VDX-111 targets proliferative pathways in canine cancer cell lines.

VDX-111 (also identified as AMPI-109) is a vitamin D derivative which has shown anticancer activity. To further assess the function of this compound against multiple cancer types, we examined the efficacy of VDX-111 against a panel of 30 well characterized canine cancer cell lines. Across a variety of cancer types, VDX-111 induced widely variable growth inhibition, cell death, and migration inhibition, at concentrations ranging from 10 nM to 1 µM. Growth inhibition sensitivity did not correlate strongly with tumor cell histotype; however, it was significantly correlated with the expression of genes in multiple cell signaling pathways, including the MAPK and PI3K-AKT pathways. We confirmed inhibition of these signaling pathways as likely participants in the effects of VDX-111. These results suggest that a subset of canine tumors may be sensitive to treatment with VDX-111, and suggests possible predictive markers of drug sensitivity and pharmacodynamic biomarkers of drug exposure that could be employed in future clinical trials.

Sensitivity of osteosarcoma cell lines to autophagy inhibition as determined by pharmacologic and genetic manipulation.

Pharmacologic inhibition of autophagy can be achieved using lysosomotropic agents such as hydroxychloroquine (HCQ) that interfere with fusion of the autophagosome to the lysosome thus preventing completion of the recycling process. The goal of the present study is to determine the sensitivity of eight canine (cOSA) and four human (hOSA) osteosarcoma tumour cell lines to antiproliferative and cytotoxic effects of lysosomal autophagy inhibitors, and to compare these results to the autophagy-dependence measured using a CRISPR/Cas9 live-cell imaging assay in OSA and other tumour cell lines. Antiproliferative and cytotoxic response to HCQ and Lys05 was determined using live cell imaging and YOYO-1 staining. CRISPR/Cas9 live cell imaging screen was done using species specific guide RNA's and transfection of reagents into cells. Response to autophagy core genes was compared to response to an essential (PCNA) and non-essential (FOXO3A) gene. cOSA and hOSA cell lines showed similar antiproliferative and cytotoxic responses to HCQ and Lys05 with median lethal dose (Dm ) values ranging from 4.6-15.8 µM and 2.1-5.1 µM for measures of anti-proliferative response, respectively. A relationship was observed between antiproliferative responses to HCQ and Lys05 and VPS34 CRISPR score with Dm values correlating with VPS34 response (r = 0.968 and 0.887) in a species independent manner. The results show that a subset of cOSA and hOSA cell lines are autophagy-dependent and sensitive to HCQ at pharmacologically-relevant exposures.

Integrated analysis of canine soft tissue sarcomas identifies recurrent mutations in TP53, KMT genes and PDGFB fusions.

Soft tissue sarcomas (STS) are a heterogenous group of mesenchymal tumors representing over 50 distinct types with overlapping histological features and non-specific anatomical locations. Currently, localized sarcomas are treated with surgery + / - radiation in both humans and dogs with few molecularly targeted therapeutic options. However, to improve precision-based cancer therapy through trials in pet dogs with naturally occurring STS tumors, knowledge of genomic profiling and molecular drivers in both species is essential. To this purpose, we sought to characterize the transcriptomic and genomic mutation profiles of canine STS subtypes (fibrosarcoma, undifferentiated pleomorphic sarcoma, and peripheral nerve sheath tumors), by leveraging RNAseq, whole exome sequencing, immunohistochemistry, and drug assays. The most common driver mutations were in cell cycle/DNA repair (31%, TP53-21%) and chromatin organization/binding (41%, KMT2D-21%) genes. Similar to a subset of human sarcomas, we identified fusion transcripts of platelet derived growth factor B and collagen genes that predict sensitivity to PDGFR inhibitors. Transcriptomic profiling grouped these canine STS tumors into 4 clusters, one PNST group (H1), and 3 FSA groups selectively enriched for extracellular matrix interactions and PDFGB fusions (H2), homeobox transcription factors (H3), and elevated T-cell infiltration (H4). This multi-omics approach provides insights into canine STS sub-types at a molecular level for comparison to their human counterparts, to improve diagnosis, and may provide additional targets for chemo- and immuno-therapy.

Integrated analysis of canine soft tissue sarcomas identifies recurrent mutations in TP53, KMT genes and PDGFB fusions.

Canine soft tissue sarcomas (STS) are a heterogenous group of malignant tumors arising from mesenchymal cells of soft tissues. This simplified collective of tumors most commonly arise from subcutaneous tissues, are treated similar clinically, and conventionally exclude other sarcomas with more definitive anatomical, histological, or biological features. Histologically, canine STS sub-types are difficult to discern at the light microscopic level due to their overlapping features. Thus, genomic, and transcriptomic profiling of canine STS may prove valuable in differentiating the diverse sub-types of mesenchymal neoplasms within this group. To this purpose we sought to characterize the transcript expression and genomic mutation profiles of canine STS. To delineate transcriptomic sub-types, hierarchical clustering was used to identify 4 groups with district expression profiles. Using the RNAseq data, we identified three samples carrying driver fusions of platelet derived growth factor B ( PDGFB ) and collagen genes. Sensitivity to imatinib was evaluated in a canine STS cell line also bearing a PDGFB fusion. Using whole exome sequencing, recurrent driver variants were identified in the cancer genes KMT2D (21% of the samples) and TP53 (21%) along with copy number losses of RB1 and CDKN2A. Gene amplifications and resulting transcript increases were identified in genes on chromosomes 13, 14, and 36. A subset of STS was identified with high T-cell infiltration. This multi-omics approach has defined canine STS sub-types at a molecular level for comparison to their human counterparts, to improve diagnosis, and may provide additional targets for therapy.

Characterizing the molecular and immune landscape of canine bladder cancer.

Transitional cell carcinoma (TCC), also known as urothelial carcinoma, is the most common bladder cancer in humans and dogs. Approximately one-quarter of human TCCs are muscle-invasive and associated with a high risk of death from metastasis. Canine TCC (cTCC) tumours are typically high-grade and muscle-invasive. Shared similarities in risk factors, histopathology, and clinical presentation suggest that cTCC may serve as a model for the assessment of novel therapeutics that may inform therapies for human muscle-invasive TCC. The goal of this study was to characterize cTCC at the molecular level to identify drivers of oncogenesis and druggable targets. We performed whole exome sequencing (WES) of 11 cTCC tumours and three matched normal samples, identifying 583 variants in protein-coding genes. The most common variant was a V-to-E missense mutation in BRAF, identified in 4 out of 11 samples (36%) via WES. Sanger sequencing identified BRAF variants in 8 out of the same 11 cTCC samples, as well as in 22 out of 32 formalin-fixed paraffin embedded (FFPE) cTCC samples, suggesting an overall prevalence of 70%. RNA-Seq was performed to compare the gene expression profiles of cTCC tumours to normal bladder tissue. cTCC tumours exhibited up-regulation of genes involved in the cell cycle, DNA repair, and antiviral immunity. We also analysed the immune landscape of cTCC using immune gene signatures and immunohistochemical analysis. A subset of tumours had characteristics of a hot tumour microenvironment and exhibited high expression of signatures associated with complete response to PD-1/PD-L1 blockade in human bladder cancer.

Losartan Blocks Osteosarcoma-Elicited Monocyte Recruitment, and Combined With the Kinase Inhibitor Toceranib, Exerts Significant Clinical Benefit in Canine Metastatic Osteosarcoma.

PURPOSE: There is increasing recognition that progress in immuno-oncology could be accelerated by evaluating immune-based therapies in dogs with spontaneous cancers. Osteosarcoma (OS) is one tumor for which limited clinical benefit has been observed with the use of immune checkpoint inhibitors. We previously reported the angiotensin receptor blocker losartan suppressed metastasis in preclinical mouse models through blockade of CCL2-CCR2 monocyte recruitment. Here we leverage dogs with spontaneous OS to determine losartan's safety and pharmacokinetics associated with monocyte pharmacodynamic endpoints, and assess its antitumor activity, in combination with the kinase inhibitor toceranib. PATIENTS AND METHODS: CCL2 expression, monocyte infiltration, and monocyte recruitment by human and canine OS tumors and cell lines were assessed by gene expression, ELISA, and transwell migration assays. Safety and efficacy of losartan-toceranib therapy were evaluated in 28 dogs with lung metastatic OS. Losartan PK and monocyte PD responses were assessed in three dose cohorts of dogs by chemotaxis, plasma CCL2, and multiplex cytokine assays, and RNA-seq of losartan-treated human peripheral blood mononuclear cells. RESULTS: Human and canine OS cells secrete CCL2 and elicit monocyte migration, which is inhibited by losartan. Losartan PK/PD studies in dogs revealed that a 10-fold-higher dose than typical antihypertensive dosing was required for blockade of monocyte migration. Treatment with high-dose losartan and toceranib was well-tolerated and induced a clinical benefit rate of 50% in dogs with lung metastases. CONCLUSIONS: Losartan inhibits the CCL2-CCR2 axis, and in combination with toceranib, exerts significant biological activity in dogs with metastatic osteosarcoma, supporting evaluation of this drug combination in patients with pediatric osteosarcoma. See related commentary by Weiss et al., p. 571.

Immune pathways and TP53 missense mutations are associated with longer survival in canine osteosarcoma.

Osteosarcoma affects about 2.8% of dogs with cancer, with a one-year survival rate of approximately 45%. The purpose of this study was to characterize mutation and expression profiles of osteosarcoma and its association with outcome in dogs. The number of somatic variants identified across 26 samples ranged from 145 to 2,697 with top recurrent mutations observed in TP53 and SETD2. Additionally, 47 cancer genes were identified with copy number variations. Missense TP53 mutation status and low pre-treatment blood monocyte counts were associated with a longer disease-free interval (DFI). Patients with longer DFI also showed increased transcript levels of anti-tumor immune response genes. Although, T-cell and myeloid cell quantifications were not significantly associated with outcome; immune related genes, PDL-1 and CD160, were correlated with T-cell abundance. Overall, the association of gene expression and mutation profiles to outcome provides insights into pathogenesis and therapeutic interventions in osteosarcoma patients.

Prospective clinical trial testing COXEN-based gene expression models of chemosensitivity in dogs with spontaneous osteosarcoma.

BACKGROUND: This study is a prospective clinical trial in dogs with osteosarcoma testing a gene expression model (GEM) predicting the chemosensitivity of tumors to carboplatin (CARBO) or doxorubicin (DOX) developed using the COXEN method. PATIENTS AND METHODS: Sixty dogs with appendicular osteosarcoma were enrolled in this trial. RNA isolation and gene expression profiling were conducted with 2 biopsies for 54/63 screened tumors, and with a single biopsy for 9 tumors. Resulting gene expression data were used for calculation of a COXEN score for CARBO and DOX based on a previous study showing the significance of this predictor on patient outcome utilizing retrospective data (BMC Bioinformatics 17:93). Dogs were assigned adjuvant CARBO, DOX or the combination based on the results of the COXEN score following surgical removal of the tumor via amputation and were monitored for disease progression by chest radiograph every 2 months. RESULTS: The COXEN predictor of chemosensitivity to CARBO or DOX was not a significant predictor of progression-free interval or overall survival for the trial participants. The calculation of DOX COXEN score using gene expression data from two independent biopsies of the same tumor were highly correlated (P < 0.0001), whereas the calculated CARBO COXEN score was not (P = 0.3039). CONCLUSION: The COXEN predictor of chemosensitivity to CARBO or DOX is not a significant predictor of outcome when utilized in this prospective study. This trial represents the first prospective trial of a GEM predictor of chemosensitivity and establishes pet dogs with cancer as viable surrogates for prospective trials of prognostic indicators.

Identifying Candidate Druggable Targets in Canine Cancer Cell Lines Using Whole-Exome Sequencing.

Cancer cell culture has been a backbone in cancer research, in which analysis of human cell line mutational profiles often correlates with oncogene addiction and drug sensitivity. We have conducted whole-exome sequence analyses on 33 canine cancer cell lines from 10 cancer types to identify somatic variants that contribute to pathogenesis and therapeutic sensitivity. A total of 66,344 somatic variants were identified. Mutational load ranged from 15.79 to 129.37 per Mb, and 13.2% of variants were located in protein-coding regions (PCR) of 5,085 genes. PCR somatic variants were identified in 232 genes listed in the Cancer Gene Census (COSMIC). Cross-referencing variants with human driving mutations on cBioPortal identified 61 variants as candidate cancer drivers in 30 cell lines. The most frequently mutated cancer driver was TP53 (15 mutations in 12 cell lines). No drivers were identified in three cell lines. We identified 501 non-COSMIC genes with PCR variants that functionally annotate with COSMIC genes. These genes frequently mapped to the KEGG MAPK and PI3K-AKT pathways. We evaluated the cell lines for ERK1/2 and AKT(S473) phosphorylation and sensitivity to the MEK1/2 inhibitor, trametinib. Twelve of the 33 cell lines were trametinib-sensitive (IC50 < 32 nmol/L), all 12 exhibited constitutive or serum-activated ERK1/2 phosphorylation, and 8 carried MAPK pathway cancer driver variants: NF1(2), BRAF(3), N/KRAS(3). This functionally annotated database of canine cell line variants will inform hypothesis-driven preclinical research to support the use of companion animals in clinical trials to test novel combination therapies.

Transcriptomic analysis of differentially expressed genes in the molting gland (Y-organ) of the blackback land crab, Gecarcinus lateralis, during molt-cycle stage transitions.

A transcriptome of the Gecarcinus lateralis molting gland (Y-organ or YO) contained 48,590 contiguous sequences (contigs) from intermolt (IM), early premolt (EP), mid premolt (MP), late premolt (LP), and postmolt (PM) stages. The YO is kept in the basal state in IM by molt-inhibiting hormone (MIH)/cyclic nucleotide-dependent signaling. YO activation in EP requires down-regulation of MIH signaling and activation of mechanistic target of rapamycin (mTOR)-dependent protein synthesis. Transition of the YO to the committed state in MP requires activin/transforming growth factor-beta (TGFß) signaling. YO repression occurs at the end of LP. A total of 28,179 contigs (58%) showed molt stage-specific changes in gene expression. The largest number of differentially-expressed genes (DEGs) were at the IM/EP (16,142 contigs), LP/PM (18,161 contigs), and PM/IM (8290 contigs) transitions. By contrast, the numbers of DEGs were 372 and 1502 contigs for the EP/MP and MP/LP transitions, respectively. DEG analysis of 23 signal transduction pathways showed significant changes in MIH, mTOR, activin/TGFß, Notch, MAP kinase, and Wnt signaling. Down-regulation of MIH signaling genes in premolt is consistent with reduced MIH sensitivity in MP and LP. Up-regulation of mTOR signaling genes in IM and premolt stages is consistent with its role in YO activation and sustained ecdysteroidogenesis. Up-regulation of activin/TGFß signaling genes in EP and MP is consistent with the role of a myostatin/activin-like factor in YO commitment. Notch, MAP kinase, and Wnt DEG analysis may indicate possible crosstalk with the MIH, mTOR, and activin/TGFß pathways to integrate other inputs to control YO ecdysteroidogenesis.

Analysis of Annotation and Differential Expression Methods used in RNA-seq Studies in Crustacean Systems.

In the field of crustacean biology, usage of RNA-seq to study gene expression is rapidly growing. Major advances in sequencing technology have contributed to the ability to examine complex patterns of genome activity in a wide range of organisms that are extensively used for comparative physiology, ecology and evolution, environmental monitoring, and commercial aquaculture. Relative to insect and vertebrate model organisms, however, information on the organization of crustacean genomes is virtually nonexistent, making de novo transcriptome assembly, annotation and quantification problematic and challenging. We present here a summary of the methodologies and software analyses employed in 23 recent publications, which describe de novo transcriptome assembly, annotation, and differential gene expression in a variety of crustacean experimental systems. We focus on establishing a series of best practices that will allow for investigators to produce datasets that are understandable, reproducible, and of general utility for related analyses and cross-study comparisons.

A Comparison of Resources for the Annotation of a De Novo Assembled Transcriptome in the Molting Gland (Y-Organ) of the Blackback Land Crab, Gecarcinus lateralis.

Next-generation sequencing technologies are revolutionizing crustacean biology. De novo assembly of RNA sequencing (RNA-seq) data allows researchers to catalog and quantify genes expressed in tissues of a species that lacks a complete genome sequence. RNA-seq has become an important tool for understanding phenotypic plasticity and the responses of organisms to environmental cues. However, there are challenges with identification of assembled contiguous sequences (contigs) without a reference genome. Thus, the selection of resources for annotating contigs is critical for the downstream analysis of gene functions. A de novo-assembled transcriptome of the Gecarcinus lateralis molting gland, or Y-organ (YO), was used to compare two functional annotation packages, Trinotate and Blast2GO. The assembled transcriptome contained 229,278 contigs derived from YOs from animals in intermolt, premolt (early, mid, and late), and postmolt stages. Gene identification using BLAST against four databases and functional annotation using Gene Ontologies were conducted. The analysis revealed two major limitations of de novo assembly: (1) assembly using Trinity generates redundant contigs and (2) transcripts that encode protein isoforms are not distinguished with current computational tools. It is recommended that the NCBI Non-Redundant, SwissProt, TrEMBL, and Uniref90 databases be used to maximize gene identification. Trinotate is preferred for assigning functions to identified genes, as the package uses multiple databases for annotation. The differences between packages to generate Gene Ontology (GO) terms are attributed to the databases used for inputs: Trinotate uses both Pfam and BLAST databases, while Blast2GO uses only the BLAST database. InterProScan was used to verify the GO terms assigned via BLAST. A comprehensive annotation of de novo assembled transcriptome is necessary for the downstream analysis of differentially expressed transcripts in the YO over the molt cycle.

Transcriptome analysis of the molting gland (Y-organ) from the blackback land crab, Gecarcinus lateralis.

In decapod crustaceans, arthropod steroid hormones or ecdysteroids regulate molting. These hormones are synthesized and released from a pair of molting glands called the Y-organs (YO). Cyclic nucleotide, mTOR, and TGFß/Smad signaling pathways mediate molt cycle-dependent phase transitions in the YO. To further identify the genes involved in the regulation of molting, a YO transcriptome was generated from three biological replicates of intermolt blackback land crab, Gecarcinus lateralis. Illumina sequencing of cDNA libraries generated 227,811,829 100-base pair (bp) paired-end reads; following trimming, 90% of the reads were used for further analyses. The trimmed reads were assembled de novo using Trinity software to generate 288,673 contigs with a mean length of 872 bp and a median length of 1842 bp. Redundancy among contig sequences was reduced by CD-HIT-EST, and the output constituted the baseline transcriptome database. Using Bowtie2, 92% to 93% of the reads were mapped back to the transcriptome. Individual contigs were annotated using BLAST, HMMER, TMHMM, SignalP, and Trinotate, resulting in assignments of 20% of the contigs. Functional and pathway annotations were carried out via gene ontology (GO) and KEGG orthology (KO) analyses; 58% and 44% of the contigs with BLASTx hits were assigned to GO and KO terms, respectively. The gene expression profile was similar to a crayfish YO transcriptome database, and the relative abundance of each contig was highly correlated among the three G. lateralis replicates. Signal transduction pathway orthologs were well represented, including those in the mTOR, TGFß, cyclic nucleotide, MAP kinase, calcium, VEGF, phosphatidylinositol, ErbB, Wnt, Hedgehog, Jak-STAT, and Notch pathways.

Morphological, Molecular, and Hormonal Basis of Limb Regeneration across Pancrustacea.

Regeneration is a developmental process that allows an organism to re-grow a lost body part. Historically, the most studied aspect of limb regeneration across Pancrustacea is its morphological basis and its dependence on successful molting. Although there are distinct morphological differences in regeneration processes between insects and crustaceans, in both groups the phenomenon is initiated via formation of a blastema, followed by proliferation, dedifferentiation, and redifferentiation of blastemal cells to generate a functional limb. In recent years, with the availability of sequence data and tools to manipulate gene expression, the emphasis of this field has shifted toward the genetic basis of limb regeneration. Among insects this focus is on genes that are known to be required during the development of legs in embryos. RNA interference-mediated functional studies conducted during regeneration of imaginal discs of Drosophila melanogaster, and nymphal legs of Gryllus bimaculatus reveal that several conserved pathways and transcription factors (Wingless, Decapentaplegic, Hedgehog, Dachshund) are required for successful regeneration. In contrast to studies on the regeneration of insects' limbs, work on crustaceans has focused on the hormonal basis of the re-growth of limbs. Regeneration in decapods, like Uca pugilator and Gecarcinus lateralis, occurs in discrete phases of growth in tandem with the stages of the molt cycle. Recent studies have shown that ecdysteroid hormone signaling is necessary for blastemal proliferation. Although the current research emphases of limb regeneration in insect and crustacean are fairly distinct, the results generated by functional studies of a wide array of regeneration genes will be beneficial for generating testable regeneration models.

Alternative splicing in the fiddler crab cognate ecdysteroid receptor: variation in receptor isoform expression and DNA binding properties in response to hormone.

RXR cDNA cloning from three Uca species led to the identification of 4 conserved isoforms, indicative of alternative splicing in the hinge and ligand binding domains (LBD). Sequencing of overlapping clones from a Ucapugilator genomic library identified EcR isoforms matching previously identified cDNA variants; in addition, a cryptic exon in the LBD was detected and evidence for expression of this new isoform was obtained from next-generation sequencing. RNA-seq analysis also identified a new amino terminal EcR variant. EcR and RXR transcript abundance increases throughout ovarian maturation in U. pugilator, while cognate receptor transcript abundance remains constant in a related Indo-Pacific species with a different reproductive strategy. To examine if crab RXR LBD isoforms have different physical properties in vitro, electromobility shift assays were performed with different EcR isoforms. The cognate crab and fruit fly receptors differ in their responses to hormone. Ecdysteroids did not increase DNA binding for the crab heterodimers, while ecdysteroids stimulate binding for Drosophilamelanogaster EcR/USP heterodimers. In swapping experiments, UpEcR/USP heterodimers did not show ligand-responsive differences in DNA binding; both crab RXR LBD isoforms, however, conferred ligand-responsive increases in DNA binding with DmEcRs. These data indicate that both UpRXR LBD isoforms can heterodimerize with the heterologous DmEcR receptors and promote ligand and DNA binding. Unresponsiveness of the cognate receptors to ecdysteroid, however, suggest additional factors may be required to mediate endogenous, perhaps isoform-specific, differences in EcR conformation, consistent with previously reported effects of UpRXR isoforms on UpEcR ligand-binding affinities.

Ecdysteroid receptor signaling disruption obstructs blastemal cell proliferation during limb regeneration in the fiddler crab, Uca pugilator.

To study ecdysteroid signaling during limb regeneration, we have applied RNAi (dsRNA) mediated silencing to EcR/RXR, the genes encoding the ecdysteroid receptor heterodimer, in the fiddler crab Uca pugilator. We injected RNAi into the blastemal chamber during early limb regeneration. Silencing was evaluated by knockdown in receptor transcript abundance, and disruption was evaluated by changes in growth rate and morphology of limb regenerates. q-PCR results indicated a 50% drop in transcript abundance 48h post injection in both RNAi (dsEcR/dsRXR) injected ipsilateral and uninjected contralateral blastemas in experimental animals relative to controls. EcR/RXR transcript levels further decreased over time. Several phenotypes were associated with knockdown. The experimental blastema failed to develop; microscopic examination of the arrested blastema revealed an absence of the cuticular ingrowths characteristic of the beginnings of limb segmentation and cell proliferation assays revealed that the arrested blastema had few dividing cells. Ecdysteroid levels were also lowered in experimental animals; given the bilateral effects of RNAi on limb buds in experimental animals, these results suggest RNAi had a systemic effect. Although hormone titers in experimental animals rose to comparable control levels during the late proecdysial phase of limb regeneration, most experimental crabs failed to molt and died. The overall failure to molt indicates that RNAi receptor knockdown has long-term effects. The combined effects of receptor knockdown indicate that, although circulating ecdysteroid titers are normally low during basal limb bud growth, signaling via the ecdysteroid receptor pathway is necessary for establishment of blastemal cell proliferation and development in the regenerating limbs of U. pugilator.