Relocalizing transcriptional kinases to activate apoptosis

Abstract

Kinases are critical regulators of cellular function that are commonly implicated in the mechanisms underlying disease. Most drugs that target kinases are molecules that inhibit their catalytic activity, but here we used chemically induced proximity to convert kinase inhibitors into activators of therapeutic genes. We synthesized bivalent molecules that link ligands of the transcription factor B cell lymphoma 6 (BCL6) to inhibitors of cyclin-dependent kinases (CDKs). These molecules relocalized CDK9 to BCL6-bound DNA and directed phosphorylation of RNA polymerase II. The resulting expression of pro-apoptotic, BCL6-target genes caused killing of diffuse large B cell lymphoma cells and specific ablation of the BCL6-regulated germinal center response. Genomics and proteomics corroborated a gain-of-function mechanism in which global kinase activity was not inhibited but rather redirected. Thus, kinase inhibitors can be used to context-specifically activate transcription.

Fig. 1.. Development of CDK-TCIPs.

(A) Schematic of a CDK-TCIP targeting BCL6-regulated loci. (B) Structure of lead compound CDK-TCIP1. (C) Structures of negative controls containing small chemical modifications abolishing BCL6 binding (Neg1) or CDK9 binding (Neg2). (D) Cell-killing potencies of CDK-TCIP1 compared with negative controls and the additive effect of both BCL6 and CDK9 inhibitors combined at 72 hours in SUDHL5 cells. Data are shown as means ± SE. n = 3 biological replicates for CDK-TCIP1, Neg1, Neg2, and n = 2 biological replicates for BI3812+SNS-032. (E) Correlation between genetic dependencies as measured by CRISPR knockout and sensitivity to CDK-TCIP1 among 859 cancer cell lines in PRISM (61). (F) Toxicity of CDK-TCIP1 (EC50 ~1.8 mM) compared with CDK9 inhibitors (SNS-032 EC50 ~150 nM, NVP2 EC50 ~7.1 nM) or degraders (THAL-SNS-032 EC50 ~15 nM) in primary human tonsillar lymphocytes at 72 hours. Data are shown as means ± SD. n = 2 biological replicates for CDK-TCIP1 and NVP2, and n = 1 with 3 technical replicates for SNS-032 and THAL-SNS-032.

Fig. 2.. CDK-TCIP1 functions by ternary complex formation and relocalization of CDK9 activity to BCL6 on chromatin.

(A) Correlation of ternary complex formation with cell-killing potency (72 hours in SUDHL5 cells) for CDK-TCIPs constructed from the CDK9 inhibitor SNS-032 and three different BCL6^BTB inhibitors. The adjacent “C#” denominates the number of carbon atoms in the linear alkyl linker. Points represent a mean of n = 3 technical replicates for TR-FRET. (B) Activation of BCL6-repressed GFP reporter construct integrated into KARPAS422 cells after compound treatment for 24 hours. Data are shown as means ± SD. n = 3 biological replicates. (C) Whole-proteome profiling of SUDHL5 cells treated with 30 nM CDK-TCIP1 for 2 hours plotted with cutoffs of |log2(fold change)| ≥ 0.75 and adjusted P ≤ 0.01 using a moderated t test and Benjamini-Hochberg adjustment. n = 4 biological replicates. (D) ChIP-seq measurement of CDK9 at BCL6 summits genome wide after 30 nM CDK-TCIP1 treatment for 2 hours in SUDHL5 cells. Summits of BCL6 enrichment computed from peaks were reconstructed from ChIP-seq in (62). (E) ChIP-seq measurement of CDK9 to genes that have BCL6 summits at their promoters. (F) Changes in local RNA Pol II Ser 2 phosphorylation as measured by ChIP-seq after 2 hours of 30 nM CDK-TCIP1 addition in SUDHL5 cells; colors indicate adjusted P ≤ 0.05 and |log2(drug/DMSO)| ≥ 0.5, n = 2 biological replicates. P values were computed by two-sided Wald test and adjusted for multiple comparisons using the Benjamini- Hochberg procedure. Labeled are known BCL6-target, cell cycle arrest, and pro-apoptotic genes. (G) ChIP-seq measurement of CDK9 after 30 nM CDK-TCIP1 treatment for 2 hours at differentially induced Pol II Ser 2 phos peaks classified in (F). (H) ChIP-seq tracks of CDK9, Pol II Ser 2 phos, Pol II Ser 5 phos, and Pol II at the BCL6-target gene CDKN1B. In (D), (E), and (G), hours, meta profiles, and tracks for CDK9 measured with Abcam’s ab239364 anti-CDK9 antibody are shown with two biological replicates merged, spike-in normalized, and input subtracted. For Pol II Ser 2 phos and Pol II Ser 5 phos, two biological replicates were merged, sequence depth normalized, and input subtracted. For Pol II, three biological replicates were merged, sequence-depth normalized, and input subtracted. The BCL6 track is from (62).

Fig. 3.. Activation of apoptotic signaling.

(A) Time-dependent changes in gene expression measured by mRNA sequencing after 30 nM CDK-TCIP1 addition in SUDHL5 cells compared with controls. Plotted are differential genes with adjusted P ≤ 0.05 and |log2(fold change)| ≥ 1. n = 3 to 4 biological replicates. P values were computed by two-sided Wald test and adjusted for multiple comparisons using the Benjamini-Hochberg procedure. (B) Gene expression programs enriched in up-regulated genes (adjusted P ≤ 0.05, fold change ≥1.5) at each time point defined from (A). P value of enrichment was computed by hypergeometric test and adjusted for multiple comparisons using the Benjamini- Hochberg procedure for FDR. (C) Acute effects on gene expression and known BCL6-target genes (labeled) caused by the addition of 30 nM CDK-TCIP1 for 2 hours. n = 3 to 4 biological replicates. P values were computed by two-sided Wald test and adjusted for multiple comparisons using the Benjamini-Hochberg procedure. (D) Dose-dependent apoptosis in SUDHL5 cells measured by annexin V staining after 24 hours of compound. n = 3 biological replicates. Data are shown as means ± SD.

Fig. 4.. Ablation of GC B cells in immunized mice.

(A) Structure of CDK-TCIP2. (B) Dose-dependent change in percentage of splenic GC (B220+Fas+GL7+) B cells. (C) Change in total frequency of splenic B220+ B cells. (D) Change in total frequency of splenic memory B cells (MB). In (B) to (D), representative flow cytometry is shown on the left and quantification on the right from five biological replicates (five different mice) for each condition, median, and range shown. P values were computed by unpaired Student’s t test. QD, once daily; BID, twice daily. Vehicle was given twice daily. (E) Body weight changes over time.

Fig. 5.. Extension of the CDK-TCIP concept to CDK12 and CDK13.

(A) Schematic of recruiting CDK12 and CDK13 to BCL6-bound loci. (B) Structure of CDK-TCIP3. (C) Activation of BCL6-repressed GFP reporter constructs after 24 hours of compound addition to lymphoma cells. Data are shown as means ± SD. n = 4 biological replicates. (D) Comparison of cell-killing potency in DLBCL cells at 72 hours between CDK-TCIP3 and the additive effect of both BCL6 and CDK12/13 inhibitors. n = 3 biological replicates. Data are shown as means ± SE.