Rewiring cancer drivers to activate apoptosis

Abstract

Genes that drive the proliferation, survival, invasion and metastasis of malignant cells have been identified for many human cancers^1-4. Independent studies have identified cell death pathways that eliminate cells for the good of the organism^5,6. The coexistence of cell death pathways with driver mutations suggests that the cancer driver could be rewired to activate cell death using chemical inducers of proximity (CIPs). Here we describe a new class of molecules called transcriptional/epigenetic CIPs (TCIPs) that recruit the endogenous cancer driver, or a downstream transcription factor, to the promoters of cell death genes, thereby activating their expression. We focused on diffuse large B cell lymphoma, in which the transcription factor B cell lymphoma 6 (BCL6) is deregulated⁷. BCL6 binds to the promoters of cell death genes and epigenetically suppresses their expression⁸. We produced TCIPs by covalently linking small molecules that bind BCL6 to those that bind to transcriptional activators that contribute to the oncogenic program, such as BRD4. The most potent molecule, TCIP1, increases binding of BRD4 by 50% over genomic BCL6-binding sites to produce transcriptional elongation at pro-apoptotic target genes within 15 min, while reducing binding of BRD4 over enhancers by only 10%, reflecting a gain-of-function mechanism. TCIP1 kills diffuse large B cell lymphoma cell lines, including chemotherapy-resistant, TP53-mutant lines, at EC₅₀ of 1-10 nM in 72 h and exhibits cell-specific and tissue-specific effects, capturing the combinatorial specificity inherent to transcription. The TCIP concept also has therapeutic applications in regulating the expression of genes for regenerative medicine and developmental disorders.

Extended Data Fig. 1 |. Potency of TCIP1 in cancer cell lines and correlation with BCL6 level.

a. Comparison of TCIP1 effect on cell viability to effect of negative controls Neg1 and Neg2, or single-sided molecules JQ1 and BI3812, or the additive effect of JQ1+BI3812. mean±s.d., 72 h drug treatment. b. TCIP1 EC₅₀ of cell viability is anti-correlated with BCL6 content across 14 different cancer cell lines, p-values computed by Students’ t-test, two-sided, not adjusted for multiple comparisons. For a,b: n = 4 biological replicates with 3 technical replicates each, mean±s.d 72 h drug treatment. c. Measurement of BCL6, BRD4, p53 and BCL2 status of DLBCL cell lines ranked from left to right from high to low-BCL6 protein content. d. Unbiased screen of the effect of TCIP1 on the viability of 906 barcoded cancer cell lines (PRISM). Drug was dosed for 120 h in triplicate (Methods).

Extended Data Fig. 2 |. Rescue of TCIP1-induced cell death by competitive titration of BCL6 inhibitors.

a. Rescue of TCIP1-induced cell death across cancer cell lines that are highly sensitive to TCIP1, b. moderately sensitive, or c. not at all sensitive. d. Comparison of JQ1, TCIP1, and Neg2, which contains a functional BRD4 inhibitor but very low-affinity BCL6 binder (K_D ~ 10 μM) in e. cell lines that have low or no BCL6. For a,b,c, e: n = 3 biological replicates, mean±s.d. Viability curves in a, b, c, and e are after 72 h drug treatment.

Extended Data Fig. 3 |. Biochemical studies of ternary complex binding affinities of TCIPs.

a. Ternary complex formation by TCIPs with related chemistries. TCIP1 plotted on every graph as a comparison. Each point represents an independent replicate which is the mean of 3 technical repeats, mean value line drawn. b. Isothermal calorimetry experiments to measure binary affinities of TCIP1 to BRD4^BD1, BCL6^BTB, and associated controls. Representative data from 1-2 independent experiments shown. c. Representative biolayer interferometry measurements (BLI) of ternary complex kinetics from 3 independent replicates shown with biotinylated BCL6^BTB on the tip and excess BRD4^BD1 in the well with titration of TCIP1. d. Off-rate and e. half-life of TCIP1 calculated from BLI dissociation curve measurements, 7-8 different doses for each of n = 3 independent replicates, mean±s.d. f. Area under curve of TR-FRET correlates with potency of TCIPs on cell death (KARPAS422 cells, viability at 72 h). Representative cellular EC₅₀s labeled, mean of 4 biological replicates. Each area under the curve point represents an independent replicate which is the mean of 3 technical repeats of the TR-FRET experiment.

Extended Data Fig. 4 |. TCIP1 induces apoptosis at every stage of the cell cycle.

a. Dose-dependent induction of apoptosis at 24 h by TCIP1 as measured by AnnexinV-positive cells. b. Kinetics of TCIP1-induced apoptosis in KARPAS422 cells. For a, b: n = 2-6 biological replicates, mean(±s.d) shown as appropriate. c. Design of assay to measure cell cycle progression simultaneously with apoptosis using Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. d. TCIP1 induction of cell cycle arrest compared to controls, representative of 2 biological replicates, see Extended Data Fig. 5 for both replicates’ flow cytometry graphs. e. 100 nM TCIP1 induction of apoptosis as measured by DNA fragmentation at each stage of the cell cycle, n = 2 biological replicates, mean shown. f. Measurement of cell viability after cell cycle arrest in G0/G1 by serum starvation in SUDHL5 cells and TCIP1 addition, n = 3 biological replicates, mean±s.d.

Extended Data Fig. 5 |. Cell-cycle block and apoptosis induction by TCIP1.

a. 100 nM TCIP1 addition at 24 h and simultaneous measurement of cell cycle block and apoptosis in KARPAS422 cells, two separate experiments on different passages of cells shown. Gates were set based on no-stain controls detailed in the Supplementary information.

Extended Data Fig. 6 |. Robust and dose-dependent gene regulation by TCIP1.

a. Principal component analysis of RNA-seq data after addition of TCIP1 for 20 h in 2 biological replicates of KARPAS422 cells. b. Gene expression changes after addition of 100 nM TCIP1 for 20 h in KARPAS422 cells. Adjusted p-values computed by two-sided Wald test and adjusted for multiple comparisons by Benjamini-Hochberg. Significance cutoffs were p_adj ≤ 0.05 and |log₂(Drug/DMSO)| ≥ 1), n = 2 biological replicates. c. Dose-dependent change in gene expression. d. Enrichment analysis of upregulated genes (MSigDB Hallmark Pathways). e. Analysis of TF binding at the top upregulated genes in over 4,500 public transcription factor ChIP-seq datasets from blood-lineage cells. For d, e: adjusted p-values computed by two-sided Fisher’s exact test and adjusted for multiple comparisons by Benjamini-Hochberg.

Extended Data Fig. 7 |. Specific activation of gene expression by TCIP1 but not related controls.

a. Gene expression changes after 1 h or 4 h addition of 10 nM TCIP1 in KARPAS422 cells. Changes at 2 h was shown in Fig. 4a. Adjusted p-values computed by two-sided Wald test and adjusted for multiple comparisons by Benjamini-Hochberg. Significance cutoffs were p_adj ≤ 0.05 and |log₂(Drug/DMSO)| ≥ 0.5), n = 3 biological replicates. b. Specific effects of TCIP1 across transcriptome. For Neg1 and Neg2, n = 2 biological replicates. For TCIP1, n = 3 biological replicates. c. Enrichment analysis of upregulated and downregulated genes (MSigDB Hallmark Pathways). Adjusted p-values computed by two-sided Fisher’s exact test and adjusted for multiple comparisons by Benjamini-Hochberg.

Extended Data Fig. 8 |. ChIP-seq analyses of BRD4, H3K27ac, and RNA Pol II in response to TCIP1.

a. PCA plots of each ChIP-seq experiment at indicated timepoints of 10 nM TCIP1 addition: 0hr (DMSO), 15 min, 1 h, 2 h, and 4 h. b. Browser tracks of Pol II Ser2 phos, Pol II Ser5 phos, H3K27ac, and BRD4 at BCL6-target genes and TCIP1-upregulated genes FOXO3 and BCL2L11/BIM. c. Volcano plots of Pol II ser 2 phos, Pol II ser 5 phos, and H3K27ac after 2 h 10 nM TCIP1 addition. Adjusted p-values computed by two-sided Wald test and adjusted for multiple comparisons by Benjamini-Hochberg. Peaks were classified as differential after reads in peaks-based regulative log expression (RLE) normalization and cutoffs p_adj ≤ 0.05 and |log₂(Drug/DMSO)|≥0.5. d. Enhancer and super-enhancer classification in KARPAS422 cells based on H3K27ac ChIP-seq and the ROSE algorithm (Methods). e. BRD4 and H3K27ac ChIP-seq track at the known OCA-B super-enhancer after TCIP1 addition for indicated timepoints. In b, e, Pol II Ser2 phos, Pol II Ser5 phos, and H3K27ac tracks in are spike-in- and input-normalized, BRD4 tracks are sequence-depth- and input-normalized.

Extended Data Fig. 9 |. Conversion of BCL6 auto-inhibitory pathway to feedforward loop.

a. Control Neg1 and Neg2 effect on BCL6 protein levels at 20 h treatment in KARPAS422 cells. b. Effect on BRD4 levels at 20 h treatment with TCIP1 in KARPAS422 cells. c. Kinetics of BCL6 upregulation in two separate DLBCL cell lines, KARPAS422 and SUDHL5, after addition of 10 nM TCIP1. Blots in a–c representative of 2 biological replicates (for SUDHL5) or 3 (for KARPAS422). d. Model for conversion of BCL6 auto-inhibitory circuit to a positive feedback loop.

Fig. 1 |. Production of TCIPs.

a, An endogenous target gene is activated or repressed using a bivalent molecule binding one endogenous transcription factor (TF) or epigenetic regulator on one side, chemically linked to a moiety that binds to a second transcription factor that binds to the regulatory region of a target gene, which might induce production of a therapeutic gene. b, A specific TCIP that recruits a transcriptional activator (BRD4) or cancer driver to the BCL6 repressor on cell death genes, thereby derepressing transcription and inducing transcription driven by BCL6. c, Chemical structures of the most potent BCL6–BRD4 TCIP, TCIP1 and the negative controls Neg1 (BRD4 non-binding) and Neg2 (BCL6 non-binding). d, TCIP1 effect on cell viability of the chemotherapy-resistant, TP53-mutant DLBCL cell line KARPAS422, as well as three other DLBCL cell lines with high levels of BCL6. n = 4 biological replicates, mean ± s.d. e, Design and activation of a BCL6 reporter with TCIP1 in KARPAS422 cells at 8 h after drug addition. n = 4 biological replicates, mean ± s.d. minP, minimal promoter. f, Comparison of TCIP1 effect on cell viability with the effect of BRD4 or BCL6 degraders (n = 3 biological replicates, mean ± s.d). Viability curves in d and f are after 72 h of drug treatment.

Fig. 2 |. TCIP1 functions by inducing ternary complex formation.

a, Competitive titration of BI3812 against TCIP1. TCIP1 was added at concentrations from 2 to 64 nM that killed 90% of SUDHL5 DLBCL cells at the same time as addition of the indicated concentrations of BI3812. n = 3 biological replicates, mean ± s.d., 72 h of drug treatment. b, Competitive titration of JQ1 against TCIP1. n = 3 biological replicates, mean ± s.d, 72 h of drug treatment. c, TR-FRET assay to measure molecule-dependent ternary complex formation between BRD4(BD1) and BCL6(BTB). Plotted are a representative set of TCIPs that were the most potent (in cell viability assays) within each category of linker structure. TCIP1 had the highest potency of all designed molecules. Each point represents an independent replicate, which is the mean of three technical repeats; the mean value line is drawn. d, Analysis of cooperative binding induced by TCIP1 and the BRD4(BD1) and BCL6(BTB) domains. A representative ternary complex K_d measurement by isothermal calorimetry is shown. For binary measurements, see Extended Data Fig. 3b. n = 3 independent replicates, mean ± s.d. Isothermal calorimetry parameters shown are 20:1 BRD4(BD1):TCIP1 in the cell and titration of BCL6(BTB). For biolayer interferometry, measurements were with 50 μM excess BRD4(BD1) in the well, nanomolar titrations of TCIP1 and biotinylated BCL6(BTB) on the tip. n = 3 independent replicates. The points and error bars are mean ± s.e. The K_d value is mean ± s.d. ΔH, enthalpy; ΔS, entropy; k_on, on-rate of binding; k_off, off-rate of binding. e, Multiple BRD4–BCL6 TCIPs synthesized with different linkers to test the structure–activity relationship. f, Effect of favourable in vitro ternary complex formation (represented by TR-FRET area under the curve) on the transcriptional activation of the BCL6 reporter in DLBCL cells. g, Effect of favourable intracellular ternary complex formation (represented by nanoBRET EC₅₀) on the transcriptional activation of the BCL6 reporter in DLBCL cells.

Fig. 3 |. TCIP1 represses MYC and its targets while activating pro-apoptotic genes.

a, Gene activation (median change: fourfold up) and repression after addition of 10 nM TCIP1 in KARPAS422 cells for 20 h, with well-known BCL6 targets labelled. Significance cut-offs were P_adjusted ≤ 0.05 and |log₂(drug/DMSO)| ≥ 1); n = 2 biological replicates. b, Downregulated genes are significantly enriched for MYC targets (MSigDB hallmark pathways). c, Analysis of transcription factor binding of the top 100 downregulated genes in 4,500 or more public ChIP–seq datasets in blood-lineage cells. For b,c, the adjusted P values were computed by two-sided Fisher’s exact test and adjusted for multiple comparisons by Benjamini–Hochberg. d, Kinetics of protein changes in MYC, p21 and FOXO3 in DLBCL cell lines after treatment with 10 nM TCIP1. e, Dose-dependent changes in protein levels of target genes selected from the RNA sequencing results in two separate DLBCL cell lines, KARPAS422 and SUDHL5. f, Negligible effect of the negative controls Neg1 and Neg2 on protein levels of TCIP1 targets. For d–f, blots are representative of two biological replicates, except KARPAS422 in d and in e where data represent three biological replicates. g, Rescue of p21 and FOXO3 upregulation and MYC downregulation by competitive titration of the BTB binder BI3812 against constant 10 nM TCIP1 treatment for 8 h. Representative of two biological replicates. In all blots in d–g, any markers immunoblotted for the same gel are followed immediately by the loading control GAPDH run on that same gel.

Fig. 4 |. Rapid activation of BCL6 target genes by recruitment of BRD4.

a, Gene expression changes after 10 nM TCIP1 for 2 h in KARPAS422, with well-known BCL6 targets labelled. P values were computed by a two-sided Wald test and adjusted for multiple comparisons by Benjamini–Hochberg. Differential gene cut-offs: P_adjusted ≤ 0.05 and |log₂(drug/DMSO)| ≥ 0.5; n = 3 biological replicates. b, Changes in gene expression after 1, 2 and 4 h of 10 nM TCIP1 compared with Neg1 and Neg2. c, Enrichment analysis of transcription factor binding in promoters of genes upregulated at 2 h after TCIP1 treatment in more than 4,500 public ChIP–seq datasets in blood-lineage cells. P values were computed by a two-sided Fisher’s exact test and adjusted for multiple comparisons by Benjamini–Hochberg. d, BRD4 density in KARPAS422 cells at BCL6 summits after 1 h of 100 nM TCIP1. e, Time-dependent density of Pol II Ser2 phos, Pol II Ser5 phos and H3K27ac along gene bodies that are ±3 kb after 10 nM TCIP1, at differential genes identified by 2 h of RNA sequencing in a. TES, transcription end site; TSS, transcription start site. f, BRD4 density at differential genes, as in e, and enhancers and super-enhancers identified by H3K27ac (Methods). Metaprofiles and shading in e represent mean ± s.e. from spike-in-normalized and input-normalized ChIP–seq data, and in f represent mean ± s.e. from sequence-depth-normalized and input-normalized ChIP–seq data. g, ChIP–seq tracks at PMAIP1 after addition of 10 nM TCIP1. h, Tracks at the BCL6 locus, with alternative transcripts shown. SE, super-enhancer. Pol II Ser2 phos, Pol II Ser5 phos and H3K27ac tracks in g,h are spike-in- and input-normalized, and BRD4 tracks are sequence-depth- and input-normalized. i, Structures of BCL6 isoforms. ZF, zinc finger. j, mRNA of long and short isoforms of BCL6 (BCL6L and BCL6S, respectively), measured by quantitative PCR with reverse transcription by primers specific to isoform-unique exon–exon junctions (shown by arrowheads in i). n = 3 biological replicates, mean ± s.d. P values were calculated by a two-tailed, unpaired Student’s t-test. k, Induction of the BCL6L isoform by 1 nM or less TCIP1. l, Simultaneous treatment of 10 nM of TCIP1 and 100 nM of the CDK9 inhibitor (CDK9i) NVP2 to block elongation. m, Competitive titration of BI3812 against 10 nM TCIP1. The blots in k–m are representative of two biological replicates.

Fig. 5 |. Toxicity of TCIP1 in mice and primary human cells and generalization to ER-positive cancers.

a, Tissue-specific transcriptomic effects of TCIP1, treated at 10 mg kg⁻¹ intraperitoneal (i.p.) once daily for 5 days. b, Quantification of transcriptome changes in the liver, lung and spleen and associated accumulated tissue concentrations of TCIP1. Treatment at 10 mg kg⁻¹ TCIP1 intraperitoneal once daily, with measurement on day 5. n = 3 mice per treatment. c, Pharmacokinetic parameters of TCIP1. t_1/2, half-life; t_max, time to max serum concentration; C_max, maximum serum concentration; AUC_0-last, area under the curve from dosing to last measured concentration. d, Comparison of key gene targets upregulated by TCIP1 in both cultured DLBCL cells (KARPAS422) and in the spleen. e, Body weight of treated mice. No adverse effects or behavioural abnormalities were noticed. f, Haematoxylin and eosin staining of the lung and spleen from representative mice treated with vehicle and drug. Scale bars, 50 μm (lung images) and 100 μm (spleen images). n = 3 mice each for treatment and vehicle for a–f. g, Effect of TCIP1 on cell viability of primary human tonsillar lymphocytes. h, Effect of TCIP1 on cell viability of primary human fibroblasts. i, ER-BCL6 TCIP2 designed to induce cell death in oestrogen-positive, BCL6-overexpressing DLBCLs. j, Chemical structure of TCIP2. k, Effect on cell viability of TCIP2 compared with controls: oestrone, BI3812 (a BCL6(BTB) inhibitor) and BI3802 (a BCL6 degrader) in KARPAS422 cells with high ERβ (encoded by ESR2) levels. l, Measurement of the selective effect on cell viability by TCIP2 in DLBCL cells with coincident overexpression of ER and BCL6 (KARPAS422) compared with primary human lymphocytes, a triple-negative breast cancer cell line (HS578T) and ER-driven but BCL6-low breast cancer cells (HCC1428). CCLE, Cancer Cell Line Encyclopedia. n = 3 biological replicates, mean ± s.d. for g,h,k,l. Viability curves in g,h,k,l are after 72 h of drug treatment.