TK216 targets microtubules in Ewing sarcoma cells.
TK216 targets microtubules in Ewing sarcoma cells.
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DOI：
10.1016/j.chembiol.2022.06.002
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发表时间：
2022-08-18
影响因子：
8.6
通讯作者：
McFadden, David G.
中科院分区：
文献类型：
--
作者：
Povedano, Juan Manuel;Li, Vicky;Lake, Katherine E.;Bai, Xin;Rallabandi, Rameshu;Kim, Jiwoong;Xie, Yang;De Brabander, Jef K.;McFadden, David G.
Ewing肉瘤（EWS）是一种由染色体易位t形成的EWSR1-FLI1融合蛋白驱动的儿科恶性肿瘤（11;22）。小分子TK216是一种新型EWSR1-FLI1直接抑制剂，目前正在与新碱联合用于EWS患者的II期临床试验中。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，其细胞毒性机制尚不清楚。我们在EWS细胞系中应用了一个利用工程超突变的前向遗传学筛选平台，并鉴定了编码<s:1>微管蛋白的TUBA1B的复发突变，这些突变足以驱动对TK216的抗性。通过体外重组微管（MT）聚合和基于细胞的化学探针竞争分析，我们证明了TK216是一种微管不稳定剂。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，并呼吁对正在进行的TK216临床试验进行重新审查。在TK216耐药的尤因肉瘤细胞中发现复发性TUBA1B突变TUBA1B突变独立驱动对TK216的耐药TK216在体外以立体选择的方式抑制微管蛋白聚合TK216和vincristine通过不同的结合机制作用于微管TK216 （ONCT-216）被开发用于抑制驱动尤因肉瘤的融合蛋白EWSR1-FLI1。然而，tk216诱导细胞毒性的机制尚不清楚。在这里，Povedano等人利用正向遗传学、生化重构和化学探针竞争证明了TK216是一种微管不稳定剂。
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. 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 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.
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