Click chemistry enables preclinical evaluation of targeted epigenetic therapies

Abstract

The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.

Fig. 1. Clickable compounds phenocopy the parental compounds.

(A) Molecular structures of BET inhibitors and clickable and biotinylated surrogates. (B) Proliferation of MV4;11 cells after 72 hours of treatment, comparing JQ1 versus JQ1–PA. Mean ± SD (error bars) (n = 3 xxxxxxxxxxx). IC₅₀, median inhibitory concentration. (C) Apoptosis assessed by FACS (fluorescence-activated cell sorting) analysis after 72 hours of incubation with dimethyl sulfoxide (DMSO), JQ1 (1 μM), or JQ1–PA (1 μM). PI, propidium iodide. (D) Cell cycle profile of MV4;11 cells after 48 hours of treatment with DMSO, JQ1, JQ1–PA, or JQ1–TCO (all compounds used at 500 nM). Mean ± SD (error bars) (n = 3). (E) qPCR analysis of BRD4 ChIP from MV4;11 cells treated with JQ1 (1 μM) compared with JQ1–PA (1 μM) or JQ1–TCO (1 μM), with primers against MYC. Mean ± SD (error bars), representative graph from three independent experiments. (F) RNA sequencing (RNA-seq) performed in MV4;11 cells treated with JQ1 or JQ1–PA compared with DMSO control. Correlation of log2 fold change of JQ1–PA with log2 fold change of JQ1 is shown. Significantly up- and down-regulated genes are depicted in red and blue, respectively. LFC, xxxxxxxxxxxxxxxxxx.

Fig. 2. Click chemistry reveals insights into the binding of BRD4 to chromatin.

(A) Schematic illustration of click-seq and click-proteomics experiments with modified JQ1 and IBET molecules. (B) Heat map of BRD4 ChIP-seq and JQ1–PA click-seq sequencing reads in the 5 kb around the TSS. JQ1 is included as a negative control. (C) Genome browser view of the MYC enhancer, comparing BRD4 ChIP-seq with click-seq using IBET-762–TCO and JQ1–TCO molecules, with competition from unmodified IBET-151 and JQ1. (D) Genes down-regulated or up-regulated after BET inhibitor treatment for 6 hours, assessed for drug occupancy with JQ1-PA click-seq. RPM, reads per million. (E) Genome browser view of two genomic regions with low and high levels of JQ1–PA relative to BRD4 with C/EBPα and C/EBPβ ChIP-seq. (F) Quantitative mass spectrometry of proteins from the lysate of K562 cells captured by click-probes (IBET-762–TCO and JQ1–TCO) in the presence or absence of the respective competitor (IBET-151 and JQ1). Correlation of log2 fold change of abundance of protein captured in the presence of inhibitor relative to vehicle. Circle size represents the quantity of protein from the mass spectrometer.

Fig. 3. Clickable compounds can be visualized and quantified in vitro.

(A) Schematic illustration of the methodology for click fluorescence and flow cytometry. (B) Confocal microscopy of HeLa cells treated with IBET-762–TCO or JQ1–TCO. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), drug was labeled with Cy5–tetrazine, and BRD4 was stained with an anti-BRD4 antibody. Scale bars, 20 μm. (C) MV4;11 cells treated with JQ1 or increasing concentrations of JQ1–PA, followed by click labeling with 488–azide. Flow cytometry analysis is represented as a histogram.

Fig. 4. Preclinical assessment of clickable compounds in vivo.

(A) Schematic illustration of the methodology to detect the clickable small molecules in vivo. (B) Flow cytometry analysis of Venus⁺ sorted cells from the bone marrow and spleen of a mouse treated with JQ1–TCO for 3.5 hours. (C) Flow cytometry analysis of drug levels within normal hematopoietic cells (Venus⁻) and leukemia cells (Venus⁺). (D) Confocal microscopy of mouse femur tissue treated with 100 mg/kg of JQ1–TCO. Leukemia cells (LC) are identified by Venus reporter. The high magnification of the merged image (at right) reveals the ability to discern individual leukemia cells containing drug in situ. Scale bars, 187 μm. All data demonstrated here are representative examples of experiments performed in biological duplicate.