Tongue orthotopic xenografts to study fusion-negative rhabdomyosarcoma invasion and metastasis in live animals.

PAX3/7 fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood mesodermal lineage malignancy with a poor prognosis for metastatic or relapsed cases. Limited understanding of advanced FN-RMS is partially attributed to the absence of sequential invasion and dissemination events and the challenge in studying cell behavior, using, for example, non-invasive intravital microscopy (IVM), in currently used xenograft models. Here, we developed an orthotopic tongue xenograft model of FN-RMS to study cell behavior and the molecular basis of invasion and metastasis using IVM. FN-RMS cells are retained in the tongue and invade locally into muscle mysial spaces and vascular lumen, with evidence of hematogenous dissemination to the lungs and lymphatic dissemination to lymph nodes. Using IVM of tongue xenografts reveals shifts in cellular phenotype, migration to blood and lymphatic vessels, and lymphatic intravasation. Insight from this model into tumor invasion and metastasis at the tissue, cellular, and subcellular level can guide new therapeutic avenues for advanced FN-RMS.

Fusion-negative rhabdomyosarcoma orthotopic tongue xenografts for study of invasion, intravasation and metastasis in live animals.

PAX3/7 Fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood mesodermal lineage malignancy with a poor prognosis for metastatic or relapsed cases. Towards achieving a more complete understanding of advanced FN-RMS, we developed an orthotopic tongue xenograft model for studies of molecular basis of FN-RMS invasion and metastasis. The behavior of FN-RMS cells injected into murine tongue was examined using in vivo bioluminescence imaging, non-invasive intravital microscopy (IVM), and histopathology and compared to the prevailing hindlimb intramuscular and subcutaneous xenografts. FN-RMS cells were retained in the tongue and invaded locally into muscle mysial spaces and vascular lumen. While evidence of hematogenous dissemination to the lungs occurred in tongue and intramuscular xenografts, evidence of local invasion and lymphatic dissemination to lymph nodes only occurred in tongue xenografts. IVM and RNA-seq of tongue xenografts reveal shifts in cellular phenotype and differentiation state in tongue xenografts. IVM also shows homing to blood and lymphatic vessels, lymphatic intravasation, and dynamic membrane protrusions. Based on these findings, the tongue orthotopic xenograft of FN-RMS is a valuable model for tumor progression studies at the tissue, cellular and subcellular levels providing insight into kinetics and molecular bases of tumor invasion and metastasis and, hence, new therapeutic avenues for advanced FN-RMS.

Drug combinations identified by high-throughput screening promote cell cycle transition and upregulate Smad pathways in myeloma.

Drug resistance and disease progression are common in multiple myeloma (MM) patients, underscoring the need for new therapeutic combinations. A high-throughput drug screen in 47 MM cell lines and in silico Huber robust regression analysis of drug responses revealed 43 potentially synergistic combinations. We hypothesized that effective combinations would reduce MYC expression and enhance p16 activity. Six combinations cooperatively reduced MYC protein, frequently over-expressed in MM and also cooperatively increased p16 expression, frequently downregulated in MM. Synergistic reductions in viability were observed with top combinations in proteasome inhibitor-resistant and sensitive MM cell lines, while sparing fibroblasts. Three combinations significantly prolonged survival in a transplantable Ras-driven allograft model of advanced MM closely recapitulating high-risk/refractory myeloma in humans and reduced viability of ex vivo treated patient cells. Common genetic pathways similarly downregulated by these combinations promoted cell cycle transition, whereas pathways most upregulated were involved in TGFß/SMAD signaling. These preclinical data identify potentially useful drug combinations for evaluation in drug-resistant MM and reveal potential mechanisms of combined drug sensitivity.

Host CLIC4 expression in the tumor microenvironment is essential for breast cancer metastatic competence.

The TGF-ß-regulated Chloride Intracellular Channel 4 (CLIC4) is an essential participant in the formation of breast cancer stroma. Here, we used data available from the TCGA and METABRIC datasets to show that CLIC4 expression was higher in breast cancers from younger women and those with early-stage metastatic disease. Elevated CLIC4 predicted poor outcome in breast cancer patients and was linked to the TGF-ß pathway. However, these associations did not reveal the underlying biological contribution of CLIC4 to breast cancer progression. Constitutive ablation of host Clic4 in two murine metastatic breast cancer models nearly eliminated lung metastases without reducing primary tumor weight, while tumor cells ablated of Clic4 retained metastatic capability in wildtype hosts. Thus, CLIC4 was required for host metastatic competence. Pre- and post-metastatic proteomic analysis identified circulating pro-metastatic soluble factors that differed in tumor-bearing CLIC4-deficient and wildtype hosts. Vascular abnormalities and necrosis increased in primary tumors from CLIC4-deficient hosts. Transcriptional profiles of both primary tumors and pre-metastatic lungs of tumor-bearing CLIC4-deficient hosts were consistent with a microenvironment where inflammatory pathways were elevated. Altogether, CLIC4 expression in human breast cancers may serve as a prognostic biomarker; therapeutic targeting of CLIC4 could reduce primary tumor viability and host metastatic competence.

CCL5 production in lung cancer cells leads to an altered immune microenvironment and promotes tumor development.

Current immunotherapies for lung cancer are only effective in a subset of patients. Identifying tumor-derived factors that facilitate immunosuppression offers the opportunity to develop novel strategies to supplement and improve current therapeutics. We sought to determine whether expression of driver oncogenes in lung cancer cells affects cytokine secretion, alters the local immune environment, and influences lung tumor progression. We demonstrate that oncogenic EGFR and KRAS mutations, which are early events in lung tumourigenesis, can drive cytokine and chemokine production by cancer cells. One of the most prominent changes was in CCL5, which was rapidly induced by KRASG12V or EGFRL858R expression, through MAPK activation. Immunocompetent mice implanted with syngeneic KRAS-mutant lung cancer cells deficient in CCL5 have decreased regulatory T cells (Tregs), evidence of T cell exhaustion, and reduced lung tumor burden, indicating tumor-cell CCL5 production contributes to an immune suppressive environment in the lungs. Furthermore, high CCL5 expression correlates with poor prognosis, immunosuppressive regulatory T cells, and alteration to CD8 effector function in lung adenocarcinoma patients. Our data support targeting CCL5 or CCL5 receptors on immune suppressive cells to prevent formation of an immune suppressive tumor microenvironment that promotes lung cancer progression and immunotherapy insensitivity.

Pharmacokinetics and tolerability of the dual TORC1/2 inhibitor sapanisertib in combination with the MEK inhibitor trametinib in dogs.

Activation of one or both the Ras/MAPK and PI3K/Akt/mTOR signal transduction pathways are known to mediate oncogenicity of several canine and human cancers, including mucosal melanomas. Reciprocal cross activation between the two pathways can be a source of drug resistance. Consequently, oral dosing for plasma pharmacokinetic (PK) analysis and tolerability to a combination of sapanisertib, a dual TORC1/2 inhibitor, and trametinib, a MEK inhibitor, was evaluated in nontumor-bearing laboratory dogs for its potential application in parallel pathway targeting. Twelve dogs, divided into three equal cohorts, received either the combination or single agents. Animals were monitored for PK following single dose and 17-day repeat dosing, and by clinical observations, hematology, serum biochemistry, coagulation studies and urinalyses. A single trametinib dose (0.025 mg/kg), sulfated as dimethyl sulfoxide which enhanced its absorption, reached mean maximum concentration (Cmax) 0.64 ng/mL [18% coefficient of variation (CV)] at a median time to maximum concentration (Tmax) of 1.5 h (hr), and mean area under the concentration-time curve (AUC) 16.8 hr*ng/mL (14%CV), which were similar when given alone or in combination with sapanisertib. A prolonged half-life afforded 3-4-fold plasma accumulation of trametinib with daily dosing, analogous to humans. Trametinib PK mirrored previous regulatory data in dogs, while exposure approximated some published human values but generally not all patients. Sapanisertib-alone in canine plasma following single 0.1 mg/kg dose [mean Cmax 26.3 ng/mL (21%CV), median Tmax 2.0 hr, and mean AUC 248 hr*ng/mL (41%CV)] resembled levels in human therapeutic trials; whereas canine sapanisertib exposure was reduced when combined with trametinib, a known cytochrome P450 CYP3A4 inducer. Sex differences were not observed for either drug. Side effects upon repeat dosing with either or both drugs may include body weight loss, maldigestion, and cutaneous discoloration. The combination was tolerated without dose limiting toxicity, although clinical laboratory analyses revealed drug-induced acute-phase inflammation, proteinuria, and decreased blood reticulocytes, mild changes not necessitating intervention. Short-term results in dogs with this combination would appear to hold translational promise for clinical trial evaluation to target canine and possibly human melanoma, as well as other cancers having one or both signal transduction pathway activations.

Nuclear pore protein NUP210 depletion suppresses metastasis through heterochromatin-mediated disruption of tumor cell mechanical response.

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis.

International Guidelines for Veterinary Tumor Pathology: A Call to Action.

Standardization of tumor assessment lays the foundation for validation of grading systems, permits reproducibility of oncologic studies among investigators, and increases confidence in the significance of study results. Currently, there is minimal methodological standardization for assessing tumors in veterinary medicine, with few attempts to validate published protocols and grading schemes. The current article attempts to address these shortcomings by providing standard guidelines for tumor assessment parameters and protocols for evaluating specific tumor types. More detailed information is available in the Supplemental Files, the intention of which is 2-fold: publication as part of this commentary, but more importantly, these will be available as "living documents" on a website (www.vetcancerprotocols.org), which will be updated as new information is presented in the peer-reviewed literature. Our hope is that veterinary pathologists will agree that this initiative is needed, and will contribute to and utilize this information for routine diagnostic work and oncologic studies. Journal editors and reviewers can utilize checklists to ensure publications include sufficient detail and standardized methods of tumor assessment. To maintain the relevance of the guidelines and protocols, it is critical that the information is periodically updated and revised as new studies are published and validated with the intent of providing a repository of this information. Our hope is that this initiative (a continuation of efforts published in this journal in 2011) will facilitate collaboration and reproducibility between pathologists and institutions, increase case numbers, and strengthen clinical research findings, thus ensuring continued progress in veterinary oncologic pathology and improving patient care.

Inter-Institutional Partnerships to Develop Veterinarian-Investigators through the NIH Comparative Biomedical Scientist Training Program Benefit One Health Goals.

Limitations in workforce size and access to resources remain perennial challenges to greater progress in academic veterinary medicine and engagement between human and veterinary medicine (One Health). Ongoing resource constraints occur in part due to limited public understanding of the role veterinarians play in improving human health. One Health interactions, particularly through interdisciplinary collaborations in biomedical research, present constructive opportunities to inform resource policies and advance health care. To this end, inter-institutional partnerships between individual veterinary medical education programs (VMEPs) and several National Institutes of Health (NIH) intramural research programs have created synergies beyond those provided by individual programs. In the NIH Comparative Biomedical Scientist Training Program (CBSTP), interdisciplinary cross-training of veterinarians consisting of specialty veterinary medicine coupled with training in human disease research leading to a PhD, occurs collaboratively on both VMEP and NIH campuses. Pre-doctoral veterinary student research opportunities have also been made available. Through the CBSTP, NIH investigators and national biomedical science policy makers gain access to veterinary perspective and expertise, while veterinarians obtain additional opportunities for NIH-funded research training. CBSTP Fellows serve as de facto ambassadors enhancing visibility for the profession while in residence at NIH, and subsequently through a variety of university, industry, and government research appointments, as graduates. Thus, the CBSTP represents an inter-institutional opportunity that not only addresses critical needs for veterinarian-scientists in the biomedical workforce, but also simultaneously exposes national policy makers to veterinarian-scientists' specialized training, leading to more effective realization of One Health goals to benefit human and animal health.

Efficacy, Tolerability, and Pharmacokinetics of Combined Targeted MEK and Dual mTORC1/2 Inhibition in a Preclinical Model of Mucosal Melanoma.

Melanomas arising in the mucous membranes are a rare and aggressive subtype. New treatment approaches are needed, yet accumulating sufficient evidence to improve patient outcomes is difficult. Clinical and pathological correlates between human and canine mucosal melanomas are substantial, and the relatively greater incidence of spontaneous naturally occurring mucosal melanoma in dogs represents a promising opportunity for predictive modeling. The genomic landscapes of human and canine mucosal melanoma appear highly diverse and generally lack recurring hotspot mutations associated with cutaneous melanomas. Although much remains to be determined, evidence indicates that Ras/MAPK and/or PI3K/AKT/mTOR signaling pathway activations are common in both species and may represent targets for therapeutic intervention. Sapanisertib, an mTORC1/2 inhibitor, was selected from a PI3K/mTOR inhibitor library to collaborate with MEK inhibition; the latter preclinical efficacy was demonstrated previously for canine mucosal melanoma. Combined inhibition of MEK and mTORC1/2, using trametinib and sapanisertib, produced apoptosis and cell-cycle alteration, synergistically reducing cell survival in canine mucosal melanoma cell lines with varying basal signaling activation levels. Compared with individual inhibitors, a staggered sapanisertib dose, coupled with daily trametinib, was optimal for limiting primary mucosal melanoma xenograft growth in mice, and tumor dissemination in a metastasis model, while minimizing hematologic and renal side effects. Inhibitors downmodulated respective signaling targets and the combination additionally suppressed pathway reciprocal crosstalk. The combination did not significantly change plasma sapanisertib pharmacokinetics; however, trametinib area under the curve was increased in the presence of sapanisertib. Targeting Ras/MAPK and PI3K/AKT/mTOR signal transduction pathways appear rational therapies for canine and human mucosal melanoma.

Hypomorphic mTOR Downregulates CDK6 and Delays Thymic Pre-T LBL Tumorigenesis.

PI3K/AKT/mTOR pathway hyperactivation is frequent in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL). To model inhibition of mTOR, pre-T-cell lymphoblastic leukemia/lymphoma (pre-T LBL) tumor development was monitored in mice with T lymphocyte-specific, constitutively active AKT (Lck-MyrAkt2) that were either crossed to mTOR knockdown (KD) mice or treated with the mTOR inhibitor everolimus. Lck-MyrAkt2;mTOR KD mice lived significantly longer than Lck-MyrAkt2;mTOR wild-type (WT) mice, although both groups ultimately developed thymic pre-T LBL. An increase in survival was also observed when Lck-MyrAkt2;mTOR WT mice were treated for 8 weeks with everolimus. The transcriptional profiles of WT and KD thymic lymphomas were compared, and Ingenuity Pathway Upstream Regulator Analysis of differentially expressed genes in tumors from mTOR WT versus KD mice identified let-7 and miR-21 as potential regulatory genes. mTOR KD mice had higher levels of let-7a and miR-21 than mTOR WT mice, and rapamycin induced their expression in mTOR WT cells. CDK6 was one of the most downregulated targets of both let-7 and miR21 in mTOR KD tumors. CDK6 overexpression and decreased expression of let-7 in mTOR KD cells rescued a G1 arrest phenotype. Combined mTOR (rapamycin) and CDK4/6 (palbociclib) inhibition decreased tumor size and proliferation in tumor flank transplants, increased survival in an intravenous transplant model of disseminated leukemia compared with single agent treatment, and cooperatively decreased cell viability in human T-ALL/LBL cell lines. Thus, mTOR KD mice provide a model to explore drug combinations synergizing with mTOR inhibitors and can be used to identify downstream targets of inhibition.

The Outcome of TGFß Antagonism in Metastatic Breast Cancer Models In Vivo Reflects a Complex Balance between Tumor-Suppressive and Proprogression Activities of TGFß.

PURPOSE: TGFßs are overexpressed in many advanced cancers and promote cancer progression through mechanisms that include suppression of immunosurveillance. Multiple strategies to antagonize the TGFß pathway are in early-phase oncology trials. However, TGFßs also have tumor-suppressive activities early in tumorigenesis, and the extent to which these might be retained in advanced disease has not been fully explored. EXPERIMENTAL DESIGN: A panel of 12 immunocompetent mouse allograft models of metastatic breast cancer was tested for the effect of neutralizing anti-TGFß antibodies on lung metastatic burden. Extensive correlative biology analyses were performed to assess potential predictive biomarkers and probe underlying mechanisms. RESULTS: Heterogeneous responses to anti-TGFß treatment were observed, with 5 of 12 models (42%) showing suppression of metastasis, 4 of 12 (33%) showing no response, and 3 of 12 (25%) showing an undesirable stimulation (up to 9-fold) of metastasis. Inhibition of metastasis was immune-dependent, whereas stimulation of metastasis was immune-independent and targeted the tumor cell compartment, potentially affecting the cancer stem cell. Thus, the integrated outcome of TGFß antagonism depends on a complex balance between enhancing effective antitumor immunity and disrupting persistent tumor-suppressive effects of TGFß on the tumor cell. Applying transcriptomic signatures derived from treatment-naïve mouse primary tumors to human breast cancer datasets suggested that patients with breast cancer with high-grade, estrogen receptor-negative disease are most likely to benefit from anti-TGFß therapy. CONCLUSIONS: Contrary to dogma, tumor-suppressive responses to TGFß are retained in some advanced metastatic tumors. Safe deployment of TGFß antagonists in the clinic will require good predictive biomarkers.

Agreement in Histological Assessment of Mitotic Activity Between Microscopy and Digital Whole Slide Images Informs Conversion for Clinical Diagnosis.

Validating digital pathology as substitute for conventional microscopy in diagnosis remains a priority to assure effectiveness. Intermodality concordance studies typically focus on achieving the same diagnosis by digital display of whole slide images and conventional microscopy. Assessment of discrete histological features in whole slide images, such as mitotic figures, has not been thoroughly evaluated in diagnostic practice. To further gauge the interchangeability of conventional microscopy with digital display for primary diagnosis, 12 pathologists examined 113 canine naturally occurring mucosal melanomas exhibiting a wide range of mitotic activity. Design reflected diverse diagnostic settings and investigated independent location, interpretation, and enumeration of mitotic figures. Intermodality agreement was assessed employing conventional microscopy (CM40×), and whole slide image specimens scanned at 20× (WSI20×) and at 40× (WSI40×) objective magnifications. An aggregate 1647 mitotic figure count observations were available from conventional microscopy and whole slide images for comparison. The intraobserver concordance rate of paired observations was 0.785 to 0.801; interobserver rate was 0.784 to 0.794. Correlation coefficients between the 2 digital modes, and as compared to conventional microscopy, were similar and suggest noninferiority among modalities, including whole slide image acquired at lower 20× resolution. As mitotic figure counts serve for prognostic grading of several tumor types, including melanoma, 6 of 8 pathologists retrospectively predicted survival prognosis using whole slide images, compared to 9 of 10 by conventional microscopy, a first evaluation of whole slide image for mitotic figure prognostic grading. This study demonstrated agreement of replicate reads obtained across conventional microscopy and whole slide images. Hence, quantifying mitotic figures served as surrogate histological feature with which to further credential the interchangeability of whole slide images for primary diagnosis.

Automated Computational Detection, Quantitation, and Mapping of Mitosis in Whole-Slide Images for Clinically Actionable Surgical Pathology Decision Support.

BACKGROUND: Determining mitotic index by counting mitotic figures (MFs) microscopically from tumor areas with most abundant MF (hotspots [HS]) produces a prognostically useful tumor grading biomarker. However, interobserver concordance identifying MF and HS can be poorly reproducible. Immunolabeling MF, coupled with computer-automated counting by image analysis, can improve reproducibility. A computational system for obtaining MF values across digitized whole-slide images (WSIs) was sought that would minimize impact of artifacts, generate values clinically relatable to counting ten high-power microscopic fields of view typical in conventional microscopy, and that would reproducibly map HS topography. MATERIALS AND METHODS: Relatively low-resolution WSI scans (0.50 µm/pixel) were imported in grid-tile format for feature-based MF segmentation, from naturally occurring canine melanomas providing a wide range of proliferative activity. MF feature extraction conformed to anti-phospho-histone H3-immunolabeled mitotic (M) phase cells. Computer vision image processing was established to subtract key artifacts, obtain MF counts, and employ rotationally invariant feature extraction to map MF topography. RESULTS: The automated topometric HS (TMHS) algorithm identified mitotic HS and mapped select tissue tiles with greatest MF counts back onto WSI thumbnail images to plot HS topographically. Influence of dye, pigment, and extraneous structure artifacts was minimized. TMHS diagnostic decision support included image overlay graphics of HS topography, as well as a spreadsheet and plot of tile-based MF count values. TMHS performance was validated examining both mitotic HS counting and mapping functions. Significantly correlated TMHS MF mapping and metrics were demonstrated using repeat analysis with WSI in different orientation (R 2 = 0.9916) and by agreement with a pathologist (R 2 = 0.8605) as well as through assessment of counting function using an independently tuned object counting algorithm (OCA) (R 2 = 0.9482). Limits of agreement analysis support method interchangeability. MF counts obtained led to accurate patient survival prediction in all (n = 30) except one case. By contrast, more variable performance was documented when several pathologists examined similar cases using microscopy (pair-wise correlations, rho range = 0.7597-0.9286). CONCLUSIONS: Automated TMHS MF segmentation and feature engineering performance were interchangeable with both observer and OCA in digital mode. Moreover, enhanced HS location accuracy and superior method reproducibility were achieved using the automated TMHS algorithm compared to the current practice employing clinical microscopy.

High-throughput screens identify HSP90 inhibitors as potent therapeutics that target inter-related growth and survival pathways in advanced prostate cancer.

The development of new treatments for castrate resistant prostate cancer (CRPC) must address such challenges as intrinsic tumor heterogeneity and phenotypic plasticity. Combined PTEN/TP53 alterations represent a major genotype of CRPC (25-30%) and are associated with poor outcomes. Using tumor-derived, castration-resistant Pten/Tp53 null luminal prostate cells for comprehensive, high-throughput, mechanism-based screening, we identified several vulnerabilities among >1900 compounds, including inhibitors of: PI3K/AKT/mTOR, the proteasome, the cell cycle, heat shock proteins, DNA repair, NFκB, MAPK, and epigenetic modifiers. HSP90 inhibitors were one of the most active compound classes in the screen and have clinical potential for use in drug combinations to enhance efficacy and delay the development of resistance. To inform future design of rational drug combinations, we tested ganetespib, a potent second-generation HSP90 inhibitor, as a single agent in multiple CRPC genotypes and phenotypes. Ganetespib decreased growth of endogenous Pten/Tp53 null tumors, confirming therapeutic activity in situ. Fifteen human CRPC LuCaP PDX-derived organoid models were assayed for responses to 110 drugs, and HSP90 inhibitors (ganetespib and onalespib) were among the select group of drugs (<10%) that demonstrated broad activity (>75% of models) at high potency (IC50 <1 µM). Ganetespib inhibits multiple targets, including AR and PI3K pathways, which regulate mutually compensatory growth and survival signals in some forms of CRPC. Combined with castration, ganetespib displayed deeper PDX tumor regressions and delayed castration resistance relative to either monotherapy. In all, comprehensive data from near-patient models presents novel contexts for HSP90 inhibition in multiple CRPC genotypes and phenotypes, expands upon HSP90 inhibitors as simultaneous inhibitors of oncogenic signaling and resistance mechanisms, and suggests utility for combined HSP90/AR inhibition in CRPC.

Naturally Occurring Canine Melanoma as a Predictive Comparative Oncology Model for Human Mucosal and Other Triple Wild-Type Melanomas.

Melanoma remains mostly an untreatable fatal disease despite advances in decoding cancer genomics and developing new therapeutic modalities. Progress in patient care would benefit from additional predictive models germane for human disease mechanisms, tumor heterogeneity, and therapeutic responses. Toward this aim, this review documents comparative aspects of human and naturally occurring canine melanomas. Clinical presentation, pathology, therapies, and genetic alterations are highlighted in the context of current basic and translational research in comparative oncology. Somewhat distinct from sun exposure-related human cutaneous melanomas, there is growing evidence that a variety of gene copy number alterations and protein structure/function mutations play roles in canine melanomas, in circumstances more analogous to human mucosal melanomas and to some extent other melanomas with murine sarcoma viral oncogene homolog B (BRAF), Neuroblastoma RAS Viral (V-Ras) Oncogene Homolog (NRAS), and neurofibromin 1 tumor suppressor NF1 triple wild-type genotype. Gaps in canine genome annotation, as well as an insufficient number and depth of sequences covered, remain considerable barriers to progress and should be collectively addressed. Preclinical approaches can be designed to include canine clinical trials addressing immune modulation as well as combined-targeted inhibition of Rat Sarcoma Superfamily/Mitogen-activated protein kinase (RAS/MAPK) and/or Phosphatidylinositol-3-Kinase/Protein Kinase B/Mammalian target of rapamycin (PI3K/AKT/mTOR) signal transduction, pathways frequently activated in both human and canine melanomas. Future investment should be aimed towards improving understanding of canine melanoma as a predictive preclinical surrogate for human melanoma and for mutually benefiting these uniquely co-dependent species.

cFOS-SOX9 Axis Reprograms Bone Marrow-Derived Mesenchymal Stem Cells into Chondroblastic Osteosarcoma.

Bone marrow-derived mesenchymal stem cells (BMSCs) are proposed as the cells of origin of several subtypes of osteosarcoma (OS). However, signals that direct BMSCs to form different subtypes of OS are unclear. Here we show that the default tumor type from spontaneously transformed p53 knockout (p53_KO) BMSCs is osteoblastic OS. The development of this default tumor type caused by p53 loss can be overridden by various oncogenic signals: RAS reprograms p53_KO BMSCs into undifferentiated sarcoma, AKT enhances osteoblastic OS, while cFOS promotes chondroblastic OS formation. We focus on studying the mechanism of cFOS-induced chondroblastic OS formation. Integrated genome-wide studies reveal a regulatory mechanism whereby cFOS binds to the promoter of a key chondroblastic transcription factor, Sox9, and induces its transcription in BMSCs. Importantly, SOX9 mediates cFOS-induced cartilage formation in chondroblastic OS. In summary, oncogenes determine tumor types derived from BMSCs, and the cFOS-SOX9 axis is critical for chondroblastic OS formation.

Synergistic targeted inhibition of MEK and dual PI3K/mTOR diminishes viability and inhibits tumor growth of canine melanoma underscoring its utility as a preclinical model for human mucosal melanoma.

Human mucosal melanoma (MM), an uncommon, aggressive and diverse subtype, shares characteristics with spontaneous MM in dogs. Although BRAF and N-RAS mutations are uncommon in MM in both species, the majority of human and canine MM evaluated exhibited RAS/ERK and/or PI3K/mTOR signaling pathway activation. Canine MM cell lines, with varying ERK and AKT/mTOR activation levels reflective of naturally occurring differences in dogs, were sensitive to the MEK inhibitor GSK1120212 and dual PI3K/mTOR inhibitor NVP-BEZ235. The two-drug combination synergistically decreased cell survival in association with caspase 3/7 activation, as well as altered expression of cell cycle regulatory proteins and Bcl-2 family proteins. In combination, the two drugs targeted their respective signaling pathways, potentiating reduction of pathway mediators p-ERK, p-AKT, p-S6, and 4E-BP1 in vitro, and in association with significantly inhibited solid tumor growth in MM xenografts in mice. These findings provide evidence of synergistic therapeutic efficacy when simultaneously targeting multiple mediators in melanoma with Ras/ERK and PI3K/mTOR pathway activation.

In Vivo Bioluminescent Imaging of ATP-Binding Cassette Transporter-Mediated Efflux at the Blood-Brain Barrier.

We provide a detailed protocol for imaging ATP-binding cassette subfamily G member 2 (ABCG2) function at the blood-brain barrier (BBB) of transgenic mice. D-Luciferin is specifically transported by ABCG2 found on the apical side of endothelial cells at the BBB. The luciferase-luciferin enzymatic reaction produces bioluminescence, which allows a direct measurement of ABCG2 function at the BBB. Therefore bioluminescence imaging (BLI) correlates with ABCG2 function at the BBB and this can be measured by administering luciferin in a mouse model that expresses luciferase in the brain parenchyma. BLI allows for a relatively low-cost alternative for studying transporter function in vivo compared to other strategies such as positron emission tomography. This method for imaging ABCG2 function at the BBB can be used to investigate pharmacokinetic inhibition of the transporter.

Creation of an NCI comparative brain tumor consortium: informing the translation of new knowledge from canine to human brain tumor patients.

On September 14-15, 2015, a meeting of clinicians and investigators in the fields of veterinary and human neuro-oncology, clinical trials, neuropathology, and drug development was convened at the National Institutes of Health campus in Bethesda, Maryland. This meeting served as the inaugural event launching a new consortium focused on improving the knowledge, development of, and access to naturally occurring canine brain cancer, specifically glioma, as a model for human disease. Within the meeting, a SWOT (strengths, weaknesses, opportunities, and threats) assessment was undertaken to critically evaluate the role that naturally occurring canine brain tumors could have in advancing this aspect of comparative oncology aimed at improving outcomes for dogs and human beings. A summary of this meeting and subsequent discussion are provided to inform the scientific and clinical community of the potential for this initiative. Canine and human comparisons represent an unprecedented opportunity to complement conventional brain tumor research paradigms, addressing a devastating disease for which innovative diagnostic and treatment strategies are clearly needed.