Drug repurposing for gastrointestinal stromal tumor

Abstract

Despite significant treatment advances over the past decade, metastatic gastrointestinal stromal tumor (GIST) remains largely incurable. Rare diseases, such as GIST, individually affect small groups of patients but collectively are estimated to affect 25 to 30 million people in the United States alone. Given the costs associated with the discovery, development, and registration of new drugs, orphan diseases such as GIST are often not pursued by mainstream pharmaceutical companies. As a result, "drug repurposing" or "repositioning," has emerged as an alternative to the traditional drug development process. In this study, we screened 796 U.S. Food and Drug Administration (FDA)-approved drugs and found that two of these compounds, auranofin (Ridaura) and fludarabine phosphate, effectively and selectively inhibited the proliferation of GISTs, including imatinib-resistant cells. One of the most notable drug hits, auranofin, an oral, gold-containing agent approved by the FDA in 1985 for the treatment of rheumatoid arthritis, was found to inhibit thioredoxin reductase activity and induce reactive oxygen species (ROS) production, leading to dramatic inhibition of GIST cell growth and viability. Importantly, the anticancer activity associated with auranofin was independent of imatinib-resistant status, but was closely related to the endogenous and inducible levels of ROS. Coupled with the fact that auranofin has an established safety profile in patients, these findings suggest for the first time that auranofin may have clinical benefit for patients with GIST, particularly in those suffering from imatinib-resistant and recurrent forms of this disease.

Figure 1

Post-HTS analysis. A, data acquisition are shown as a 3-dimensional representation. Plot was generated from GIST T1-10R cell line. B, C and D, examples of drug concentration-response curves. Detailed information of curves are summarized in Table S3. E and F, IC₅₀ curves for auranofin derived from the primary screen and derived from the subsequent validation assays.

Figure 2

In vitro effects of auranofin or IM treatment on TrxR/Trx system. A and B, auranofin inhibits thioredoxin reductase (TrxR) activity in GIST cells. IM does not alter TrxR activity. GIST cells were treated for 6 h with the indicated concentration of auranofin before cells were harvested and lysed. For each sample, 50 μg of protein cell lysate was assessed for TrxR activity by measuring NADPH-dependent reduction of DTNB. C and D, auranofin increases reactive oxygen species (ROS) activity in GIST cells. IM does not increase the production of ROS. GIST cells in a 96-well plate were pretreated with 1 mM DCFH-DA for 60 minutes at 37 °C. Cells were then treated for 6 h with the indicated concentration of auranofin. ROS activity was measured using DCF fluorescence. (* p value < 0.05)

Figure 3

Effect of auranofin or IM treatments on signaling pathways and cell viability. A, GIST 882. B, GIST-T1; and C, GIST T1-10R cells were treated with auranofin or IM at indicated concentration for 6 h. For each sample, 50 μg of total protein cell lysate was loaded for western blots. D, E, and F, Bar graph shows combined densitometric quantification (Image J) of western blot results. Each bar represents results from two independent experiments. G, proposed mechanism of auranofin action in GIST cells. Auranofin inhibits TrxR system and results in increased ROS production which finally leads to apoptosis and cell growth inhibition. H, Auranofin induces apoptosis in GIST cells. GIST cells in a 384-well plate were treated for 72 h with or without 0.01 μM or 1 μM of auranofin. Caspase 3/7 activity was measured by luminescence. F, Auranofin inhibits proliferation of GIST cells. GIST cells in a 384-well plate were treated for 72 h with or without 0.01 μM or 1 μM of auranofin. (* p value < 0.05)