Cancer Therapy: Preclinical CUDC-305, a Novel Synthetic HSP90 Inhibitor with Unique Pharmacologic Properties for Cancer Therapy

Purpose: We designed and synthesized CUDC-305, an HSP90 inhibitor of the novel imidazopyridine class. Here, we report its unique pharmacologic properties and antitumor activities in a variety of tumor types. Experimental Design: The potency of the compound was analyzed by fluorescence polarization competition binding assay. Its antiproliferative activities were assessed in 40 human cancer cell lines. Its pharmacologic properties and antitumor activities were evaluated in a variety of tumor xenograft models. Results: CUDC-305 shows high affinity for HSP90α/β (IC50, ∼100 nmol/L) and HSP90 complex derived from cancer cells (IC50, 48.8 nmol/L). It displays potent antiproliferative activity against a broad range of cancer cell lines (mean IC50, 220 nmol/L). CUDC305 exhibits high oral bioavailability (96.0%) and selective retention in tumor (half-life, 20.4 hours) compared with normal tissues. Furthermore, CUDC-305 can cross bloodbrain barrier and reach therapeutic levels in brain tissue. CUDC-305 exhibits dosedependent antitumor activity in an s.c. xenograft model of U87MG glioblastoma and significantly prolongs animal survival in U87MG orthotopic model. CUDC-305 also displays potent antitumor activity in animal models of erlotinib-resistant non–small cell lung cancer and induces tumor regression in animal models of MDA-MB-468 breast cancer and MV4-11 acute myelogenous leukemia. Correlating with its efficacy in these various tumor models, CUDC-305 robustly inhibits multiple signaling pathways, including PI3K/AKT and RAF/MEK/ERK, and induces apoptosis. In combination studies, CUDC-305 enhances the antitumor activity of standard-of-care agents in breast and colorectal tumor models. Conclusion: CUDC-305 is a promising drug candidate for the treatment of a variety of cancers, including brain malignancies. Recent advances in understanding the molecular biology of cancer have resulted in the development of drugs that target known molecular pathways (1). For a limited number of cancer subtypes, these drugs exploit the dependence of the tumor on dysregulated signaling pathways to achieve therapeutic selectivity for cancer over normal cells. Molecularly targeted agents that have been approved for clinical use include imatinib (Gleevec), a small-molecule inhibitor of BCR/ABL kinase in chronic myelogenous leukemia; trastuzumab (Herceptin), an antibody against ERBB2 (HER2) in breast cancer; bevacizumab (Avastin), an antibody against vascular endothelial growth factor in solid tumors; and erlotinib (Tarceva), a small-molecule inhibitor of epidermal growth factor receptor (EGFR) in non–small cell lung cancer (NSCLC; refs. 2–5). However, it has been increasingly recognized that, in each individual tumor, there are a large number of mutated genes that disrupt multiple pathways, which normally exhibit extensive biological cross-talk and redundancy (6). Therefore, interfering with a single target and/or pathway may not abrogate the malignant phenotype of most tumors. For example, among the roughly 20% of breast cancer patients with HER2 overexpression, only one third respond to trastuzumab treatment. The remaining two thirds of patients fail to respond, which is likely due to other distinct molecular abnormalities within their tumors, such as activation of the insulin-like growth factor signaling pathway (7). Furthermore, resistance to molecularly targeted agents can develop through secondary target gene mutation or compensatory activation of alternative pathways, socalled “oncogenic switching.” This problem is exemplified by erlotinib, an EGFR inhibitor that has been approved for use in NSCLC. An activating mutation in EGFR (exon 19 deletions or exon 21 point mutation Authors' Affiliation: Curis, Inc., Cambridge, Massachusetts Received1/21/09; revised3/16/09; accepted3/19/09; publishedOnlineFirst 6/9/09. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be herebymarked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: R. Bao and C. Lai contributed equally to this work. Requests for reprints: Rudi Bao, Oncology, Curis, Inc., 45 Moulton Street, Cambridge, MA 02138. Phone: 617-503-6540; Fax: 617-503-6501; E-mail: [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-09-0152 4046 Clin Cancer Res 2009;15(12) June 15, 2009 www.aacrjournals.org Research. on May 28, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from L858R) renders cancer cells sensitive to the EGFR inhibitor. However, subsequent resistance to erlotinib emerges as a result of an additional EGFR mutation (T790M) or amplification and/or activation of parallel signaling pathways, including ERBB3 (HER3), insulin-like growth factor receptor, and c-MET (8–10). A promising strategy for mitigating such acquired drug resistance is to simultaneously inhibit multiple molecular pathways, either by using several agents in combination or by using a single agent that concurrently blocks multiple targets or pathways. One approach to inhibiting multiple pathways with one single agent is to target the heat shock protein HSP90. Among the “client proteins” that HSP90 chaperones are many oncogenic proteins, such as the estrogen receptor, androgen receptor, HER2, ERBB1 (EGFR), MEK, c-MET, AKT, MAPK (ERK), CDK, RAF, BCR/ABL, HIF1-α, and hTERT (11). These oncoproteins, ranging from transcription factors and kinases to antiapoptotic molecules, are involved in cancer cell proliferation, survival, invasion, metastasis, and angiogenesis (11). It has been shown that pharmacologic inhibition of HSP90 function can trigger proteasomal degradation of multiple oncoproteins, thereby reducing cancer cell proliferation/survival and tumor angiogenesis and promoting apoptosis (12–14). The chaperone function of HSP90s is highly dependent on its ATPase activity. HSP90 inhibitors compete with ATP at the NH2 terminal nucleotide-binding site to neutralize the intrinsic ATPase activity of the protein. In preclinical tumor models, HSP90 inhibitors have been shown to deplete oncoproteins and inhibit tumor growth (15). The most advanced class of HSP90 inhibitors, including tanespimycin and other 17-AAG derivatives, are now in phase II/phase III clinical trials for solid and hematologic malignancies. A combination of tanespimycin and trastuzumab has shown encouraging results in a phase II trial for trastuzumab refractory breast cancer (16). However, significant clinical limitations of these 17-AAG derivatives have been reported, including poor solubility, potential liver toxicity, substrate for the P-glycoprotein multidrug resistance efflux pump, quinine reductase NQO1 dependence, and limited oral bioavailability (17–19). To overcome the limitations of the 17-AAG class of HSP90 inhibitors, several synthetic HSP90 inhibitors have recently been discovered (20–22) and are now being tested in phase I/phase II clinical trials. Importantly, these synthetic HSP90 inhibitors, including purine (BIIB-021), isoxazole (VER-52296, NVP-AUY922), and indazole (SNX-5422) classes exhibit more favorable pharmacologic properties than the 17-AAG class inhibitors (20–22). Here, we describe CUDC-305, a leading HSP90 inhibitor of the imidazopyridine class. In addition to potent antitumor efficacy against a broad range of cancers in preclinical tumor models, we report that CUDC-305 exhibits enhanced pharmacologic features in several areas, including high oral bioavailability, selectivity, blood-brain barrier penetration, and extended tumor retention. Materials and Methods Reagents and chemicals. CUDC-305 and other reference HSP90 inhibitors were synthesized in house. For in vitro assays, compounds were dissolved in DMSO as stock and stored at −20°C. For in vivo studies, CUDC-305 was formulated in 30% Captisol (Cydex Pharmaceuticals, Inc.) with 2 molar equivalents of HCl. Paclitaxel (Taxol, 6 mg/mL) was purchased fromMayne Pharma, Inc. Camptothecin-11 (20 mg/mL) was purchased from Pfizer, Inc. All other reagents including culture medium, unless otherwise stated, were purchased from Invitrogen. Assay for HSP90 binding. COOH terminal His-tagged human HSP90α and HSP90β proteins were expressed in Escherichia coli. Fluorescence polarization competition binding assays were done with purified HSP90α or HSP90β and FITC-labeled geldanamycin (InvivoGen) in the presence of different concentrations of test articles. The final reaction contained 10 and 50 nmol/L of labeled geldanamycin and purified HSP90 protein, respectively. The assay buffer contained 20 mmol/L HEPES (pH 7.3), 50 mmol/L KCl, 1 mmol/L DTT, 50 mmol/L MgCl2, 20 mmol/L Na2MoO4, and 0.01% NP40 with 0.1 mg/mL bovine γ-globulin. Polarization degree (mP) values were determined using a Synergy II plate reader (BioTek Instruments, Inc.) with background subtraction after 24 h of incubation at 4°C. For binding assay with HSP90 complex from cancer, cancer cell lines were cultured in flasks. Total protein was extracted with radioimmunoprecipitation assay buffer (Sigma-Aldrich Corp.) following manufacturer's instructions. Fluorescence polarization competition binding assay was done as described above. Final protein concentration was adjusted to achieve the same FITC-geldanamycin binding level as with purified HSP90α or HSP90β without test articles. Cell growth and viability assay. Human cancer cell lines were purchased from American Type Culture Collection and plated at 5,000 to 10,000 per well in 96-well plates with culture medium, as suggested by the provider. The cells were then incubated with compounds at various concentrations for 120 h. Growth inhibition was assessed by ATP content assay using the Perkin-Elmer ATPlite kit. Briefly, a 25-μL cell lysis solution was added to the 50-μL phenol red–free culture medium per well to lyse cells and stabilize ATP. Then 25-μL substrate solutions were added to the wells, and subsequently, luminescence was measured with a TopCount liquid scintillation analyzer (Perkin-Elmer). Values were expressed as a percentage relative to those obtained in untreated controls. IC50 values were calculated using PRISM software (GraphPad Software) with sigmoidal dose-response curve fitting. Western blot analysis of cells in culture. Cancer cells grown in culture were treated with compounds at 1 μmol/L for 24 h and then harvested in 1 sample loading buffer (Sigma-Aldrich Corp.). Cell lysates were resolved on NuPAGE Novex 4-12% bis-Tris gels (Invitrogen) and then Translational Relevance CUDC-305 is a novel HSP90 inhibitor of the imidazopyridine class that displays more favorable pharmacolog ic proper t ies , inc lud ing high ora l bioavailability, sustained tumor retention, bloodbrain barrier penetration, and potentially a better therapeutic window, compared with the HSP90 inhibitors currently in clinical development. Most importantly, it displays potent antitumor activity against a variety of tumor types in vitro and in vivo, including epidermal growth factor receptor inhibitor–resistant, non–small cell lung cancer, glioblastoma, triplenegative breast cancer, and acute myelogenous leukemia. Remarkably, all of these tumors represent crucial unmet medical needs. Mechanistically, CUDC-305 robustly inhibits multiple signaling pathways and induces apoptosis in cancer cells. Taken together, these original findings may have a significant effect on future clinical practice in cancer the‐ rapy, particularly in the above-mentioned cancer types. 4047 Clin Cancer Res 2009;15(12) June 15, 2009 www.aacrjournals.org Antitumor Activity of HSP90 Inhibitor CUDC-305 Research. on May 28, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from transferred to nitrocellulose membranes (Bio-Rad Laboratories). The blots were incubated first with a primary antibody overnight at 4°C. Antibodies to detect HSP70, AKT, phosphorylated AKT (p-AKT), c-MET, phosphorylated MET, EGFR, FLT3, phosphorylated FLT3, ERK1/2, phosphorylated ERK1/2 (p-ERK1/2), HER2, phosphorylated HER2, phosphorylated HER3, estrogen receptor α, androgen receptor, CDK4, MEK1, survivin, activated CDC42-associated kinase, signal transducers and activators of transcription 5 (STAT5), cleaved poly(ADPribose) polymerase, and c-RAF (1:1,000-2,000) were obtained from Cell Signaling Technology. Antibody against cyclin D1 (1:2,000) was obtained from Santa Cruz Biotechnology. Glyceraldehyde-3-phosphate dehydrogenase (1:30,000; Abcam) or tubulin (1:5,000; Sigma-Aldrich) was used as an internal control for each assay. Membranes were then incubated with an IR-labeled secondary antibody (1:10,000): conjugated IR Dye-800 (Rockland Immunochemicals, Inc.) or conjugated Alexa Fluor-680 (Invitrogen). Membranes were imaged with the Odyssey IR Imaging System (Li-Cor Biotechnology). Animals and tumor implantation. Female athymic nude (CD-1 nu/nu) or severe combined immunodeficient mice (6-8 wk of age) were obtained from Charles River Laboratories for in-house studies. For efficacy studies conducted in Crown Biosciences, Inc., BALB/c nu/nu mice were used. Animals were housed in ventilated microisolator cages in the animal facilities conditioned at a temperature of 23 ± 1°C, humidity of 50% to 70%, and a 12-h light/12-h dark cycle. The mice were provided with sterile laboratory rodent diet and water ad libitum. The animal procedures and protocols were approved by the Institutional Animal Care and Use Committee of Curis and Crown Biosciences, Inc., respectively. Before tumor implantation, various cancer cell lines of human origin were cultured in the medium suggested by the provider. When cultured cells reached ∼70% to 90% confluence. They were harvested by treatment with trypsin-EDTA (0.25% trypsin, 1 mmol/L EDTA). The cell pellet was suspended in HBSS for implantation after medium was removed. For s.c. tumor implantation, various numbers (3-20 10) of cancer cells were injected into the right hind flank region of each mouse. For orthotopic implantation of breast cancer, a small incision (5 mm) was made in the skin over the lateral thorax to expose the mammary fat pad. MDA-MB-468 cancer cells (20 10) suspended in 100 μL HBSS were injected into the mammary fat pad. Tumor size was measured with an electronic caliper. The following formula was used to calculate the