High-throughput screening of FDA-approved drugs identifies colchicine as a potential therapeutic agent for atypical teratoid/rhabdoid tumors (AT/RTs)

Abstract

Atypical teratoid/rhabdoid tumor (AT/RT) is a rare and aggressive tumor of the primary central nervous system primarily affecting children. It typically originates in the cerebellum and brain stem and is associated with a low survival rate. While standard chemotherapy has been used as a primary treatment for AT/RTs, its success rate is unsatisfactory, and patients often experience severe side effects. Therefore, there is an urgent need to develop new and effective treatment strategies. One promising approach for identifying new therapies is drug repurposing. Although many FDA-approved drugs have been repurposed for various cancers, there have been no reports of such applications for AT/RTs. In this study, a library of 2130 FDA-approved drugs was screened using a high-throughput screening system against 2D traditional cultures and 3D spheroid cultures of AT/RT cell lines (BT-12 and BT-16). From this screening, colchicine, a non-chemotherapeutic agent, was identified as a promising candidate. It exhibited IC₅₀ values of 0.016 and 0.056 μM against 2D BT-12 and 2D BT-16 cells, respectively, and IC₅₀ values of 0.004 and 0.023 μM against 3D BT-12 and BT-16 spheroid cultures. Additionally, the cytotoxic effects of colchicine on human brain endothelial cells and human astrocytes were evaluated, and CC₅₀ > 20 μM was observed, which is over two orders of magnitude higher than its effective concentrations in AT/RT cells, indicating considerably lower toxicity to normal brain cells and brain endothelial cells. In conclusion, colchicine shows significant potential to be repurposed as a treatment for AT/RTs, providing a safer and more effective therapeutic option for this rare and challenging disease.

Fig. 1. High-throughput screening of 2130 FDA-approved drugs against AT/RT cells. The primary screening was conducted in BT-12 cells (A) and BT-16 cells (B) using FDA-approved drugs at a concentration of 10 μM. Hit compounds were identified based on their ability to inhibit cancer cell viability by over 80% indicated by the orange dotted line. A total of 104 hit compounds were effective against BT-12 cells and BT-16 cells as shown in (C). Amongst these, 62 compounds were classified as chemotherapeutic drugs (orange dots), while 42 compounds were identified as non-chemotherapeutic drugs (blue dots) (C). Doxorubicin served as the positive control (red dot), and DMSO served as the negative control (green dot).

Fig. 2. Dose–response effects of three FDA-approved drug candidates on AT/RT cells. (A) IC₅₀ comparison graph between BT-12 cells and BT-16 cells highlighting the three drug candidates in orange dots. (B) IC₅₀ values of three FDA-approved drugs affect AT/RT cell viability corresponding to the numbered orange dots in (A). (C–F) Dose-dependent effects on BT-12 cell viability for colchicine (C), digoxin (D), emetine (E), with doxorubicin as a control (F). (G–J) Dose-dependent effects on BT-16 cell viability for colchicine (G), digoxin (H), emetine (I), and doxorubicin (J).

Fig. 3. Cytotoxicity testing (CC₅₀) of the hit compounds against the normal cells. (A–D) Dose-dependent cytotoxicity effects of colchicine (A), digoxin (B), emetine (C), and doxorubicin (D) on the viability of human cerebral microvascular endothelial cells (hCMEC/D3 cells). (E–H) Dose-dependent cytotoxicity effects of colchicine (E), digoxin (F), emetine (G), and doxorubicin (H) on the viability of primary astrocytes.

Fig. 4. Effect of colchicine on three-dimensional (3D) AT/RT spheroid growth models. BT-12 spheroids were treated with colchicine (A) or doxorubicin (B) at varying concentrations. The percentage effect of colchicine (red line) and doxorubicin (dark line) on BT-12 spheroid viability is shown in (C). Similarly, BT-16 spheroids were exposed to colchicine (D) or doxorubicin (E) at the stated concentrations. The percentage effect of colchicine (red line) and doxorubicin (black line) on BT-16 spheroid viability is presented in (F). Hoechst (green) and ethidium homodimer (orange) staining were used to distinguish live and dead cells, respectively.

Fig. 5. Proposed antimitotic mechanism of colchicine. Colchicine disrupts microtubule stability by binding to two tubulin heterodimers near the exchangeable GTP-binding site, forming a tubulin-colchicine (TC) complex. This interaction destabilizes tubulin, preventing proper microtubule assembly and arresting mitotic cells at the G₂/M phase. As a result, AT/RT cell proliferation is inhibited, particularly in the proliferative zone of 3D AT/RT spheroids, ultimately triggering programmed cell death, often known as apoptosis. The cell cycle consists of four main phases: G₁ (first growth phase), where the cell grows and carries out normal metabolic functions; S (synthesis phase), during which DNA replication occurs; G₂ (second growth phase), preparing the cell for mitosis; and M (mitotic phase), where nuclear division results in two genetically identical daughter cells. The M phase is further divided into prophase, metaphase, anaphase, and telophase. (Created by BioRender.com/Mahidol University.)