When CDK12/13 stalls, healthy prostate cells take a malignant turn

By Pooja Toshniwal PahariaReviewed by Danielle Ellis, B.Sc.Oct 7 2024

Breakthrough research sheds light on the genetic mutation behind aggressive prostate cancer.

In a recent study published in Cell Reports Medicine, researchers investigated whether loss of the cyclin-dependent kinase 12 (CDK12) gene drives prostate cancer (PCa) development.

Background

Cyclin-dependent kinase enzymes (CDKs) are of two types: cell cycle-regulating CDKs that participate in the progression of the cell cycle and transcriptional ones that control gene expression. CDK12, a transcriptional enzyme, binds to the deoxyribonucleic acid (DNA) in enhancer and protein-coding regions.

Genetic and pharmacologic targeting have shown that the CDK12 transcriptionally regulates DNA damage response (DDR) genes. Clinical and preclinical data suggest that CDK12 acts as a tumor suppressor in serous ovarian carcinoma and triple-negative breast cancer.

About the study

In the present study, researchers investigated CDK12 as a prostate tumor suppressor gene using murine models where inactivation could drive tumorigenesis. They also investigated whether loss of CDK12 might render prostate tumors vulnerable to synthetic lethal paralog enzymes.

The researchers tested the effects of CDK12 loss, combined with other canonical metastatic castration-resistant PCa (mCRPC)-associated mutations, in vivo and in vitro. Investigators used genetically engineered mice lacking functional CDK12 in prostate epithelial cells to assess whether CDK12 inactivation promotes prostate tumorigenesis. Immunoblotting and immunohistochemistry confirmed the loss of CDK12 protein.

To describe more explicitly how the loss of CDK12 promoted tumorigenesis, investigators generated organoids from purified populations of prostate epithelial cells lacking CDK12. Utilizing the organoid model of prostate cancer, investigators performed clustered regularly interspaced short palindromic repeats (CRISPR) screening to identify mutations that either interacted positively or negatively with the loss of CDK12.

The researchers hypothesized that CDK12 loss increases DNA damage in prostate epithelial cells and enhances enhanced tumorigenic potential by mimicking some aspects of transformation-related protein 53 (TRP53) deletion. They used CRISPR-associated protein 9 (Cas9) to ablate TRP53 in CDK12 wild-type (WT) and knockout (KO) organoids to test the hypothesis.

Researchers then implanted organoids into immunocompromised mice as subcutaneous allografts. They investigated whether allografts are sensitive to immune checkpoint blockade (ICB) using antibodies against programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Moreover, they investigated whether loss of CDK12 influences tumor progression driven by the inactivation of phosphatase and tensin homolog (PTEN), a tumor suppressor gene.

Given the link between CDK12 loss and castration resistance in human PCa, investigators analyzed androgen receptor (AR) signaling in CDK12KO organoids.

The organoids were treated with 5,6-dichloro-1-beta-D-ribofurano sylbenzimidazole (DRB), a ribonucleic acid polymerase II inhibitor, to analyze the consequences of CDK12 loss on DNA. To determine whether the cell cycle synthesis (S)-phase-specific DNA damage caused by CDK12 loss is associated with transcription-replication conflicts (TRCs), investigators conducted proximity ligation assays (PLA).

To identify candidates with synthetic lethal effects of CDK12, the authors performed small-interfering ribonucleic acid (siRNA) screening of Henrietta Lacks (HeLa) cells through a library targeting 297 genes with potentially shared functions with CDK12. The team validated Cdk12KO organoids for the efficacy of pharmacological targeting of CDK13 with the CDK12/13 degrader, YJ9069.

Results

CDK12 loss characterizes a mCRPC subtype. The study demonstrated that CD12 suppresses prostate cancers, and its loss drives AR and myelocytomatosis proto-oncogene (MYC)-regulated hypertranscription. CDK12 loss causes TRCs that induce DNA damage, as demonstrated by DRB treatment and γH2AX immunohistochemistry.

Loss of CDK12 enhances tumor development in situations of loss of the TRP53 tumor suppressor gene but prevents the growth of tumors devoid of PTEN. Loss of CDK12 inactivated TRP53 but showed negative correlations with the inactivation of PTEN. Moreover, concurrent deletion of TRP53 and CDK12 enhanced the growth of prostate-derived organoids, and deletion of CDK12 among PTEN-null murine animals negates PCa development. CDK12/TRP53 double knockout allografts showed a lymphocytic immune response and sensitivity to ICB treatment.

CDK12 loss in the murine prostate epithelium led to preneoplastic prostate lesions with enhanced cellular proliferation and immune infiltration by T cells with elevated cytokine levels. CDK12-deficient prostate-derived organoids were hyperplastic with basal-luminal disorganization, confirmed through single-cell RNA sequencing.

Murine tumor tissues lacking CDK12 showed sensitivity to CDK13 inhibitors and degraders, a vulnerability that could be exploited to treat CDK12-mutant tumors. The data establish the status of CDK12 and CDK13 as synthetic paralog enzymes. The team noted lower cell viability in the CDK12KO organoids after treatment with the CD12/13 degrader, YJ9069. CDK12/13 degradation inhibited the protein kinase B (AKT) pathway. The CDK12/13 inhibitors, YJ5118 and THZ53, showed stronger effects.

Based on the study findings, CDK12 is a tumor suppressor gene, and YJ1206, a CDK12/13 degrader, is a potential therapeutic candidate for preclinical models of advanced prostate cancer.