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TK216 targets microtubules in Ewing sarcoma cells

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.

 

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The passage describes a study that investigated the mechanism of action of TK216, a potential treatment for Ewing sarcoma (EWS). Initially designed as a direct inhibitor of the EWSR1-FLI1 fusion protein, TK216 has shown efficacy against cancer cells and xenografts that do not express EWSR1-FLI1, raising questions about its cytotoxic mechanism.

The researchers employed a forward-genetics screening platform, utilizing hypermutation in EWS cell lines, to identify mutations associated with resistance to TK216. They discovered recurrent mutations in TUBA1B, which encodes the α-tubulin protein. These mutations were found to confer resistance to TK216, suggesting a role for α-tubulin in the drug's effectiveness.

To further understand the mode of action, the researchers conducted in vitro experiments involving reconstituted microtubule (MT) polymerization and cell-based chemical probe competition assays. The results indicated that TK216 functions as an MT destabilizing agent, disrupting the polymerization of microtubules.

The findings from this study shed light on the cytotoxicity mechanism of TK216, explaining why it exhibits anti-cancer activity beyond EWSR1-FLI1-expressing cells. The study also elucidates the synergy observed when TK216 is combined with vincristine, another drug that affects microtubule dynamics. Consequently, the researchers suggest reevaluating ongoing clinical trials with TK216 in light of these mechanistic insights.