cdk13 inhibitor  (TargetMol)

Journal: Cell Reports Medicine

Article Title: Development of an orally bioavailable CDK12/13 degrader and induction of synthetic lethality with AKT pathway inhibition

doi: 10.1016/j.xcrm.2024.101752

Figure Lengend Snippet: YJ9069, a specific degrader of CDK12/13, exhibits preferential cytotoxicity in multiple cancers (A) Chemical structure of CDK12/13 degrader YJ9069. CDK12/13 warhead is in red, and E3 ligand thalidomide is in blue. (B) Docking model of YJ9069 (cyan sticks) with CDK12 (PDB: 6CKX) and CRBN (PDB: 6R1A) complex. (C) Immunoblots of CDK12, CDK13, and pSer2 of RNAPII CTD in VCaP cells treated with YJ9069 at increasing concentrations or time durations. Tubulin is used as a loading control. (D) Effects of YJ9069 (500 nM, 5 h) on the proteome of 22Rv1 cells. Data plotted Log2 of the fold change (FC) versus DMSO (dimethyl sulfoxide) control against −Log10 of the p value per protein (FDR, false discovery rate) from n = 3 independent experiments. All tests performed were two-tailed t test assuming equal variances. CDK12, CDK13, and CCNK are highlighted. (E) Growth curves of VCaP, 22Rv1, RWPE, and BPH-1 cells upon treatment with increasing concentrations of YJ9069. Data are presented as mean ± standard deviation from n = 3 independent experiments. (F) IC 50 of YJ9069 and THZ531 in a panel of human-derived prostate and breast cell lines after 5 days of treatment. WPMY-1 is a human-derived prostate stromal cell line, while the others are human epithelial cell lines. All benign immortalized cell lines are highlighted as dark red. IC 50 values are calculated from n = 3 independent experiments. (G) IC 50 of YJ9069 in a panel of human-derived cancer or normal cell lines after 5 days of treatment. AR, androgen receptor. IC 50 values are calculated from n = 3 independent experiments. See also Figure S1 and .

Article Snippet: Crystal structures of CDK12 and CDK13 with the inhibitors (PDB codes: 7NXK and 7NXJ , respectively) were sourced from the Protein DataBank ( http://www.pdb.org ).

Techniques: Western Blot, Control, Two Tailed Test, Standard Deviation, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Development of an orally bioavailable CDK12/13 degrader and induction of synthetic lethality with AKT pathway inhibition

doi: 10.1016/j.xcrm.2024.101752

Figure Lengend Snippet: CDK12/13 degradation leads to gene-length-dependent elongation defects in transcription, loss of long gene expression, DNA damage, and cell-cycle arrest (A) Scatterplot showing Log2 fold changes in gene expression vs. Log2 scale in gene length for each protein-coding gene in VCaP cells following treatment with YJ9069 at 500 nM for 12 h ( p < 2.2e−16, F-test). Differentially expressed genes are indicated (FDR < 0.05 and Log2 FC > 1) from n = 3 independent experiments. (B) Number of genes up- or downregulated relative to gene length. The genome was ranked from smaller to longer genes and fractioned into 4 groups containing the same number of genes. (C) Representative images of comet assay in VCaP cells after treatment with vehicle or YJ9069 (100 nM) for 12 h (scale, 50 μm) (left panel), and quantification of tail moments (right panel). Boxplots represent interquartile ranges; horizontal bars denote the median. For each condition, 50 cells were analyzed. (D) Analysis of indicated gene expression by real-time qPCR at 4 h, 8 h, and 15 h with YJ9069 (50 nM) or vehicle in VCaP cells. Data are presented as mean values ± standard deviation of triplicate experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by t test. (E) Cell-cycle analyses by flow cytometry of VCaP cells treated with 100 nM and 500 nM of YJ9069 for 15 h. The bar graph (right) demonstrates the quantification of the cell-cycle phase data. See also Figure S2 .

Article Snippet: Crystal structures of CDK12 and CDK13 with the inhibitors (PDB codes: 7NXK and 7NXJ , respectively) were sourced from the Protein DataBank ( http://www.pdb.org ).

Techniques: Gene Expression, Single Cell Gel Electrophoresis, Standard Deviation, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Development of an orally bioavailable CDK12/13 degrader and induction of synthetic lethality with AKT pathway inhibition

doi: 10.1016/j.xcrm.2024.101752

Figure Lengend Snippet: YJ9069 suppresses tumor growth in multiple in vivo models of castration-resistant prostate cancer (A) Dose-response curves and IC 50 of cells treated with YJ9069. Data are presented as mean ± standard deviation ( n = 3) from one of three independent experiments. (B) Immunoblot of CDK12, CDK13, and cleaved PARP for castrated VCaP in vivo xenografted tumors after 5 days treatment with YJ9069 (i.v., 30 mg/kg, 3x/week). Tubulin is the loading control. (C) Representative H&E staining and immunohistochemistry for CDK12, cleaved PARP, and TUNEL from the PD5 study in (B) (scale, 50 μm). (D) Tumor volume (measured twice weekly using calipers) in the castrated VCaP model treated with YJ9069 (i.v., 30 mg/kg, 3x/week) (two-sided t test). Data are mean ± standard error of the mean (SEM) (vehicle: n = 10; YJ9069: n = 11). (E) Tumor weights for vehicle and YJ9069 groups from castrated VCaP study (two-sided t test). Data are presented as mean ± SEM. (F) Waterfall plot depicting the change in tumor volume after 18 days of treatment. Response evaluation criteria in solid tumors (RECIST) was used to stratify tumors: progressive disease (PD), at least a 20% increase in tumor size; stable disease (SD), an increase of <20% to a decrease of <30%; partial response (PR), at least a 30% decrease. The vehicle group has 100% PD; the YJ9069 group has 18% SD and 82% PR. (G) Percent body weight measurement showing the effect of vehicle and YJ9069 in the castrated VCaP model throughout the treatment period. Data are presented as mean ± SEM. (H) Representative H&E staining for vehicle- and YJ9069-treated tumors from castrated VCaP xenograft at the endpoint (scale, 200 μm). The inset scale, 50 μm. (I–M) as in (D–H), except in the WA74 patient-derived xenograft (PDX) model with YJ9069 treatment (i.v., 30 mg/kg, 2x/week or 3x/week) ( n = 8 per condition). In the waterfall plot, the YJ9069 2x/week group has 14% PD, 43% SD, and 43% PR; the YJ9069 3x/week group has 100% PR. (N–R) as in (D–H), except in the PC310 PDX model with YJ9069 (i.v., 30 mg/kg, 3x/week). In the waterfall plot, the YJ9069 group has 63% PD, 31% SD, and 6% PR. (S) Genitourinary tract measurement for vehicle and YJ9069 groups at noted doses in CD-1 male mouse (two-sided t test). Data are presented as mean ± SEM ( n = 4, biological replicates). (T) Representative photographs with matched H&E staining of the genitourinary region from vehicle and YJ9069 (i.v., 30 mg/kg, 3x/week) groups in CD-1 male mouse (scale, 50 μm). See also Figure S3 .

Article Snippet: Crystal structures of CDK12 and CDK13 with the inhibitors (PDB codes: 7NXK and 7NXJ , respectively) were sourced from the Protein DataBank ( http://www.pdb.org ).

Techniques: In Vivo, Standard Deviation, Western Blot, Control, Staining, Immunohistochemistry, TUNEL Assay, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Development of an orally bioavailable CDK12/13 degrader and induction of synthetic lethality with AKT pathway inhibition

doi: 10.1016/j.xcrm.2024.101752

Figure Lengend Snippet: Development of YJ1206, an orally bioavailable CDK12/13 degrader analog of YJ9069 (A) Chemical design of the oral CDK12/13 degrader YJ1206. (B) Immunoblots of CDK12 and CDK13 in VCaP cells treated with YJ1206 at increasing concentrations for 4 h. Tubulin is used as a loading control. (C) Dose-response curves and IC 50 of VCaP cells treated with YJ9069 and YJ1206 for 5 days. Data are presented as mean ± standard deviation from n = 3 independent experiments. (D) Effects of YJ1206 (500 nM, 8 h) on the proteome of 22Rv1 cells. Data plotted Log2 of the fold change (FC) versus DMSO against −Log10 of the p value per protein (FDR, false discovery rate) from n = 3 independent experiments. All tests performed were two-tailed t test assuming equal variances. CDK12, CDK13, and CCNK are highlighted in red. (E) Plasma concentration-time curve of YJ1206 with intravenous (i.v., 2.5 mg/kg) and oral administration (p.o., 10 mg/kg) injection in CD-1 mice ( n = 3 per condition). (F) Pharmacokinetic profile of YJ9069 and YJ1206 following intravenous (i.v., 2.5 mg/kg) and oral (p.o., 10 mg/kg) injection in CD-1 mouse. (G) Immunoblots of CDK12, CDK13, CCNK, cleaved PARP, and γ-H2AX for the castrated VCaP tumors after 5 days treatment with YJ1206 (p.o., 100 mg/kg, 3x/week). Tubulin is the loading control. (H) Representative H&E staining and immunohistochemistry of CDK12, cleaved PARP, and TUNEL for PD5 study in panel G (scale, 50 μm). (I) Histology score for immunohistochemistry of cleaved PARP and TUNEL in (G) (two-sided t test). Data are presented as mean ± standard deviation. (J) Genitourinary tract measurement of the vehicle and YJ1206 group at noted doses in CD-1 male mouse (two-sided t test). Data are presented as mean ± SEM ( n = 4, biological replicates). (K) Representative photographs with matched H&E staining of the genitourinary region from vehicle and YJ1206 group in CD-1 male mouse (scale, 50 μm). (L) Tumor volume (measured twice weekly using calipers) in the WA74 PDX model with YJ1206 (p.o., 100 mg/kg, 3x/week) (two-sided t test). Data are mean ± SEM ( n = 16 per condition). (M) Waterfall plot depicting the change in tumor volume. The evaluation criteria are the same as Figure 3 F. The YJ1206 group has 56% PD, 25% SD, and 19% PR. (N) Tumor weights for vehicle and YJ1206 groups from WA74 PDX study (two-sided t test). Data are presented as mean ± SEM. (O) Percent body weight measurement showing the effect of vehicle and YJ1206 in WA74 PDX study throughout the treatment period. Data are presented as mean ± SEM. (P) Representative H&E staining for WA74 PDX tumors at the endpoint treatment (scale, 200 μm). The inset scale, 50 μm See also Figures S4 and and .

Article Snippet: Crystal structures of CDK12 and CDK13 with the inhibitors (PDB codes: 7NXK and 7NXJ , respectively) were sourced from the Protein DataBank ( http://www.pdb.org ).

Techniques: Western Blot, Control, Standard Deviation, Two Tailed Test, Clinical Proteomics, Concentration Assay, Injection, Staining, Immunohistochemistry, TUNEL Assay

Journal: Cell Reports Medicine

Article Title: Development of an orally bioavailable CDK12/13 degrader and induction of synthetic lethality with AKT pathway inhibition

doi: 10.1016/j.xcrm.2024.101752

Figure Lengend Snippet: CDK12/13 degradation induces synthetic lethality in conjunction with AKT pathway inhibition in vitro (A) Human phosphorylation pathway profiling array analysis of VCaP cells treated with YJ1206 (500 nM) for 15 h. (B) The PI3K/AKT signaling pathway. The top phosphorylated proteins identified in Figure 5 A are highlighted in red. (C) Immunoblot of the noted proteins in VCaP cells treated with siRNA targeting CDK12 and/or CDK13 or YJ1206 at increasing concentrations and time durations. Tubulin is the loading control probed on all immunoblots. (D) Real-time growth curves of VCaP cells upon treatment with siCDK12/13 and/or uprosertib. Data are presented as mean ± standard deviation from three independent experiments. (E) Cell viability of VCaP cells treated with siCDK12/13 and/or uprosertib by CellTiter-Glo assay (two-sided t test, n = 3 independent experiments). (F and G) Real-time growth curves of 22Rv1 cells upon treatment with YJ1206 and/or uprosertib/capivasertib. Data are presented as mean ± standard deviation from n = 3 independent experiments. (H and I) PC310 PDX organoids were treated with YJ1206 and/or uprosertib/capivasertib at varied concentrations to determine the effect on cell growth and drug synergism, with assessments using the Bliss independence method. Red peaks in the 3D plots denote synergy, and the average synergy scores are noted above the plots. See also Figure S6 .

Article Snippet: Crystal structures of CDK12 and CDK13 with the inhibitors (PDB codes: 7NXK and 7NXJ , respectively) were sourced from the Protein DataBank ( http://www.pdb.org ).

Techniques: Inhibition, In Vitro, Phospho-proteomics, Western Blot, Control, Standard Deviation, Glo Assay

Journal: Cell Reports Medicine

Article Title: Development of an orally bioavailable CDK12/13 degrader and induction of synthetic lethality with AKT pathway inhibition

doi: 10.1016/j.xcrm.2024.101752

Figure Lengend Snippet: The combination regimen of CDK12/13 degraders with AKT inhibitors suppresses tumor growth in vivo (A) Tumor volume in castrated VCaP xenograft model with YJ1206 (p.o., 100 mg/kg, 3x/week) alone or combined with uprosertib (p.o., 15 mg/kg, 5x/week) treatment ( n = 20 per condition; two-sided t test). Data are mean ± SEM. (B) Waterfall plot depicting the change in tumor volume after 31 days of treatment. The evaluation criteria are the same as Figure 3 F. The vehicle and uprosertib groups have 100% PD; the YJ1206 group has 80% PD, 15% SD, and 5% PR; and the YJ1206 + uprosertib group has 62% PD, 24% SD, and 14% PR. (C) Tumor weights from VCaP-CRPC xenograft study (two-sided t test). Data are presented as mean ± SEM. (D–F) same as in (A–C), except in PC310 PDX ( n = 12 per condition). In the waterfall plot, the vehicle, uprosertib, and YJ1206 groups have 100% PD; the YJ1206 + uprosertib group has 50% PD and 50% SD. See also Figure S7 .

Article Snippet: Crystal structures of CDK12 and CDK13 with the inhibitors (PDB codes: 7NXK and 7NXJ , respectively) were sourced from the Protein DataBank ( http://www.pdb.org ).

Techniques: In Vivo

Journal: Cell Reports Medicine

Article Title: Development of an orally bioavailable CDK12/13 degrader and induction of synthetic lethality with AKT pathway inhibition

doi: 10.1016/j.xcrm.2024.101752

Figure Lengend Snippet: Mechanism of action of CDK12/13 degrader-induced AKT phosphorylation in prostate cancer The CDK12/13 degrader inhibits Ser2 phosphorylation on RNAPII, disrupting gene expression and leading to DNA damage and instability. This DNA damage triggers AKT phosphorylation, promoting synthetic lethality with AKT inhibition.

Article Snippet: Crystal structures of CDK12 and CDK13 with the inhibitors (PDB codes: 7NXK and 7NXJ , respectively) were sourced from the Protein DataBank ( http://www.pdb.org ).

Techniques: Phospho-proteomics, Gene Expression, Inhibition

Journal: Cell Reports Medicine

Article Title: Development of an orally bioavailable CDK12/13 degrader and induction of synthetic lethality with AKT pathway inhibition

doi: 10.1016/j.xcrm.2024.101752

Figure Lengend Snippet:

Article Snippet: Crystal structures of CDK12 and CDK13 with the inhibitors (PDB codes: 7NXK and 7NXJ , respectively) were sourced from the Protein DataBank ( http://www.pdb.org ).

Techniques: Recombinant, Transfection, Cell Viability Assay, Purification, One Step RT-PCR, SYBR Green Assay, Gene Expression, Single Cell Gel Electrophoresis, Phospho-proteomics, Software

Journal: BMC Biology

Article Title: ARID1A loss promotes RNA editing of CDK13 in an ADAR1-dependent manner

doi: 10.1186/s12915-024-01927-9

Figure Lengend Snippet: The CDK13 gene is edited by ADAR1 and ARID1A deficiency increased sensitivity to SR4835. A Comparison of HCT116 shARID1A to ARID1A_Scramble cell lines. The differential genes in “RNA editing” category consists of ARL6IP4, BLCAP, COPA, CDK13, and NEIL1. B The RNA editing frequency at different sites of COPA, BLCAP, ARL6IP4 genes in HCT116 ARID1A_WT, ARID1A_KO, ARID1A_KO + shADAR#1, and ARID1A_KO + shADAR#2 cell lines. C The RNA editing frequency at three different sites of CDK13 genes in HCT116 ARID1A_WT, ARID1A_KO, ARID1A_KO + shADAR#1, and ARID1A_KO + shADAR#2 cell lines. D RT-qPCR results show ADAR mRNA expression in HCT116 ARID1A_KO, ARID1A_KO + shADAR#1, and ARID1A_KO + shADAR#2 cell lines. n = 3; mean ± SD; *, P < 0.05; ns, not significant. E Diagram of the CDK13 kinase, showing the serine/threonine protein kinase active region. Intron regions and three RNA editing sites (K21R, K117R, and Q113R) are marked. K21R:7_39990302; K117R:7_39990590; Q113R: 7_39990578. Exported by "SMART" software, the red line represents intron Phase 2 and the blue line represents intron Phase 1. F Representative images of A375 Scramble and shARID1A cells treated with SR-4835 at a concentration of 1 nM and 10 nM. G Quantitative results represent the mean ± SD of three independent experiments. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. H Representative images of HCT116 Scramble and shARID1A cells treated with SR-4835 at a concentration of 1 nM and 10 nM. I Quantitative results represent the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: The CDK13 inhibitor SR-4835 was purchased from Selleckchem (Houston, USA).

Techniques: Comparison, Quantitative RT-PCR, Expressing, Software, Concentration Assay

Journal: BMC Biology

Article Title: ARID1A loss promotes RNA editing of CDK13 in an ADAR1-dependent manner

doi: 10.1186/s12915-024-01927-9

Figure Lengend Snippet: ADAR1 edits CDK13 at position Q113R and K117R, ADAR1 knockdown rescues the sensitivity to SR4835. A Sanger sequencing chromatograms using a reverse primer illustrate editing of CDK13 K21R position in ARID1A_WT, ARID1A_KO and genomic DNA (gDNA) in HCT116 cell lines. B Sanger sequencing chromatograms using a reverse primer illustrate editing of CDK13 Q113R position in ARID1A_WT, ARID1A_KO and genomic DNA (gDNA) in HCT116 cell lines. C Sanger sequencing chromatograms using a reverse primer illustrate editing of CDK13 K117R position in ARID1A_WT, ARID1A_KO and genomic DNA (gDNA) in HCT116 cell lines. D Representative images of HCT116_ARID1A_KO cell lines transfected with siNC and siADAR and treatment with SR-4835 at a concentration of 5 nM and 10 nM. E Quantitative results represent the mean ± SD of three independent experiments. **, P < 0.01; ***, P < 0.001. F Representative images of A375_ARID1A_KO cell lines transfected with siNC and siADAR and treatment with SR-4835 at a concentration of 5 nM and 10 nM. G Quantitative results represent the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01. H Representative images of DLD-1_ARID1A_KO cell lines transfected with siNC and siADAR and treatment with SR-4835 at a concentration of 5 nM and 10 nM. I Quantitative results represent the mean ± SD of three independent experiments. ***, P < 0.001.****, P < 0.0001

Article Snippet: The CDK13 inhibitor SR-4835 was purchased from Selleckchem (Houston, USA).

Techniques: Knockdown, Sequencing, Transfection, Concentration Assay