(P 02) A PRECLINICAL ASSAY PIPELINE TO IDENTIFY AND OPTIMIZE BRACHYURY-TARGETED THERAPIES IN CHORDOMA AND OTHER SOLID TUMORS

Objective: Chordoma is a rare bone cancer with no approved systemic therapy. A nearly universal hallmark of chordoma is the expression of TBXT, which encodes the T-box transcription factor Brachyury. Functional genomics studies identified TBXT as the most selectively essential gene in chordoma cells, and TBXT appears to be minimally expressed in healthy adult tissues – making it an attractive therapeutic target. Indeed, the Structural Genomics Consortium (SGC) recently solved the structure of Brachyury bound to DNA, identified new potential binding pockets, and generated multiple structure-guided small molecule series that bind to Brachyury with low micromolar affinity. Furthermore, Brachyury has been associated with tumor progression, chemotherapy resistance, and poor patient outcomes in other cancers including lung, colon, breast, and prostate – positioning Brachyury as an attractive drug target beyond chordoma. The objectives of this study are to establish a preclinical assay pipeline that enables the identification and optimization of Brachyury-targeted therapies and assess the role of Brachyury in the progression and response to standard of care therapy in other solid tumor models.

Methods: Biotinylated Brachyury was purified by nickel affinity and gel filtration using a HiLoad 16/600 Superdex column on an Akta Prime Plus FPLC. Brachyury function was assessed using a DNA gel-shift assay with DNA oligomers containing the Brachyury response element in tandem. Small molecule binding was measured using surface plasmon resonance on a Biacore 8K. UM-Chor1 and Brachyury-positive H460 cells were transduced with a lentivector that expresses luciferase and/or GFP downstream of the Brachyury response element. U-CH1 chordoma cells and Brachyury-positive lung, colon, prostate, and triple-negative breast cancer cells were transduced with a lentivector expressing a doxycycline-inducible TBXT shRNA. Doxycycline-induced Brachyury knockdown was validated, along with two of its targets (YAP1, KRT8), using western blot, immunofluorescence, and/or RT-PCR. Cells were treated with or without doxycycline plus standard of care therapy, and EC50 values were determined using a four-parameter logistical model.

Results: We obtained highly-pure, biotinylated Brachyury that binds to DNA in a dose-dependent manner and validated a subset of the open-source SGC compounds that bind to Brachyury with low micromolar affinity. To assess whether Brachyury ligands are functional inhibitors, we developed a Brachyury reporter assay in UM-Chor1 and H460 cells that exhibit a density-dependent increase in luciferase secretion over time. An orthogonal RT-PCR assay to directly assess the expression of Brachyury targets showed that Brachyury knockdown in U-CH1 cells significantly reduces the expression of YAP1 and KRT8. Furthermore, Brachyury knockdown in other preclinical tumor models does not significantly impact cell proliferation but sensitizes non-small cell lung cancer cells to docetaxel and pemetrexed treatments.

Conclusion: Our preclinical assay pipeline enables the identification, optimization, and validation of Brachyury-targeted molecules to catalyze drug discovery for chordoma and other Brachyury-positive tumors. The biochemical and cell-based functional assays will prioritize ligands that directly inhibit Brachyury activity and cell proliferation, which can be tested further across a panel of chordoma cells and patient-derived xenograft models. Furthermore, our studies in other preclinical tumor models show that Brachyury knockdown in other indications such as non-small cell lung cancer can enhance their sensitivity to standard of care therapy and may provide a larger market size for Brachyury-targeted therapies.