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.

尤文肉瘤 (EWS) 是一种由染色体易位 t(11;22) 形成的 EWSR1-FLI1 融合蛋白驱动的儿科恶性肿瘤。小分子 TK216 被开发为一流的直接 EWSR1-FLI1 抑制剂，与长春新碱联合治疗 EWS 患者正在进行 II 期临床试验。然而，TK216 对不表达 EWSR1-FLI1 的癌细胞系和异种移植物表现出抗癌活性，并且细胞毒性的机制仍未解决。我们在 EWS 细胞系中应用了利用工程超突变的正向遗传学筛选平台，并鉴定了编码 ⍺-微管蛋白的TUBA1B中的反复突变，这些突变证明足以驱动对 TK216 的耐药性。使用体外重构微管 (MT) 聚合和基于细胞的化学探针竞争测定，我们证明 TK216 可以作为 MT 不稳定剂。这项工作定义了 TK216 的细胞毒性机制，解释了与长春新碱观察到的协同作用，并呼吁重新审查正在进行的 TK216 临床试验。