Heather L. Gardner

Preclinical Evaluation of the Novel BTK Inhibitor Acalabrutinib in Canine Models of B-Cell Non-Hodgkin Lymphoma.

Acalabrutinib (ACP-196) is a second-generation inhibitor of Bruton agammaglobulinemia tyrosine kinase (BTK) with increased target selectivity and potency compared to ibrutinib. In this study, we evaluated acalabrutinib in spontaneously occurring canine lymphoma, a model of B-cell malignancy similar to human diffuse large B-cell lymphoma (DLBCL). First, we demonstrated that acalabrutinib potently inhibited BTK activity and downstream effectors in CLBL1, a canine B-cell lymphoma cell line, and primary canine lymphoma cells. Acalabrutinib also inhibited proliferation in CLBL1 cells. Twenty dogs were enrolled in the clinical trial and treated with acalabrutinib at dosages of 2.5 to 20mg/kg every 12 or 24 hours. Acalabrutinib was generally well tolerated, with adverse events consisting primarily of grade 1 or 2 anorexia, weight loss, vomiting, diarrhea and lethargy. Overall response rate (ORR) was 25% (5/20) with a median progression free survival (PFS) of 22.5 days. Clinical benefit was observed in 30% (6/20) of dogs. These findings suggest that acalabrutinib is safe and exhibits activity in canine B-cell lymphoma patients and support the use of canine lymphoma as a relevant model for human non-Hodgkin lymphoma (NHL).

Impact of Toceranib/Piroxicam/ Cyclophosphamide Maintenance Therapy on Outcome of Dogs with Appendicular Osteosarcoma following Amputation and Carboplatin Chemotherapy: A Multi- Institutional Study

Background We hypothesized that the addition of toceranib to metronomic cyclophosphamide/piroxicam therapy would significantly improve disease-free interval (DFI) and overall survival (OS) in dogs with appendicular osteosarcoma (OSA) following amputation and carboplatin chemotherapy. Methods and Findings This was a randomized, prospective clinical trial in which dogs with OSA free of gross metastatic disease (n = 126) received carboplatin chemotherapy (4 doses) following amputation. On study entry, dogs were randomized to receive piroxicam/cyclophosphamide with or without toceranib (n = 63 each) after completing chemotherapy. Patient demographics were not significantly different between both groups. During or immediately following carboplatin chemotherapy, 32 dogs (n = 13 toceranib; n = 19 control) developed metastatic disease, and 13 dogs left the study due to other medical conditions or owner preference. Following carboplatin chemotherapy, 81 dogs (n = 46 toceranib; n = 35 control) received the metronomic treatment; 35 dogs (n = 20 toceranib; n = 15 control) developed metastatic disease during the maintenance therapy, and 26 dogs left the study due to other medical conditions or owner preference. Nine toceranib-treated and 11 control dogs completed the study without evidence of metastatic disease 1-year following amputation. Toceranib-treated dogs experienced more episodes of diarrhea, neutropenia and weight loss than control dogs, although these toxicities were low-grade and typically resolved with supportive care. More toceranib-treated dogs (n = 8) were removed from the study for therapy-associated adverse events compared to control dogs (n = 1). The median DFI for control and toceranib treated dogs was 215 and 233 days, respectively (p = 0.274); the median OS for control and toceranib treated dogs was 242 and 318 days, respectively (p = 0.08). The one year survival rate for control dogs was 35% compared to 38% for dogs receiving toceranib. Conclusions The addition of toceranib to metronomic piroxicam/cyclophosphamide therapy following amputation and carboplatin chemotherapy did not improve median DFI, OS or the 1-year survival rate in dogs with OSA.

Dogs as a Model for Cancer

Spontaneous cancers in client-owned dogs closely recapitulate their human counterparts with respect to clinical presentation, histological features, molecular profiles, and response and resistance to therapy, as well as the evolution of drug-resistant metastases. In several instances the incorporation of dogs with cancer into the preclinical development path of cancer therapeutics has influenced outcome by helping to establish pharmacokinetic/pharmacodynamics relationships, dose/regimen, expected clinical toxicities, and ultimately the potential for biologic activity. As our understanding regarding the molecular drivers of canine cancers has improved, unique opportunities have emerged to leverage this spontaneous model to better guide cancer drug development so that therapies likely to fail are eliminated earlier and therapies with true potential are optimized prior to human studies. Both pets and people benefit from this approach, as it provides dogs with access to cutting-edge cancer treatments and helps to insure that people are given treatments more likely to succeed.

Abstract 1744: ACP-196: A second generation Btk inhibitor demonstrates biologic activity in a canine model of B-cell non-Hodgkin lymphoma

Recent recognition of B-cell receptor (BCR) signaling as a critical factor in the progression of B-cell malignancies, including non-Hodgkin lymphoma (NHL), has resulted in the development of numerous targeted therapeutics that inhibit this signaling pathway. Ibrutinib, a small molecule inhibitor of Bruton tyrosine kinase (Btk) a key signaling molecule in the BCR pathway, has demonstrated significant clinical activity in a broad range of B-cell cancers. ACP-196 is a second generation Btk inhibitor with increased target selectivity and enhanced in vivo potency compared with ibrutinib and, thus, may represent an improvement over its predecessor. With the following studies, we sought to evaluate ACP-196 in canine models of B-cell NHL with the ultimate goal of providing the preclinical data necessary to move ACP-196 into human clinical trials. Using two immunophenotypically confirmed canine B-cell lymphoma cell lines, CLBL-1 and 17-71, we demonstrate potent in vitro inhibition of activation of Btk and the downstream effectors ERK 1/2 and PLCγ2 following 1 hour of treatment with ACP-196 at concentrations as low as 10nM. In vivo studies were performed in companion dogs as part of an ongoing clinical trial. Twelve dogs with immunophenotypically confirmed, spontaneously occurring B-cell NHL were orally administered ACP-196 at dosages of 2.5mg/kg every 24 hours (6 dogs), 5mg/kg every 24 hours (5 dogs), or 10mg/kg every12 hours (1 dog). Btk occupancy in peripheral blood and lymphoma cells was assessed using a biotin-tagged probe derived from ACP-196. Using this assay we found that at 2.5mg/kg full Btk occupancy was achieved in peripheral B cells 3h after dosing for all dogs, except for a single dog with high peripheral B-cell count. At 24 hours after dosing, 83-99% Btk target occupancy was observed for all dogs. Partial response, as assessed by a modified RECIST scheme, was achieved in 2 dogs in the 2.5mg/kg group and the dog in the 10mg/kg group. Upon relapse, one of the responders in the 2.5mg/kg group was dose escalated to 10mg/kg q12 on day 42 and partial response from relapse was reestablished. Of the remaining 9 dogs, 3 achieved stable disease for > 28 days and 6 discontinued the study after developing progressive disease within 28 days of starting treatment. In total, to date, 3 dogs achieved a partial response, 3 dogs stable disease, and 6 dogs progressive disease. ACP-196 was well tolerated with only mild anorexia noted in some dogs. These data demonstrate that ACP-196 has single agent biologic activity in a spontaneous large animal model of human NHL. Studies in dogs with NHL are ongoing to define regimens prior to initiation of human phase I clinical trials. Additional cohorts are planned combining ACP-196 with a phosphatidylinositide 3-kinase (PI3K) delta-specific inhibitor. Citation Format: Heather L. Gardner*, Bonnie K. Harrington*, Raquel Izumi, Ahmed Hamdy, Allard Kaptein, Bart Van Lith, Cheryl A. London, John C. Byrd, Amy J. Johnson, William C. Kisseberth. ACP-196: A second generation Btk inhibitor demonstrates biologic activity in a canine model of B-cell non-Hodgkin lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1744. doi:10.1158/1538-7445.AM2014-1744

ΔNp63 mediates cellular survival and metastasis in canine osteosarcoma

p63 is a structural homolog within the 53 family encoding two isoforms, ΔNp63 and TAp63. The oncogenic activity of ΔNp63 has been demonstrated in multiple cancers, however the underlying mechanisms that contribute to tumorigenesis are poorly characterized. Osteosarcoma (OSA) is the most common primary bone tumor in dogs, exhibiting clinical behavior and molecular biology essentially identical to its human counterpart. The purpose of this study was to evaluate the potential contribution of ΔNp63 to the biology of canine OSA. As demonstrated by qRT-PCR, nearly all canine OSA cell lines and tissues overexpressed ΔNp63 relative to normal control osteoblasts. Inhibition of ΔNp63 by RNAi selectively induced apoptosis in the OSA cell lines overexpressing ΔNp63. Knockdown of ΔNp63 upregulated expression of the proapoptotic Bcl-2 family members Puma and Noxa independent of p53. However the effects of ΔNp63 required transactivating isoforms of p73, suggesting that ΔNp63 promotes survival in OSA by repressing p73-dependent apoptosis. In addition, ΔNp63 modulated angiogenesis and invasion through its effects on VEGF-A and IL-8 expression, and STAT3 phosphorylation. Lastly, the capacity of canine OSA cell lines to form pulmonary metastasis was directly related to expression levels of ΔNp63 in a murine model of metastatic OSA. Together, these data demonstrate that ΔNp63 inhibits apoptosis and promotes metastasis, supporting continued evaluation of this oncogene as a therapeutic target in both human and canine OSA.

Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma

Background STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5. Methods and findings An in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing’s sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice. Conclusions LY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified in vitro were not mediated through STAT3 inhibition.

KTN0158, a Humanized Anti-KIT Monoclonal Antibody, Demonstrates Biologic Activity against both Normal and Malignant Canine Mast Cells

Purpose: KTN0158 is a novel anti-KIT antibody that potently inhibits wild-type and mutant KIT. This study evaluated the safety, biologic activity, and pharmacokinetic/pharmacodynamics profile of KTN0158 in dogs with spontaneous mast cell tumors (MCT) as a prelude to human clinical applications. Experimental Design: Cell proliferation, KIT phosphorylation, and mast cell degranulation were evaluated in vitro. KTN0158 was administered to 4 research dogs to assess clinical effects and cutaneous mast cell numbers. Thirteen dogs with spontaneous MCT were enrolled into a prospective phase I dose-escalating open-label clinical study of KTN0158 evaluating 3 dose levels and 2 schedules and with weekly assessments for response and clinical toxicities. Results: KTN0158 was a potent inhibitor of human and dog KIT activation and blocked mast cell degranulation in vitro. In dogs, KTN0158 was well tolerated and reduced cutaneous mast cell numbers in a dose-dependent manner. Clinical benefit of KTN0158 administration in dogs with MCT (n = 5 partial response; n = 7 stable disease) was observed regardless of KIT mutation status, and decreased KIT phosphorylation was demonstrated in tumor samples. Histopathology after study completion demonstrated an absence of neoplastic cells in the primary tumors and/or metastatic lymph nodes from 4 dogs. Reversible hematologic and biochemical adverse events were observed at doses of 10 and 30 mg/kg. The MTD was established as 10 mg/kg. Conclusions: KTN0158 inhibits KIT phosphorylation, demonstrates an acceptable safety profile in dogs, and provides objective responses in canine MCT patients with and without activating KIT mutations, supporting future clinical evaluation of KTN0158 in people. Clin Cancer Res; 23(10); 2565–74. ©2016 AACR.

Phase I/II evaluation of RV1001, a novel PI3Kδ inhibitor, in spontaneous canine lymphoma

Background RV1001 is a novel, potent, and selective PI3Kδ inhibitor. The purpose of this study was to evaluate the safety and efficacy of RV1001 in canine Non-Hodgkin lymphoma (NHL). Methods and results Inhibition of endogenous pAKT by RV1001 in primary canine NHL cells was determined by Western blotting. A phase I study of RV1001 was performed in 21 dogs with naïve and drug resistant T and B-cell NHL to assess safety, pharmacokinetic profile, and response to therapy. The objective response rate was 62% (complete response (CR) n = 3; partial response (PR) n = 10), and responses were observed in both naïve and chemotherapy-resistant B and T cell NHL. This study provided the recommended starting dose for a phase II, non-pivotal, exploratory, open label multi-centered clinical trial in 35 dogs with naïve and drug resistant T and B-cell NHL, to further define the efficacy and safety profile of RV1001. The objective response rate in the phase II study was 77% (CR n = 1; PR n = 26). Clinical toxicities were primarily hepatobiliary and gastrointestinal, and were responsive to dose modifications and/or temporary drug discontinuation. Hepatotoxicity was the primary dose limiting toxicity. Conclusions RV1001 exhibits good oral bioavailability, an acceptable safety profile, and biologic activity with associated inhibition of pAKT in dogs with B and T cell NHL. Data from these studies can be leveraged to help inform the design of future studies involving isoform-selective PI3K inhibitors in humans.

The addition of metronomic chemotherapy does not improve outcome for canine splenic haemangiosarcoma: Adjuvant metronomic chemotherapy for dogs with splenic haemangiosarcoma

OBJECTIVES To determine whether the addition of metronomic chemotherapy improved outcome for dogs with splenic haemangiosarcoma treated with splenectomy and adjuvant maximum tolerated dose chemotherapy. MATERIALS AND METHODS Medical records were examined retrospectively for dogs with splenic haemangiosarcoma that had undergone splenectomy followed by anthracycline-based chemotherapy. Thirty-nine dogs underwent splenectomy followed by maximum tolerated dose chemotherapy with an anthracycline, cyclophosphamide, or both (Group 1). Twenty-two dogs underwent splenectomy followed by adjuvant maximum tolerated dose chemotherapy with an anthracycline, cyclophosphamide, or both, plus metronomic chemotherapy (Group 2). Dogs in both groups were further separated into those treated with either maximum tolerated dose anthracycline or maximum tolerated dose anthracycline and cyclophosphamide. RESULTS Median progression-free survival was 165 days and median overall survival time was 180 days in Group 1. Median progression-free survival was 185 days and median overall survival time was 212 days in Group 2. In both groups, the overall survival was shorter in dogs that had received maximum tolerated dose cyclophosphamide. CLINICAL SIGNIFICANCE The addition of metronomic to maximum tolerated dose chemotherapy protocols does not appear to improve outcome in dogs with splenic haemangiosarcoma treated with splenectomy and maximum tolerated dose chemotherapy.

Consecutive day HSP90 inhibitor administration improves efficacy in murine models of KIT-driven malignancies and canine mast cell tumors

Purpose: STA-1474, prodrug of the heat shock protein 90 inhibitor (HSP90i) ganetespib, previously demonstrated activity in canine preclinical models of cancer; interestingly, prolonged infusions were associated with improved biologic activity. The purpose of this study was to identify the ideal treatment schedule for HSP90i in preclinical models of KIT-driven malignancies and in dogs with spontaneous mast cell tumors (MCT), where KIT is a known driver. Experimental Design: In vitro and murine xenograft experiments and clinical studies in dogs with MCTs were used to define the effects of HSP90i-dosing regimen on client protein downregulation and antitumor activity. Results: Continuous HSP90 inhibition led to durable destabilization of client proteins in vitro; however, transient exposure required >10× drug for comparable effects. In vivo, KIT was rapidly degraded following a single dose of HSP90i but returned to baseline levels within a day. HSP90 levels increased and stabilized 16 hours after HSP90i and were not elevated following a subsequent near-term exposure, providing a functional pool of chaperone to stabilize proteins and a means for greater therapeutic activity upon HSP90i reexposure. HSP90i administered on days 1 and 2 (D1/D2) demonstrated increased biologic activity compared with D1 treatment in KIT or EGFR-driven murine tumor models. In a trial of dogs with MCT, D1/D2 dosing of HSP90i was associated with sustained KIT downregulation, 50% objective response rate and 100% clinical benefit rate compared with D1 and D1/D4 schedules. Conclusions: These data provide further evidence that prolonged HSP90i exposure improves biologic activity through sustained downregulation of client proteins.