A Synergetic Screening Approach with Companion Effector for Combination Therapy: Application to Retinoblastoma

<div><p>For many cancers, the lack of potency and the toxicity of current drugs limits the dose achievable in patients and the efficacy of treatment. Among them, retinoblastoma is a rare cancer of the eye for which better chemotherapeutic options are needed. Combination therapy is a compelling approach to enhance the efficacy of current treatment, however clinical trials to test rationally designed combinations of approved drugs are slow and expensive, and limited by our lack of in-depth knowledge of drug specificity. Since many patients already turn to nutraceuticals in hopes of improving their condition, we hypothesized that certain approved drugs could potentially synergize with widely consumed supplements. Following this hypothesis, we devised an alternative screening strategy aimed at taking advantage of a bait compound such as a nutraceutical with potential therapeutic benefits but low potency, by screening chemical libraries for approved drugs that synergize with this companion effector. As a proof of concept, we sought to identify approved drugs with synergetic therapeutic effects toward retinoblastoma cells in combination with the antioxidant resveratrol, popular as a supplement. We systematically tested FDA-approved drugs and known bioactives seeking to identify such pairs, which led to uncovering only a few additive combinations; but to our surprise, we identified a class of anticancer drugs widely used in the clinic whose therapeutic effect is antagonized with resveratrol. Our observations could explain in part why some patients do not respond well to treatment. Our results validate this alternative approach, and we expect that our companion effector strategy could significantly impact both drug discovery and the nutraceutical industry.</p> </div

Summary of IC₅₀ in the Alamar Blue viability assay readout for eight resupplied agonists and 13 antagonists toward the Y79 and RB355 human retinoblastoma cell lines.

<p>Summary of IC₅₀ in the Alamar Blue viability assay readout for eight resupplied agonists and 13 antagonists toward the Y79 and RB355 human retinoblastoma cell lines.</p

Dose response curves for confirmation of selected positives.

<p>Potency assessment of confirmed positives toward Y79 and RB355 cells in absence of resveratrol or with 30 or 80 µM resveratrol co-treatment in an Alamar Blue-based viability assay. Dose response curves of the confirmed agonist berberine chloride toward <b>A)</b> Y79 and <b>B)</b> RB355 cells inducing inhibition of cell viability with IC₅₀ values of 24, 15 and 8.6 µM toward Y79 and 59, 23 and 6.8 µM toward RB355 cells in absence of resveratrol or with 30 or 80 µM resveratrol co-treatment. Dose response curves of the confirmed antagonist nocodazole toward <b>C)</b> Y79 and <b>D)</b> RB355 cells inducing inhibition of cell viability with IC₅₀ values of 34, 21 and 5.6 µM toward Y79 and 1.1, 0.53 and greater than 100 µM toward RB355 cells in absence of resveratrol or with 30 or 80 µM resveratrol co-treatment. For partial dose response curves leading to a maximum of 50% inhibition or less, an IC₅₀ greater than 100 µM was reported, since an accurate IC₅₀ cannot be calculated.</p

Assessment of screen reproducibility.

<p>Scatter plot analysis of four screens against 6,912 compounds performed in duplicate. The percentage inhibition (%I) for each compound and for each set of data is plotted as a scatter plot for <b>A)</b> Y79 in absence of resveratrol <b>B)</b> Y79 in presence of 80 µM resveratrol <b>C)</b> RB355 in absence of resveratrol and <b>D)</b> RB355 in presence of 80 µM resveratrol.</p

Selection and confirmation of positives. A)

<p>Scatter plot analysis of the average percentage inhibition (%I) of each compound toward Y79 cells, in absence of resveratrol (−RES) or with 80 µM resveratrol co-treatment (+RES). Initial positives with agonistic activity are selected as those compounds inducing greater than or equal to 50% inhibition in presence of resveratrol and inducing less than or equal to 50% inhibition in absence of resveratrol (green). Initial positives with antagonistic activity are selected as those compounds inducing less than or equal to 50% inhibition in presence of resveratrol and inducing greater than or equal to 50% inhibition in absence of resveratrol (red). <b>B)</b> Scatter plot analysis of the average percentage inhibition (%I) of each compound toward RB355 cells, in absence of resveratrol (−RES) or with 80 µM resveratrol co-treatment (+RES). Positives from the Y79 screen with agonistic activity in both screens are selected as those compounds inducing greater than or equal to 50% inhibition in presence of resveratrol and inducing less than or equal to 50% inhibition in absence of resveratrol (green). Positives from the Y79 screen with antagonistic activity in both screens are selected as those compounds inducing less than or equal to 50% inhibition in presence of resveratrol and inducing greater than or equal to 50% inhibition in absence of resveratrol (red). <b>C)</b> Heatmap representation of the IC₅₀ values of the selected 21 positives (8 agonists and 13 antagonists) toward Y79 and RB355 cells in absence of resveratrol or with 30 or 80 µM resveratrol co-treatment. The triangle symbolizes the increasing concentration in resveratrol.</p

20X images of vincristine treated MDA-MB231.

<p>INCA 2000 images of triple negative human breast cancer cells MDA-MB-231 at 20X objective magnification showing Hoechst-stained nuclei (blue) and rhodamine phalloidin-stained actin (red) for high control 1% DMSO and 0.2 µM vincristine in absence of resveratrol or with 30 or 80 µM resveratrol co-treatment.</p

Activity profiling of confirmed positives toward a panel of cancer cell lines.

<p>Summary of potency assessment of 21 confirmed positives toward a panel of 13 cancer cell lines in absence of resveratrol or with 30 or 80 µM resveratrol co-treatment. Heat map representation of IC₅₀ values in Alamar Blue-based viability assay of <b>A)</b> 8 agonists and <b>B)</b> 13 antagonists in absence of resveratrol or with 30 or 80 µM resveratrol co-treatment. The triangle symbolizes the increasing concentration in resveratrol.</p

Summary of IC₅₀ in the nuclei count readout for eight resupplied agonists, 13 antagonists and two additional compounds toward the human uveal melanoma cell line OCM290.

<p>Summary of IC₅₀ in the nuclei count readout for eight resupplied agonists, 13 antagonists and two additional compounds toward the human uveal melanoma cell line OCM290.</p

20X images of colchicine treated MDA-MB231.

<p>INCA 2000 images of triple negative human breast cancer cells MDA-MB-231 at 20X objective magnification showing Hoechst-stained nuclei (blue) and rhodamine phalloidin-stained actin (red) for high control 1% DMSO and 12.5 µM colchicine in absence of resveratrol or with 30 or 80 µM resveratrol co-treatment.</p