UTI, and enumerated UPEC burden in the urine, bladder, and kidneys., Results: Compared with controls, study participants with a UTI history had 1.5-fold lower urinary RNase 7 concentrations. When RNase 7 was silenced in vitro , the percentage of UPEC binding or invading human urothelial cells increased; when cells overexpressed RNase 7, UPEC attachment and invasion decreased. In the transgenic mice, we detected RNase 7 expression in the kidney's intercalated cells and bladder urothelium. RNase 7 humanized mice exhibited marked protection from UPEC., Conclusions: These findings provide evidence that RNase 7 has a role in kidney and bladder host defense against UPEC and establish a foundation for investigating RNase 7 as a UTI prognostic marker or nonantibiotic-based therapy., (Copyright © 2019 by the American Society of Nephrology.)

Although limited by severe side effects and development of resistance, platinum-based therapies still represent the most common first-line treatment for non-small cell lung cancer (NSCLC). However, a crucial need in the clinical management of NSCLC is represented by the identification of cases sensitive to DNA damage response (DDR)-targeting drugs, such as cisplatin or PARP inhibitors. Here, we provide a molecular rationale for the stratification of NSCLC patients potentially benefitting from platinum compounds based on the expression levels of RANBP9, a recently identified player of the cellular DDR. RANBP9 was found overexpressed by immunohistochemistry (IHC) in NSCLC compared to normal adjacent tissues (NATs) (n = 147). Moreover, a retrospective analysis of 132 platinum-treated patients from the multi-centric TAILOR trial showed that RANBP9 overexpression levels are associated with clinical response to platinum compounds [Progression Free Survival Hazard Ratio (RANBP9 high vs low) 1.73, 95% CI 1.15-2.59, p = 0.0084; Overall Survival HR (RANBP9 high vs low) 1.99, 95% CI 1.27-3.11, p = 0.003]. Accordingly, RANBP9 KO cells showed higher sensitivity to cisplatin in comparison with WT controls both in vitro and in vivo models. NSCLC RANBP9 KO cells were also more sensitive than control cells to the PARP inhibitor olaparib alone and in combination with cisplatin, due to defective ATM-dependent and hyper-activated PARP-dependent DDR. The current investigation paves the way to prospective studies to assess the clinical value of RANBP9 protein levels as prognostic and predictive biomarker of response to DDR-targeting drugs, leading to the development of new tools for the management of NSCLC patients.

IWS1 is an RNA-polymerase II (RNAPII)-associated transcription elongation factor whose biological functions are poorly characterized. To shed some light on the function of this protein at the organismal level, we performed a systematic tissue analysis of its expression and generated Iws1-deficient mice. A thorough immunohistochemical characterization shows that IWS1 protein is present in the nucleus of all cells in most of the examined tissues, with few notable exceptions. We also report that ablation of Iws1 consistently causes lethality at the pre-implantation stage with high expression of the gene in fertilized oocytes. In summary, we are providing evidence that Iws1 is expressed in all adult organs and it is an essential gene for mouse embryonic development., Competing Interests: The authors have declared that no competing interests exist.

Background: Thyroid cancer is an emerging health problem in the United States and worldwide. With incidence rates of thyroid cancer rapidly rising, the need to develop new treatment options is becoming a priority, and understanding the molecular mechanisms of this disease is crucial to furthering these efforts. Thyroid growth is driven by the TSH/cAMP/PKA signaling pathway, and it has previously been shown that activation of PKA through genetic ablation of the regulatory subunit Prkar1a (Prkar1a KO) is sufficient to cause follicular thyroid cancer in mouse models. cAMP also activates the Epac proteins and their downstream effectors, Rap1a and Rap1b., Methods: Previously, the authors' laboratory generated a mouse model of follicular thyroid cancer by conferring thyroid-specific deletion of Prkar1a (R1a-TpoKO). To probe the roles of other components of the PKA signaling system in the development of thyroid cancer, this study deleted Rap1 and Epac1 in the setting of the Prkar1a knockout., Results: Deletion of Rap1 significantly decreases thyroid size and cancer incidence in Prkar1a KO thyroids. Further, isoform-specific ablation of Rap1a and Rap1b implicates Rap1b as the downstream effector of PKA during thyroid carcinogenesis. In vivo modeling provides definitive evidence that Epac1 plays little role in thyroid proliferation and is dispensable for thyroid carcinogenesis arising from the deletion of Prkar1a., Conclusions: This study demonstrate that PKA signaling to Rap1b is a key signaling node for follicular thyroid carcinogenesis, while Epac1 activity is not required for tumor development. This work sheds new light on the pathways involved in FTC development and identifies a possible target for the development of new therapies in the treatment of FTC.

Neuroblastoma, the most common extracranial solid tumor in childhood, remains a therapeutic challenge. However, one promising patient treatment strategy is the delivery of anti-tumor therapeutic agents via mesenchymal stromal cell (MSC) therapy. MSCs have been safely used to treat genetic bone diseases such as osteogenesis imperfecta, cardiovascular diseases, autoimmune diseases, and cancer. The pro-inflammatory cytokine interferon-gamma (IFNγ) has been shown to decrease tumor proliferation by altering the tumor microenvironment (TME). Despite this, clinical trials of systemic IFNγ therapy have failed due to the high blood concentration required and associated systemic toxicities. Here, we developed an intra-adrenal model of neuroblastoma, characterized by liver and lung metastases. We then engineered MSCs to deliver IFNγ directly to the TME. In vitro, these MSCs polarized murine macrophages to the M1 phenotype. In vivo, we attained a therapeutically active TME concentration of IFNγ without increased systemic concentration or toxicity. The TME-specific IFNγ reduced tumor growth rate and increased survival in two models of T cell deficient athymic nude mice. Absence of this benefit in NOD SCID gamma (NSG) immunodeficient mouse model indicates a mechanism dependent on the innate immune system. IL-17 and IL-23p19, both uniquely M1 polarization markers, transiently increased in the tumor interstitial fluid. Finally, the MSC vehicle did not promote tumor growth. These findings reveal that MSCs can deliver effective cytokine therapy directly to the tumor while avoiding systemic toxicity. This method transiently induces inflammatory M1 macrophage polarization, which reduces tumor burden in our novel neuroblastoma murine model. Stem Cells 2018;36:915-924., (© AlphaMed Press 2018.)

Mutations in genes encoding enzymes in the tricarboxylic acid cycle (TCA, also known as the Krebs cycle) have been implicated as causative genetic lesions in a number of human cancers, including renal cell cancers, glioblastomas and pheochromocytomas. In recent studies, missense mutations in the succinate dehydrogenase (SDH) complex have also been proposed to cause differentiated thyroid cancer. In order to gain mechanistic insight into this process, we generated mice lacking the SDH subunit D (Sdhd) in the thyroid. We report that these mice develop enlarged thyroid glands with follicle hypercellularity and increased proliferation. In vitro , human thyroid cell lines with knockdown of SDHD exhibit an enhanced migratory capability, despite no change in proliferative capacity. Interestingly, these cells acquire stem-like features which are also observed in the mouse tumors. The stem-like characteristics are reversed by α-ketoglutarate, suggesting that SDH-associated tumorigenesis results from dedifferentiation driven by an imbalance in cellular metabolites of the TCA cycle. The results of this study reveal a metabolic vulnerability for potential future treatment of SDH-associated neoplasia., (© 2017 Society for Endocrinology.)

Mutated mitogen-activated protein kinase (MAPK) pathway components promote tumor survival, proliferation, and immune evasion in solid tumors. MAPK mutations occur in hematologic cancers as well, but their role is less clear and few models are available to study this. We developed an in vivo model of disseminated BRAF V600E B-cell leukemia to determine the effects of this mutation on tumor development and immune evasion. Mice with B-cell-restricted BRAF V600E expression crossed with the Eµ-TCL1 model of chronic lymphocytic leukemia (CLL) developed leukemia significantly earlier (median, 4.9 vs 8.1 months; P < .001) and had significantly shorter lifespan (median, 7.3 vs 12.1 months; P < .001) versus BRAF wild-type counterparts. BRAF V600E expression did not affect B-cell proliferation but reduced spontaneous apoptosis. BRAF V600E -mutant leukemia produced greater T-cell effects, evidenced by exhaustion immunophenotype and CD44 + T-cell percentage, as well as increased expression of PD-L1 on CD11b + cells. Results were confirmed in syngeneic mice engrafted with BRAF V600E leukemia cells. Furthermore, a BRAF V600E -expressing CLL cell line more strongly inhibited anti-CD3/CD28-induced T-cell proliferation, which was reversed by BRAF V600E inhibition. These results demonstrate the immune-suppressive impact of BRAF V600E in B-cell leukemias and introduce a new model to develop rational combination strategies targeting both tumor cells and tumor-mediated immune evasion., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.

Purpose: KTN0158 is a novel anti-KIT antibody that potently inhibits wild-type and mutant KIT. This study evaluated the safety, biologic activity, and pharmacokinetic/pharmacodynamics profile of KTN0158 in dogs with spontaneous mast cell tumors (MCT) as a prelude to human clinical applications. Experimental Design: Cell proliferation, KIT phosphorylation, and mast cell degranulation were evaluated in vitro KTN0158 was administered to 4 research dogs to assess clinical effects and cutaneous mast cell numbers. Thirteen dogs with spontaneous MCT were enrolled into a prospective phase I dose-escalating open-label clinical study of KTN0158 evaluating 3 dose levels and 2 schedules and with weekly assessments for response and clinical toxicities. Results: KTN0158 was a potent inhibitor of human and dog KIT activation and blocked mast cell degranulation in vitro In dogs, KTN0158 was well tolerated and reduced cutaneous mast cell numbers in a dose-dependent manner. Clinical benefit of KTN0158 administration in dogs with MCT ( n = 5 partial response; n = 7 stable disease) was observed regardless of KIT mutation status, and decreased KIT phosphorylation was demonstrated in tumor samples. Histopathology after study completion demonstrated an absence of neoplastic cells in the primary tumors and/or metastatic lymph nodes from 4 dogs. Reversible hematologic and biochemical adverse events were observed at doses of 10 and 30 mg/kg. The MTD was established as 10 mg/kg. Conclusions: KTN0158 inhibits KIT phosphorylation, demonstrates an acceptable safety profile in dogs, and provides objective responses in canine MCT patients with and without activating KIT mutations, supporting future clinical evaluation of KTN0158 in people. Clin Cancer Res; 23(10); 2565-74. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Metastasis suppressors are key regulators of tumor growth, invasion, and metastases. Loss of metastasis suppressors has been associated with aggressive tumor behaviors and metastatic progression. We previously showed that regulator of calcineurin 1, isoform 4 (RCAN1-4) was upregulated by the KiSS1 metastatic suppression pathway and could inhibit cell motility when overexpressed in cancer cells. To test the effects of endogenous RCAN1-4 loss on thyroid cancer in vivo, we developed RCAN1-4 knockdown stable cells. Subcutaneous xenograft models demonstrated that RCAN1-4 knockdown promotes tumor growth. Intravenous metastasis models demonstrated that RCAN1-4 loss promotes tumor metastases to the lungs and their subsequent growth. Finally, stable induction of RCAN1-4 expression reduced thyroid cancer cell growth and invasion. Microarray analysis predicted that nuclear factor, erythroid 2-like 3 (NFE2L3) was a pivotal downstream effector of RCAN1-4. NFE2L3 overexpression was shown to be necessary for RCAN1-4-mediated enhanced growth and invasiveness and NEF2L3 overexpression independently increased cell invasion. In human samples, NFE2L3 was overexpressed in TCGA thyroid cancer samples versus normal tissues and NFE2L3 overexpression was demonstrated in distant metastasis samples from thyroid cancer patients. In conclusion, we provide the first evidence to our knowledge that RCAN1-4 is a growth and metastasis suppressor in vivo and that it functions in part through NFE2L3., Competing Interests: Conflict of interest: The authors have declared that no conflict of interest exists.

The FHIT gene, encompassing an active common fragile site, FRA3B, is frequently silenced in preneoplasia and cancer, through gene rearrangement or methylation of regulatory sequences. Silencing of Fhit protein expression causes thymidine kinase 1 downregulation, resulting in dNTP imbalance, and spontaneous replication stress that leads to chromosomal aberrations, allele copy number variations, insertions/deletions, and single-base substitutions. Thus, Fhit, which is reduced in expression in the majority of human cancers, is a genome "caretaker" whose loss initiates genome instability in preneoplastic lesions. To follow the early genetic alterations and functional changes induced by Fhit loss that may recapitulate the neoplastic process in vitro, we established epithelial cell lines from kidney tissues of Fhit-/- and +/+ mouse pups early after weaning, and subjected cell cultures to nutritional and carcinogen stress, which +/+ cells did not survive. Through transcriptome profiling and protein expression analysis, we observed changes in the Trp53/p21 and survivin apoptotic pathways in -/- cells, and in expression of proteins involved in epithelial-mesenchymal transition. Some Fhit-deficient cell lines showed anchorage-independent colony formation and increased invasive capacity in vitro. Furthermore, cells of stressed Fhit-/- cell lines formed s.c. and metastatic tumors in nude mice. Collectively, we show that Fhit loss and subsequent thymidine kinase 1 inactivation, combined with selective pressures, leads to neoplasia-associated alterations in genes and gene expression patterns in vitro and in vivo., (© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Unlabelled: Cutaneous T-cell lymphoma (CTCL) is the most common type of primary cutaneous lymphoma. Here, we report that patients with CTCL show increased IL15 in a clinical stage-dependent manner. Mechanistically, we show that ZEB1 is a transcriptional repressor of IL15 in T cells and that hypermethylation of the ZEB1 binding region within the IL15 promoter, as seen in patients with CTCL, prevents ZEB1 binding and causes increased transcription of IL15 Using a transgenic mouse model of IL15, we provide evidence that overexpression of IL15 induces a spontaneous CTCL that mimics the human neoplasm. Excessive autocrine production of IL15 in T cells inhibits an HDAC1-mediated negative autoregulatory loop, resulting in the upregulation of HDAC1 and HDAC6 and transcriptional induction of the onco-miR-21. Interruption of IL15 downstream signaling with isotype-specific HDAC inhibitors halts (HDAC1) or significantly delays (HDAC6) the progression of CTCL in vivo and provides preclinical evidence supporting a hierarchical model of oncogenic signaling in CTCL., Significance: To date, CTCL pathogenesis remains unknown, and there are no curative therapies. Our findings not only demonstrate a critical role for IL15-mediated inflammation in cutaneous T-cell lymphomagenesis, but also uncover a new oncogenic regulatory loop in CTCL involving IL15, HDAC1, HDAC6, and miR-21 that shows differential sensitivity to isotype-specific HDAC inhibitors. Cancer Discov; 6(9); 986-1005. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 932., (©2016 American Association for Cancer Research.)

XPO1/CRM1 is a key nuclear exporter protein that mediates translocation of numerous cellular regulatory proteins. We investigated whether XPO1 is a potential therapeutic target in melanoma using novel selective inhibitors of nuclear export (SINE). In vitro effects of SINE on cell growth and apoptosis were measured by MTS assay and flow cytometry [Annexin V/propidium iodide (PI)], respectively in human metastatic melanoma cell lines. Immunoblot analysis was used to measure nuclear localization of key cellular proteins. The in vivo activity of oral SINE was evaluated in NOD/SCID mice bearing A375 or CHL-1 human melanoma xenografts. SINE compounds induced cytostatic and pro-apoptotic effects in both BRAF wild type and mutant (V600E) cell lines at nanomolar concentrations. The cytostatic and pro-apoptotic effects of XPO1 inhibition were associated with nuclear accumulation of TP53, and CDKN1A induction in the A375 cell line with wild type TP53, while pMAPK accumulated in the nucleus regardless of TP53 status. The orally bioavailable KPT-276 and KPT-330 compounds significantly inhibited growth of A375 (p<0.0001) and CHL-1 (p = 0.0087) human melanoma cell lines in vivo at well tolerated doses. Inhibition of XPO1 using SINE represents a potential therapeutic approach for melanoma across cells with diverse molecular phenotypes by promoting growth inhibition and apoptosis.

No treatment is currently available for mucopolysaccharidosis (MPS) IIIB, a neuropathic lysosomal storage disease caused by autosomal recessive defect in α-N-acetylglucosaminidase (NAGLU). In anticipation of a clinical gene therapy treatment for MPS IIIB in humans, we tested the rAAV9-CMV-hNAGLU vector administration to cynomolgus monkeys (n=8) at 1E13 vg/kg or 2E13 vg/kg via intravenous injection. No adverse events or detectable toxicity occurred over a 6-month period. Gene delivery resulted in persistent global central nervous system and broad somatic transduction, with NAGLU activity detected at 2.9-12-fold above endogenous levels in somatic tissues and 1.3-3-fold above endogenous levels in the brain. Secreted rNAGLU was detected in serum. Low levels of preexisting anti-AAV9 antibodies (Abs) did not diminish vector transduction. Importantly, high-level preexisting anti-AAV9 Abs lead to reduced transduction in liver and other somatic tissues, but had no detectable impact on transgene expression in the brain. Enzyme-linked immunoabsorbent assay showed Ab responses to both AAV9 and rNAGLU in treated animals. Serum anti-hNAGLU Abs, but not anti-AAV9 Abs, correlated with the loss of circulating rNAGLU enzyme. However, serum Abs did not affect tissue rNAGLU activity levels. Weekly or monthly peripheral blood interferon-γ enzyme-linked immunospot assays detected a CD4(+) T-cell (Th-1) response to rNAGLU only at 4 weeks postinjection in one treated subject, without observable correlation to tissue transduction levels. The treatment did not result in detectable CTL responses to either AAV9 or rNAGLU. Our data demonstrate an effective and safe profile for systemic rAAV9-hNAGLU vector delivery in nonhuman primates, supporting its clinical potential in humans.

Disruption of adenylyl cyclase type 5 (AC5) knockout (KO) is a novel model for longevity. Because malignancy is a major cause of death and reduced lifespan in mice, the goal of this investigation was to examine the role of AC5KO in protecting against cancer. There have been numerous discoveries in genetically engineered mice over the past several decades, but few have been translated to the bedside. One major reason is that it is difficult to alter a gene in patients, but rather a pharmacological approach is more appropriate. The current investigation employs a parallel construction to examine the extent to which inhibiting AC5, either in a genetic knockout (KO) or by a specific pharmacological inhibitor protects against cancer. This study is unique, not only because a combined genetic and pharmacological approach is rare, but also there are no prior studies on the extent to which AC5 affects cancer. We found that AC5KO delayed age-related tumor incidence significantly, as well as protecting against mammary tumor development in AC5KO × MMTV-HER-2 neu mice, and B16F10 melanoma tumor growth, which can explain why AC5KO is a model of longevity. In addition, a Food and Drug Administration approved antiviral agent, adenine 9-β-D-arabinofuranoside (Vidarabine or AraAde), which specifically inhibits AC5, reduces LP07 lung and B16F10 melanoma tumor growth in syngeneic mice. Thus, inhibition of AC5 is a previously unreported mechanism for prevention of cancers associated with aging and that can be targeted by an available pharmacologic inhibitor, with potential consequent extension of lifespan., (© 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

miR-122, an abundant liver-specific microRNA (miRNA), regulates cholesterol metabolism and promotes hepatitis C virus (HCV) replication. Reduced miR-122 expression in hepatocellular carcinoma (HCC) correlates with metastasis and poor prognosis. Nevertheless, the consequences of sustained loss of function of miR-122 in vivo have not been determined. Here, we demonstrate that deletion of mouse Mir122 resulted in hepatosteatosis, hepatitis, and the development of tumors resembling HCC. These pathologic manifestations were associated with hyperactivity of oncogenic pathways and hepatic infiltration of inflammatory cells that produce pro-tumorigenic cytokines, including IL-6 and TNF. Moreover, delivery of miR-122 to a MYC-driven mouse model of HCC strongly inhibited tumorigenesis, further supporting the tumor suppressor activity of this miRNA. These findings reveal critical functions for miR-122 in the maintenance of liver homeostasis and have important therapeutic implications, including the potential utility of miR-122 delivery for selected patients with HCC and the need for careful monitoring of patients receiving miR-122 inhibition therapy for HCV.

This report describes a spontaneous prostate fibromyxoid sarcoma with smooth muscle differentiation in an approximately 136-week-old intact male F344xBNF1 rat on a diet study for 2 weeks. At necropsy, the prostate was markedly distorted and enlarged by a firm white multinodular mass (6.0 × 4.5 × 3.5 cm). Histopathologically, the mass consisted of solid sheets of interlacing mesenchymal spindle cells with indistinct cell borders. Nuclei were separated by variable amounts of hyaline to fibrillar eosinophilic and/or myxomatous material. The extracellular myxomatous material tended to form whorls and stained positively with alcian blue. The mass stained strongly with Masson trichrome and vimentin throughout. Approximately 5% of the neoplastic cells were positive for smooth muscle actin, and none stained for desmin and pancytokeratin. To the authors' knowledge, this fibromyxoid sarcoma with smooth muscle differentiation is the first such described prostatic sarcoma in a rat.

Germline mutations in the tumor suppressor gene PTEN (phosphatase and tensin homology deleted on chromosome 10) cause Cowden and Bannayan-Riley-Ruvalcaba (BRR) syndromes, two dominantly inherited disorders characterized by mental retardation, multiple hamartomas, and variable cancer risk. Here, we modeled three sentinel mutant alleles of PTEN identified in patients with Cowden syndrome and show that the nonsense Pten(4-5) and missense Pten(C124R) and Pten(G129E) alleles lacking lipid phosphatase activity cause similar developmental abnormalities but distinct tumor spectra with varying severity and age of onset. Allele-specific differences may be accounted for by loss of function for Pten(4-5), hypomorphic function for Pten(C124R), and gain of function for Pten(G129E). These data demonstrate that the variable tumor phenotypes observed in patients with Cowden and BRR syndromes can be attributed to specific mutations in PTEN that alter protein function through distinct mechanisms.

We developed a new approach to bioluminescent T cell imaging using a membrane-anchored form of the Gaussia luciferase (GLuc) enzyme, termed extGLuc, which we could stably express in both mouse and human primary T cells. In vitro, extGLuc+ cells emitted significantly higher bioluminescent signal when compared to cells expressing GLuc, Renilla luciferase (RLuc) or membrane-anchored RLuc (extRLuc). In vivo, mouse extGLuc+ T cells showed higher bioluminescent signal when compared to GLuc+ and RLuc+ T cells. Application of this imaging approach to human T cells genetically modified to express tumor-specific chimeric antigen receptors (CARs) enabled us to show in vivo CAR-mediated T cell accumulation in tumor, T cell persistence over time and concomitant imaging of T cells and tumor cells modified to express firefly luciferase. This sensitive imaging technology has application to many in vivo cell-based studies in a wide array of mouse models.

Purpose: Human T cells targeted to the B cell-specific CD19 antigen through retroviral-mediated transfer of a chimeric antigen receptor (CAR), termed 19z1, have shown significant but partial in vivo antitumor efficacy in a severe combined immunodeficient (SCID)-Beige systemic human acute lymphoblastic leukemia (NALM-6) tumor model. Here, we investigate the etiologies of treatment failure in this model and design approaches to enhance the efficacy of this adoptive strategy., Experimental Design: A panel of modified CD19-targeted CARs designed to deliver combined activating and costimulatory signals to the T cell was generated and tested in vitro to identify an optimal second-generation CAR. Antitumor efficacy of T cells expressing this optimal costimulatory CAR, 19-28z, was analyzed in mice bearing systemic costimulatory ligand-deficient NALM-6 tumors., Results: Expression of the 19-28z CAR, containing the signaling domain of the CD28 receptor, enhanced systemic T-cell antitumor activity when compared with 19z1 in treated mice. A treatment schedule of 4 weekly T-cell injections, designed to prolong in vivo T-cell function, further improved long-term survival. Bioluminescent imaging of tumor in treated mice failed to identify a conserved site of tumor relapse, consistent with successful homing by tumor-specific T cells to systemic sites of tumor involvement., Conclusions: Both in vivo costimulation and repeated administration enhance eradication of systemic tumor by genetically targeted T cells. The finding that modifications in CAR design as well as T-cell dosing allowed for the complete eradication of systemic disease affects the design of clinical trials using this treatment strategy.

Brains from subjects who have Alzheimer's disease (AD) express inducible nitric oxide synthase (iNOS). We tested the hypothesis that iNOS contributes to AD pathogenesis. Immunoreactive iNOS was detected in brains of mice with AD-like disease resulting from transgenic expression of mutant human beta-amyloid precursor protein (hAPP) and presenilin-1 (hPS1). We bred hAPP-, hPS1-double transgenic mice to be iNOS(+/+) or iNOS(-/-), and compared them with a congenic WT strain. Deficiency of iNOS substantially protected the AD-like mice from premature mortality, cerebral plaque formation, increased beta-amyloid levels, protein tyrosine nitration, astrocytosis, and microgliosis. Thus, iNOS seems to be a major instigator of beta-amyloid deposition and disease progression. Inhibition of iNOS may be a therapeutic option in AD.

Carney complex (CNC) is a familial multiple neoplasia syndrome characterized by cardiac and extracardiac myxomas in the setting of spotty skin pigmentation and endocrinopathy. We previously identified PRKAR1A (regulatory subunit 1alpha of protein kinase A) mutations in CNC. Mutational analyses of the PRKAR1A gene in 51 unrelated CNC probands now detect mutations in 65%. All mutations, except for one unique missense mutation, lead to PRKAR1A haploinsufficiency. Therefore, we studied the consequences of prkar1a haploinsufficiency in mice. Although we did not observe cardiac myxomas or altered pigmentation in prkar1a(+/-) mice, we did observe some phenotypes similar to CNC, including altered heart rate variability. Moreover, prkar1a(+/-) mice exhibited a marked propensity for extracardiac tumorigenesis. They developed sarcomas and hepatocellular carcinomas. Sarcomas were frequently associated with myxomatous differentiation. Tumors from prkar1a(+/-) mice did not exhibit prkar1a loss of heterozygosity. Thus, we conclude that although PRKAR1A haploinsufficiency does predispose to tumorigenesis, distinct secondary genetic events are required for tumor formation.

Antemortem diagnosis of generalized ulcerative and pyogranulomatous dermatitis with numerous intralesional tachyzoites was made from skin biopsy specimens from 2 adult dogs on chronic immunosuppressive therapy. A 9-year-old Italian Greyhound was on long-term corticosteroid therapy for the treatment of a lupus-like systemic autoimmune disorder, and a 7-year-old Labrador Retriever had received several months of chemotherapy for lymphosarcoma. The tachyzoites were identified as Neospora caninum by immunoperoxidase immunohistochemistry. Both dogs were treated with clindamycin. Lesions in the Greyhound resolved; however, the Labrador Retriever was euthanized because of evidence of neuromuscular disease, despite improvement of the skin lesions. These 2 cases indicate that cutaneous neosporosis can occur in adult dogs on chronic immunosuppressive therapy. The disease may result from reactivation of a congenital infection and/or a recently acquired primary infection.

Papillary thyroid carcinomas in humans are associated with the ret/PTC oncogene and, following loss of p53 function, may progress to anaplastic carcinomas. Mice with thyroid-targeted expression of ret/PTC1 developed papillary thyroid carcinomas that were minimally invasive and did not metastasize. These mice were crossed with p53-/- mice to investigate whether loss of p53 would promote anaplasia and metastasis of ret/PTC1-induced thyroid tumors. The majority of p53-/- mice died or were euthanized by 17 weeks of age due to the development of thymic lymphomas, soft tissue sarcomas, and testicular teratomas. All ret/PTC1 mice developed thyroid carcinomas, but tumors in p53-/- mice were more anaplastic, larger in diameter, more invasive, and had a higher mitotic index than tumors in p53+/+ and p53+/- mice. Thyroid tumors did not metastasize in any of the experimental p53+/+ and p53+/- mice

Quantitation in histopathology generates numerical or rank-order data amenable to statistical analysis, but can be very labor-intensive. The advent and coupling of computerized image analysis systems and optimized immunostaining procedures has not only decreased the laborious aspect of counting, but has also improved reproducibility and reliability by overcoming inter- and intra-observer variation. Still, a certain degree of manual intervention is required and a good understanding of the underlying biology is mandatory for proper design of the sampling system. Here, we focus on subcutaneous tumor models to illustrate the use and benefits of computer-assisted image analysis in quantitative histopathology. In particular, we discuss the quantitation of biological parameters frequently assessed in biology and toxicology: cell proliferation, apoptotic and oncotic necrosis, and angiogenesis. For each of these parameters, specific sampling procedures and labeling techniques are discussed based on current molecular and cellular concepts.

Disseminated eosinophilic myositis was diagnosed in an alpaca that had been imported to the USA from Peru 5 years earlier. The myositis was associated with macroscopically visible large sarcocysts that were characterized histologically by septate compartments containing bradyzoites, and ultrastructurally by cyst walls composed of anastomosing villous protrusions. Two hours before death, the alpaca aborted an 8-month-gestation fetus, but no lesions were found in the uterus, placenta or fetus. Additional macroscopical findings included haemoabdomen and myofibre haemorrhage, degeneration and necrosis. It is believed that this is the first described case of clinical disease associated with a Sarcocystis sp. (probably S. aucheniae) in camelids., (Copyright 1999 Harcourt Publishers Ltd.)

Hodgkin's-like lymphoma involving the lung, mediastinum, liver, kidneys and mesenteric lymph nodes was diagnosed in a ferret. The diagnosis was based on the presence of an admixture of CD3+ small lymphocytes with smaller numbers of macrophages, eosinophils, and large, pleomorphic, frequently multinucleated, Reed-Sternberg-like cells which were immunoreactive to BLA.36 monoclonal antibody. In addition, the liver, pancreas, small intestine and lungs were infiltrated with moderate to large numbers of eosinophils, forming eosinophilic granulomas with occasional deposition of Splendore-Hoeppli material, supporting a diagnosis of hypereosinophilic syndrome. The concurrent diagnosis of hypereosinophilic syndrome and Hodgkin's-like lymphoma in this ferret provides further support to the concept that, in animals, multisystemic eosinophilic infiltrates may be caused by the abnormal proliferation of T lymphocytes, as has been demonstrated in man.

Systemic candidiasis, with involvement of the spleen, liver, kidneys, and lymph nodes, was diagnosed in a geriatric captive cheetah (Acinonyx jubatus). The animal had a long clinical history of intermittent chronic gastritis associated with Helicobacter acinonyx and chronic renal failure, both of which were repeatedly treated with broad-spectrum antimicrobial therapy. Following euthanasia, a postmortem examination showed numerous microabscesses and granulomas composed of degenerate eosinophils and containing asteroids or Splendore-Hoeppli material throughout the body. Yeast, pseudohyphae, and infrequently branching septate hyphae, demonstrated with special stains, were identified as a Candida sp. by fluorescent antibody testing. Low genetic variation in cheetahs may increase their susceptibility to infectious agents. Additional factors contributing to the overgrowth and dissemination of Candida sp. in this case may have included changes in the bacterial flora of the alimentary tract as a result of repeated antimicrobial therapy and alterations in the topography of the alimentary mucosa caused by chronic gastritis.