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

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

Supplementary Figure from 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

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Tumor draining lymph nodes (TDLNs) function as an anatomic niche which primes immune cells to generate anti-cancer responses. While tumors are frequently staged based on the presence or absence of TDLN metastasis, mechanisms for how tumors impact the TDLN microenvironment and immunologic responses are incompletely understood. Dogs with spontaneously arising tumors provide an opportune model to study immuno-oncology, sharing many similarities to human patients with their environment, clinical care, and pathobiology. To evaluate how tumors impact TDLN responses, we conducted single cell RNA sequencing (scSeq) on cryopreserved TDLNs of dogs with osteosarcoma (n=4) and healthy controls (n=2). We obtained an average of 3,984 cells per sample (range 1,603-5,259) with a total of 4,780 cells from healthy samples and 19,121 cells from TDLN samples. All samples were integrated into one dataset, then unsupervised clustering was completed. Each unique cell cluster was assigned an identity using canonical markers and reference mapping to human datasets. Following cell classification, we observed notable changes in gene expression across all cells isolated from the LNs, with most differences arising from changes to the transcriptome of myeloid cells. Although still preliminary, the data also demonstrate differences in the abundance of T, B, and myeloid cells between healthy and tumor draining LNs. Taken together, this dataset provides indications of the changes TDLNs undergo in dogs with osteosarcoma. Citation Format: Samuel A. Brill, Dylan T. Ammons, Anne C. Avery, Douglas H. Thamm. Evaluation of tumor draining lymph nodes in dogs with spontaneously arising osteosarcoma using single-cell sequencing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4465.

Omacetaxine mepesuccinate (OMX), also known as homoharringtonine, is a protein translation inhibitor approved for use in chronic myeloid leukemia. Recent studies suggest OMX may be effective against other cancer types with high rates of protein translation, and may be successful against protein targets that are difficult to inhibit by reducing their translation rates. Using both canine and human osteosarcoma (OS) cell lines, we have investigated the efficacy of OMX against osteosarcoma and expression of c-MYC. OMX provides significant growth inhibition of OS cells with IC50 values in the low nanomolar range. OMX also reduces migration and invasion capabilities of OS cells. Strikingly, we observed reduction in levels of c-MYC protein in most canine and human OS cell lines after OMX exposure. This reduction was both dose and time dependent. We also investigated the efficacy of OMX against OS in an orthotopic mouse OS model. OMX is a promising candidate for treatment of OS in both dogs and humans, and potentially additional cancers dependent on c-MYC. Citation Format: Kristen B. Farrell, Douglas H. Thamm. Omacetaxine reduces c-MYC expression and demonstrates antitumor effects in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6233.

The goals of this work are biomarker and drug discovery to advance cancer diagnostic and therapeutic strategies in humans through the study of naturally-occurring canine cancers. Many factors, including shared environment, intact host immunity, and greater cancer gene family homology between dogs and humans than mice and humans, make spontaneous canine cancers valuable complementary models of human cancer. Transcriptomic profiling utilizing RNA sequencing (RNA SEQ) of 5 canine cancers, including osteosarcoma, melanoma, B cell lymphoma, T cell lymphoma, and pulmonary carcinoma, have revealed five distinct gene co-expression models. From these unique module expression profiles, cancer-specific gene panels were derived. A similar analysis performed on existing RNA-SEQ data from human tumor samples produced cancer-specific human gene panels. Comparison of the canine and human gene panels found significant correlation (Spearman correlation > 0.6) which supports the translational relevance of naturally-occurring canine cancers to their human counterparts. Further, proteomic profiles derived from matched tumor tissue and peripheral blood samples mirror those of the tumor transcriptome, demonstrating these cancer-specific gene panels and their encoded proteins may represent robust canine diagnostic biomarker and/or therapeutic target candidates. Therapeutic hypotheses associated with each cancer specific gene panel were derived through matching of drugs documented to alter expression of panel genes in opposition to that exhibited by each cancer type. We identified 60 candidate drugs and screened them against a panel of canine cancer cell lines, finding 40 drugs that inhibited cell growth by 75% or more. Three or more active compounds were found for each cell line. From these 40 active compounds we then derived 30 synergistic drug combinations with the requirement that that they alter two or more panel genes in opposition to that exhibited by each cancer type. Additional studies to document drug synergism are underway and those confirmed as such will be considered good drug combination candidates for future canine clinical trials. Biomarker and drug combination candidates that perform well in canine clinical trials will then be considered for human trials. This work exemplifies the type of approach meant to further establish the comparative relevance of canine to human cancer and provide opportunities to explore hypotheses related to detection and treatment in both species. Citation Format: Amy Kathleen LeBlanc, Christina N. Mazcko, Matthew Breen, Rachael Thomas, Douglas H. Thamm. A comparative oncology approach to biomarker and drug discovery for cancer diagnosis and treatment in dogs and humans [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6134.

Background: ITK is a non-receptor tyrosine kinase that modifies T cell receptor (TCR) signaling. Mice deficient in ITK, but not resting lymphocyte kinase (RLK), exhibit defects in Th2 differentiation while retaining the ability to differentiate into Th1 cells and secrete IFNγ. Combined disruption of ITK and RLK in mice leads to more severe T cell functional defects compared to disrupting ITK alone, and paradoxically allows for normal Th2 responses. Thus, selective pharmacologic inhibition of ITK versus RLK is necessary to inhibit Th2 responses without affecting Th1-dependent immunity. ITK is widely expressed in T cell malignancies, and activation of ITK upregulates GATA-3, a transcription factor that drives Th2 differentiation and is associated with poor survival. Here we report the discovery and characterization of CPI-818, an irreversible inhibitor of ITK. CPI-818 is highly selective for ITK over RLK allowing for an assessment of pure ITK inhibition on normal and malignant T cells. Results: CPI-818 irreversibly inhibited ITK (IC502.3 nM) with >100-fold selectivity over RLK (430 nM) and BTK (850 nM). The mechanism of ITK inhibition involves covalent binding to CYS-442 confirmed by mass spectrometry. Irreversible inhibition of ITK in vitroand in vivowas demonstrated using an active site competitive probe. CPI-818 inhibited anti-CD3/28 induced phosphorylation of ERK (T202/Y204) and PLCγ (Y783) in PMBCs, and inhibited IL2 secretion in Jurkat T cells (IC5075 nM). CPI-818 demonstrated dose dependently inhibition of TCR-induced proliferation of malignant T cells from Sezary Syndrome patients. In mice orally treatedwith CPI-818 an increase in the ratio of IFNγ/IL-4 (p Citation Format: James W. Janc, Craig M. Hill, Patrick P. Ng, Andrew N. Hoston, Antonett Madriaga, Trang P. Dao-Pick, Kitman S. Yeung, Ryan Hudson, Anne-Marie Beausoleil, Erin Bradley, Erik Verner, Douglas H. Thamm, Richard A. Miller, Joseph J. Buggy. CPI-818: A selective inhibitor of interleukin-2-inducible T-cell kinase (ITK) that inhibits T-cell receptor signaling, promotes Th1 skewing, and achieves objective tumor responses when administered to dogs with T cell lymphomas [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 1313.

Analysis of circulating tumor biomarkers, designated “liquid biopsies” are less invasive and lower risk to the patient and might replace or complement standard tissue biopsies to determine prognosis, predict benefit from specific therapies, or monitor patients with cancer. Circulating tumor cells (CTCs) are extremely rare, with only 1-10 CTCs per mL of blood. Current technologies typically process 1-10 mL of blood ex vivo. We report an in vivo, indwelling CTC-capture device that is analogous to a Holter monitor used to interrogate cardiac rhythm abnormalities over long periods of time. The system incorporates a dual lumen intravenous catheter that directs blood from the vein into a small (~ the size of a wristwatch) capture carriage by virtue of a peristaltic pump. The carriage contains a CTC capture chip which separates CTCs from whole blood based on flow differences and solid phase anti-EpCAM capture, while the remaining whole blood is returned to the venous system via the dual lumen catheter. The chip is then removed from the carriage for further enumeration and characterization of the CTCs. In a proof of principle set of experiments, the system was placed into investigational canines into which fluorescently labeled human breast cancer cells were injected intravenously. Although the vast majority of these xeno-cancer cells were rapidly removed from circulation, the system captured “CTCs” over a 2 hour time period more successfully than was seen in simultaneous blood draws for ex vivo enumeration over the same time. No adverse effects were observed in the subjects at the time of the experiments or over the succeeding 48 hours after removal of the IV system.Our system permits interrogation of a much larger volume of blood than can be processed with standard blood draws. The larger processed volume will drastically increase the number of cells isolated which in turn increases the number of downstream assays that can be performed. This study is focused on developing a high throughput microfluidic device that can detect CTCs and implementing it in a canine model. Citation Format: Kaylee Smith, Tae Hyun Kim, Costanza Paoletti, Douglas H. Thamm, Daniel F. Hayes, Sunitha Nagrath. Continuous, high throughput microfluidic device to monitor circulating tumor cells in cancer patients [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 444.

Osteosarcoma is the second-highest cause of cancer-related death in children and adolescents. Despite advances in chemotherapy and surgery, the survival rate for metastatic osteosarcoma remains below 30% for the last three decades. Discovery of new chemotherapy agents would be crucial for improving outcomes of osteosarcoma patients. Here, using high-throughput screening, we have found a new compound, called 2-{2-[(3,5-dimethoxybenzyl)sulfanyl]-1,3-thiazol-4-yl}-N-(2-thienylmethyl)acetamide (referred to as KU0171032), as an inducer of apoptosis in various canine and human osteosarcoma cells. This compound shows minimal effects on non-transformed osteoblast and fibroblast cells. Intriguingly, KU0171032-induced apoptosis is more robust in osteosarcoma cells having mutant p53 or null for p53, as compared to cells with wild-type p53. Knockdown of wild-type p53 in U2OS and SJSA-1 cells significantly enhances sensitivity to KU0171032 with increase in DNA damage and caspase-3 cleavage. Moreover, KU0171032 significantly reduces in vivo tumor growth of osteosarcoma cells with p53 knockdown or carrying mutant p53. Our results strongly suggest that KU0171032 shows synthetic lethality with p53 mutations in osteosarcoma cells. Given that loss of p53 activity is frequent event in many cancer types including osteosarcoma while normal cells usually retain wild-type p53, this compound could be used to develop novel therapeutic strategies that capitalize on vulnerabilities in osteosarcoma and other types of cancer. Citation Format: Tomoo Iwakuma, Alejandro Parrales, Peter McDonald, Anuradha Roy, Mitchell W. Braun, Frank J. Schoenen, Jenna Wang, Steve Rogers, Melinda Broward, Tyce Bruns, Shrikant Anant, Dan A. Dixon, Fred Meyer, Katherine M. Chastain, Douglas H. Thamm, Scott J. Weir, Kathleen Neville, Joy M. Fulbright. A novel compound induces synthetic lethality for p53 mutations in osteosarcoma cells [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 2903.

Purpose: Tumor necrosis factor-α (TNF) is a cytokine with potent antitumor activity; however, toxicity and short half-life have limited its utility. Polyethylene glycol (PEG) conjugation of biotherapeutics can decrease immunogenicity while improving bioactivity and half-life. PEGylation of TNF (PEG-TNF) significantly improved half-life and toxicity in mice, resulting in enhanced antitumor activity. This study characterized toxicity, biological effect, and antitumor activity of PEG-TNF in pet dogs with spontaneous cancer. Experimental Design: A phase I clinical trial enrolled dogs with measurable tumors in which standard therapy had failed or been declined. Physiologic, hematologic, and biochemical parameters were evaluated and tumor biopsies obtained serially. A subset of patients underwent serial dynamic contrast-enhanced magnetic resonance imaging. Results: Fifteen dogs were enrolled at doses from 20.0 to 30.0 μg/kg. Dose-limiting toxicity at 30.0 μg/kg consisted of vascular leak in one and hypotension/coagulopathy in one, establishing 26.7 μg/kg as the maximum tolerated dose. Mean elimination half-life was 15.3 ± 4.9 hours. Biological activity (transient fever and leukopenia, increased tumor inflammation, and necrosis) was observed at all dosages. A significant increase in tumor blood flow was observed with dynamic contrast-enhanced magnetic resonance imaging. Minor/transient antitumor responses were observed in dogs with melanoma, squamous cell carcinoma, and mammary carcinoma, and a partial response was observed in a dog with angiosarcoma. Conclusions: Using a clinically relevant, spontaneous large animal model of neoplasia, we have shown that biologically effective doses of PEG-TNF can be administered safely, and that PEG-TNF administration is associated with encouraging biological activity. These results justify the clinical evaluation of PEG-TNF in human cancer. Clin Cancer Res; 16(5); 1498–508

Purpose: To assess, in dogs with naturally occurring non-Hodgkin's lymphoma, pharmacokinetics, safety, and activity of GS-9219, a prodrug of the nucleotide analogue 9-(2-phosphonylmethoxyethyl) guanine (PMEG), which delivers PMEG and its phosphorylated metabolites to lymphoid cells with preferential cytotoxicity in cells with a high proliferation index such as lymphoid malignancies. Experimental Design: To generate proof-of-concept, a phase I/II trial was conducted in pet dogs (n = 38) with naturally occurring non-Hodgkin's lymphoma using different dose schedules of GS-9219. A subset of dogs was further evaluated with 3′-deoxy-3′-18F-fluorothymidine positron emission tomography/computed tomography imaging before and after treatment. Results: The prodrug had a short plasma half-life but yielded high and prolonged intracellular levels of the cytotoxic metabolite PMEG diphosphate in peripheral blood mononuclear cells in the absence of detectable plasma PMEG. Dose-limiting toxicities were generally manageable and reversible and included dermatopathy, neutropenia, and gastrointestinal signs. Antitumor responses were observed in 79% of dogs and occurred in previously untreated dogs and dogs with chemotherapy-refractory non-Hodgkin's lymphoma. The median remission durations observed compare favorably with other monotherapies in dogs with non-Hodgkin's lymphoma. High 3′-deoxy-3′-18F-fluorothymidine uptake noted in lymphoid tissues before treatment decreased significantly after treatment (P = 0.016). Conclusions: GS-9219 was generally well tolerated and showed significant activity against spontaneous non-Hodgkin's lymphoma as modeled in pet dogs and, as such, supports clinical evaluation in humans.

Purpose: Genetically modified bacteria are a potentially powerful anticancer therapy due to their tumor targeting capacity, inherent antitumor activity, and ability to serve as efficient vectors for gene delivery. This study sought to characterize the acute and short-term toxicities and tumor colonization rates of a genetically modified Salmonella typhimurium (VNP20009) in dogs with spontaneous tumors, in the context of a phase I dose escalation trial. Experimental Design: Forty-one pet dogs with a variety of malignant tumors received weekly or biweekly i.v. infusions of VNP20009, at doses ranging from 1.5 × 105 to 1 × 108 cfu/kg. Vital signs and clinicopathologic variables were monitored regularly. Incisional biopsies were obtained before and 1 week following the first infusion for histopathology and bacterial culture. Results: The nominal maximum tolerated dose was 3 × 107 cfu/kg, with refractory fever and vomiting being the dose-limiting toxicities. One treatment-related acute death occurred. Bacteria were cultured from tumor tissue in 42% of cases. Thirty-five patients were evaluable for antitumor response. Major antitumor responses were seen in 15% (4 complete response and 2 partial response), and disease stabilization for at least 6 weeks in 10%. Conclusions: Administration of VNP20009 at doses with acceptable toxicity results in detectable bacterial colonization of tumor tissue and significant antitumor activity in tumor-bearing dogs.

While extremely rare in humans, hemangiosarcoma (HSA) accounts for nearly 2% of canine neoplasia, and is characterized by both aggressive local growth/invasion and a high rate of metastasis. Both canine and human HSA exhibit sustained aberrant PI3K/Akt/mTOR pathway signaling. The purpose of this study was to examine the in vitro effects of a novel dual PI3K/mTOR inhibitor, VDC-597, in canine HSA cells. Three canine HSA cell lines (DEN-HSA, CIN-HSA, and SB-HSA) were employed in multiple in vitro assays. Western analysis evaluated activation (phosphorylation) of key downstream pro-survival proteins in the PI3K/mTOR pathway. Changes in tumor cell growth/apoptosis were assessed using both bioreductive assays (Alamar Blue) and in vitro live-cell imaging (IncuCyte), in the presence and absence of doxorubicin, a standard-of-care cytotoxic drug for canine and human sarcomas, as well as in the presence and absence of U-0126, an inhibitor of the MEK pathway. Migration was assessed using both Boyden chamber and scratch assays, via live-cell imaging (IncuCyte). Matrigel invasion was assessed using traditional Boyden chambers. Finally, ELISA was utilized to quantify relative expression of vascular endothelial growth factor (VEGF). VDC-597 suppressed activation of both Akt and 4eBP1 in canine HSA cells in a dose- and time-dependent fashion, with an IC50 of approximately 0.3 uM, a concentration predicted to be clinically achievable based on preliminary early-phase canine and human studies. VDC-597 dose-dependently reduced proliferation, migration, invasion, and VEGF expression in HSA cells, while promoting tumor cell apoptosis. VDC-597 demonstrated additive antiproliferative effects when combined with doxorubicin and U-0126. Together, these results suggest that inhibitors of the PI3K/Akt/mTOR pathway may act against multiple components of the neoplastic process, including proliferation/apoptosis, chemosensitivity, invasion/migration and angiogenesis, and justify the evaluation of PI3K/mTOR inhibitors in canine, and eventually human, HSA. Experiments to examine the effect of VDC-597 in reducing tumor burden and metastasis in a rodent model are ongoing. Citation Format: Alex A. Pyuen, Douglas H. Thamm. In vitro effects of PI3K/mTOR inhibition in canine hemangiosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 383.

Purpose: Mammalian cell tissue culture has been a critical tool leading to our current understanding of cancer including many aspects of cellular transformation, growth, and response to therapies. The current use of large panels of human cancer cell lines (i.e. NCI60, GDSC) with associated phenotypic and genotypic information allows for informatics approaches and in silico screens to rapidly test hypotheses based on simple as well as complex relationships. We have assembled a panel of canine cancer cell lines to facilitate translational studies in canine cancer and report here the characteristics and some applications of this panel in comparative oncology. Methods: Canine cancer cell lines isolated from tumors have been accumulated from a number of sources over the years. Our current panel consists of 29 cell lines that have been validated as being of canine origin and “fingerprinted” by microsatellite analysis. The tumor types represented include 10 osteosarcomas, 5 melanomas, 2 mammary carcinomas, 1 hemangiosarcoma, 2 bladder carcinomas, 4 lymphoma/leukemias, 1 mast cell tumor, 2 histiocytic sarcomas, 1 thyroid carcinoma and 1 soft-tissue sarcoma. Drug sensitivity analysis was carried out using a resazurin-based bioreductive fluorometric assay. Gene expression microarray analysis was carried out using the Affymetrix GeneChip Canine 2.0 at the Genomics and Microarray Core (University of Colorado Cancer Center). Results: Drug sensitivity to the cytotoxic chemotherapeutic agents carboplatin, cisplatin, doxorubicin, lomustine, paclitaxel and vinblastine were assessed and compared to responses in the human NCI-60 panel. Results show that the human and canine cell lines show similar ranges of sensitivity to these agents. The mean GI50 values for the human and canine cell lines are significantly different for DOX, VBL, CARBO, CCNU as well as PTX. The variances in the data were also significantly different for CARBO and PTX. Overall, however, the drug sensitivity/resistance patterns in the canine and human tumor cell line panels shared general trends with regard to sensitivity and variance observed to the specific agents with the mean Log GI50 values for each agent showing a cross-species correlation (r = 0.88, p = 0.0194, Pearson). Cluster analysis of the gene expression data using the top 100 most variant genes as well as the 100 top most variant cancer genes largely separates the panel into groups with similar histiotypes. Cluster analysis of cancer genes also highlights alterations in gene expression that may contribute to pathogenesis in some cell lines such as loss of the PTEN tumor suppressor or elevated expression of receptor tyrosine kinase genes. Conclusions: The FACC canine tumor cell line panel represents a validated panel of representative cancers from dogs that can be utilized for comparative oncology studies as well as applications in drug development where use of a companion animal model is appropriate. Citation Format: Daniel L. Gustafson, Jared S. Fowles, Douglas H. Thamm, Rodney L. Page, Dawn L. Duval. The Flint Animal Cancer Center (FACC) canine tumor cell line panel: A resource for comparative and translational oncology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5136. doi:10.1158/1538-7445.AM2015-5136

Background: (Macro) Autophagy is the bulk, lysosomal degradation process of organelles and long-lived cytosolic proteins via sequestration into double membraned vesicles called autophagosomes. In recent years, autophagy inhibition has become an attractive, anti-cancer therapy as numerous studies have provided evidence for autophagy as a mechanism of survival and resistance. The most widely used autophagy inhibitors in clinical trials are chloroquine (CQ) or hydroxychloroquine (HCQ), due to their well-established safety profiles after decades long use as anti-malarial therapeutics. However, since measuring autophagy modulation is difficult in vivo, there is still uncertainty as to whether autophagy inhibition by CQ or HCQ is achievable in tumors. In order to determine the efficacy of HCQ in combination with doxorubicin (DOX) and to obtain measures of pharmacodynamic (PD) response, a clinical trial for canine patients with lymphoma was undertaken. Dogs present a unique model for studying human cancer as they inhabit the same environment and their cancers also arise spontaneously. In addition, canine clinical trials progress more rapidly than human trials, and samples can be collected more readily in dogs. Material and Methods: After an initial phase I dose escalation study, twelve canine patients presenting with lymphoma were enrolled into the study. Patients received 12.5 mg/kg daily HCQ, orally, and received their first round of 25 mg/m2 DOX treatment 3 days later. This dose is a 20% reduction from the standard 30 mg/m2, as dose-limiting neutropenia was observed at the full DOX dose in the dose escalation study. DOX was delivered IV every 21 days, for five treatments. Whole blood, serum, plasma, lymph node aspirates, and two punch biopsies, were collected prior to HCQ administration and 3 days after HCQ, but before DOX delivery. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and were immediately processed for flow cytometry, Western blot (WB), or electron microscopy (EM) analysis. Lymph node aspirates and PBMCs were stained with an LC3 antibody, a marker for autophagosomes, and analyzed by flow cytometry. Western analysis for LC3 and p62, a selective substrate for autophagy degradation, was performed on PBMCs and punch biopsies. Mass spectrometry analysis was used to determine HCQ levels in the plasma and remaining biopsy. Results: HCQ was detectable in both plasma and tumor after administration, with tumor levels about 300 times higher than that of plasma. However, HCQ levels varied widely and no correlation was observed between plasma and tumor levels. Patients with at least a 1.5 fold increase in LC3 I or II, as determined by WB, had significantly higher tumor HCQ levels (p=0.028). Increases in LC3 expression, indicative of inhibited autophagy, also correlated to increased p62 expression (p=0.034 Pearson r = 0.745). Yet, there was no correlation of LC3 expression in PBMCs compared with tumor, nor was there a correlation between plasma HCQ levels and LC3 expression. Additionally, there was no correlation between LC3 expression in PBMCs, as determined by flow cytometry, to expression determined by WB. Autophagosomes were visible by EM in the PBMCs after HCQ treatment. Conclusions: Autophagy inhibition by HCQ was only achieved in those patients with the highest tumor HCQ levels. Flow cytometry does not appear to accurately reflect PD response and it is recommended to use WB or EM analysis instead. Lastly, plasma does not appear to be a good surrogate for tumor PK/PD assessment, and thus, should not be used in lieu of tumor assessment. Citation Format: Rebecca A. Barnard, Luke A. Wittenburg, Daniel L. Gustafson, Andrew Thorburn, Douglas H. Thamm. Measuring autophagy inhibition as a pharmacodynamic response to hydroxychloroquine in a spontaneous canine lymphoma clinical trial. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A04.

Introduction: Treatment of cancer based on molecular characteristics of individual tumors is a promising area of research that can lead to improved outcomes. Validation of these new genomic methods is crucial for their success in future clinical trials. The co-expression extrapolation (COXEN) method has been shown to extrapolate data from a reference dataset to accurately predict drug sensitivity in vitro and in vivo in human cancer. Canine cancer models have great translational potential because of their biologic and genetic similarities with humans. Implementing genomic strategies across species allows dogs to take advantage of the wealth of available human data, and humans to take advantage of data from veterinary clinical trials. Our purpose is to explore the ability of COXEN for interspecies extrapolation of human cancer cell line data to generate gene expression models for the prediction of chemosensitivity in canine cancer cell lines and tumors. Methods: Publicly available gene expression and drug sensitivity data for the human NCI60 panel was obtained for doxorubicin (DOX), vinblastine (VBL), carboplatin (CAR), paclitaxel (PTX), lomustine (LOM), and cisplatin (CIS). Gene expression data was publicly obtained or generated via microarray gene expression analysis after RNA extraction of archived tumor samples for canine osteosarcoma (COS49) or from 16 canine cancer cell lines (ACC16). Alamar blue assays were performed to calculate drug sensitivity in the ACC16. Gene signatures predictive of drug sensitivity were identified by comparing the 12 least and most sensitive cell lines from the NCI60 via Significance Analysis of Microarrays (SAM). Human probesets were matched with canine orthologs according to highest sequence homology. A subset of genes from the signatures that shared strong co-expression with a second dataset (ACC16 or COS49) was identified through correlation matrices followed by percentile cutoffs from a permutation-based null distribution. The co-expressed genes were used in the Missclassification Penalized Posterior (MiPP) algorithm to generate models using the NCI60 panel with or without the second dataset for external cross-validation. Drug sensitivity in cell lines or disease free interval (DFI) in tumor patients was predicted from final models. Results: NCI60 models with co-expressed genes with the ACC16 were on average 66% accurate in predicting sensitivity to 6 drugs when built on the NCI60 panel alone (significant by binomial test for LOM (P = 0.0327)) and 82% accurate when using the ACC16 for external cross-validation (significant by binomial test for DOX, CAR, and LOM (P = 0.0461, 0.0327, 0.0005), and by spearman rank correlation for VBL, PTX, and LOM (P = 0.0202, 0.0105, 0.0306)). NCI60 models with co-expressed genes with the COS49 were 70% and 50% accurate for DOX and CAR when built on NCI60 panel and 75% and 66% accurate when using the COS49 for external cross-validation (significant by binomial test for DOX (P = 0.0414)). Conclusions: The results show the potential of the COXEN method in translating known human in vitro chemosensitivity data to both canine cancer cells and tumors. NCI60 models that used canine datasets for external cross-validation were more accurate in most cases, suggesting the need for a canine component during the model building process. Future expansion of the canine cancer panels may lead to improvement with accuracy and robustness in predicting chemosensitivity in dogs. Likewise, incorporating canine models with these genomic strategies in the veterinary clinical trial setting could provide needed validation for personalized medicine in human cancer. Citation Format: Jared S. Fowles, Ann M. Hess, Dawn L. Duval, Douglas H. Thamm, Daniel L. Gustafson. Interspecies applications of gene expression-based prediction of chemosensitivity. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A30.

Canine lymphoma (LSA) and osteosarcoma (OS) have high mortality rates and remain in need of more effective therapeutic approaches. Due to the strong similarities between canine and human LSA and OS, canine LSA and OS are excellent models for the human disease. Survivin, an IAP family member protein that inhibits apoptosis and drives cell proliferation, is commonly elevated in human and canine cancer. Survivin expression is a negative prognostic factor in dogs and humans with LSA and OS. The objective of our research was to determine the effects of survivin inhibition using a locked nucleic acid antisense oligonucleotide (EZN-3042) on canine LSA and canine OS cell lines, with respect to cell proliferation, apoptosis, and chemosensitivity in vitro. Furthermore, we sought to determine the efficacy of EZN-3042 on inhibition of survivin transcription, survivin protein production, and tumor growth in subcutaneous and orthotopic canine OS xenografts. We inhibited survivin using EZN-3042 in two canine LSA and two canine OS cell lines. Survivin inhibition was confirmed by qRT-PCR and western blot. Percent dead and total cell number was assessed by manual cell counting with trypan blue. Growth inhibition was confirmed with a bioreductive fluorometric assay. A caspase-3/7 assay was used to determine levels of apoptosis in EZN-3042 treated cells compared to controls. Cells were then treated with antineoplastic drug doxorubicin (DOX) +/- EZN-3042 and assays repeated. In vivo, nude mice with subcutaneous and orthotopic OS xenografts were given 100 mg/kg EZN3042 intraperitoneally. Survivin inhibition was confirmed with Immunohistochemistry and qRT-PCR analysis. Survivin inhibition in canine LSA and OS cells via EZN-3042 resulted in 34-72% decrease in survivin protein expression and a 1.3-3.4 fold decrease in endogenous survivin mRNA expression. When EZN-3042 treated cells were compared to controls, total and viable cell numbers were decreased, and apoptosis was increased. Survivin inhibition via EZN-3042 enhanced canine LSA and OS cell lines sensitivity to DOX. IHC and qRT-PCR analysis of subcutaneous and orthotopic canine OS xenografts confirmed decreased tumor survivin expression in EZN-3042 treated mice. Mice treated with EZN-3042 in combination with DOX had significantly decreased tumor growth when compared to single agent treatment and control groups. These results demonstrate that survivin inhibition via EZN-3042 decreased LSA and OS cell proliferation, and increased cellular apoptosis and chemosensitivity to DOX, and that EZN-3042 treatment inhibited tumor survivin expression in vivo and significantly decreased tumor growth when combined with DOX. Survivin-directed therapies may be highly effective in treatment of both canine and human LSA and OS, and spontaneous canine cancer may be a valuable model for the evaluation of survivin-targeted treatment. Citation Format: Jenette K. Shoeneman, Eugene J. Ehrhart, III, Joseph B. Charles, Douglas H. Thamm. Survivin inhibition via EZN-3042 in canine models of lymphoma and osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B35.

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 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.