Pharmacological perturbation of CDK9 using selective CDK9 inhibition or degradation

Abstract

Cyclin-dependent kinase 9 (CDK9), an important regulator of transcriptional elongation, is a promising target for cancer therapy, particularly for cancers driven by transcriptional dysregulation. We characterized NVP-2, a selective ATP-competitive CDK9 inhibitor, and THAL-SNS-032, a selective CDK9 degrader consisting of a CDK-binding SNS-032 ligand linked to a thalidomide derivative that binds the E3 ubiquitin ligase Cereblon (CRBN). To our surprise, THAL-SNS-032 induced rapid degradation of CDK9 without affecting the levels of other SNS-032 targets. Moreover, the transcriptional changes elicited by THAL-SNS-032 were more like those caused by NVP-2 than those induced by SNS-032. Notably, compound washout did not significantly reduce levels of THAL-SNS-032-induced apoptosis, suggesting that CDK9 degradation had prolonged cytotoxic effects compared with CDK9 inhibition. Thus, our findings suggest that thalidomide conjugation represents a promising strategy for converting multi-targeted inhibitors into selective degraders and reveal that kinase degradation can induce distinct pharmacological effects compared with inhibition.

Figure 1. THAL-SNS-032 selectively induces degradation of CDK9

a. Chemical structures of NVP-2, SNS-032, THAL-SNS-032 where R=H for SNS-032 and R is the shown linked thalidomide derivative for THAL-SNS-032. b. Kinativ™ data from MOLT4 lysates showing CMGC family targets engaged by SNS-032 or THAL-SNS-032 at a concentration of 1uM. Full data set in Supplementary Dataset 1. c. Immunoblot of proteins shown after 6-hour treatment with the indicated concentrations of THAL-SNS-032 in wildtype or CRBN −/− MOLT4 cells. d. Log fold-change in abundance of proteins with 2 peptide thresholds as measured using multiplexed quantitative-mass spectrometry-based proteomics following a 2-hour treatment of MOLT4 cells with 250nM THAL-SNS-032 vs p-value (n=3, biological replicates). Full data set in Supplementary Dataset 2. e. Immunoblot for proteins shown after treatment of MOLT4 cells with 250nM THAL-SNS-032 for the indicated times. f. Immunoblot for CDK9 and Tubulin after 4-hour pre-treatment with MG132 (5μM), SNS-032 (10μM), thalidomide (10μM), or DMSO vehicle followed by a 2 hour THAL-SNS-032 treatment (250nM) in MOLT4 cells. Uncut western blots are in Supplementary Figure 6.

Figure 2. NVP-2 selectively inhibits CDK9

a. Kinase trees represent kinome-wide selectivity at S(1) of NVP-2 through Kinomescan™ profiling at 1μM (CDK9 labeled in blue, while all other inhibited kinases appear in red). S(1) indicates the kinases with binding inhibited 99% or greater by the compound. Full dataset in Supplementary Dataset 3. b. Kinativ™ data from MOLT4 lysates showing CMGC family targets engaged by NVP-2 at a concentration of 1uM c. Bio-AT7519 pulldown of CDK2, CDK7 or CDK9 (PD) or input in MOLT4 wildtype at concentrations indicated for each compound. d. Bio-AT7519 pulldown of CDK2, CDK7 or CDK9 (PD) or input in MOLT4 CRBN−/−) cells at concentrations indicated for each compound. Uncut western blots are in Supplementary Figure 6.

Figure 3. THAL-SNS-032 exhibits CRBN-dependent anti-proliferative and pro-apoptotic effects

a. Cell viability IC50s for THAL-SNS-032, SNS-032, or NVP-2 on wild-type and CRBN−/− MOLT4 after 72 hours as approximated by using CellTiter Glo (data are presented as means ± s.d. (n=4) biological replicates). b. Immunoblot of proteins shown for the indicated times after treatment with THAL-SNS-032 at 250 nM in wildtype MOLT4 cells. c. Immunoblot of proteins shown for the indicated times after treatment with 250nM NVP-2 in wildtype MOLT4 cells. d. Immunoblot of proteins shown for the indicated times after treatment with 250nM SNS-032 in wildtype MOLT4 cells. e. Viability effect of THAL-SNS-032, SNS-032 or NVP-2 of MOLT4 cells at 250nM concentration of inhibitors comparing 6-hour treatment followed by removal of compound (Washout) or 72 hour of prolonged exposure to compound (no washout) (data are presented as means ± s.d. (n=3) biological replicates) using CellTiter Glo f. Quantification of Annexin V-positive MOLT4 cells treated with compounds at 250 nM after 24 hours with and without washout (data represented as means ± s.d. (n=3) biological replicates). g. Immunoblot of proteins shown after 24-hour treatment of MOLT4 cells with 250nM THAL-SNS-032, NVP-2, or SNS-032 with and without washout after 6 hours. Uncut western blots are in Supplementary Figure 7.

Figure 4. THAL-SNS-032 exhibits transcriptional effects consistent with a selective CDK9 inhibitor

a. Immunoblot of proteins shown after treatment of wildtype and CRBN−/− MOLT4 cells with THAL-SNS-032 at the concentrations indicated for 6 hours. b. Immunoblot of proteins shown after treatment of wildtype MOLT4 cells with NVP-2 at the concentrations indicated for 6 hours. c. Immunoblot of proteins shown after treatment of wildtype MOLT4 cells with SNS-032 at the concentrations indicated for 6 hours. d. Box plot analysis of changes to gene expression levels in MOLT4 cells after treatment with 250nM THAL-SNS-032, SNS-032 or NVP-2 for 6 hours e. Scatter plots showing the correlation of transcriptional changes in MOLT4 cells when treated with THAL-SNS-032 or SNS-032 at concentrations of 250 nM. f. Scatter plots showing the correlation of transcriptional changes in MOLT4 cells when treated with 250nM THAL-SNS-032 or 250nM NVP-2. g. p-value resulting from GO term analysis of genes downregulated by treatment with NVP-2 (1018 genes), THAL-SNS-032 (1026 genes) or SNS-032 (1013 genes). h. Heatmap of gene expression changes to the core regulatory circuitry genes after treatment with NVP-2, THAL-SNS-032, or SNS-032. Uncut western blots are in Supplementary Figure 7.

Figure 5. THAL-SNS-032 diminishes elongating polymerase II

a. Metagene representation of global Pol II (left) or Spt5 (right) occupancy at gene bodies after 250 nM treatment with THAL-SNS-032 (red), SNS-032 (blue), NVP-2 (orange), or DMSO vehicle (black) in MOLT4 cells. b. Pol II (left) or Spt5 (right) occupancy at PRCC gene after treatment with THAL-SNS-032, NVP-2 and SNS-032 normalized to spike-in controls. c. Box plots of Pol II signal at transcriptional start sites (TSS) as normalized to DMSO treatment. Boxes in depicted boxplots represent the first to third quartiles and whiskers represent 1.5X the interquartile range. Data points outside this range are identified by individual points. d. Box plots of Spt5 signal at transcription start sites (TSS) as normalized to DMSO treatment. Boxes in depicted boxplots represent the first to third quartiles and whiskers represent 1.5X the interquartile range. Data points outside this range are identified by individual points. e. Distribution of the Pol II bound genes with a given Traveling Ratio.