PT  - JOURNAL ARTICLE
AU  - Juan Manuel Povedano
AU  - Vicky Li
AU  - Katherine E. Lake
AU  - Xin Bai
AU  - Rameshu Rallabandi
AU  - Jiwoong Kim
AU  - Yang Xie
AU  - Jef K. De Brabander
AU  - David G. McFadden
TI  - TK216 targets microtubules in Ewing sarcoma cells
AID  - 10.1101/2021.11.19.469182
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.11.19.469182
4099  - http://biorxiv.org/content/early/2021/11/19/2021.11.19.469182.short
4100  - http://biorxiv.org/content/early/2021/11/19/2021.11.19.469182.full
AB  - 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 EWS patients. 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 α-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.Competing Interest StatementThe authors have declared no competing interest.