The activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression, and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high-throughput screening led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87-2243 was found to inhibit HIF-1α and HIF-2α protein accumulation under hypoxic conditions in non-small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF-1α protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87-2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel–Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase-2. Antitumor activity of BAY 87-2243, suppression of HIF-1α protein levels, and reduction of HIF-1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors.

Mesothelin is a tumor differentiation antigen frequently overexpressed in tumors such as mesothelioma, ovarian, pancreatic, and lung adenocarcinomas while showing limited expression in nonmalignant tissues. Mesothelin is therefore an attractive target for cancer therapy using antibody-drug conjugates (ADC). This study describes the detailed characterization of anetumab ravtansine, here referred to as BAY 94-9343, a novel ADC consisting of a human anti-mesothelin antibody conjugated to the maytansinoid tubulin inhibitor DM4 via a disulfide-containing linker. Binding properties of the anti-mesothelin antibody were analyzed using surface plasmon resonance, immunohistochemistry, flow cytometry, and fluorescence microscopy. Effects of BAY 94-9343 on cell proliferation were first studied in vitro and subsequently in vivo using subcutaneous, orthotopic, and patient-derived xenograft tumor models. The antibody binds to human mesothelin with high affinity and selectivity, thereby inducing efficient antigen internalization. In vitro, BAY 94-9343 demonstrated potent and selective cytotoxicity of mesothelin-expressing cells with an IC 50 of 0.72 nmol/L, without affecting mesothelin-negative or nonproliferating cells. In vivo, BAY 94-9343 localized specifically to mesothelin-positive tumors and inhibited tumor growth in both subcutaneous and orthotopic xenograft models. In addition, BAY 94-9343 was able to induce a bystander effect on neighboring mesothelin-negative tumor cells. Antitumor efficacy of BAY 94-9343 correlated with the amount of mesothelin expressed and was generally superior to that of standard-of-care regimen resulting in complete tumor eradication in most of the models. BAY 94-9343 is a selective and highly potent ADC, and our data support its development for the treatment of patients with mesothelin-expressing tumors. Mol Cancer Ther; 13(6); 1537-48. Ó2014 AACR.

BackgroundThe transcription factor hypoxia-inducible factor-1 (HIF-1) pathway plays an important role in tumor response to cytotoxic treatments. We investigated the effects of a novel small molecule inhibitor of mitochondrial complex I and hypoxia-induced HIF-1 activity BAY-87-2243, on tumor microenvironment and response of human squamous cell carcinoma (hSCC) to clinically relevant fractionated radiotherapy (RT) with and without concomitant chemotherapy.MethodsWhen UT-SCC-5 hSCC xenografts in nude mice reached 6 mm in diameter BAY-87-2243 or carrier was administered before and/or during RT or radiochemotherapy with concomitant cisplatin (RCT). Local tumor control was evaluated 150 days after irradiation and the doses to control 50% of tumors (TCD50) were compared between treatment arms. Tumors were excised at different time points during BAY-87-2243 or carrier treatment for western blot and immunohistological investigations.ResultsBAY-87-2243 markedly decreased nuclear HIF-1α expression and pimonidazole hypoxic fraction already after 3 days of drug treatment. BAY-87-2243 prior to RT significantly reduced TCD50 from 123 to 100 Gy (p=0.037). Additional BAY-87-2243 application during RT did not decrease TCD50. BAY-87-2243 before and during radiochemotherapy did not improve local tumor control.ConclusionsPronounced reduction of tumor hypoxia by application of BAY-87-2243 prior to RT improved local tumor control. The results demonstrate that radiosensitizing effect importantly depends on treatment schedule. The data support further investigations of HIF-1 pathway inhibitors for radiotherapy and of predictive tests to select patients who will benefit from this combined treatment.

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Purpose We describe a non-invasive PET imaging method that monitors early therapeutic efficacy of BAY 87-2243, a novel small molecule inhibitor of mitochondrial complex I as a function of hypoxia-inducible factor-1α (HIF-1α) activity Experimental Design Four PET tracers (18F-FDG, 18F-Fpp(RGD)2, 18F-FLT and 18F-FAZA) were assessed for uptake into tumor xenografts of drug-responsive (H460, PC3) or drug-resistant (786-0) carcinoma cells. Mice were treated with BAY 87-2243 or vehicle. At each point, RNA from treated and vehicle H460 tumor xenografts (n=3 each) was isolated and analyzed for target genes. Results Significant changes in uptake of 18F-FAZA, 18F-FLT and 18F-Fpp(RGD)2 (p<0.01) occurred with BAY 87-2243 treatment with 18F-FAZA being the most prominent. 18F-FDG uptake was unaffected. 18F-FAZA tumor uptake declined by 55–70% (1.21±0.10 %ID/g to 0.35±0.1 %ID/g, n=6, vehicle vs. treatment) in both H460 (p<0.001) and PC3 (p<0.05) xenografts 1–3 days post-drug. 18F-FAZA uptake in 786-0 xenografts was unaffected. Decline occurred before significant differences in tumor volume, thus that suggesting 18F-FAZA decrease reflected early changes in tumor metabolism. BAY 87-2243 reduced expression of hypoxia-regulated genes CA IX, ANGPTL4 and EGLN-3 by 99 %, 93% and 83%, respectively (p<0.001 for all), which corresponds with reduced 18F-FAZA uptake upon drug treatment. Heterogeneous expression of genes associated with glucose metabolism, vessel density and proliferation was observed. Conclusions Our studies suggest suitability of 18F-FAZA-PET as an early pharmacodynamic monitor on the efficacy of anti-cancer agents that target the mitochondrial complex I and intra-tumor oxygen levels (e.g. BAY 87-2243).