Canine Mastocytoma Cell Line Research Articles

O51 | Algal extracts as potential anticancer agents: Preliminary investigations in C2 canine mastocytoma cell line

Journal of Veterinary Pharmacology and TherapeuticsVolume 46, Issue S1 p. 52-53 ORAL PRESENTATIONS O51 | Algal extracts as potential anticancer agents: Preliminary investigations in C2 canine mastocytoma cell line First published: 11 June 2023 https://doi.org/10.1111/jvp.13217Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume46, IssueS1Special Issue: 15th International Congress of the European Association for Veterinary Pharmacology and Toxicology held Bruges, Belgium, July 2–5, 2023June 2023Pages 52-53 RelatedInformation

Characterization of WWOX expression and function in canine mast cell tumors and malignant mast cell lines

BackgroundThe WW domain-containing oxidoreductase (WWOX) tumor suppressor gene is frequently lost in a variety of solid and hematopoietic malignancies in humans. Dysregulation of WWOX has been implicated as playing a key role in tumor cell survival, DNA damage repair, and genomic stability. The purpose of this study was to characterize WWOX expression in spontaneous canine mast cell tumors (MCTs) and malignant cell lines and investigate the potential contribution of WWOX loss on malignant mast cell behavior.Methods/resultsWWOX expression is decreased in primary canine MCTs and malignant mast cell lines compared to normal canine bone marrow-cultured mast cells. In transformed canine mastocytoma cell lines, overexpression of WWOX or WWOX knockdown had no effect on mast cell viability. Inhibition of WWOX enhanced clonogenic survival following treatment with ionizing radiation in the C2 mast cell line. Lastly, immunohistochemistry for WWOX was performed using a canine MCT tissue microarray, demonstrating that WWOX staining intensity and percent of cells staining for WWOX is decreased in high-grade MCTs compared to low-grade MCTs.ConclusionsThese data suggest that WWOX expression is attenuated or lost in primary canine MCTs and malignant mast cell lines. Given the observed increase in clonogenic survival in WWOX-deficient C2 mast cells treated with ionizing radiation, further investigation of WWOX and its role in mediating the DNA damage response in malignant mast cells is warranted.

The Effect of Co0.2Mn0.8Fe2O4 Ferrite Nanoparticles on the C2 Canine Mastocytoma Cell Line and Adipose-Derived Mesenchymal Stromal Stem Cells (ASCs) Cultured Under a Static Magnetic Field: Possible Implications in the Treatment of Dog Mastocytoma

Cobalt manganese ferrite nanoparticles have application potential in the biomedical field, however there is limited information concerning the biological response. The aim of this work was to investigate the cytotoxic potential of cobalt-manganese ferrite nanoparticles in canine mastocytoma tumor cells (C2) and adipose-derived mesenchymal stromal stem cells (ASCs) cultured under a static magnetic field (MF). In this study, we investigated the viability and proliferation rate of ASC and C2 cells cultured with Co0.2Mn0.8Fe2O4 nanoparticles under 0.5T MF. We observed cells morphology and measured intracellular ROS generation. Thermal observations were used to characterize the thermotrophic cell behavior in different condition and RNA level of heat shock proteins and apoptotic genes was measured. Nanoparticles reduced cell viability, caused cell damage, i.e., through the formation of reactive oxygen species (ROS) and increased transcriptional level of apoptotic genes (Bcl-2, Bax, p53, p21). In addition, we have found that C2 mastocytoma cells cultured with metal oxide nanoparticles under MF exhibited unexpected biological responses, including thermotolerance and apoptotic response induced by the expression of heat shock proteins and ROS produced under a MF. Our results suggest that stimulation using MF and Co0.2Mn0.8Fe2O4 nanoparticles is involved in mechanisms associated with controlling cell proliferative potential signaling events. We can state that significant differences between normal and cancer cells in response to nanoparticles and MF are apparent. Our results show that nanoparticles and MF elevate the temperature in vitro in tumor cells, thereby increasing the expression of ROS as well as heat shock proteins.

IL-4 downregulates expression of the target receptor CD30 in neoplastic canine mast cells.

CD30 is a novel therapeutic target in human mast cell (MC) neoplasms. In this 'comparative oncology' study, we examined CD30 expression and regulation in neoplastic canine MC using a panel of immunomodulatory cytokines [interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-13 and stem cell factor (SCF)] and the canine mastocytoma cell lines NI-1 and C2. Of all cytokines tested IL-4 was found to downregulate expression of CD30 in NI-1 and C2 cells. We also found that the CD30-targeting antibody-conjugate brentuximab vedotin induces growth inhibition and apoptosis in both MC lines. Next, we asked whether IL-4-induced downregulation of CD30 interferes with brentuximab vedotin-effects. Indeed, pre-incubation of NI-1 cells with IL-4 decreased responsiveness towards brentuximab vedotin. To overcome IL-4-mediated resistance, we applied drug combinations and found that brentuximab vedotin synergizes with the Kit-targeting drugs masitinib and PKC412 in inhibiting growth of NI-1 and C2 cells. In summary, CD30 is a new marker and IL-4-regulated target in neoplastic canine MC.

Development of an in vitro model of acquired resistance to toceranib phosphate (Palladia®) in canine mast cell tumor

BackgroundMast cell tumors (MCTs) are the most common skin tumors in dogs and exhibit variable biologic behavior. Mutations in the c-kit proto-oncogene are associated with the tumorigenesis of MCTs, resulting in growth factor-independent and constitutive phosphorylation of the KIT receptor tyrosine kinase (RTK). Toceranib (TOC) phosphate (Palladia®) is a KIT RTK inhibitor that has biological activity against MCTs. Despite these benefits, patients ultimately develop resistance to TOC. Therefore, there is a need to identify distinguishing clinical and molecular features of resistance in this population.ResultsThe canine C2 mastocytoma cell line contains an activating mutation in c-kit. Three TOC-resistant C2 sublines (TR1, TR2, TR3) were established over seven months by growing cells in increasing concentrations of TOC. TOC inhibited KIT phosphorylation and cell proliferation in a dose-dependent manner in the treatment-naïve, parental C2 line (IC50 < 10 nM). In contrast, the three sublines were resistant to growth inhibition by TOC (IC50 > 1,000 nM) and phosphorylation of the KIT receptor was less inhibited compared to the TOC-sensitive C2 cells. Interestingly, sensitivity to three structurally distinct KIT RTK inhibitors was variable among the sublines, and all 3 sublines retained sensitivity to the cytotoxic agents vinblastine and lomustine. Sequencing of c-kit revealed secondary mutations in the juxtamembrane and tyrosine kinase domains of the resistant sublines. These included point mutations in TR1 (Q574R, M835T), TR2 (K724R), and TR3 (K580R, R584G, A620S). Additionally, chronic TOC exposure resulted in c-kit mRNA and KIT protein overexpression in the TOC-resistant sublines compared to the parental line. C2, TR1, TR2, and TR3 cells demonstrated minimal P-glycoprotein (P-gp) activity and no functional P-gp.ConclusionsThis study demonstrates the development of an in vitro model of acquired resistance to targeted therapy in canine MCTs harboring a c-kit-activating mutation. This model may be used to investigate the molecular basis of and strategies to overcome TOC resistance.

Bromodomain-Containing Protein 4 (BRD4): A Novel Marker and Drug Target Expressed In Neoplastic Cells In Advanced Mast Cell Neoplasms

Advanced mast cell (MC) neoplasms are characterized by uncontrolled growth and rapid expansion of neoplastic MC in various organ systems and a poor prognosis. In human patients, advanced systemic mastocytosis (SM) is rare and usually presents as aggressive SM (ASM) or mast cell leukemia (MCL). In canines, advanced MC tumors (MCT) are considered one of the most frequent skin tumors. Hence, so far, no effective treatment concept has been established for advanced MC neoplasms in humans or canines. This is due to the fact that neoplastic MC in advanced SM usually are resistant against various cytoreductive agents. Therefore, research is currently focusing on potential drug targets and the effects of novel targeted drugs on neoplastic MC. We have recently identified the bromodomain-containing protein-4 (BRD4) as a promising new drug target expressed by neoplastic cells in acute myeloid leukemia (Zuber et al, Nature 2011;478:524-528). In the present study, we examined BRD4 as a potential therapeutic target in human ASM and MCL, and canine MCT. As assessed by immunohistochemistry, neoplastic MC expressed substantial amounts of cytoplasmic and nuclear BRD4 in ASM and MCL, whereas in human indolent SM (ISM), MC expressed lower amounts of BRD4 or did not express cytoplasmic BRD4. The human MCL lines HMC-1.1 and HMC-1.2 as well as the canine mastocytoma cell lines NI-1 and C2, also expressed BRD4. In order to study the potential biological function of BRD4, we applied a BRD4-specific shRNA. The shRNA-induced knockdown of BRD4 was found to be associated with a markedly reduced proliferation of HMC-1 cells. Correspondingly, the BRD4-targeting drug JQ1 induced dose-dependent growth inhibition in all MC lines examined, with higher IC50 values obtained in HMC-1.1 cells (IC50: 1.0 µM) and HMC-1.2 cells (IC50: 1.0 µM) compared to NI-1 cells (0.2 µM) and C2 cells (0.5 µM). As assessed by morphology and AnnexinV/PI staining, the growth-inhibitory effects of JQ1 on neoplastic human and canine MC lines were accompanied by apoptosis (Figure). The apoptosis-inducing effects of JQ1 was also demonstrable by staining for active caspase 3. Furthermore, JQ1 was found to downregulate the expression of the activation-related MC antigen CD63 and the proliferation-related antigen CD71 (transferrin receptor) in HMC-1 cells. In addition, JQ1 was found to inhibit proliferation in primary neoplastic MC obtained from patients with ASM or MCL (IC50 0.1-0.5 µM). We next screened for potential drug partners that would potentiate the anti-neoplastic effects of JQ1. Of all drugs tested, all-trans retinoic acid (ATRA) and PKC412 were identified as most potent drug partners of JQ1. These drugs were found to act highly synergistic with JQ1 in producing apoptosis and growth inhibition in HMC-1.1 and HMC-1.2 cells (Figure). Together, BRD4 is a novel marker and promising target in advanced MC neoplasms. Whether JQ1 or other BET bromodomain inhibitors are effective in vivo in patients with advanced SM or canine MCT remains to be elucidated. [Display omitted] HMC-1.2 cells were incubated in control medium (Co) or in various concentrations of JQ1 (black bars), ATRA (gray bars), or a combination of both agents at a ratio of 1:5 (white bars) at 37°C for 48 hours. Then, AnnexinV/propidium iodide (PI) staining was performed. The percentage of AnnexinV+ cells was determined by flow cytometry. Results represent the mean±S.D. of 3 independent experiments. Asterisk (*): p<0.05 compared to control (Co). Disclosures:Bradner:Tensha Therapeutics: Dr. Bradner is a scientific founder of Tensha Therapeutics, which is developing drug-like derivatives of the JQ1 bromodomain inhibitor as cancer therapeutics, through a license from the Dana-Farber Cancer Institute. Other. Horny:Novartis : Consultancy, Honoraria.

The pan-Bcl-2 blocker obatoclax promotes the expression of Puma, Noxa, and Bim mRNA and induces apoptosis in neoplastic mast cells

Advanced SM is an incurable neoplasm with short survival time. So far, no effective therapy is available for these patients. We and others have shown recently that neoplastic MC in ASM and MCL express antiapoptotic Mcl-1, Bcl-2, and Bcl-xL. In this study, we examined the effects of the pan-Bcl-2 family blocker obatoclax (GX015-070) on primary neoplastic MC, the human MC leukemia cell line HMC-1, and the canine mastocytoma cell line C2. Obatoclax was found to inhibit proliferation in primary human neoplastic MC (IC₅₀: 0.057 μM), in HMC-1.2 cells expressing KIT D816V (IC₅₀: 0.72 μM), and in HMC-1.1 cells lacking KIT D816V (IC₅₀: 0.09 μM), as well as in C2 cells (IC₅₀: 0.74 μM). The growth-inhibitory effects of obatoclax in HMC-1 cells were accompanied by an increase in expression of Puma, Noxa, and Bim mRNA, as well as by apoptosis, as evidenced by microscopy, TUNEL assay, and caspase cleavage. Viral-mediated overexpression of Mcl-1, Bcl-xL, or Bcl-2 in HMC-1 cells was found to introduce partial resistance against apoptosis-inducing effects of obatoclax. We were also able to show that obatoclax synergizes with several other antineoplastic drugs, including dasatinib, midostaurin, and bortezomib, in producing apoptosis and/or growth arrest in neoplastic MC. Together, obatoclax exerts major growth-inhibitory effects on neoplastic MC and potentiates the antineoplastic activity of other targeted drugs. Whether these drug effects can be translated to application in patients with advanced SM remains to be determined.

NI‐1: a novel canine mastocytoma model for studying drug resistance and IgER‐dependent mast cell activation

BackgroundAdvanced mast cell (MC) disorders are characterized by uncontrolled growth of neoplastic MC in various organs, mediator-related symptoms, and a poor prognosis. Kit mutations supposedly contribute to abnormal growth and drug resistance in these patients.MethodsWe established a novel canine mastocytoma cell line, NI-1, from a patient suffering from MC leukemia.ResultsNI-1 cells were found to form mastocytoma lesions in NOD/SCID IL-2Rgammanull mice and to harbor several homozygous Kit mutations, including missense mutations at nucleotides 107(C→T) and 1187(A→G), a 12-bp duplication (nucleotide 1263), and a 12-bp deletion (nucleotide 1550). NI-1 cells expressed several MC differentiation antigens, including tryptase, Kit, and a functional IgE receptor. Compared to the C2 mastocytoma cell line harboring a Kit exon 11 mutation, NI-1 cells were found to be less responsive against the Kit tyrosine kinase inhibitors (TKI) masitinib and imatinib, but were even more sensitive against proliferation-inhibitory effects of the mammalian target of rapamycin (mTOR) blocker RAD001 and PI3-kinase/mTOR blocker NVP-BEZ235. The Kit-targeting multikinase inhibitors PKC412 and dasatinib were also found to override TKI resistance in NI-1 cells, and produced growth inhibition with reasonable IC50 values (<0.1 μM).ConclusionNI-1 may serve as a useful tool to investigate IgE-dependent reactions and mechanisms of abnormal growth and drug resistance in neoplastic MC in advanced mastocytosis.

Abstract A56: Canine Mast Cell Tumor and Toceranib Phosphate (Palladia™): Development of a Spontaneous Canine Model of Kinase Inhibitor Resistance

Abstract Mast cell tumors (MCTs) are one of the most common skin tumors in dogs, accounting for up to 21% of all canine cutaneous tumors, and exhibit extremely variable biologic behavior. Mutations in the juxtamembrane, kinase and ligand binding domains of the c-kit proto-oncogene have been associated with the tumorigenesis of canine MCTs, resulting in growth factor-independent and constitutive phosphorylation of the Kit receptor tyrosine kinase (RTK). Approximately one-third of canine MCTs carry a c-kit mutation. As such, small molecule inhibitors of Kit are an attractive therapeutic strategy for MCTs in dogs. Toceranib (TOC) phosphate (Palladia) is one such RTK inhibitor of Kit that has biological activity against canine MCTs. Approximately 40% of dogs experience an objective response to TOC while the remaining 60% have intrinsic resistance. Two-thirds of the responders are positive for an activating mutation in c-kit. The time to tumor progression in all responders is 18 weeks; therefore, essentially all dogs with MCTs either have intrinsic TOC resistance or eventually develop resistance. Therefore, there is a need to identify distinctive clinical and molecular features of resistance in this population. The canine C2 mastocytoma cell line, established from a spontaneously occurring MCT, contains the previously described c-kit internal tandem duplication in the juxtamembrane domain involving exon 11. TOC-resistant C2 cells were selected by growing cells in concentrations of TOC ranging from 0.02 uM to 0.3 uM and increasing in 0.025–0.05 uM increments. Three resistant sublines were established over a period of 7 months. All three resistant sublines as well as the original naïve C2 cells were grown in the presence of increasing concentrations of TOC, 3 other KIT kinase inhibitors, or the cytotoxic agents vinblastine (VBL) or lomustine (CCNU) for 72 hours, followed by determination of relative viable cell number using a fluorometric bioreductive assay (Alamar Blue). To evaluate drug effects on Kit autophosphorylation, parental C2 cells and the resistant sublines were incubated for 24 hours with increasing concentrations of TOC (0 nM–100 nM) and phosphorylated and total Kit were analyzed by western blot. Western analysis of the C2 line demonstrated constitutive tyrosine phosphorylation of Kit in the absence of exogenous ligand. TOC inhibited Kit phosphorylation in a dose-dependent manner. Proliferation of the naïve C2 cells was inhibited by TOC in a dose-dependent manner with an IC50 of &amp;lt;10 nM. In contrast, all three resistant sublines were resistant to inhibition by TOC (IC50 &amp;gt; 1,000 nM). Interestingly, sensitivity to the other 3 Kit RTK inhibitors was variable between the sublines, and all 3 sublines retained sensitivity to the cytotoxic agents VBL and CCNU. To determine whether this lack of growth inhibition was due to a lack of inhibition of autophosphorylation by TOC, the resistant sublines were incubated with increasing concentrations of TOC for 24 hours and western analysis for phophorylated and total Kit was performed. Phosphorylation of the Kit receptor was less inhibited in all three resistant sublines compared to the TOC-sensitive C2 cells, suggesting that acquisition of secondary mutations in the c-kit gene is likely responsible for the observed TOC resistance. Canine MCTs and their intrinsic or acquired resistance to the TKI TOC (Palladia) demonstrate an excellent in vivo model of a spontaneous, Kit driven neoplastic disease. This study demonstrates the development of a canine model to study drug-resistant tumor clones with a c-kit activating mutation. This model may be better utilized to study the molecular basis of and strategies to overcome drug resistance.

Polyunsaturated fatty acid supplements modulate mast cell membrane microdomain composition

In the present study, the lipid raft composition of a canine mastocytoma cell line (C2) was analyzed. Lipid rafts were well separated from non-raft plasma membranes using a detergent-free isolation technique. To study the influence of n-3 and n-6 polyunsaturated fatty acids (PUFA) on raft fatty acid composition in comparison to non-raft cell membrane, C2 were supplemented with one of the following: α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid or arachidonic acid. Enrichment of the culture medium with a specific PUFA resulted in an increase in the content of this fatty acid both in rafts and non-raft membranes. Contents of cholesterol and protein were found not to be affected by the changes in the fatty acid profiles. In conclusion, our data provide strong evidence that PUFA modulate lipid composition and physiological properties of membrane micro domains of mast cells which in turn may have effects on mast cell function.

Polo-like kinase-1 as a novel target in neoplastic mast cells: demonstration of growth-inhibitory effects of small interfering RNA and the Polo-like kinase-1 targeting drug BI 2536

In advanced systemic mastocytosis the response of neoplastic mast cells to conventional drugs is poor and the prognosis is bad. Current research is, therefore, attempting to identify novel drug targets in neoplastic mast cells. Polo-like kinase-1 is a serine/threonine kinase that plays an essential role in mitosis and has recently been introduced as a new target in myeloid leukemias and solid tumors. In the present study, we analyzed the expression and function of Polo-like kinase-1 in neoplastic mast cells in systemic mastocytosis. As determined by immunostaining, primary neoplastic mast cells as well as the human mast cell leukemia cell line HMC-1 displayed phosphorylated Polo-like kinase-1. In addition, neoplastic mast cells expressed Polo-like kinase-1 mRNA. Polo-like kinase-1-specific small interfering RNA induced apoptosis in neoplastic mast cells, whereas no effect was seen with a control small interfering RNA. BI 2536, a drug targeting Polo-like kinase-1, was found to inhibit proliferation in HMC-1 cells in a dose-dependent manner. BI 2536 also inhibited the growth of primary neoplastic mast cells and cells of the canine mastocytoma cell line C2. The growth-inhibitory effects of BI 2536 on neoplastic mast cells were found to be associated with mitotic arrest and subsequent apoptosis. Finally, BI 2536 was found to synergize with the KIT-targeting kinase inhibitor midostaurin (PKC412) in inhibiting the growth of neoplastic mast cells. In control experiments, BI 2536 did not induce apoptosis in normal cultured mast cells. Collectively, our data show that Polo-like kinase-1 is a potential therapeutic target in neoplastic mast cells. Targeting Polo-like kinase-1 may be an attractive pharmacological concept in the management of advanced systemic mastocytosis.

PUFA-dependent alteration of oxidative parameters of a canine mastocytoma cell line

Mast cells play a key role in the immune response. Thereby, the balance of oxidative metabolism is of importance in mast cell mediator synthesis and release. Fatty acids may modify mast cell function in several ways. In this study, we investigated the influence of polyunsaturated fatty acids (PUFAs) on oxidative parameters of a canine mastocytoma cell line. C2 cells were cultured in media supplemented with linoleic acid, arachidonic acid, alpha-linolenic acid and eicosapentaenoic acid, respectively. Production of reactive oxygen species (ROS) as well as lipid peroxides was tested. Furthermore, stressor-induced DNA damage was measured. Exposure of the cells to PUFAs resulted in a significant increase in the synthesis of both ROS and lipid peroxides. Distinct differences between the PUFAs tested underline the impact of the unsaturation degree of fatty acids as well as the position of double bonds on mast cells.

Establishment of a Novel Canine Mastocytoma Cell Line, NI-1: a Model for Studying Resistance Against KIT Tyrosine Kinase Inhibitors In Canine Neoplastic Mast Cells

AbstractAbstract 4936Advanced mast cell (MC) disorders are characterized by uncontrolled growth of neoplastic MC in various organ systems, resistance to conventional cytoreductive drugs, and a poor prognosis. In most patients, transforming mutations in the KIT proto-oncogene are detectable and are considered to contribute to resistance. MC lines are an important model for analyzing drug resistance in neoplastic MC. We have established a novel canine mastocytoma cell line, NI-1 from a canine patient suffering from mast cell leukemia. NI-1 cells were found to harbour several homozygous KIT mutations including two single nucleotide mutations, one at nucleotide 107 (C to T, leading to a missense mutation, P36L) and one at nucleotide 1187 (A to G, leading to a missense mutation, Q396R), a 12 bp duplication at nucleotide 1263, and a 12 bp deletion at nucleotide 1550, the latter reflecting a transcriptional variant. NI-1 cells contained histamine and formed tumors in NOD-SCID IL-2Rgammanull (NSG) mice. As assessed by 3H-thymidine uptake, a number of targeted drugs were found to inhibit the proliferation of NI-1 cells at pharmacologically relevant concentrations. Among these drugs were the KIT kinase blockers midostaurin (IC50 <0.1 μM), dasatinib (IC50 <0.1 μM), sunitinib (IC50 <0.1 μM), tozasertib (0.1-0.5 μM), imatinib (IC50 0.25–0.37 μM), sorafenib (IC50 0.25–0.5 μM), masatinib (0.1-1.0 μM) as well as drugs targeting important KIT-downstream signaling molecules (PI3 kinase, mTOR), i.e. RAD001 (IC50 <0.1 μM) and NVP-BEZ235 (IC50 0.01–0.05 μM). In most instances, drug-induced growth inhibition was found to be accompanied by apoptosis. No clear effects were seen with drugs that are not capable of blocking KIT or KIT-downstream kinases in MC, i.e. bosutinib, erlotinib, gefitinib, and lapatinib (IC50 >2 μM). However, the HDAC inhibitor vorinostat was found to block growth of NI-1 cells (0.1-0.5 μM). Drug response profiling also revealed that NI-1 cells differ markedly from canine C2 mastocytoma cells harbouring an exon 11 KIT mutation, and the human mast cell lines HMC-1.1 (exon 11 mutation in KIT) and HMC-1.2 (exon 11 and exon 17 KIT mutations). In contrast to C2 cells, NI-1 cells were found to be largely resistant against the growth-inhibitory effects of masatinib, sorafenib, and sunitinib. By contrast, NI-1 cells were found to be even more sensitive against the growth-inhibitory effects of the mTOR blocker RAD001 and the PI3-kinase/mTOR inhibitor NVP-BEZ235. Together, our data suggest that certain KIT mutations may be associated with relative resistance of neoplastic MC against KIT-targeting drugs, a phenomenon that has also been described for human MC and the KIT mutant D816V that mediates resistance against masatinib and imatinib. The novel MC line NI-1 may serve as a novel tool to investigate the mechanisms of resistance against TKI in neoplastic MC. Disclosures:Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

The Aurora-Kinase Inhibitor R763/AS703569 Exerts Major Growth-Inhibitory and Apoptosis-Inducing Effects on Neoplastic Mast Cells

AbstractAbstract 3972Systemic mastocytosis (SM) is a myeloid neoplasm defined by abnormal growth and accumulation of neoplastic mast cells (MC) in one or more internal organs. In most patients, the D816V-mutated variant of KIT is detectable. This mutant supposedly confers resistance against several tyrosine kinase inhibitors including imatinib and masitinib. In aggressive SM (ASM) or mast cell leukemia (MCL) the response to conventional drugs is poor and the prognosis is grave. In these patients, additional KIT-independent signalling pathways and molecules, such as BTK and LYN may play an important role in disease evolution and MC proliferation. R763/AS703569 is a multikinase inhibitor that blocks the kinase activity of KIT, BTK, LYN, Aurora-Kinase-A, Aurora-Kinase-B, ABL, AKT, and FLT3. We analyzed the effects of R763/AS703569 on growth and survival of the human mast cell leukemia cell line HMC-1 and the canine mastocytoma cell line C2. Two subclones of HMC-1 were used, one expressing KIT D816V (HMC-1.2) and one lacking KIT D816V (HMC-1.1). Both HMC-1 subclones were found to express Aurora-Kinase-A mRNA and Aurora-Kinase-B mRNA in RT-PCR experiments. As assessed by 3H-thymidine uptake, R763/AS703569 was found to inhibit proliferation of HMC-1 cells in a dose-dependent manner, with lower IC50 values obtained in HMC-1.2 cells (1-5 nM) compared to HMC-1.1 cells (10-10-50 nM). Moreover, R763/AS703569 produced growth inhibition in C2 cells (IC50: 1–5 nM). As assessed by light microscopy and Tunel assay, the growth-inhibitory effects of R763/AS703569 were found to be accompanied by apoptosis in all three cell lines. Correspondingly, R763/AS703569 was found to induce cleavage of caspase-3, caspase-8, and caspase-9 in HMC-1 cells. Moreover, R763/AS703569 was found to induce a G2/M cell cycle arrest in HMC-1 cells and C2 cells after 24 hours. In order to define the target spectrum for R763/AS703569 in HMC-1 cells, Western blot experiments were performed. In these experiments, R763/AS703569 was found to inhibit the phosphorylation of KIT, Aurora-Kinase-A, and BTK in HMC-1.1 cells, whereas no effects of R763/AS703569 on phosphorylation of LYN were seen. We then combined R763/AS703569 with dasatinib, a drug known to block LYN activation in HMC-1 cells. In these experiments, we were able to show that both drugs cooperate with each other in inducing apoptosis in HMC-1.1 cells and HMC-1.2 cells. In summary, our data suggest that R763/AS703569 is a novel promising drug that should be tested for its anti-neoplastic effects in patients with ASM and MCL in clinical trials. Complete inhibition of growth of neoplastic MC may require drug combinations employing R763/AS703569 and other targeted or cytotoxic drugs. Disclosures:Sarno:Merck-Serono: Employment. Valent:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Merck-Serono: Research Funding.

KIT polymorphisms and mutations determine responses of neoplastic mast cells to bafetinib (INNO-406)

Advanced systemic mastocytosis (SM) is characterized by uncontrolled growth of neoplastic mast cells (MC) and drug resistance. The tyrosine kinase receptor KIT is often mutated and activated and thus contributes to malignant growth of MC. Therefore, KIT-targeting drugs are currently tested for their ability to block growth of malignant MC. We determined the effects of the multikinase inhibitor INNO-406 (bafetinib) on primary neoplastic MC, the canine mastocytoma cell line C2, the human MC leukemia cell line HMC-1.1 bearing the KIT mutant V560G, and HMC-1.2 cells harboring KIT V560G and KIT D816V. INNO-406 was found to inhibit proliferation in HMC-1.1 cells (IC(50): 30-40 nM), but not in HMC-1.2 cells or primary neoplastic cells in patients with KIT D816V-positive SM. In canines, growth-inhibitory effects of INNO-406 were seen in C2 cells (IC(50): 50-100 nM) exhibiting a KIT exon 11 internal tandem-duplication and in primary neoplastic MC harboring wild-type exon 11, whereas no effects were seen in MC exhibiting a polymorphism at amino acid 581 in exon 11. INNO-406 was found to block KIT phosphorylation and expression in HMC-1.1 cells and C2 cells, but not in HMC-1.2 cells, whereas Lyn-phosphorylation was blocked by INNO-406 in all types of MC. In neoplastic MC, the major target of INNO-406 appears to be KIT. Drug responses may depend on the presence and type of KIT mutation. In human MC, the KIT D816V mutant introduces resistance, and in canine mastocytomas, an exon 11 polymorphism may be indicative of resistance against INNO-406.

Effect of radiation on vascular endothelial growth factor expression in the C2 canine mastocytoma cell line

To establish the radiosensitivity and effect of irradiation on vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) expression in the canine mastocytoma cell line C2. Canine mastocytoma cell line C2. C2 cells were irradiated with single doses of 2, 4, 6, and 8 Gy. The 3-(4, 5-di-methyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and proliferation assays with (methyl-hydrogen 3) thymidine were used for radiosensitivity experiments. Expression of VEGFR was determined via flow cytometry and apoptotic rate via annexin assay. Human and canine VEGF ELISA kits were evaluated in crossover assay experiments, and the canine kit was used thereafter. C2 cells secreted VEGF constitutively. Radiation did not induce a significant increase in VEGF secretion, regardless of radiation dose. Consistently, radiation did not up-regulate VEGFR. Cell survival rates decreased in a dose-dependent manner. The apoptotic cell fraction had a dose-dependent increase that reached its maximum 24 to 48 hours after radiation. The C2 cell line was radiosensitive, and a fraction (up to 40%) of cells died via apoptosis in a dose-dependent manner. In response to radiation, C2 cells did not upregulate VEGF production or VEGFR. Further studies are needed to determine whether tumor control could be improved by combining radiotherapy with VEGFR inhibitors or apoptosis-modulating agents.

Characterization of an early signaling defect following FcɛRI activation in the canine mastocytoma cell line, C2

A comparison of IgE recognition by cognate receptors expressed on the C2 canine mastocytoma cell line with analogous events in a rat basophilic leukemia cell line transfected with the α-chain subunit of the canine high-affinity IgE receptor using flow cytometry show that canine IgE recognizes the α-chain of its cognate receptor on both cell lines. Our study confirms the expression of functional IgE receptors in both cell lines, but receptor-mediated signaling in the C2 line only supports the early stages of downstream signaling as shown by the phosphorylation of the γ-chain and the failure to effect the phosphorylation of Syk. In contrast RBL-2H3 cells respond to sensitization with IgE and challenge with cognate antigen with tyrosine phosphorylation of the γ-subunits of the receptor complex followed by downstream phosphorylation of Syk and Ca2+ mobilization, culminating in β-hexosaminidase release. We propose that the identification of the precise signaling defect in C2 cells will yield useful information regarding the pathway leading to mast cell exocytosis and facilitate the restoration of the complete signaling cascade through complementation of the missing/defective signal transducer since signaling events downstream of Ca2+ mobilization are intact as demonstrated by β-hexosaminidase release following non-immunologic stimulation with the calcium ionophore, A23187.

Susceptibility of the C2 canine mastocytoma cell line to the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family, which preferentially induces apoptosis in cells that have undergone malignant transformation. In humans, non-neoplastic cells are normally protected from the effects of TRAIL by expressing decoy receptors, lacking death domains. In contrast, neoplastic cells tend to downregulate their decoy receptor expression, increasing their susceptibility to the pro-apoptotic effects of TRAIL, via the functional TRAIL receptors. The aim of the current study was to investigate the effect of TRAIL on the canine C2 mastocytoma cell line to determine whether this agent might be a suitable treatment for mast cell tumors in dogs. C2 and MDCK cells were cultured with recombinant human TRAIL. Apoptosis was assessed using a Caspase 3 and 7 chemiluminescence assay and flow cytometry following Annexin V:FITC labelling. Cell metabolism was assessed using a colorimetric MTT-based assay. C2 cells demonstrated greater sensitivity to TRAIL-induced apoptosis compared to MDCK cells by all assessment methods. The dog genome assembly was searched for orthologs of TRAIL and its receptors using published sequences from other species for reference. Although a canine ortholog for TRAIL was identified, only one TRAIL receptor ortholog (TNFRSF11B) could be found. C2, but not MDCK, cells expressed mRNA for TNFRSF11B, detected by RT-PCR. In other species, TNFRSF11B is a decoy receptor, as even though it has a death domain it is secreted due to its lack of a transmembrane domain. The effect of TRAIL on the C2 cell line suggests that this cytokine might be suitable for treatment of mast cell tumors in dogs.

Effects of the Mcl-1/Bcl-2 Inhibitor GX015-070 (Obatoclax®) on Growth and Viability of Canine and Human Neoplastic Mast Cells

Mcl-1 is a Bcl-2 family-member that has been described to act anti-apoptotic in various myeloid neoplasms. We and others have recently shown that neoplastic mast cells (MC) in patients with systemic mastocytosis (SM) display Mcl-1, Bcl-2, and Bcl-xL. In the present study, we examined the effects of the Mcl-1/Bcl-2-targeting drug GX015-070 (obatoclax®; GeminX, Montréal, Quebéc, Canada) on growth and viability of primary neoplastic MC obtained from patients with SM (n=3), the human MC leukemia cell line HMC-1, and the canine mastocytoma cell line C2. Two HMC-1 subclones, one lacking KIT D816V (HMC- 1.1) and one expressing KIT D816V (HMC-1.2) were examined. As assessed by RT-PCR and immunostaining, primary neoplastic MC as well as HMC-1 cells (both subclones) were found to express Mcl-1 mRNA and the Mcl-1 protein in a constitutive manner, but did not express significant amounts of proapoptotic Bim. Transfection of HMC-1 cells with Mcl-1-specific siRNA resulted in reduced proliferation and increased apoptosis compared to cells transfected with a control siRNA. GX015-070 was found to inhibit 3H-thymidine uptake and thus proliferation in HMC-1 cells in a dose-dependent manner, with higher IC50 values obtained in HMC-1.2 cells (0.5 μM) compared to HMC-1.1 cells (0.05 μM). GX015-070 also inhibited the growth and survival in the canine mastocytoma cell line C2 (IC50: 0.5-1 μM). Moreover, GX015-070 was found to inhibit the proliferation of primary human neoplastic MC in all SM patients tested (IC50: 0.05-0.1 μM). We next attempted to combine obatoclax with a modulator of Mcl-1/Bim expression in MC, in order to enhance drug effects. Since Bim is degraded via the proteasome, we applied the proteasome inhibitor bortezomib. Whereas GX015-07 did not modulate the production/expression of Mcl-1 or Bim in HMC-1 cells, bortezomib was found to promote the expression of Bim in our Western blot experiments. In addition, bortezomib was found to suppress 3H-thymidine uptake in both HMC-1 subclones. Finally, bortezomib was found to cooperate with GX015-070 in producing apoptosis in HMC-1.1 cells, HMC-1.2 cells, and C2 cells. Together, our data show that the Mcl-1/Bcl-2-targeting drug GX015-070 is a potent inhibitor of in vitro growth and survival of canine and human neoplastic MC. Targeting of Mcl-1 in neoplastic MC alone or in combination with a Bim-regulator may be an interesting pharmacologic approach in advanced SM.

Synergistic antiproliferative effects of KIT tyrosine kinase inhibitors on neoplastic canine mast cells

Aggressive mast cell (MC) tumors are hematopoietic neoplasms characterized by uncontrolled growth of MC and resistance to conventional drugs. In most cases, the tyrosine kinase (TK) receptor KIT is involved in malignant cell growth. Therefore, several KIT TK-targeting drugs are currently being tested for their ability to block growth of neoplastic MC. We examined the effects of four TK inhibitors (imatinib, midostaurin, nilotinib, and dasatinib) on C2 canine mastocytoma cells, as well as primary neoplastic canine MC. As assessed by (3)H-thymidine incorporation experiments, all TK inhibitors produced dose-dependent inhibition of proliferation in C2 cells with the following IC(50) values: imatinib: 269 +/- 180 nM, midostaurin: 157 +/- 35 nM, nilotinib: 55 +/- 24 nM, dasatinib: 12 +/- 3 nM. Growth-inhibitory effects of TK inhibitors were also observed in primary neoplastic mast cells, although IC(50) values for each drug varied from patient to patient, with midostaurin being the most potent agent in all samples tested. In consecutive experiments, we were able to show that TK inhibitors cooperate with each other in producing growth inhibition in C2 cells with synergistic effects observed with most drug combinations. In flow cytometry and TUNEL assay experiments, growth-inhibitory effects of TK inhibitors were found to be associated with cell-cycle arrest and apoptosis. Together, these data show that several TK-targeting drugs induce apoptosis and inhibit proliferation in canine mastocytoma cells in vitro, and that synergistic drug interactions can be obtained. Clinical trials are now warranted to explore whether these TK inhibitors also counteract growth of neoplastic cells in vivo in patients with aggressive MC tumors.