The Design and Characterization of Bifunctional Degraders of TRIM24, a Novel Transcriptional Dependency in Hematologic Malignancies
Gechijian, Lara N.
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AbstractTranscriptional control is critical to the proper functioning of cells, their growth, differentiation, and survival. Because genomic and epigenomic alterations can deregulate transcription and promote an oncogenic state, targeting transcription has become a rich area for therapeutic research. There has been limited success targeting transcription factors with small organic molecules because many transcriptional regulators are not amenable to conventional therapeutic approaches, as their ligandable domain may not be functionally relevant in disease. Targeted protein degradation has recently emerged as a strategy to inactivate the entire multifunctional protein by inducing the association of a protein of interest with the cellular E3 ubiquitin ligase machinery, promoting proteasome-mediated degradation of the target. In this study, we employ the strategy of protein degradation to target TRIM24, a multifunctional transcriptional regulator, because potent ligands of its bromodomain do not exert antiproliferative effects in contexts of TRIM24 genetic dependence. We probed the Project Achilles, a genome-wide loss-of-function screen, and discovered a novel dependence on TRIM24 in hematologic malignancies. By employing CRISPR-Cas9 mutagenesis across the TRIM24 locus, we found that the RING domain, and not the chemically tractable bromodomain, appeared responsible for this dependency. We repurposed the potent ligands of the TRIM24 bromodomain as the TRIM24 targeting-ligand component of heterobifunctional degraders to orchestrate the recruitment of TRIM24 to the E3 ubiquitin ligase machinery. We explored chemical parameters to optimize conditions for degradation, including varying the TRIM24 targeting ligands, E3 ligase recruited, and conjugation strategy between the two targeting ligands. We achieved robust VHL-dependent degradation of TRIM24, selectively across the proteome. With both a TRIM24 degrader and a bromodomain inhibitor, we performed comparative studies to probe the function of TRIM24 in leukemia. We found that the degrader induces marked transcriptional deregulation in the leukemia lines sensitive to TRIM24 depletion, and chromatin displacement of TRIM24 from its target genes. Importantly, dTRIM24 induced chemical depletion of TRIM24 that mimics genetic depletion in several cancer contexts, providing our community with a robust tool that provides temporal precision and a method for chemical inactivation of this transcriptional regulator. More broadly, we delineate a therapeutic route to repurpose selective yet ineffectual ligands.
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