Misty D. Bear

The novel HSP90 inhibitor STA-9090 exhibits activity against Kit-dependent and -independent malignant mast cell tumors

OBJECTIVE Mutations of the receptor tyrosine kinase Kit occur in several human and canine cancers. While Kit inhibitors have activity in the clinical setting, they possess variable efficacy against particular forms of mutant Kit and drug resistance often develops over time. Inhibitors of heat shock protein 90 (HSP90), a chaperone for which Kit is a client protein, have demonstrated activity against human cancers and evidence suggests they downregulate several mutated and imatinib-resistant forms of Kit. The purpose of this study was to evaluate a novel HSP90 inhibitor, STA-9090, against wild-type (WT) and mutant Kit in canine bone marrow-derived cultured mast cells (BMCMCs), malignant mast cell lines, and fresh malignant mast cells. MATERIALS AND METHODS BMCMCs, cell lines, and fresh malignant mast cells were treated with STA-9090, 17-AAG, and SU11654 and evaluated for loss in cell viability, cell death, alterations in HSP90 and Kit expression/signaling, and Kit mutation. STA-9090 activity was tested in a canine mastocytoma xenograft model. RESULTS Treatment of BMCMCs, cell lines, and fresh malignant cells with STA-9090 induced growth inhibition, apoptosis that was caspase-3/7-dependent, and downregulation of phospho/total Kit and Akt, but not extracellular signal-regulated kinase (ERK) or phosphoinositide-3 kinase (PI-3K). Loss of Kit cell-surface expression was also observed. Furthermore, STA-9090 exhibited superior activity to 17-AAG and SU11654, and was effective against malignant mast cells expressing either WT or mutant Kit. Lastly, STA-9090 inhibited tumor growth in a canine mastocytoma mouse xenograft model. CONCLUSIONS STA-9090 exhibits broad activity against mast cells expressing WT or mutant Kit, suggesting it may be an effective agent in the clinical setting against mast cell malignancies.

Characterization of STAT3 activation and expression in canine and human osteosarcoma

BackgroundDysregulation of signal transducer and activator of transcription 3 (STAT3) has been implicated as a key participant in tumor cell survival, proliferation, and metastasis and is often correlated with a more malignant tumor phenotype. STAT3 phosphorylation has been demonstrated in a subset of human osteosarcoma (OSA) tissues and cell lines. OSA in the canine population is known to exhibit a similar clinical behavior and molecular biology when compared to its human counterpart, and is often used as a model for preclinical testing of novel therapeutics. The purpose of this study was to investigate the potential role of STAT3 in canine and human OSA, and to evaluate the biologic activity of a novel small molecule STAT3 inhibitor.MethodsTo examine STAT3 and Src expression in OSA, we performed Western blotting and RT-PCR. OSA cells were treated with either STAT3 siRNA or small molecule Src (SU6656) or STAT3 (LLL3) inhibitors and cell proliferation (CyQUANT), caspase 3/7 activity (ELISA), apoptosis (Western blotting for PARP cleavage) and/or viability (Wst-1) were determined. Additionally, STAT3 DNA binding after treatment was determined using EMSA. Expression of STAT3 targets after treatment was demonstrated with Western blotting, RT-PCR, or gel zymography.ResultsOur data demonstrate that constitutive activation of STAT3 is present in a subset of canine OSA tumors and human and canine cell lines, but not normal canine osteoblasts. In both canine and human OSA cell lines, downregulation of STAT3 activity through inhibition of upstream Src family kinases using SU6656, inhibition of STAT3 DNA binding and transcriptional activities using LLL3, or modulation of STAT3 expression using siRNA, all resulted in decreased cell proliferation and viability, ultimately inducing caspase-3/7 mediated apoptosis in treated cells. Furthermore, inhibition of either Src or STAT3 activity downregulated the expression of survivin, VEGF, and MMP2, all known transcriptional targets of STAT3.ConclusionThese data suggest that STAT3 activation contributes to the survival and proliferation of human and canine OSA cells, thereby providing a potentially promising target for therapeutic intervention. Future investigational trials of LLL3 in dogs with spontaneous OSA will help to more accurately define the role of STAT3 in the clinical setting.

Oncostatin M promotes STAT3 activation, VEGF production, and invasion in osteosarcoma cell lines

BackgroundWe have previously demonstrated that both canine and human OSA cell lines, as well as 8 fresh canine OSA tumor samples, exhibit constitutive phosphorylation of STAT3, and that this correlates with enhanced expression of matrix metalloproteinase-2 (MMP2). While multiple signal transduction pathways can result in phosphorylation of STAT3, stimulation of the cytokine receptor gp130 through either IL-6 or Oncostatin M (OSM) is the most common mechanism through which STAT3 is activated. The purpose of this study was to evaluate the role of IL-6 and OSM stimulation on both canine and human OSA cell lines to begin to determine the role of these cytokines in the biology of OSA.MethodsRT-PCR and Western blotting were used to interrogate the consequences of OSM and IL-6 stimulation of OSA cell lines. OSA cells were stimulated with OSM and/or hepatocyte growth factor (HGF) and the effects on MMP2 activity (gel zymography), proliferation (CyQUANT), invasion (Matrigel transwell assay), and VEGF production (Western blotting, ELISA) were assessed. The small molecule STAT3 inhibitor LLL3 was used to investigate the impact of STAT3 inhibition following OSM stimulation of OSA cells.ResultsOur data demonstrate that the OSM receptor (OSMR), but not IL-6 or its receptor, is expressed by all human and canine OSA cell lines and canine OSA tumor samples; additionally, OSM expression was noted in all tumor samples. Treatment of OSA cell lines with OSM induced phosphorylation of STAT3, Src, and JAK2. OSM stimulation also resulted in a dose dependent increase in MMP2 activity and VEGF expression that was markedly reduced following treatment with the small molecule STAT3 inhibitor LLL3. Lastly, OSM stimulation of OSA cell lines enhanced invasion through Matrigel, particularly in the presence of rhHGF. In contrast, both OSM and HGF stimulation of OSA cell lines did not alter their proliferative capacity.ConclusionsThese data indicate OSM stimulation of human and canine OSA cells induces STAT3 activation, thereby enhancing the expression/activation of MMP2 and VEGF, ultimately promoting invasive behavior and tumor angiogenesis. As such, OSM and its receptor may represent a novel target for therapeutic intervention in OSA.

AR-42, a novel HDAC inhibitor, exhibits biologic activity against malignant mast cell lines via down-regulation of constitutively activated Kit

Histone hypoacetylation occurs in many cancers and inhibition of histone deacetylation is a promising approach to modulate these epigenetic changes. Our laboratory previously demonstrated that the histone deacetylase inhibitors (HDACis) vorinostat and AR-42 reduced the viability of a canine malignant mast cell line. The purpose of this study was to further investigate the mechanisms of pan-HDAC inhibition in normal and malignant mast cells. Mouse and canine malignant mast cell lines expressing various Kit mutations, normal canine mast cells, and primary canine malignant mast cells were treated with AR-42 (a novel HDACi) and effects on cell viability, cycling, and signaling were evaluated. Treatment with AR-42 induced growth inhibition, cell- cycle arrest, apoptosis, and activation of caspases-3/7. AR-42 promoted hyperacetylation of H3, H4, and alpha-tubulin, and up-regulation of p21. Down-regulation of Kit occurred after AR-42 treatment via inhibition of Kit transcription. Disassociation between Kit and heat shock protein 90 (HSP90) and up-regulation of HSP70 were observed after AR-42 treatment, suggesting potential loss of HSP90 chaperone function. Lastly, AR-42 down-regulated the expression of p-Akt, total Akt, phosphorylated STAT3/5 (pSTAT3/5), and total STAT3/5. In summary, AR-42 exhibits in vitro and ex vivo biologic activity against malignant mast cells, representing a promising therapeutic approach for malignant mast cell disease.

The novel HSP90 inhibitor STA‐1474 exhibits biologic activity against osteosarcoma cell lines

Osteosarcoma (OSA), the most common malignant bone tumor in dogs and children, exhibits a similar clinical presentation and molecular biology in both species. Unfortunately, 30–40% of children and 90% of dogs still die of disease despite aggressive therapy. The purpose of this study was to test the biologic activity of a novel heat shock protein 90 (HSP90) inhibitor, STA‐1474, against OSA. Canine and human OSA cell lines and normal canine osteoblasts were treated with STA‐1474 and evaluated for effects on proliferation (CyQuant), apoptosis (Annexin V, PARP cleavage, caspase 3/7 activation) and known HSP90 client proteins. HSP90 was immunoprecipitated from normal and malignant osteoblasts and Western blotting for co‐chaperones was performed. Mice bearing canine OSA xenografts were treated with STA‐1474, and tumors samples were evaluated for caspase‐3 activation and loss of p‐Akt/Akt. Treatment with STA‐1474 promoted loss of cell viability, inhibition of cell proliferation and induction of apoptosis in OSA cell lines. STA‐1474 and its active metabolite STA‐9090 also demonstrated increased potency compared to 17‐AAG. STA‐1474 exhibited selectivity for OSA cells versus normal canine osteoblasts, and HSP90 co‐precipitated with co‐chaperones p23 and Hop in canine OSA cells but not in normal canine osteoblasts. Furthermore, STA‐1474 downregulated the expression of p‐Met/Met, p‐Akt/Akt and p‐STAT3. Finally, STA‐1474 induced tumor regression, caspase‐3 activation and downregulation of p‐Met/Met and p‐Akt/Akt in OSA xenografts. Together, these data suggest that HSP90 represents a relevant target for therapeutic intervention in OSA.

The novel curcumin analog FLLL32 decreases STAT3 DNA binding activity and expression, and induces apoptosis in osteosarcoma cell lines

BackgroundCurcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities; however, it does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin. FLLL32 possesses superior biochemical properties and more specifically targets STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. In our previous work, we found that several canine and human osteosarcoma (OSA) cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3. Compared to curcumin, we hypothesized that FLLL32 would be more efficient at inhibiting STAT3 function in OSA cells and that this would result in enhanced downregulation of STAT3 transcriptional targets and subsequent death of OSA cells.MethodsHuman and canine OSA cells were treated with vehicle, curcumin, or FLLL32 and the effects on proliferation (CyQUANT®), apoptosis (SensoLyte® Homogeneous AMC Caspase- 3/7 Assay kit, western blotting), STAT3 DNA binding (EMSA), and vascular endothelial growth factor (VEGF), survivin, and matrix metalloproteinase-2 (MMP2) expression (RT-PCR, western blotting) were measured. STAT3 expression was measured by RT-PCR, qRT- PCR, and western blotting.ResultsOur data showed that FLLL32 decreased STAT3 DNA binding by EMSA. FLLL32 promoted loss of cell proliferation at lower concentrations than curcumin leading to caspase-3- dependent apoptosis, as evidenced by PARP cleavage and increased caspase 3/7 activity; this could be inhibited by treatment with the pan-caspase inhibitor Z-VAD-FMK. Treatment of OSA cells with FLLL32 decreased expression of survivin, VEGF, and MMP2 at both mRNA and protein levels with concurrent decreases in phosphorylated and total STAT3; this loss of total STAT3 occurred, in part, via the ubiquitin-proteasome pathway.ConclusionsThese data demonstrate that the novel curcumin analog FLLL32 has biologic activity against OSA cell lines through inhibition of STAT3 function and expression. Future work with FLLL32 will define the therapeutic potential of this compound in vivo.

Preclinical evaluation of the novel, orally bioavailable Selective Inhibitor of Nuclear Export (SINE) KPT-335 in spontaneous canine cancer: Results of a phase i study

Background The purpose of this study was to evaluate the activity of Selective Inhibitors of Nuclear Export (SINE) compounds that inhibit the function of the nuclear export protein Exportin 1 (XPO1/CRM1) against canine tumor cell lines and perform a Phase I clinical trial of KPT-335 in dogs with spontaneous cancer to provide a preliminary assessment of biologic activity and tolerability. Methods and Findings Canine tumor cell lines derived from non-Hodgkin lymphoma (NHL), mast cell tumor, melanoma and osteosarcoma exhibited growth inhibition and apoptosis in response to nanomolar concentrations of SINE compounds; NHL cells were particularly sensitive with IC50 concentrations ranging from 2–42 nM. A Phase I clinical trial of KPT-335 was performed in 17 dogs with NHL (naive or relapsed), mast cell tumor or osteosarcoma. The maximum tolerated dose was 1.75 mg/kg given orally twice/week (Monday/Thursday) although biologic activity was observed at 1 mg/kg. Clinical benefit (CB) including partial response to therapy (PR, n = 2) and stable disease (SD, n = 7) was observed in 9/14 dogs with NHL with a median time to progression (TTP) for responders of 66 days (range 35–256 days). A dose expansion study was performed in 6 dogs with NHL given 1.5 mg/kg KPT-335 Monday/Wednesday/Friday; CB was observed in 4/6 dogs with a median TTP for responders of 83 days (range 35–354 days). Toxicities were primarily gastrointestinal consisting of anorexia, weight loss, vomiting and diarrhea and were manageable with supportive care, dose modulation and administration of low dose prednisone; hepatotoxicity, anorexia and weight loss were the dose limiting toxicities. Conclusions This study provides evidence that the novel orally bioavailable XPO1 inhibitor KPT-335 is safe and exhibits activity in a relevant, spontaneous large animal model of cancer. Data from this study provides critical new information that lays the groundwork for evaluation of SINE compounds in human cancer.

Phase I Evaluation of STA-1474, a Prodrug of the Novel HSP90 Inhibitor Ganetespib, in Dogs with Spontaneous Cancer

Background The novel water soluble compound STA-1474 is metabolized to ganetespib (formerly STA-9090), a potent HSP90 inhibitor previously shown to kill canine tumor cell lines in vitro and inhibit tumor growth in the setting of murine xenografts. The purpose of the following study was to extend these observations and investigate the safety and efficacy of STA-1474 in dogs with spontaneous tumors. Methods and Findings This was a Phase 1 trial in which dogs with spontaneous tumors received STA-1474 under one of three different dosing schemes. Pharmacokinetics, toxicities, biomarker changes, and tumor responses were assessed. Twenty-five dogs with a variety of cancers were enrolled. Toxicities were primarily gastrointestinal in nature consisting of diarrhea, vomiting, inappetence and lethargy. Upregulation of HSP70 protein expression was noted in both tumor specimens and PBMCs within 7 hours following drug administration. Measurable objective responses were observed in dogs with malignant mast cell disease (n = 3), osteosarcoma (n = 1), melanoma (n = 1) and thyroid carcinoma (n = 1), for a response rate of 24% (6/25). Stable disease (>10 weeks) was seen in 3 dogs, for a resultant overall biological activity of 36% (9/25). Conclusions This study provides evidence that STA-1474 exhibits biologic activity in a relevant large animal model of cancer. Given the similarities of canine and human cancers with respect to tumor biology and HSP90 activation, it is likely that STA-1474 and ganetespib will demonstrate comparable anti-cancer activity in human patients.

Met interacts with EGFR and Ron in canine osteosarcoma

The receptor tyrosine kinase (RTK) Met is known to be over-expressed in canine osteosarcoma (OSA). In human cancers, the RTKs Met, epidermal growth factor receptor (EGFR) and Ron are frequently co-expressed and engage in heterodimerization, altering signal transduction and promoting resistance to targeted therapeutics. We found that EGFR and Ron are expressed in canine OSA cell lines and primary tissues, EGFR and Ron are frequently phosphorylated in OSA tumour samples, and Met is co-associated with EGFR and Ron in canine OSA cell lines. Transforming growth factor alpha (TGFα) and hepatocyte growth factor (HGF) stimulation induced amplification of ERK1/2 and STAT3 phosphorylation in OSA cells and Met was phosphorylated following TGFα stimulation providing evidence for receptor cross-talk. Lastly, treatment of OSA cells with combined gefitinib and crizotinib inhibited cell proliferation in an additive manner. Together, these data support the notion that Met, EGFR and Ron interact in OSA cells and as such, may represent viable targets for therapeutic intervention.

Biologic activity of the novel small molecule STAT3 inhibitor LLL12 against canine osteosarcoma cell lines.

BackgroundSTAT3 [1] has been shown to be dysregulated in nearly every major cancer, including osteosarcoma (OS). Constitutive activation of STAT3, via aberrant phosphorylation, leads to proliferation, cell survival and resistance to apoptosis. The present study sought to characterize the biologic activity of a novel allosteric STAT3 inhibitor, LLL12, in canine OS cell lines.ResultsWe evaluated the effects of LLL12 treatment on 4 canine OS cell lines and found that LLL12 inhibited proliferation, induced apoptosis, reduced STAT3 phosphorylation, and decreased the expression of several transcriptional targets of STAT3 in these cells. Lastly, LLL12 exhibited synergistic anti-proliferative activity with the chemotherapeutic doxorubicin in the OS lines.ConclusionLLL12 exhibits biologic activity against canine OS cell lines through inhibition of STAT3 related cellular functions supporting its potential use as a novel therapy for OS.