Canine Tumor Cell Research Articles

Corrigendum: The Flint Animal Cancer Center (FACC) canine tumor cell line panel: a resource for veterinary drug discovery, comparative oncology and translational medicine

Corrigendum: The Flint Animal Cancer Center (FACC) canine tumor cell line panel: a resource for veterinary drug discovery, comparative oncology and translational medicine

Reovirus changes the expression of anti-apoptotic and proapoptotic proteins with the c-kit downregulation in canine mast cell tumor cell lines

Although reovirus has reached phase II and III clinical trials in human cancers, the exact mechanism of reovirus oncolysis is still not completely understood. Previously, we have shown that canine mast cell tumor (MCT) cell lines were highly susceptible to reovirus, as compared with other kinds of canine cancer cell lines. In this study, we showed that reovirus infection not only led to the dephosphorylation but also downregulation of c-kit in four canine MCT cell lines, where c-kit activation is required for proliferation. Consistent with c-kit dysregulation, downstream signaling of c-kit, the level of Ras-GTP and phosphorylation of all the downstream effectors of Ras (Raf, MEK, and ERK) and Akt decreased in all the cell lines after reovirus infection, except for Akt in one of cell lines. Pro-apoptotic and anti-apoptotic proteins such as Bim, Bad and Mcl-1 were also altered by reovirus infection in these cell lines. In short, reovirus infection degraded c-kit in all the canine MCT cell lines, leading to the downregulation of downstream signaling of c-kit, which may relate to the cell death induced by reovirus.

Abstract 4778: Identification of promoters for targeted gene expression in tumors

Abstract A variety of novel therapeutic approaches to cancer have been proposed in the past several decades. One of the most compelling is the use of gene therapy to treat tumors. Restricting the expression of these genes to tumor avoids toxicity in normal cells and the concurrent side effects that this activity engenders. Promoter sequences can be used as a tool for transcriptional targeting, by restricting the expression of the therapeutic genes, or, in the case of oncolytic virotherapy, the replication of the viral agent. Several tumor-upregulated promoters have been identified in a wide variety of human tumors. Given the recognition of the validity of canine tumors as comparative models of human disease, it is critical to determine the activities of these promoters in the canine system. To achieve this goal, endogenous cSurvivin, cTERT, and cCXCR4 promoter activity was evaluated in a panel of canine normal and tumor cell lines/tissues and primary tumors by RT-Q-PCR. These three promoters, along with an E2F modified Canine Adenovirus 2 E1a promoter (E2F-E1a), were also assessed for their activity when supplied exogenously to tumor cells in an in vitro GFP reporter transfection assay. Endogenous expression of cTERT showed negligible differences between normal canine cells/tissues and tumor cell/tissues. cSurvivin showed elevated endogenous activity in non-hematopoietic tumor cells and moderately elevated activity in some normal tissues and hematopoietic tumors. cCXCR4 activity was elevated exclusively in a T-lymphoma cell line and a primary T-cell lymphoma. Exogenous expression in most cancer cell lines showed enhanced activity with most of the promoters tested. The percentage of cells expressing GFP was elevated in tumor compared to normal with all four tested promoters, when normalized to CMV-GFP expression. However, the relative fluorescence intensity varied significantly between cell lines, while the fluorescence intensity varied less between promoters within a specific cell line. Exogenous expression results did not correlate well with endogenous expression in tumor cells, but they did correlate well in normal cells. This disparity implies that promoter selection for transcriptional targeting of tumors should account for both endogenous and exogenous expression patterns. Additionally, these findings indicate that identification of a pan-cancer promoter is complex and that expression targeting may need to rely on selecting patient-specific promoters to drive the activity of therapeutic genes. Citation Format: Abdul Mohin Sajib, Maninder Sandey, Samantha Morici, Bradley Schuler, Payal Agarwal, Bruce Smith. Identification of promoters for targeted gene expression in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4778.

Effect of a two-base insertion mutation of the TP53 gene on expression of p53 protein in canine histiocytic sarcoma cells.

To examine effects of a common mutation (2-base insertion in exon 5) of the TP53 gene on biological function of p53 protein in canine histiocytic sarcoma cells. Canine histiocytic tumor cell lines DH82 with deletion of TP53 and CHS-3 with the wild-type TP53 and canine wild-type and mutant TP53 fragments. Wild-type or mutant TP53 with a polyprotein peptide tag at the N-terminus was transduced into DH82 and CHS-3 cells. Expression of p53 protein, changes in function as a transcription factor, and susceptibility to doxorubicin and nimustine were compared. Transduced p53 protein was detected in wild-type TP53-transduced DH82 and CHS-3 cells, whereas expression was not detected in mutant TP53-transduced cells. There were significant increases in expression of target genes of p53 protein, including p21 and MDM2, in wild-type TP53-transduced cells, compared with results for native and mock-transfected cells, but not in mutant TP53-transduced cells. There was no significant difference in drug susceptibilities among native and derivative cells of CHS-3. However, cell viabilities of wild-type TP53-transduced DH82 cells incubated with doxorubicin were significantly lower than viabilities of native, mock-transfected, and AT insertion mutation-TP53-transduced DH82 cells; susceptibility to nimustine did not differ significantly among cells. Expression of p53 protein and its function as a transcription factor were lost after addition of a 2-base insertion in the TP53 gene in canine histiocytic tumor cells. Additional studies are needed to investigate the clinical relevance of this mutation in histiocytic sarcomas of dogs.

Abstract 1446: Characterization, evaluation and safety studies of the oncolytic Vaccinia virus TG6002 for canine cancer therapy

Abstract Introduction: Oncolytic virotherapy with tumor selective viruses, such as Vaccinia viruses (VV), offers a promising treatment modality for cancer. TG6002 is a recombinant oncolytic VV deleted in two viral genes (thymidine kinase and ribonucleotide reductase) and armed with the suicide gene FCU1 that encodes a bifunctional chimeric protein which efficiently catalyses the direct conversion of the nontoxic 5-fluorocytosine (5-FC) into the toxic metabolite 5-fluorouracil (5-FU). Canine tumors are relevant predictive preclinical surrogates for human oncology. The first objective was to evaluate the susceptibility, the replication rate and the oncolytic potency of VV in canine tumor cell lines. The second objective was to evaluate oncolytic potency of TG6002 in xenograft model and fresh canine tumor biopsies. The third objective was to assess safety and viral shedding of TG6002 in healthy dogs. Materials and Methods: Transduction efficiency, replication and oncolytic potency of TG6002 were evaluated in vitro in a variety of different canine cancer cell lines. In vivo anti-tumor effect of TG6002 was examined in a canine tumor xenograft model. TG6002 was injected intratumorally or intravenously with or without 5-FC. Three canine mammary adenocarcinoma explants were infected with TG6002 in presence of 5-FC during 6 days. Oncolytic potency was assessed by histological exams. Concentrations of 5-FC and 5-FU were monitored. TG6002 was administered intramuscularly for 7 healthy dogs and intravenously for 4 healthy dogs. Clinical exams, complete blood count and biochemistry analysis were performed. Blood, saliva, urine, feces were collected for virus detection by qPCR and plaque assay. Results: Canine cell lines were highly susceptible to VV infection. A replication factor of 106 to 107 was determined 4 days after infection and a significant reduction of cell viability was noticed 5 days after infection. In xenograft model, intratumoral or intravenous injections of TG6002 with oral 5-FC induced a significant inhibition of tumor growth compared to control groups. In canine mammary adenocarcinoma biopsies, a lysis of 90% of tubular cells was observed on histological exams. Conversion of more than 50% of 5-FC to 5-FU was noticed. In healthy dogs, a good tolerance of intramuscular and intravenous injections of TG6002 without viral shedding was assessed. Conclusion: This study demonstrates that TG6002 is able to infect and replicate in canine tumor cell lines and is oncolytic in both cell lines, xenograft model and canine mammary adenocarcinoma samples. This study also confirms that TG6002 can be safely administered in dogs. These promising results support the use of TG6002 in a clinical trial for both human and canine species. This study emphasizes the importance of a One Health approach in oncology. Citation Format: Jérémy Béguin, Johann Foloppe, Eve Laloy, Virginie Nourtier, Isabelle Farine, Murielle Gantzer, Christelle Pichon, Sandrine Cochin, Pascale Cordier, Dominique Tierny, Jean Marc Balloul, Eric Quémeneur, Christelle Maurey, Bernard Klonjkowski, Philippe Erbs. Characterization, evaluation and safety studies of the oncolytic Vaccinia virus TG6002 for canine cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1446.

Evaluation of a Canine Transmissible Venereal Tumour Cell Line with Tumour Immunity Capacity but Without Tumorigenic Property.

IntroductionCanine transmissible venereal tumour (CTVT) is a sexually transmitted tumour affecting dogs worldwide, imposing a financial burden on dog owners. A stable culture cell line in continuous passages for >18 months has only been achieved once. The present study investigated a stable CTVT cell line isolated from a bitch and its potential as a vaccine.Material and MethodsA biopsy from a 2-year-old mongrel bitch with CTVT was obtained for histopathological confirmation and isolation of tumour cells. The isolated cells were cultured to passage 55 and characterised by flow cytometry, with karyotyping by GTG-banding and by PCR detection of myc S-2 and LINE AS1. The isolated CTVT cell line was also used as a preventive vaccine in a canine model.ResultsHistopathological analysis of the isolated tumour cells revealed typical CTVT characteristics. Constant proliferation and stable morphological characteristics were observed during culture. Phenotypic analysis determined the expression of HLA-DR+, CD5.1+, CD14+, CD45+, CD83+, CD163+, and Ly-6G-Ly-6C+. GTG-banding revealed a mean of 57 chromosomes in the karyotype with several complex chromosomal rearrangements. LINE-c-myc insertion in the isolated CTVT cell line at 550 bp was not detected. However, a 340-bp band was amplified. Isolated CTVT cell line inoculation at a concentration of 1×108 did not induce tumour growth in bitches, nor did a challenge with primary CTVT cells.ConclusionThe present study successfully identified and isolated a stable CTVT cell line that may be useful in CTVT prevention.

Comprehensive analysis of miRNA and protein profiles within exosomes derived from canine lymphoid tumour cell lines.

Exosomes are small extracellular vesicles released from almost all cell types, which play roles in cell-cell communication. Recent studies have suggested that microenvironmental crosstalk mediated by exosomes is an important factor in the escape of tumour cells from the anti-tumour immune system in human haematopoietic malignancies. Here, we conducted comprehensive analysis of the miRNA and protein profiles within the exosomes released from four canine lymphoid tumour cell lines as a model of human lymphoid tumours. The results showed that the major miRNAs and proteins extracted from the exosomes were similar among the four cell lines. However, the miRNA profiles differed among the exosomes of each cell line, which corresponded to the expression patterns of the parent cells. In the comparison of the amounts of miRNAs and proteins among the cell lines, those of three miRNAs (miR-151, miR-8908a-3p, and miR-486) and CD82 protein differed between exosomes derived from vincristine-sensitive and resistant cell lines. Further investigations are needed to elucidate the biological functions of the exosomal contents in the microenvironmental crosstalk of lymphoid tumours.

In vitro Effect of Recombinant Feline Interferon-Ω (rFeIFN-Ω) on the Primary CanineTransmissible Venereal Tumor Culture.

Background: Interferons (IFNs), signaling proteins produced by host cells, are secreted in response to pathogen activity as well as to tumor cells, and display antiviral, antiproliferative, and immunomodulatory effects. Recombinant feline interferon omega (rFeIFN-ω) has in vitro growth inhibition activities on various canine and feline tumor cell lines. Canine transmissible venereal tumor (CTVT) is used as an animal model for immunotherapy due to its specific growth phase. Previous studies have usually focused on the interaction between tumor infiltrating lymphocytes (TILs) and CTVT cells. However, the specific effects of rFeIFN-ω on CTVT cells remains poorly defined.Aims: The aims of this study, therefore, were to evaluate the in vitro effect of rFeIFN-ω on primary CTVT cells and to study the mRNA expression of apoptotic genes and drug resistance genes.Materials and Methods: Purified CTVT cells were treated with various concentrations of rFeIFN-ω and the viability of the cultured cells was ascertained at 24, 48, and 72 h post treatment (hpt) and a dose-response curve plotted. The mRNA expression of apoptotic (BAX and BCL-2) and drug resistance (ABCB1 and ABCG2) genes was performed by reverse transcription quantitative real-time PCR at 72 hpt.Results: rFeIFN-ω displayed an effect against CTVT cell viability, which decreasing viability in a dose-dependent manner within 72 hpt. The relative mRNA expression of BCL-2 was upregulated only at a rFeIFN-ω concentration of 104 IU/100 μl. However, higher concentrations of rFeIFN-ω gave a higher level of relative mRNA expression of ABCB1 transporter gene.Conclusion: This study provided the information of in vitro effect of rFeIFN-ω on CTVT cell viability in a dose dependent manner, as well as, the alteration of BCL-2 and ABCB1 gene expression after treatment. These results encourage future in vivo studies to evaluate the potential efficacy of this treatment in CTVT cases.

Efficacy of 5-Aminolevulinic Acid in Photodynamic Detection and Photodynamic Therapy in Veterinary Medicine.

5-Aminolevulinic acid (5-ALA), a commonly used photosensitizer in photodynamic detection (PDD) and therapy (PDT), is converted in situ to the established photosensitizer protoporphyrin IX (PpIX) via the heme biosynthetic pathway. To extend 5-ALA-PDT application, we evaluated the PpIX fluorescence induced by exogenous 5-ALA in various veterinary tumors and treated canine and feline tumors. 5-ALA-PDD sensitivity and specificity in the whole sample group for dogs and cats combined were 89.5 and 50%, respectively. Notably, some small tumors disappeared upon 5-ALA-PDT. Although single PDT application was not curative, repeated PDT+/−chemotherapy achieved long-term tumor control. We analyzed the relationship between intracellular PpIX concentration and 5-ALA-PDT in vitro cytotoxicity using various primary tumor cells and determined the correlation between intracellular PpIX concentration and 5-ALA transporter and metabolic enzyme mRNA expression levels. 5-ALA-PDT cytotoxicity in vitro correlated with intracellular PpIX concentration in carcinomas. Ferrochelatase mRNA expression levels strongly negatively correlated with PpIX accumulation, representing the first report of a correlation between mRNA expression related to PpIX accumulation and PpIX concentration in canine tumor cells. Our findings suggested that the results of 5-ALA-PDD might be predictive for 5-ALA-PDT therapeutic effects for carcinomas, with 5-ALA-PDT plus chemotherapy potentially increasing the probability of tumor control in veterinary medicine.

Functional comparison of the human epidermal growth factor receptor and telomerase reverse transcriptase promoters in canine tumor cells.

We previously showed that the promoter region of the human epidermal growth factor receptor (hEGFR) gene elicits high transduction efficiency, with transgene expression restricted to canine breast tumor cells. However, it was unclear whether this promoter induces tumor cell-specific transgene expression in canine urothelial carcinoma cells. Furthermore, compared with studies in human cancer cells, the utility of the telomerase reverse transcriptase (TERT) gene promoter for therapeutic transgene expression in canine cancer cells has not been evaluated thus far. Here, we compared the activity of these promoters in canine mammary tumor and urothelial carcinoma cells. Our results showed that compared with the TERT promoter, the hEGFR promoter was more useful as a tumor-specific promoter to induce efficient transgene expression in canine tumor cells.

Trimer form of tumor necrosis factor-related apoptosis inducing ligand induces apoptosis in canine cell lines derived from mammary tumors.

We evaluated the cytotoxic effect of isoleucine-zipper tumor necrosis factor-related apoptosis inducing ligand (izTRAIL) against cell lines, B101592, Cha, and C090115, derived from canine mammary gland tumors. These cells were derived from three dogs diagnosed with mammary adenoma or carcinoma. All three cells were positive for vimentin, while B101592 and C090115 were positive for cytokeratin (CK) AE1/AE3 and CK CAM5.2. Treatment with izTRAIL decreased the viability of the three cell lines. The proportion of annexin V+/propidium iodide- cells increased in all three cell lines after treatment with izTRAIL. Additionally, cell cycle analysis revealed that izTRAIL treatment increased the number of cells in sub-G1 phase. Moreover, izTRAIL treatment activated caspase-8 and caspase-3 and enhanced the levels of cleaved poly (ADP-ribose) polymerase. The cytotoxic effect of izTRAIL was mitigated upon co-treatment with caspase-8 or caspase-3 inhibitor. These results indicated that izTRAIL induces apoptosis in cell lines derived from canine mammary tumor, which was also previously reported in canine hemangiosarcoma cell lines. This suggested that canine tumor cells have conserved TRAIL receptors. This study will provide the basis for further studies on TRAIL receptors and TRAIL-related molecules.

Luehea candicans Increase In Vitro Cell Cancer Metabolism Even with High Polyphenols Content

Brazil is the country with the greatest biodiversity; however, most of the plants are used empirically, without scientific evidence. Luehea candicans, popularly called as-acoita-cavalo, is a specie characteristic of the Brazilian Cerrado Biome, used for fight hemorrhages, dysenteries, diarrhea, rheumatism and tumors. On the other hands, there are practically no studies about chemical composition and assess of their biological effects. Thus, the aim of this investigation was characterize and evaluate the antitumor potential of crude hydroethanolic extract from the bare stem parts of this plant, as well as its ethyl acetate and methanolic fractions. For the characterization, the crude extract was submitted to qualitative (phytochemical screening and analysis by direct flow injection by mass spectrometry-FIA-ESI-IT-MS) and quantitative tests (determination of phenolic compounds and determination of total flavonoids and anthocyanins). In order to study the antitumor potential, MTT technique was used to evaluate the cellular metabolism of canine breast cancer tumor cells (TCM) and osteosarcoma (OST). The results demonstrated the presence of polyphenols, mainly flavonoids and tannins. In addition, were observed an increase in metabolism when TCM and OST cells were incubated with all extracts of L. candicans. Based on this, L. candicans in these tumor types should be careful use, and that in vivo studies should be performed to prove the true effects of this plant.

Tissue factor procoagulant activity in the tumor cell lines and plasma of dogs with various malignant tumors.

Hypercoagulability is a common paraneoplastic complication in dogs with various malignant tumors. Importantly, tissue factor procoagulant activity (TF-PCA) induced by TF-bearing microparticles (TF-MPs) is associated with hypercoagulability in human patients with cancer. However, TF-PCA in tumor cells and the association between circulating TF-MPs and hypercoagulability in dogs with malignant tumors remain poorly understood. Therefore, the present study was conducted to evaluate the TF-PCA in various types of canine tumor cell lines and plasma in dogs with malignant tumors. Mammary gland tumor, hemangiosarcoma, and malignant melanoma cell lines, but not lymphoma cell lines, expressed TF on their surfaces and showed cellular surface and MP-associated TF-PCA. The plasma TF-PCA was elevated in some dogs that naturally developed such tumors. No significant difference was observed in plasma TF-PCA between the disseminated intravascular coagulation (DIC) group (median: 43.40; range: 3.47–85.19; n=5) and non-DIC group (median: 7.73; range: 1.70–16.13; n=12). However, plasma TF-PCA was remarkably elevated in three of five dogs with DIC. To the best of our knowledge, this is the first study to evaluate plasma TF-PCA in dogs with malignant tumors. Further studies must be conducted to determine the cellular origin of TF-MPs and the efficacy of plasma TF-PCA as a biomarker of DIC in dogs with malignant tumors.

Immunogenic potential of three transmissible venereal tumor cell lysates to prime canine-dendritic cells for cancer immunotherapy

Whole tumor cell lysates consist of a mixture of tumor antigens and danger associated molecular patterns (DAMPs) that can be used for dendritic cell maturation and consequently for the activation of a polyclonal T cell-specific tumor response. We evaluated the in vitro efficacy of three different preparations of canine transmissible venereal tumor (TVT) cell lysates: hypochlorous acid-whole tumor cell lysates (HOCl-L), heat shock-whole tumor cell lysates (HS-L), and freeze-thaw cycles-whole tumor cell lysates (FT-L) for the maturation of canine-derived dendritic cells. Our results showed calreticulin, HSP70, and HSP90 release in the three tumor lysates preparations (HOCl-L, HS-L, and FT-L); however, HMGB1 was detected only in HOCl-L and FT-L. Additionally, the uptake by HOCl-L pulsed dendritic cell (DC) increased compared to HS-L and FT-L pulsed DC; and dendritic cell maturation was confirmed by the appropriate cell surface markers (CD11c, CD80, CD83, and MHCII). Furthermore, dendritic cells pulsed with HOCl-L, HS-L or FT-L were cultured with canine lymphocytes. There was an increase of Th1-type cytokines (IL-12, TNF-α, and IFN-γ), in all the tumor cell lysates co-cultures, this correlates with T lymphocyte activation and cytotoxic response. Our data confirm that TVT cell lysates can induce functional canine-DC and that HOCl-L is the most effective one. This preparation of TVT cell lysates with HOCl is an attractive approach that allows the recognition of neoantigens as potential tumor targets and DC priming and therefore could be used for cancer immunotherapy against TVT.

Targeting Canine KIT Promoter by Candidate DNA G-Quadruplex Ligands.

G-quadruplexes (G4) are nucleic acid secondary structures frequently assumed by G-rich sequences located mostly at telomeres and proto-oncogenes promoters. Recently, we identified, in canine KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) promoter, two G-rich sequences able to fold into G4: d_kit1 and d_kit2_A16. In this study, an anthraquinone (AQ1) and an anthracene derivative (AN6), known to stabilize the G4 structures of the corresponding human h_kit1 and h_kit2, were tested on the canine G4 and in two canine mast cell tumor (MCT) cell lines (C2 and NI-1) to verify their capability to down-regulate KIT expression. The cytotoxicity of AQ1 and AN6 was determined using the Alamar Blue test; also the constitutive expression of KIT and other proto-oncogenes containing G4 structures in their promoter (BCL2, VEGFα, VEGFR2, KRAS, and TERT) was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Then the time- and dose-dependent effects of both ligands on target gene expression were assessed by qRT-PCR. All target genes were constitutively expressed up to 96 hours of culture. Both ligands decreased KIT mRNA levels and c-kit protein amount, and AN6 was comparatively fairly more effective. DNA interaction studies and a dual-luciferase gene reporter assay performed on a noncancerous canine cell line (Madin-Darby Canine Kidney cells) proved that this down-regulation was the result of the interaction of AN6 with KIT proximal promoter. Interestingly, our results only partially overlap with those previously obtained in human cell lines, where AQ1 was found as the most effective compound. These preliminary data might suggest AN6 as a promising candidate for the selective targeting of canine KIT-dependent tumors.

Canine squamous cell carcinoma cell lines with high expression of survivin are sensitive to survivin inhibitor YM155

Treatment of unresectable canine squamous cell carcinoma (SCC) remains challenging and new therapeutic strategies are needed. Survivin is a member of the inhibitor of apoptosis protein family and its inhibitor, YM155, is a potential anti-tumour agent. In the present study, 10 canine tumour cell lines (representing eight different tumour types) were screened for sensitivity to YM155; the drug potently inhibited the growth of the HAPPY SCC cell line. The growth inhibitory properties of YM155 were then examined in more detail using a panel of seven SCC cell lines. YM155 inhibited the growth of the cell lines HAPPY and SQ4; in contrast to the other lines in the panel, these two cell lines had high levels of expression of survivin. In HAPPY cells, YM155 inhibited expression of the survivin gene at the transcriptional level. In contrast, YM155 down-regulated survivin at the post-transcriptional level in SQ4 cells. YM155 suppressed cell growth in HAPPY cells, mostly via induction of apoptosis, but this was not the case in SQ4 cells. Two canine SCC cell lines with high cellular expression of survivin were sensitive to YM155. The possible underlying mechanisms of the cytotoxic effect of YM155 in these cell lines were different. One cell line had down-regulation of survivin mRNA and protein expression, associated with induction of apoptotic cell death. The other cell line had post-transcriptional down-regulation of survivin expression and subsequent induction of non-apoptotic cell death. Targeting survivin with YM155 is a potential approach for the treatment of canine SCCs with high expression of survivin.

Checkpoint Blockade Reverses Anergy in IL-13Rα2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells’ efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor in vitro. Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested in vitro by co-culture with canine tumor cells and in vivo in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells.

Abstract 1880: Validation of protein arginine methyltransferase 5 (PRMT5) as a candidate therapeutic target in the canine model of non-Hodgkin lymphoma

Abstract Introduction: Lymphoma is a common, aggressive malignancy in dogs. While generally responsive to upfront combination chemotherapy with traditional cytotoxic drugs, remission times are short and cures rare. New treatment strategies are therefore needed. Protein arginine methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase (PRMT) enzyme whose relevance to cancer biology has become increasingly evident as more studies have shown this enzyme to be involved in the regulation of multiple major signaling pathways affecting cell death, proliferation, and malignant transformation. While the mechanism behind many of these cell-transforming capabilities of PRMT5 remains unknown, knockdown of PRMT5 expression by genetic or pharmacologic means has been shown to modulate the methylation status of target proteins and exert antitumor effects in vitro and in vivo. Consequently, PRMT5 small-molecule inhibitors are in early preclinical development. Methods: In this study, we characterized patterns of PRMT5 expression and correlated these with histologic subtype using canine lymphoma tissue microarrays (TMAs). We characterized expression of PRMT5 in canine lymphoma tissues and methylation targets in canine B-cell lymphoma cell lines and treated them with different PRMT5 inhibitors to determine effects on PRMT5, target proteins and antitumor activity. We also assessed PRMT5 inhibition ex vivo in patient B-cell lymphomas via flow cytometry and assessment of apoptosis with annexin V/PI staining. Results: Canine diffuse large B-cell lymphoma showed cytoplasmic staining for PRMT5 (48.8% strong, 50.0% weak, n = 165) compared to negative or weak staining in normal and hyperplastic lymph nodes (n = 40). Lymphoblastic T-cell lymphoma samples showed strong nuclear staining (40%, n= 10). No nuclear staining was appreciated in normal or hyperplastic lymph nodes. Similarly, canine B-cell lines showed high expression of PRMT5. PRMT5 small-molecule inhibitors inhibited growth of CLBL-1 and 17-71 canine lymphoma cell lines. Histone (H3R8 and H4R3) methylation status was suppressed at their respective IC50s. Ex vivo treatment of canine patient tumor cells showed suppression of growth at 24 hours. Conclusion: We have demonstrated that PRMT5 is expressed in canine lymphomas and that PRMT5 inhibition can suppress the growth of canine lymphoma cells, supporting the continued use of the spontaneous canine lymphoma model for the preclinical development of PRMT5 inhibitors. Citation Format: Kyle A. Renaldo, Lindsay E. Courtney, Konstantin Shilo, Robert A. Baiocchi, William C. Kisseberth. Validation of protein arginine methyltransferase 5 (PRMT5) as a candidate therapeutic target in the canine model of non-Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1880.

In vitro antineoplastic effects of auranofin in canine lymphoma cells

BackgroundThe orally available gold complex auranofin (AF) has been used in humans, primarily as an antirheumatic/immunomodulatory agent. It has been safely administered to healthy dogs to establish pharmacokinetic parameters for oral administration, and has also been used as a treatment in some dogs with immune-mediated conditions. Multiple in vitro studies have recently suggested that AF may possess antineoplastic properties. Spontaneous canine lymphoma may be a very useful translational model for the study of human lymphoma, prompting the evaluation of AF in canine lymphoma cells.MethodsWe investigated the antineoplastic activity of AF in 4 canine lymphoid tumor derived cell lines through measurements of proliferation, apoptosis, thioredoxin reductase (TrxR) activity and generation of reactive oxygen species (ROS), and detected the effects of AF when combined with conventional cytotoxic drugs using the Chou and Talalay method. We also evaluated the antiproliferative effects of AF in primary canine lymphoma cells using a bioreductive fluorometric assay.ResultsAt concentrations that appear clinically achievable in humans, AF demonstrated potent antiproliferative and proapoptotic effects in canine lymphoid tumor cell lines. TrxR inhibition and increased ROS production was observed following AF treatment. Moreover, a synergistic antiproliferative effect was observed when AF was combined with lomustine or doxorubicin.ConclusionsAuranofin appears to inhibit the growth and initiate apoptosis in canine lymphoma cells in vitro at clinically achievable concentrations. Therefore, this agent has the potential to have near-term benefit for the treatment of canine lymphoma, as well as a translational model for human lymphoma. Decreased TrxR activity and increasing ROS production may be useful biomarkers of drug exposure.

A decrease in ubiquitination and resulting prolonged life-span of KIT underlies the KIT overexpression-mediated imatinib resistance of KIT mutation-driven canine mast cell tumor cells.

Overexpression of KIT is one of the mechanisms that contributes to imatinib resistance in KIT mutation-driven tumors. Here, the mechanism underlying this overexpression of KIT was investigated using an imatinib-sensitive canine mast cell tumor (MCT) line CoMS, which has an activating mutation in KIT exon 11. A KIT-overexpressing imatinib-resistant subline, rCoMS1, was generated from CoMS cells by their continuous exposure to increasing concentrations of imatinib. Neither a secondary mutation nor upregulated transcription of KIT was detected in rCoMS1 cells. A decrease in KIT ubiquitination, a prolonged KIT life-span, and KIT overexpression were found in rCoMS1 cells. These events were suppressed by withdrawal of imatinib and were re-induced by re‑treatment with imatinib. These findings suggest that imatinib elicited overexpression of KIT via suppression of its ubiquitination. These results also indicated that imatinib-induced overexpression of KIT in rCoMS1 cells was not a permanently acquired feature but was a reversible response of the cells. Moreover, the pan deubiquitinating enzyme inhibitor PR619 prevented imatinib induction of KIT overexpression, suggesting that the imatinib-induced decrease in KIT ubiquitination could be mediated by upregulation and/or activation of deubiquitinating enzyme(s). It may be possible that a similar mechanism of KIT overexpression underlies the acquisition of imatinib resistance in some human tumors that are driven by KIT mutation.