Browsing by Author "Duval, Dawn, committee member"

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Open Access
Binding of MBNL1 to CUG repeats slows 5'-to-3' RNA decay by XRN2 in a cell culture model of type I myotonic dystrophy
(Colorado State University. Libraries, 2017) Zhang, Junzhen, author; Wilusz, Carol J., advisor; Wilusz, Jeffrey, advisor; Duval, Dawn, committee member; Di Pietro, Santiago, committee member; Yao, Tingting, committee member
Type I myotonic dystrophy (DM1) is a multi-systemic inherited disease caused by expanded CTG repeats within the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene. The encoded CUG repeat-containing mRNAs are toxic to the cell and accumulate in nuclear foci, where they sequester cellular RNA-binding proteins such as the splicing factor Muscleblind-1 (MBNL1). This leads to widespread changes in gene expression. Currently, there is no treatment or cure for this disease. Targeting CUG repeat-containing mRNAs for degradation is a promising therapeutic avenue for myotonic dystrophy, but we know little about how and where these mutant mRNAs are naturally decayed. We established an inducible C2C12 mouse myoblast model to study decay of reporter mRNAs containing the DMPK 3' UTR with 0 (CUG0) or ~700 (CUG700) CUG repeats and showed that the CUG700 cell line exhibits characteristic accumulation of repeat-containing mRNA in nuclear foci. We utilized qRT-PCR and northern blotting to assess the pathway and rate of decay of these reporter mRNAs following depletion of mRNA decay factors by RNA interference. We have identified four factors that influence decay of the repeat-containing mRNA – the predominantly nuclear 5' 3' exonuclease XRN2, the nuclear exosome containing RRP6, the RNA-binding protein MBNL1, and the nonsense-mediated decay factor, UPF1. We have discovered that the 5' end of the repeat-containing transcript is primarily degraded in the nucleus by XRN2, while the 3' end is decayed by the nuclear exosome. Interestingly, we have shown for the first time that the ribonucleoprotein complex formed by the CUG repeats and MBNL1 proteins represents a barrier for XRN2-mediated decay. We suggest that this limitation in XRN2-mediated decay and the resulting delay in degradation of the repeats and 3' region may play a role in DM1 pathogenesis. Additionally, our results support previous studies suggesting that UPF1 plays a role in initiating the degradation of mutant DMPK transcripts. This work uncovers a new role for MBNL1 in DM1 and other CUG-repeat expansion diseases and identifies the nuclear enzymes involved in decay of the mutant DMPK mRNA. Our model has numerous applications for further dissecting the pathways and factors involved in removing toxic CUG-repeat mRNAs, as well as in identifying and optimizing therapeutics that enhance their turnover.
Open Access
Cleavage of exosomal-associated transferrin receptor in dogs, cats, and horses: progress towards a soluble transferrin receptor assay
(Colorado State University. Libraries, 2018) Martinez, Caitlyn Marisa Romero, author; Olver, Christine, advisor; Santangelo, Kelly, advisor; Duval, Dawn, committee member
Iron deficiency anemia and anemia of chronic disease are two complications that patients in human medicine as well as veterinary medicine often encounter. These two diseases usually occur secondary to other primary diseases and are associated with increased morbidity and a decline in prognosis. The diagnosis and differentiation of these diseases is complicated by the fact that many of the parameters used to characterize iron deficiency are also influenced by inflammatory cytokines. Consequently, the detection of iron deficiency in the presence of inflammation or the detection of combined iron deficiency anemia and anemia of chronic disease is difficult. Differentiation of these two diseases is important as treatment for each disease is different and potentially harmful if utilized on a patient who has been misdiagnosed with one disease or the other. In human medicine, soluble transferrin receptor 1 (sTfR) has shown promise as a marker, alone or in ratio with serum ferritin, that can differentiate iron deficiency anemia, anemia of chronic disease, and combined disease. sTfR is the product of cleavage of transferrin receptor 1 (TfR1) from the surface of exosomes which are released into circulation from maturing reticulocytes. Humans cleave the majority of their exosomal-associated TfR1 yielding substantial levels of circulating sTfR for detection and quantification by clinical assays. However, the level of cleavage in many of our veterinary species, including dogs, cats, and horses, remains unknown. Additionally, no currently developed sTfR clinical assays have been found to successfully detect sTfR in our veterinary species. The purpose of this study was to first confirm the presence of exosomes and exosomal-associated TfR1 in the serum of dogs, cats, and horses. Secondly, the level of cleavage of exosomal-associated TfR1 in healthy dogs, cats, and horses was explored to indirectly characterize the anticipated levels of circulating sTfR in these species. Lastly, the level of cleavage of exosomal-associated TfR1 was compared between healthy and diseased dogs and cats to investigate any potential effect of inflammation and chronic disease on the cleavage of exosomal-associated TfR1 and thus on the anticipated levels of circulating sTfR. The results of this study demonstrated significant evidence indicating the successful isolation of exosomes and identification of exosomal-associated TfR1 from the serum of dogs, cats, and horses. The level of cleavage of exosomal-associated TfR1 in dogs was found to be greater than 50% on average with significant between-individual variation. There was also no significant difference in the means of the proportion of cleavage between healthy and diseased dogs. The level of cleavage of exosomal-associated TfR1 in cats was found to be very low at about 11% without substantial variation between individuals. However, a small but significant difference between healthy and diseased cats was detected. Healthy horses do not appear to cleave exosomal-associated TfR1. These results together would suggest that development of a clinical assay for the detection and quantification of sTfR in these veterinary species may not be successful and consequently may not be worth the time, effort, and expense.
Open Access
Effects of survivin and survivin inhibition in canine models of lymphoma and osteosarcoma
(Colorado State University. Libraries, 2014) Shoeneman, Jenette K., author; Thamm, Douglas, advisor; Gustafson, Daniel, committee member; Duval, Dawn, committee member; Biller, Barbara, committee member; Ehrhart, E. J., committee member
Canine lymphoma (LSA) and osteosarcoma (OS) have high mortality rates and remain in need of more effective therapeutic approaches. Survivin, an IAP (inhibitor of apoptosis) family member protein that inhibits apoptosis and drives cell proliferation, is commonly elevated in human and canine cancer. Survivin expression is a negative prognostic factor in both dogs and humans with LSA and OS, and canine LSA and OS cell lines express high levels of survivin. Due to the strong similarities between canine and human LSA and OS, canine LSA and OS are excellent models for the human disease. In the following research, we illustrate the potential of the canine LSA and OS models as a translational tool for evaluating survivin-directed therapies, owing to the striking similarities in gross and microscopic appearance, biologic behavior, gene expression and signaling pathway alterations compared to the respective human forms of these diseases. In this research we sought to determine the effects of survivin inhibition in canine OS and LSA cell lines in vitro, and in vivo in canine OS, and to evaluate a correlation between survivin expression and outcome in canine OS patients. We hypothesized, as observed in human OS and LSA, that survivin inhibition would decrease cell proliferation and increase apoptosis and chemosensitivity in canine OS and LSA cell lines. We further hypothesized that we would observe inhibition of survivin and reduced tumor growth in murine models of canine OS treated with EZN-3042, an inhibitor of survivin. We additionally hypothesized, as observed in human OS, that increased survivin expression would correlate with a poor prognosis in canine OS patients. Survivin attenuation in canine OS cells via siRNA was confirmed by RT-PCR and western blot analysis. Cell number and viablility was assessed via manual cell counting with trypan blue. Cellular apoptosis was confirmed via caspase-3/7 and TUNEL assays. Cell cycle analysis was performed with propidium iodide staining followed by flow cytometry. Chemosensitivity to doxorubicin (DOX) was also assessed with caspase-3/7 assay. We determined that survivin inhibition via siRNA in canine OS cells inhibited cell cycle progression, and increased apoptosis, mitotic arrest and chemosensitivity. Next we inhibited survivin using EZN-3042, a locked nucleic acid oligonucleotide targeting survivin, in two canine LSA and two canine OS cell lines. Survivin inhibition was confirmed by qRT-PCR and Immunofluorescence. Percent dead and total cell number were assessed by manual cell counting with trypan blue. Growth inhibition was confirmed with a bioreductive fluorometric assay. A caspase-3/7 assay was used to determine levels of apoptosis and chemosensitivity. Survivin inhibition in vitro using EZN-3042 resulted in decreased total and viable cell numbers and increased apoptosis and chemosensitivity to DOX. In vivo, nude mice with subcutaneous and orthotopic OS xenografts were given 100 mg/kg EZN3042 intraperitoneally. Survivin inhibition was confirmed with immunohistochemistry and qRT-PCR analysis. EZN-3042 treatment in vivo in subcutaneous and orthotopic canine OS xenografts decreased tumor survivin expression. Mice treated with EZN-3042 in combination with DOX had significantly decreased tumor growth when compared to single agent treatment and control groups. Lastly, we evaluated survivin expression in archived paraffin embedded canine OS tissue samples. Survivin expression was studied via immunohistochemistry in 67 canine OS cases. Elevated survivin protein immunoreactivity in primary canine OS tissue samples correlated with increased histologic grade and mitotic index and a decreased disease free interval (DFI). These findings strongly suggest that survivin-directed therapies may be highly effective in treatment of both canine and human LSA and OS, and spontaneous canine cancer may be a valuable model for the evaluation of survivin-targeted treatment.
Open Access
Experimental and computational analysis of Caenorhabditis elegans small RNAs
(Colorado State University. Libraries, 2019) Brown, Kristen, author; Montgomery, Tai, advisor; Duval, Dawn, committee member; Prasad, Ashok, committee member; Hess, Ann, committee member
Caenorhabditis elegans contains twenty-five Argonautes, of which, only ALG-1 and ALG-2 are known to interact with microRNAs (miRNAs). ALG-5 belongs to the AGO subfamily of Argonautes that includes ALG-1 and ALG-2, but its role in small RNA pathways is unknown. We analyzed by high-throughput sequencing the small RNAs associated with ALG-5, ALG-1, and ALG-2, as well as changes in mRNA expression in alg-5, alg-1, and alg-2 mutants. We show that ALG-5 defines a distinct branch of the miRNA pathway affecting the expression of genes involved in immunity, defense, and development. In contrast to ALG-1 and ALG-2, which associate with the majority of miRNAs and have general roles throughout development, ALG-5 interacts with only a small subset of miRNAs and is specifically expressed in the germline. alg-5 is required for optimal fertility and mutations in alg-5 lead to a precocious transition from spermatogenesis to oogenesis. Our results provide a near-comprehensive analysis of miRNA-Argonaute interactions in C. elegans and reveal a new role for miRNAs in the germline. The small RNA field has grown rapidly since miRNAs were discovered to be conserved regulators of developmental timing. This growth occurred during a time when high-throughput transcriptomic data from microarrays and next-generation sequencing became widely accessible. As a result, research projects dissecting small RNA pathways often produce sequencing data that can be complex and difficult to perform appropriate data analysis for without specialized or advanced computational knowledge. Many researchers end up only study a subset of small RNAs, outsourcing their analysis, or piecing together a pipeline using tools developed for mRNA sequencing. We aim to reduce this barrier to entry in the field and improve reproducibility by creating an open-source, user-friendly data processing pipeline for small RNA sequencing. To create a simple, reproducible pipeline, we utilized the Common Workflow Language (CWL) and Python, while otherwise minimizing dependencies. The pipeline reads a configuration file and sample sheets that can be easily modified by a user to run the complete analysis from raw fastq file to summary statistics and publication-ready plots. We present AQuATx (automated quantitative analysis of transcript expression) for small RNAs and the analysis of C. elegans germline tissue as an example data set. Our software will allow bench scientists with little to no computational knowledge to easily analyze their small RNA sequencing data. Overall, the final software will be a valuable tool for anyone interested in studying small RNAs.
Open Access
Overactive NF-KB signaling as a druggable target and evaluation of parthenolide an NF-KB inhibitor in canine cancer
(Colorado State University. Libraries, 2022) Schlein, Lisa Janelle, author; Thamm, Douglas H., advisor; Avery, Paul, committee member; Duval, Dawn, committee member; MacNeill, Amy, committee member
This study provides a unique translational research opportunity to help both humans and dogs diagnosed with diseases that carry dismal prognoses in both species: histiocytic sarcoma (HS), hemangiosarcoma (HSA), and disseminated mastocytosis (MCT). Lymphoma is one of the most common cancer types affecting dogs and humans, and therefore, novel therapeutic approaches are always needed. For all of these cancer types, dogs and human cancers share common molecular abnormalities, consistent with a conserved pathogenesis between species. Relative to traditional murine models for human cancers, dogs are genetically diverse, large mammals with heterogeneous, spontaneous tumors. Dogs generally receive good medical care and share the environmental factors with humans, and accordingly, dogs with spontaneous tumors are an excellent model for human oncology generally. Additionally, although disseminated HS, MCT and visceral HSA are exceedingly rare diseases in humans, they are more common in some dog breeds, giving us the opportunity to study this disease in a larger population than would otherwise be available. Therapeutics evaluated in dogs with these diseases stand to benefit both canine and human patients. NF-kB proteins are a family of structurally related, eukaryotic transcription factors that have 400+ genetic targets, and are involved in many vital cellular processes, including innate immunity, inflammatory responses, development, cellular growth, and survival. Not surprisingly, overactivation of NF-kB is a feature of many chronic disease processes, including cardiac disease, neurodegenerative disease, immune-mediated disease, and cancer. While NF-kB overactivation has been documented extensively in human oncology, there is a relative paucity of data documenting the same phenomenon in veterinary medicine. As part of this study, large scale validation of NF-kB overactivation was performed in canine cancer via immunohistochemistry of 215 tumor samples (lymphoma, HS, HSA, and MCT). Antibodies were validated for use via western blot, immortalized cell pellets, and evaluation of normal canine tissues. In addition to validation of NF-kB overactivation, assays were performed to assess the therapeutic potential of parthenolide (PTL), a known, canonical NF-kB signaling inhibitor with additional mechanisms of antineoplastic activity, including alteration of cellular redox balance. Growth inhibition assays were performed with canine cell lines and primary lymphoma cells isolated from canine patients, using PTL alone or in combination with redox-perturbing standard-of-care therapeutics. Cell death was assessed using flow cytometry. Immunofluorescence was used to assess NF-kB localization, western blot was used to assess NF-kB activity with and without PTL, and canine cells were transfected with a reporter gene cassette containing the NFkB consensus sequence followed by firefly luciferase gene to study the effect of PTL on NF-kB-related luminescence. PTL's effects on glutathione and reactive oxygen species generation were assessed with a colorimetric assay and a fluorescent H2DCFDA assay, respectively. Genetic expression changes were assessed with RNA sequencing of HS cells, with and without PTL treatment. A mouse model of disseminated HS was created with NF-kB luminescent cells to study the effect of PTL on this disease in vivo. Many spontaneous canine tumor samples have nuclear p65 and p100/p52 IHC staining that is of greater magnitude than observed in comparable, normal cell populations, indicating the promise of PTL and other therapeutics that target aberrant NF-kB signaling. Canine cell lines and primary cells are sensitive to PTL and undergo dose-dependent apoptosis following exposure to drug. PTL exposure also leads to glutathione depletion, reactive oxygen species generation, and NF-kB inhibition in canine cells. Standard-of-care therapeutics broadly synergize with PTL. In two canine HS cell lines, genetic expression of NF-kB pathway signaling partners is downregulated with PTL therapy. Preliminary data suggest that PTL inhibits NF-kB activity of cells in a mouse model of disseminated HS. Overall, these data support further investigation of compounds that can antagonize canonical and alternative NF-kB pathway signaling, which are overactivated in canine lymphoma, HS, HSA, and MCT disease. PTL is one promising therapeutic that acts, in part, via canonical NF-kB antagonism in canine neoplasms. Further investigation of this compound in vivo is underway in a mouse model of disseminated HS, and if this study is successful, it will provide strong justification for clinical trials with this compound in dogs.
Open Access
Regulation of local L-type calcium channel signaling in anterior pituitary gonadotropes
(Colorado State University. Libraries, 2017) Dang, An Khanh, author; Amberg, Greg, advisor; Clay, Colin, advisor; Tamkun, Michael, committee member; Navratil, Amy, committee member; Duval, Dawn, committee member
The binding of gonadotropin-releasing hormone (GnRH) to its receptor initiates signaling cascades in gonadotropes which result in enhanced luteinizing hormone (LH) and follicle stimulating hormone (FSH) biosynthesis and secretion. Most dramatic is the sharp rise in LH secretion ("LH surge") that precedes and is necessary for follicular maturation and ovulation. Ca2+ influx activates mitogen-activated protein kinases (MAPKs) which lead to increased transcription of LH and FSH genes. Interestingly, previous research suggests that two MAPK signaling pathways, ERK and JNK, are activated by either Ca2+ influx through L-type Ca2+ channels or by global Ca2+ signals originating from intracellular stores, respectively. These discrete Ca2+ sources for divergent signaling cascades provides a mechanism in which gonadotropes can decode different pathways for appropriate gonadotropin release during various stages of the ovulatory cycle. However, direct evidence supporting an underlying subplasmalemmal local Ca2+ signaling through L-type Ca2+ channels distinct from intracellular Ca2+ was lacking. Here we used a combination of electrophysiology and total internal reflection fluorescence (TIRF) microscopy to visualize discrete sites of Ca2+ influx (Ca2+ sparklets) in gonadotrope-derived αT3-1 cells in real time. These localized GnRH-induced Ca2+ influxes are mediated by L-type Ca2+ channels and important for downstream ERK activation. In addition, precise structural and molecular elements to create a microenvironment suitable for localized subplasmalemmal L-type Ca2+ channel signaling was necessary for gonadotrope function, in which GnRH-dependent stimulation of L-type Ca2+ channel influx was found to require PKC and a dynamic actin cytoskeleton. More recently, we have further elucidated molecular mechanisms modulating localized L-type Ca2+ channel influx. Reactive oxygen species (ROS) are cognate signaling molecules that mediate cell function, but their role in regulating Ca2+ in gonadotropes is unknown. We have explored GnRH regulation of both NADPH oxidase complexes and mitochondrial sources of ROS and assessed ROS modulation of L-type Ca2+ channel activity in gonadotropes. We identified GnRH-induced spatially localized ROS "puncta" in αT3-1 cells, and ROS increased local Ca2+ channel activity in both αT3-1 cells and primary mouse gonadotropes. In addition, GnRH increased mitochondrial oxidation activity at the subplasmalemmal surface and mitochondrial ROS increased localized L-type Ca2+ channel influx. Also, active L-type Ca2+ channels were associated with subplasmalemmal mitochondria. Taken together, this dissertation explored the first direct evidence for localized L-type Ca2+ channel signaling in αT3-1 cells and elucidated signaling mechanisms in gonadotropes. Specifically, cellular organization via an intact cytoskeletal platform and ROS regulated L-type Ca2+ channel sparklet activity that are important for the downstream ERK activation and gonadotropin gene expression that regulates reproduction.
Open Access
Seroanalysis of Felis catus gammaherpesvirus 1 infection in domestic cats
(Colorado State University. Libraries, 2015) Stutzman-Rodriguez, Kathryn, author; VandeWoude, Susan, advisor; Rovnak, Joel, committee member; Duval, Dawn, committee member
We recently described a novel herpesvirus of domestic cats, Felis catus gammaherpesvirus 1 (FcaGHV1). FcaGHV1 is a member of the gammaherpesvirus subfamily, which also includes the human cancer-associated herpesviruses, Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV). To determine FcaGHV1 antigens that elicit a detectable humoral-immune response in naturally-infected domestic cats, I chose to evaluate seven FcaGHV1 proteins. These proteins are conserved across the subfamily and antigenic in other gammaherpesvirus infections. I amplified and cloned each of the seven FcaGHV1 genes into a mammalian expression vector and transfected intact clones into Crandell Rees feline kidney (CRFK) cells. I developed an immunofluorescent antibody test using transfected cells exposed to sera from nine shelter cats diagnosed as FcaGHV1-positive by quantitative PCR (qPCR) of blood-cell DNA. This analysis indicated that tegument proteins ORF52 and ORF38 reacted most consistently with serum from cats with positive FcaGHV1-qPCR reactions. Based on these results, recombinant antigens were used to develop two optimized indirect ELISAs. Genes for ORF52 and ORF38 were cloned into a mammalian expression vector. Antigens were produced in a transient transfection system and purified using immunoprecipitation. Indirect ELISA conditions were optimized using known positive and negative controls. Using the two optimized ELISAs, I screened sera from 133 shelter cats that had been previously tested by FcaGHV1-qPCR. Seroprevalence of FcaGHV1 reactive antibodies was 32%, compared to the previously published 16% prevalence evaluated by qPCR. Nineteen of twenty qPCR positive cats were also seroreactive against one or both antigens on ELISA. Sera from 24 cats were seropositive based upon ELISA testing, but negative using qPCR analysis. Risk factors identified in previous publications were confirmed by ELISA, namely geographic location, male sex, adult age, and association of FcaGHV1 with several co-infections. Based on our knowledge of gammaherpesvirus latency, this ELISA provides evidence of viral exposure, while qPCR viral DNA detection likely represents reactivation from latency or primary infection. The addition of serologic analysis as a measurement of FcaGHV1 exposure will aid in determining association of FcaGHV1 with disease and routes of transmission.
Embargo
Using gene expression and mutational profiling to characterize canine acute myeloid leukemia and assess their comparative features with human acute myeloid leukemia
(Colorado State University. Libraries, 2023) Harris, Adam, author; Avery, Anne, advisor; Avery, Paul, committee member; Dow, Steven, committee member; Duval, Dawn, committee member
Acute myeloid leukemia (AML) is an aggressive heterogenous hematopoietic neoplasm that afflicts both dogs and people. Over 10,000 individuals (about the seating capacity of Cameron basketball stadium at Duke University) in the United States succumb to AML-related deaths every year. Treatment options for AML have made little progress in the past few decades and prognosis for both human and canine AML (cAML) remains dismal. However, there are large ongoing multi-institutional studies devoted to advancing medical management for human AML (hAML) by providing targeted therapeutics to patients based on their molecular characteristics. A preclinical model for testing novel therapies could accelerate the development of better treatments in people. We hypothesize that cAML will have similar underlying molecular features as human AML and dogs could be a translational model for developing therapeutics focused on treating AML. The goals of this thesis were to assess the gene expression programs and mutational profiles of cAML and compare our findings with available human AML data. First, we established diagnostic criteria for defining cAML using flow cytometry. Next, we globally assessed normal hematopoiesis in dogs using single cell transcriptomics to generate a hematopoietic tree for defining the cellular composition of cAML. Additionally, we investigated the mRNA expression and genetic variants in cAML to ultimately compare the molecular features with pediatric and adult AML subtypes. We hope this work advances our knowledge of cAML molecular characteristics and adds further credentials to the dog as spontaneous model for human AML.
Open Access
Utilization of a canine cancer cell line (FACC) panel in comparative and translational studies of gene expression and drug sensitivity
(Colorado State University. Libraries, 2015) Fowles, Jared S., author; Gustafson, Daniel, advisor; Duval, Dawn, committee member; Hess, Ann, committee member; Thamm, Douglas, committee member; Weil, Michael, committee member
Canine cancer is the leading cause of death in adult dogs. The use of the canine cancer model in translational research is growing in popularity due to the many biologic and genetic similarities it shares with human cancers. Cancer cell tissue culture has long been an established tool for expanding our understanding of cancer processes and for development of novel cancer treatments. With the high rate of genomic advancements in cancer research over the last decade human cancer cell line panels that combine pharmacologic and genomic information have proven very helpful in elucidating the complex relationships between gene expression and drug response in cancer. We have assembled a panel of canine cancer cell lines at the Flint Animal Cancer Center (FACC) at Colorado State University to be utilized in a similar fashion as a tool to advance canine cancer research. The purpose of these studies is to describe the characteristics of the FACC panel with the available genomic and drug sensitivity data we have generated, and to show its utility in comparative and translational oncology by focusing specifically on canine melanoma and osteosarcoma. We were able to confirm our panel of cell lines as being of canine origin and determined their genetic fingerprint through PCR and microsatellite analyses, creating a point of reference for validation in future studies and collaborations. Gene expression microarray analysis allowed for further molecular characterization of the panel, showing that similar tumor types tended to cluster together based on general as well as cancer specific gene expression patterns. In vitro studies that measure phenotypic differences in the panel can be coupled with genomic data, resulting in the identification of potential gene targets worthy of further exploration. We also showed that human and canine cancer cells are similarly sensitive to common chemotherapy. Next we utilized the FACC panel in a comparative analysis to determine if signaling pathways important in human melanoma were also activated and sensitive to targeted inhibition in canine melanoma. We were able to show that despite apparent differences in the mechanism of pathway activation, human and canine melanoma tumors and cell lines shared constitutive signaling of the MAPK and PI3K/AKT pathways, and responded similarly to targeted inhibition. These data suggest that studies involving pathway-targeted inhibition in either canine or human melanoma could potentially be directly translatable to each other. Evidence of genetic similarities between human and canine cancers led us to ask whether or not non-pathway focused gene expression models for predicting drug sensitivity could be developed in an interspecies manner. We were able to show that models built on canine datasets using human derived gene signatures successfully predicted response to chemotherapy in canine osteosarcoma patients. When compared to a large historical cohort, dogs that received the treatment our models predicted them to be sensitive to lived significantly longer disease-free. Taken together, these studies show that human and canine cancers share strong molecular similarities that can be used advantageously to develop better treatment strategies in both species.