Bifunctional Small Molecules That Induce Nuclear Localization and Targeted Transcriptional Regulation

Abstract

The aberrant localization of proteins in cells is a key factor in the development of various diseases, including cancer and neurodegenerative disease. To better understand and potentially manipulate protein localization for therapeutic purposes, we engineered bifunctional compounds that bind to proteins in separate cellular compartments. We show these compounds induce nuclear import of cytosolic cargoes, using nuclear-localized BRD4 as a "carrier" for co-import and nuclear trapping of cytosolic proteins. We use this system to calculate kinetic constants for passive diffusion across the nuclear pore and demonstrate single-cell heterogeneity in response to these bifunctional molecules with cells requiring high carrier to cargo expression for complete import. We also observe incorporation of cargo into BRD4-containing condensates. Proteins shown to be substrates for nuclear transport include oncogenic mutant nucleophosmin (NPM1c) and mutant PI3K catalytic subunit alpha (PIK3CA^E545K), suggesting potential applications to cancer treatment. In addition, we demonstrate that chemically induced localization of BRD4 to cytosolic-localized DNA-binding proteins, namely, IRF1 with a nuclear export signal, induces target gene expression. These results suggest that induced localization of proteins with bifunctional molecules enables the rewiring of cell circuitry, with significant implications for disease therapy.

Figure 1

Rapid import of GFP into the nucleus by AP1867-PEG2-JQ1 (NICE-01). (A) Schematic of the Nuclear Import and Control of Expression (NICE) concept. A bifunctional molecule allows import of a cytosolic protein of interest into the nucleus. (B) Structure of AP1867-PEG2-JQ1. (C) U2OS cells co-transfected with mCherry-BRD4 and FKBP^F36V-mEGFP, treated with NICE-01 (200 nM) or DMSO, and imaged for 40 min. (D) 293T cells stably expressing FKBP^F36V-mEGFP, treated with NICE-01 (250 nM) or DMSO, and imaged for 3 h. (E) 293T cells stably expressing FKBP^F36V-mEGFP and co-treated with NICE-01 (250 nM) and either JQ1 or AP1867 at the indicated concentration and imaged for 15 min. (F) 293T cells stably expressing FKBP^F36V-mEGFP and transfected with mCherry-BRD4, and either untreated or treated with NICE-01 (250 nM, 10 μM), and imaged for 15 min. White arrows indicate representative cells exhibiting the hook effect. Red arrow indicates representative cell with mCherry-BRD4 expression that does not exhibit hook effect. (G) 293T cells stably expressing FKBP^F36V-mEGFP and transfected with mCherry-BRD4 and treated with NICE-01 (250 nM) and imaged for 50 min. 100 mCherry+ and mCherry– cells were randomly selected and assessed for complete import of mEGFP (cellular nuclear/cytosolic mEGFP intensity ≥2.5). P values were calculated by Fisher’s exact test.

Figure 2

Import of cytosolically localized NPM1c into nuclear BRD4 condensates upon addition of bifunctional molecule NICE-01. (a) 293T cells co-transfected with FKBP^F36V-EGFP-NPM1c and mCherry-BRD4 before (top) and 25 min after (bottom) NICE-01 treatment (250 nM). (b) Box plot of cellular mEGFP-mCherry pixel intensity correlation, before and 25 min after NICE-01 treatment (250 nM, P values calculated from Mann–Whitney U test). Each dot represents one cell. (c) Histograms of relative log₂(integrated mEGFP cellular intensity/integrated mCherry cellular intensity) for cells with co-localized mEGFP and mCherry (mEGFP-mCherry pixel intensity correlation coefficient: R ≥ 0.75) or non-co-localized (mEGFP-mCherry pixel intensity correlation coefficient: R < 0). P values calculated from the Kolmogorov–Smirnov test. (d) High-resolution images of U2OS cells co-transfected with FKBP^F36V-EGFP-NPM1c and mCherry-BRD4 before (top) and 55 min after (bottom) NICE-01 treatment (250 nM). (e) Image of two condensates fusing from panel d; arrows highlight two separate condensates fusing into one. (f) Image of condensate undergoing fission from panel d; arrows highlight condensate fission.

Figure 3

NPM1c exports p53 out of the nucleus and fails to import p53 upon the addition of NICE-01. (a) 293T cells imaged following mono-transfection or co-transfection with FKBP^F36V-mEGFP-NPM1c and Halo-p53^R273H-mCherry. (b) RNAi co-dependency analysis of NPM1 across DepMap. (c) CRISPR and RNAi TP53 dependency score in AML cell lines across DepMap, colored by TP53/NPM1 mutation status. (d) NPM1 and TP53 comutation plot across OHSU acute myeloid leukemia cohort from cBioPortal. (e) 293T cells co-transfected with FKBP^F36V-mEGFP-NPM1c, BRD4-Flag, and Halo-p53^R273H-mCherry, treated with NICE-01 (250 nM) and imaged for 90 min. Top right cell displays FKBP^F36V-mEGFP-NPM1c import but lack of p53 co-import.

Figure 4

Import of cytosolic-localized transcription factor into the nucleus drives transcription. (A) 293T cells transfected with FKBP^F36V-mEGFP-IRF1-NES and treated with NICE-01 (250 nM) or JQ1-PEG2-NH2 + AP1867 (binders, 250 nM each) and subject to RNA sequencing. Volcano plot showing upregulated genes following treatment with 250 nM NICE-01 versus binders; genes in red are IRF1 ChIP-seq targets. (b) Hallmark preranked gene set enrichment analysis of cells treated with NICE-01 versus binders. Top ranked positively enriched gene sets are shown.