Systematic Repurposing Screening in Xenograft Models Identifies Approved Drugs with Novel Anti-Cancer Activity

<div><p>Approved drugs target approximately 400 different mechanisms of action, of which as few as 60 are currently used as anti-cancer therapies. Given that on average it takes 10–15 years for a new cancer therapeutic to be approved, and the recent success of drug repurposing for agents such as thalidomide, we hypothesized that effective, safe cancer treatments may be found by testing approved drugs in new therapeutic settings. Here, we report <i>in-vivo</i> testing of a broad compound collection in cancer xenograft models. Using 182 compounds that target 125 unique target mechanisms, we identified 3 drugs that displayed reproducible activity in combination with the chemotherapeutic temozolomide. Candidate drugs appear effective at dose equivalents that exceed current prescription levels, suggesting that additional pre-clinical efforts will be needed before these drugs can be tested for efficacy in clinical trials. In total, we suggest drug repurposing is a relatively resource-intensive method that can identify approved medicines with a narrow margin of anti-cancer activity.</p></div

Primary Screen Hits.

<p>Hits significant in primary screen survival analysis are shown; near-neighbors in the tail of the screening hit distribution are shown, additionally.</p><p>a. Mantel-Hanzel hazard ratio, combo treatment compared to 5 mg/kg temozolomide alone.</p><p>b. Chi-square, survival analysis.</p

Screen validation and confirmation.

<p><u>A. Hit validation in additional models.</u> Selected primary screen hits were tested in combination with sub-efficacious doses of temozolomide in a model of glioblastoma and melanoma xenograft models. To assess pharmacological effects terbinafine was dosed p.o in A375 model at a dose interval five times greater than its screening dose administered by i.p. route. In primary screen comparisons, risedronate was significant by survival analysis at a cut-off at 1000 mm³, but not 500 mm³. <u>B. Hypertension drug combinations.</u> Drugs were dosed in combination in the U87-MG model, at the dose in the primary screen, or as indicated in supplemental materials. Effects on tumor growth were assessed at Day 20 of the study. Means plus SEM shown (N = 10); candesartan and temozolomide 100 mg/kg significant by M-ANOVA at p<0.05.</p

Systematic Repurposing Screening in Xenograft Models Identifies Approved Drugs with Novel Anti-Cancer Activity - Figure 3

<p><u>A. Candesartan dose range confirmation in the U87-MG model.</u> Candesartan was confirmed at its screening dose and route; efficacy diminished following oral administration near and below the stated MTD dose equivalent of 14.3 mg/kg. <u>B. Risedronate dose range confirmation.</u> Risedronate exhibited modest combination effects when dosed orally at 7.5 mg/kg, which near MHRDD dose equivalent of 5 mg/kg (compare to screen result at 15 mg/kg, i.p.). The dose exposure trend was modest, and appeared equivalent at doses slightly below 5 mg/kg. <u>C. Terbinafine dose range confirmation.</u> Terbinafine was tested by i.p. administration. Efficacy diminished as drug was administered below the human dose equivalent of 4 mg/kg. Group means shown; cohort SEM not shown for clarity.</p

Epithelial-mesenchymal transition increases tumor sensitivity to COX-2 inhibition by apricoxib

Although cyclooxygenase-2 (COX-2) inhibitors, such as the late stage development drug apricoxib, exhibit antitumor activity, their mechanisms of action have not been fully defined. In this study, we characterized the mechanisms of action of apricoxib in HT29 colorectal carcinoma. Apricoxib was weakly cytotoxic toward naive HT29 cells in vitro but inhibited tumor growth markedly in vivo. Pharmacokinetic analyses revealed that in vivo drug levels peaked at 2-4 μM and remained sufficient to completely inhibit prostaglandin E 2 production, but failed to reach concentrations cytotoxic for HT29 cells in monolayer culture. Despite this, apricoxib significantly inhibited tumor cell proliferation and induced apoptosis without affecting blood vessel density, although it did promote vascular normalization. Strikingly, apricoxib treatment induced a dose-dependent reversal of epithelial-mesenchymal transition (EMT), as shown by robust upregulation of E-cadherin and the virtual disappearance of vimentin and ZEB1 protein expression. In vitro, either anchorage-independent growth conditions or forced EMT sensitized HT29 and non-small cell lung cancer cells to apricoxib by 50-fold, suggesting that the occurrence of EMT may actually increase the dependence of colon and lung carcinoma cells on COX-2. Taken together, these data suggest that acquisition of mesenchymal characteristics sensitizes carcinoma cells to apricoxib resulting in significant single-agent antitumor activity