Partitioning of cancer therapeutics in nuclear condensates

Abstract

The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy.

Fig. 1.

Nuclear condensates in human tissue and in vitro. (A) Model illustrating potential behaviors of small molecules in nuclear condensates. (B–C) Immunofluorescence of scaffold proteins of various nuclear condensates in tissue biopsies from benign and malignant human breast (B), and benign and malignant colon tissue (C), in nuclei stained with Hoechst, imaged at 100× on a fluorescent confocal microscope (see also Figures S1, S2). (D) Schematic of in vitro droplet formation assay to measure small molecule partitioning into nuclear condensates. (E) In vitro droplet assay showing the behavior of fluorescein dye in the presence of six protein condensates formed in 125mM NaCl and 10% PEG, with 10μM protein and 5μM fluorescein, imaged at 150× on a confocal fluorescent microscope (see also Figures S3–S6). Quantification of enrichment of the drug is shown to the right, error bars represent SEM.

Fig. 2.

The partitioning behavior of small molecule drugs in nuclear condensates in a droplet assay. Six nuclear condensates formed in 125mM NaCl and 10% PEG, with 10μM protein treated with either (A) 5μM Cisplatin-TMR, (B) 50 μM Mitoxantrone, (C) 100μM FLTX1, (D) 5μM THZ1-TMR, or (D) 1μM JQ1-ROX imaged at 150× on a confocal fluorescent microscope (see also Figures S7–S11). Quantification of enrichment of the drug within droplets is shown to the right of each panel, error bars represent SEM (see also S12–S14).

Fig. 3.

Small molecule concentration within condensates influences drug activity. (A) In vitro droplet assay of MED1 and HP1α condensates formed in 125mM NaCl and 10% PEG, 5nM of 450bp DNA, 10μM MED1, and 5μM cisplatin-TR, imaged at 150× on a confocal fluorescent microscope (see also Figure S15). (B) Bioanalyzer tracings of DNA contained within either MED1 or HP1α droplets exposed to the indicated concentration of cisplatin. (C) (Top) Schematic of an assay to determine the location of platinated DNA relative to various nuclear condensates. (Bottom) Co-immunofluorescence of platinated DNA and the indicated protein in HCT116 cells treated with 50μM cisplatin for 6 hours. Imaged at 100× on a confocal fluorescent microscope. Quantification of overlap shown to the right. (D) (Top) Schematic of a live cell condensate dissolution assay. (Bottom) HCT116 cells bearing endogenously mEGFP-tagged MED1, HP1α, or FIB1 treated with 50μM cisplatin for 12 hours. Quantification of MED1, HP1α, or FIB1 condensate score is shown to the right. (E) MED1 ChIP-seq in HCT116 cells treated with vehicle or 50μM cisplatin for 6 hours. (Left) Plotted are mean read density of MED1 at super-enhancers and typical-enhancers (error bars show min and max) and (Right) gene tracks of MED1 ChIP-Seq at the MYC super-enhancer and AQPEP typical-enhancer. (F) Metaplot of cisplatin-DNA-Seq in cisplatin treated Hela cells comparing super-enhancers and typical enhancers (41) (see also Figures S16–S21).

Fig. 4.

Tamoxifen action and resistance in MED1 condensates. (A) Schematic showing tamoxifen resistance by ER mutation and MED1 overexpression in breast cancer. (B) In vitro droplets assay of the indicated form of GFP-tagged ER in the presence of estrogen, +/− 100μM tamoxifen. Droplets are formed in 125mM NaCl and 10% PEG with 10μM each protein and 100μM estrogen. (C) (Left) Immunofluorescence of MED1 in tamoxifen sensitive (MCF7) and resistant (TAMR7) ER+ breast cancer cell lines imaged at 100× on a confocal fluorescent microscope. (Top right) Quantification of MED1 condensate size in breast cancer cells. (Bottom right) Relative quantities of MED1 in the indicated breast cancer cell line by western blot, error bars show SEM. (D) In vitro droplets assays of ER in the presence of 100μM estrogen, +/− 100μM tamoxifen with either 5μM (Low) or 20μM (High) MED1. Droplets are formed with 5μM ER in 125mM NaCl and 10% PEG, imaged at 150× on a confocal fluorescent microscope, error bars are SEM. (E) In vitro droplet assay with either 5μM (Low) or 20μM (High) MED1 with 100μM FLTX1 in 125mM NaCl and 10% PEG, error bars are SD. (F) Models for tamoxifen resistance due to altered drug affinity (via ER mutation) or concentration (via MED1 overexpression) (see also Figures S22–S30).