Synergistic combination of flavopiridol and carfilzomib targets commonly dysregulated pathways in adrenocortical carcinoma and has biomarkers of response

Drug repurposing is an effective approach to identify active drugs with known toxicity profiles for rare cancers such as ACC. The objective of this study was to determine the anticancer activity of combination treatment for ACC from previously identified candidate agents using quantitative high-throughput screening (qHTS). In this study, we evaluated the anticancer activity of flavopiridol and carfilzomib in three ACC cell lines in vitro and in vivo. Human ACC samples were analyzed for drug-target analysis, and cancer-related pathway arrays were used to identify biomarkers of treatment response. Because flavopiridol is a potent cyclin-dependent kinase (CDK) inhibitor, we found significantly higher CDK1 and CDK2 mRNA expression in three independent cohorts human ACC (p<0.01) and CDK1 protein by immunohistochemistry (p<0.01) in human ACC samples. In vitro treatment with flavopiridol and carfilzomib in all three ACC cell lines resulted in a dose-dependent, anti-proliferative effect, and the combination had synergistic activity as well as in three-dimensional tumor spheroids. We observed increased G2M cell-cycle arrest and apoptosis with combination treatment compared to other groups in vitro. The combination treatment decreased XIAP protein expression in ACC cell lines. Mice with human ACC xenografts treated with flavopiridol and carfilzomib had significantly lower tumor burden, compared to other groups (p<0.05). We observed increased cleaved-caspase expression and decreased XIAP in tumor xenografts of mice treated with combined agents. Our preclinical data supports the evaluation of combination therapy with flavopiridol and carfilzomib in patients with advanced ACC.

An additional 100 μl of fresh media containing the 1× concentration of the tested drugs or vehicles was added 48 hours (Day 2) after initial administration to support optimal cell growth. Based on IC50 determined by qHTS, we selected at least five concentrations of flavopiridol and carfilzomib to test the efficacy of various combinations. Each concentration was performed in quadruplicates. CyQuant Cell Proliferation Assay (Invitrogen Corp., Carlsbad, CA) was performed according to the manufacturer's protocol. The cell densities in the 96-well black plates were determined using a SpectraMax M5e 96-well fluorescence microplate reader (Molecular Devices, Sunnyvale, CA) at 485 nm/538 nm. For each drug tested, the experiments were repeated at least three times.

Three-dimensional multicellular aggregates (MCAs)
MCAs of NCI-H295R cells form compact tumor spheroids in suspension culture conditions. We generated NCI-H295R and SW-13 MCAs by plating 1.0 × 10 5 cells/well into Ultra Low Cluster, 24-well plate (Costar ® , Corning, NY) and incubated the cells at 37°C in 5% CO2 for two weeks. The media was changed twice a week. At the beginning of the third week of culture, MCAs were treated with selected combinations and their corresponding single drug and vehicle controls using concentrations based on IC50 from the monolayer proliferation assay below maximum tolerable serum levels. All experiments were performed in triplicates for each testing concentration. SW-13 and H295R MCAs were continuously treated for two weeks. Media containing respective drugs and vehicle control were changed twice a week. MCAs were photographed with a Nikon D5100 (Nikon, Inc., Melville, NY) under a 12.5× magnification microscope (Olympus SZX9 microscope with DF PLAPO 1X-2 lens, Olympus America, Inc., Center Valley, PA).

Immunohistochemistry
Sections were deparaffinized and rehydrated in serially graded alcohol. Sections were then placed in citrate buffer, and epitope retrieval was performed at 120°C using pressurized steam for 10 minutes. The endogenous peroxidase activity was blocked with 6% hydrogen peroxide. The primary antibodies were anticleaved caspase 3 (Asp175) polyclonal rabbit antibody at 1:300 dilution (#9661, Cell Signaling, Inc., Danvers, MA) and anti-XIAP (D2Z8W) rabbit monoclonal antibody at 1:50 dilution (#14334, Cell Signaling, Inc., Danvers, MA). The sections were incubated at 4°C overnight, then incubated with a biotinylated secondary antibody at 1:150 dilution (Vector Laboratories) for one hour at room temperature. The sections were developed using 3,3'-diaminobenzidine as the chromogen (Elite ABC Kit, Vector Laboratories) and hematoxylin as the counterstain. The sections were dehydrated and mounted with VectaMount Mounting Medium (Vector Laboratories). The slides were viewed under a Zeiss Axio Observer D1 microscope (Zeiss USA, North Chesterfield, VA) and photographs were taken at 200x magnification. The Quickscore (Q) method was used to quantitate the protein expression in immunohistochemistry [1].

In vivo study in mice with human ACC xenografts
A total of 5×10 6 NCI-H295R cells with luciferase reporter were injected into each flank of a Nuþ/Nuþ mouse (two xenografts per mouse). Tumors were allowed to grow and mice of both sexes were randomized into four treatment groups (nine mice per treatment group). Mice were treated with flavopiridol (6 mg/kg), carfilzomib (5 mg/kg), the combination of flavopiridol (6 mg/kg) and carfilzomib (5 mg/kg), and vehicle control. We used bioluminescence imaging to confirm a deposition of injected cells using the Xenogen in vivo imaging system. Mice were injected intraperitoneally with 3-4.5 mg of luciferin/mouse 10 minutes prior to imaging. Then the animals were anesthetized in a plastic chamber filled with a 2.5% isoflurane/oxygen/air mixture, and isoflurane anesthesia was maintained using a nose-cone delivery system during imaging. The pseudocolor image representing the spatial distribution of detected photon counts emerging from the active luciferase within each animal was collected. Signal intensity was quantified as the sum of all detected photon counts within a region of interest using IVIS Living Image software (Caliper Life Sciences Inc.). All mice were euthanized by CO 2 inhalation. Tumor xenografts were harvested, flash-frozen in liquid nitrogen, and formalin-fixed. Formalin-fixed tissues were processed into paraffin blocks for H&E and immunohistochemistry staining.
Mice were injected intraperitoneally with 3-4.5 mg of luciferin/mouse 10 minutes prior to imaging. Then the animals were anesthetized in a plastic chamber filled with a 2.5% isoflurane/oxygen/air mixture, and isoflurane anesthesia was maintained using a nose-cone delivery system during imaging. The pseudocolor image representing the spatial distribution of detected photon counts emerging from the active luciferase within each animal was collected. Signal intensity was quantified as the sum of all detected photon counts within a region of interest using IVIS Living Image software (Caliper Life Sciences Inc.). All mice were euthanized by CO 2 inhalation. Tumor xenografts were harvested, flash-frozen in liquid nitrogen, and formalin-fixed. Formalin-fixed tissues were processed into paraffin blocks for H&E and immunohistochemistry staining.