TK216 targets microtubules in Ewing sarcoma cells. Issue 8 (18th August 2022)

• Record Type: Journal Article
• Authors: Povedano, Juan Manuel; Li, Vicky; Lake, Katherine E.; Bai, Xin; Rallabandi, Rameshu; Kim, Jiwoong; Xie, Yang; De Brabander, Jef K.; McFadden, David G.
• Is Part Of: Cell chemical biology. Volume 29:Issue 8(2022)
• Page Start: 1325
• Page End: 1332.e4
• Publication Date: 2022-08-18
• Subjects: Ewing sarcoma -- TK216 -- ONCT-216 -- YK-4-279 -- microtubules -- target identification; Biochemistry -- Periodicals; 572.05
• DOI: 10.1016/j.chembiol.2022.06.002
• Languages: English
• ISSNs: 2451-9456

Abstract

Summary: Ewing sarcoma (EWS) is a pediatric malignancy driven by the EWSR1-FLI1 fusion protein formed by the chromosomal translocation t(11; 22). The small molecule TK216 was developed as a first-in-class direct EWSR1-FLI1 inhibitor and is in phase II clinical trials in combination with vincristine for patients with EWS. However, TK216 exhibits anti-cancer activity against cancer cell lines and xenografts that do not express EWSR1-FLI1, and the mechanism underlying cytotoxicity remains unresolved. We apply a forward-genetics screening platform utilizing engineered hypermutation in EWS cell lines and identify recurrent mutations in TUBA1B, encoding ⍺-tubulin, that prove sufficient to drive resistance to TK216. Using reconstituted microtubule (MT) polymerization in vitro and cell-based chemical probe competition assays, we demonstrate that TK216 acts as an MT destabilizing agent. This work defines the mechanism of cytotoxicity of TK216, explains the synergy observed with vincristine, and calls for a reexamination of ongoing clinical trials with TK216. Graphical abstract: Highlights: Recurrent TUBA1B mutations were identified in TK216-resistant Ewing sarcoma cells TUBA1B mutations independently drove resistance to TK216 TK216 suppressed tubulin polymerization in vitro in a stereo-selective manner TK216 and vincristine act on microtubules through distinct binding mechanisms Abstract : TK216 (ONCT-216) was developed to inhibit the fusion protein that drives Ewing sarcoma, EWSR1-FLI1. However, the mechanism underlying TK216-induced cytotoxicity remained elusive. Here, Povedano et al. demonstrate using forward genetics, biochemical reconstitution, and chemical probe competition that TK216 acts as a microtubule destabilizing agent.