Functional TRIM24 degrader via conjugation of ineffectual bromodomain and VHL ligands

Abstract

The addressable pocket of a protein is often not functionally relevant in disease. This is true for the multidomain, bromodomain-containing transcriptional regulator TRIM24. TRIM24 has been posited as a dependency in numerous cancers, yet potent and selective ligands for the TRIM24 bromodomain do not exert effective anti-proliferative responses. We therefore repositioned these probes as targeting features for heterobifunctional protein degraders. Recruitment of the VHL E3 ubiquitin ligase by dTRIM24 elicits potent and selective degradation of TRIM24. Using dTRIM24 to probe TRIM24 function, we characterize the dynamic genome-wide consequences of TRIM24 loss on chromatin localization and gene control. Further, we identify TRIM24 as a novel dependency in acute leukemia. Pairwise study of TRIM24 degradation versus bromodomain inhibition reveals enhanced anti-proliferative response from degradation. We offer dTRIM24 as a chemical probe of an emerging cancer dependency, and establish a path forward for numerous selective yet ineffectual ligands for proteins of therapeutic interest.

Figure 1. Design and chemical characterization of dTRIM24 as a degrader of TRIM24

(a) Crystal structure of TRIM24 PHD-BROMO bound by IACS-9571 in proximity to the crystal structure of VHL bound to VL-269 to model a strategy for building the degrader (PDB: 4YC9 and 4W9H). (b) Chemical structures of IACS-7e, IACS-9571, dTRIM24 and eTRIM24 with stereocenter of the VHL ligand component highlighted in red. (c) TRIM24 ligand displacement assay (values represent means normalized to DMSO calculated from n=2 technical replicates). n=3 independent experiments with one representative experiment shown. (d) dTRIM24 in vitro binding assay to panel of human bromodomain proteins by single point screening at 1 µM dTRIM24 in singlicate (BromoScan). (e) Cellular VHL degron displacement assay (values represent means normalized to 50 µM VL-269 calculated from n=2 technical replicates). n=2 independent experiments with one representative experiment shown. (f) Immunoblot of TRIM24 and Vinculin following 24 hours of incubation with the indicated concentrations of dTRIM24 in 293FT cells. (g) Immunoblot of TRIM24 and Vinculin following treatment of 293FT cells with 5 µM dTRIM24 for the indicated incubation times. For f–g, percentages were calculated by normalization of the band intensity to the loading control and relative to DMSO at each timepoint. n=3 independently conducted experiments with one representative experiment shown. Full immunoblots shown in Supplementary Fig. 9.

Figure 2. Characterization of the cellular mechanism of degradation of dTRIM24

(a) Immunoblot of TRIM24 and Vinculin following chemical competition with 5 µM of ligands IACS-9571 and VL-269 with co-treatment of 5 µM dTRIM24 for 24 hours in 293FT cells. (b) Immunoblot of TRIM24 and Vinculin following incubation with carfilzomib (500nM) or MLN4924 (1 µM) with co-treatment of 5 µM dTRIM24 for 24 hours in 293FT cells. (c) Immunoblot of TRIM24 and Vinculin following treatment with the indicated concentrations of dTRIM24 or eTRIM24 for 24 hours in 293FT cells. (d) Immunoblot of VHL and Vinculin in Clone 3 and Clone 10, expanded from single 293FT cells after transient transfection with a guide targeting the VHL locus for CRISPR/Cas9-mediated VHL knockout (antibody: Cell Signaling 68547). (e) Immunoblot of HIF-1α and Actin in Clone 3 and Clone 10. (f) Immunoblot of TRIM24 and Vinculin following treatment of Clone 3 or Clone 10 for 24 hours with the indicated concentrations of dTRIM24. For a–f, percentages were calculated by normalization of the band intensity to the loading control and relative to DMSO. n=3 independently conducted experiments with one representative experiment shown. Full immunoblots shown in Supplementary Fig. 9 and Supplementary Fig. 10.

Figure 3. Genetic dependency on TRIM24 in acute leukemia

(a) 216 cancer cell lines ranked by ATARiS score from TRIM24 genetic knockdown from the Achilles dataset. All hematopoietic and lymphoid tissue cell lines are indicated in red and selected cell lines are labeled (p-value determined by t-test). (b) CRISPR/Cas9-mediated mutagenesis tiling TRIM24 in MOLM-13 stably expressing Cas9. After transduction with a pool of all sgRNAs, fold change from Day 3 to Day 18 was calculated for each guide by next generation sequencing at both timepoints in singlicate.

Figure 4. Antiproliferative effect of selective TRIM24 degradation in acute leukemia

(a) Immunoblot for TRIM24 and Vinculin following 24 hours of incubation of MOLM-13 cells with the indicated concentrations of dTRIM24. (b) Immunoblot for TRIM24 and Vinculin following treatment of MOLM-13 cells with 2.5 µM dTRIM24 for the indicated incubation times. For a–b, percentages were calculated by normalization of the band intensity to the loading control and relative to DMSO at each timepoint. n=3 independently conducted experiments with one representative experiment shown. (c) Proteomic analysis in MOLM-13 cells treated for 4 hours with DMSO or 2.5 µM of IACS-9571. Fold-change in the abundance of 5897 proteins from IACS-9571 to DMSO treatment versus p-value (t-test; triplicate analysis). (d) As described in (c), with cells treated with 2.5 µM of dTRIM24. (e) Growth over time of MOLM-13 cells treated with 5 µM of indicated compounds over 7 days (values represent means +/− standard deviation, n=3 independently conducted experiments). (f) Induction of apoptosis in MOLM-13 cells indicated by PARP cleavage at 48 hours with dTRIM24, comparable to PARP cleavage induced by treatment with the positive control staurosporine (STS) for 4 hours. n=2 independently conducted experiments with one representative experiment shown. Full immunoblots shown in Supplementary Fig. 10.

Figure 5. Global displacement of TRIM24 from chromatin and transcriptional response to TRIM24 degradation

(a) Average genome-wide reads for TRIM24 centered on a ± 4 kb window around the TRIM24 peaks with the indicated treatment conditions (2.5 µM) for 24 hours. The y-axis shows ChIP-Rx signal (rpm/bp) and the x-axis indicates position. (b) ChIP-seq binding density of TRIM24 at exemplary asymmetrically loaded TRIM24 loci following the indicated treatments for 24 hours. The y-axis indicates ChIP-seq signal (rpm/bp) and the x-axis indicates genomic position. For a–b n=1 independently conducted experiment. (c) Gene expression change represented by Log2 fold change of transcripts versus Log10 p-value after 24 hours of 2.5 µM IACS-9571 treatment. Expression values were normalized to cell number using ERCC spike-in controls. (d) As described in (c), with dTRIM24 treatment. For c–d, data represents biological triplicates.