ELR510444 Inhibits Tumor Growth and Angiogenesis by Abrogating HIF Activity and Disrupting Microtubules in Renal Cell Carcinoma

Background: Hypoxia-inducible factor (HIF) is an attractive therapeutic target for renal cell carcinoma (RCC) as its high expression due to the loss of von Hippel-Lindau (VHL) promotes RCC progression. Considering this, we hypothesized that ELR510444, a novel orally available small molecule inhibitor of HIF activity, would reduce angiogenesis and possess significant activity in RCC. The mechanism of action and therapeutic efficacy of ELR510444 were investigated in in vitro and in vivo models of RCC. Principal Findings: ELR510444 decreased HIF-1a and HIF-2a levels, reduced RCC cell viability and clonogenic survival, and induced apoptosis. VHL-deficient RCC cells were more sensitive to ELR510444-mediated apoptosis and restoration of VHL promoted drug resistance. Higher concentrations of ELR51044 promoted apoptosis independently of VHL status, possibly due to the microtubule destabilizing properties of this agent. ELR510444 significantly reduced tumor burden in the 786-O and A498 RCC xenograft models. These effects were associated with increased necrosis and apoptosis and inhibition of angiogenesis. Conclusions: ELR510444 is a promising new HIF inhibitor that reduced RCC cell viability, induced apoptosis, and diminished tumor burden in RCC xenograft models. ELR510444 also destabilized microtubules suggesting that it possesses vascula

ELR510444 exhibits enhanced sensitivity in VHL-deficient RCC cell lines.

<p>(A) Quantification of VEGF levels in RCC cell lines. Equal numbers of cells were plated and media was collected 16 h later. VEGF levels were measured by ELISA assay. Mean ± SD, <i>n</i> = 3. (B) ELR510444 reduces RCC cell viability. Cells were treated with the indicated concentrations of ELR510444 for 72 h and viability was determined by MTT assay. Mean ± SD, <i>n</i> = 3. (C) ELR510444 reduces clonogenic survival. RCC cells were treated with the indicated concentrations of ELR510444 for 24 h. After treatment, cells were washed in fresh media and allowed to grow for 10 days. Colonies were fixed and stained with crystal violet and quantified using an Alpha Innotech gel imager. Mean ± SD, <i>n</i> = 3. *Indicates a significant difference compared to controls, p<0.05. (D) ELR510444 induces apoptosis. Cells were treated with ELR510444 for 24 h and apoptosis was measured by PI-FACS analysis. Mean ± SD, <i>n</i> = 3. *Denotes a significant difference compared to controls, p<0.05.</p

ELR510444 arrests RCC cells in mitosis and depolymerizes microtubules.

<p>(A) RCC cells were treated with 10 and 30 nM ELR510444 for 24 h. Cell cycle distribution was determined by PI-FACS analysis. Representative histograms and the percentage of cells in each phase of the cell cycle are shown for each experimental condition. (B) RCC4 cells were plated on chamber slides and treated for 24 h with the indicated concentrations of ELR510444 or 100 nM vincristine. Cells were stained with a β-tubulin antibody and microtubules were visualized using an Alexa Fluor 488-conjugated secondary antibody. Nuclei were counterstained using DAPI. The percent microtubule depolymerization was determined at each concentration visually in 100 cells. Representative images are shown. (C) ELR510444 interacts with the colchicine-binding site of β-tubulin. RCC4 cells were pre-treated with the indicated concentrations of ELR510444 for 2 h. 100 µM EBI was then given for an additional 1.5 h. Cells were harvested for immunoblotting and blots were probed with a β-tubulin antibody to detect native β-tubulin and EBI: β-tubulin adducts.</p

ELR510444 decreases HIF-1α and HIF-2α expression.

<p>(A) ELR510444 (chemical structure on left) inhibits HIF-1α activity. RCC4 cells were treated with the indicated concentrations of ELR510444 for 16 h. Cells were harvested, nuclei isolated and lysed, and HIF-1α activity was measured using a commercial activity kit according to the manufacturer's protocol. Mean ± SD, <i>n</i> = 3. (B) ELR510444 decreases HIF-1α and HIF-2α protein expression. RCC4 cells were treated with ELR510444 for 24 h and protein levels were measured by immunoblotting. (C) Quantitative real-time PCR analysis of <i>HIF-1α</i> and HIF-2α (<i>EPAS1</i>) levels. RCC cells were treated with 10 nM ELR510444 for 24 h and gene expression was measured by qRT-PCR. Mean ± SD, <i>n</i> = 3. *Indicates a significant difference from controls, p<0.05. Note that HIF-1α is not present 786-O and A498 cells. (D) ELR510444 decreases VEGF secretion. RCC cells were treated with 10 nM ELR510444 for 16 h. Media was harvested and VEGF levels were measured by ELISA. Mean ± SD, <i>n</i> = 3. *Represents a significant difference compared to controls, p<0.05.</p

VHL-deficient cells are hypersensitive to ELR510444-induced apoptosis.

<p>(A) HIF-2α is reduced in A498 and 786-O cells transfected with VHL. Cells were transfected with empty vector or VHL. Positively transfected cells were selected by incubation with puromycin. HIF-2α was measured by immunoblotting. (B) VEGF levels are reduced in the presence of VHL. Cells were plated and media was collected 16 h later. VEGF levels were measured by ELISA assay. Mean ± SD, <i>n</i> = 3. *Indicates a significant difference compared to controls, p<0.05. (C) A498/VHL and 786-O/VHL cells are less sensitive to ELR510444-mediated apoptosis. Cells were treated with 10 nM ELR510444 for 24 h and apoptosis was measured by PI-FACS analysis. Mean ± SD, <i>n</i> = 3. *Represents a significant difference compared to ELR510444-treated vector-only transfected cells, p<0.05.</p

ELR510444 reduces tumor burden and cell proliferation and stimulates apoptosis.

<p>(A) 786-O and A498 cells (1×10⁷/mouse) were injected into the flanks of nude mice. When tumors reached approximately 150 mm³ in size, mice were randomized into groups and treated on a QDx5 schedule with 8 mg/kg ELR510444 for 2 weeks. Tumor volume was measured twice weekly. Mean ± SEM, <i>n</i> = 10. *Indicates a significant difference compared to vehicle, p<0.05. (B) ELR510444 was well tolerated <i>in vivo</i>. Animal body weight was measured throughout the study to quantify drug-induced weight loss. Mean ± SD, <i>n</i> = 10. (C) ELR510444 reduces tumor cell proliferation. PCNA levels were determined by conducting immunohistochemistry on tumor sections. Positive cells were scored manually. Mean ± SD, <i>n</i> = 5. *Indicates a significant difference compared to controls (vehicle), p<0.05. (D) ELR510444 induces apoptosis. Apoptosis was measured by cleaved caspase-3 staining. Quantification was conducted by manually counting positive cells. Mean ± SD, <i>n</i> = 5. *Indicates a significant difference compared to controls (vehicle), p<0.05.</p