CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation

Abstract

damage response (DDR) and mRNA processing genes. Yet, the mechanism(s) by which it achieves this selectivity remains unclear. Using a highly selective CDK12/13 inhibitor, THZ531, and nascent RNA sequencing, we show that CDK12 inhibition results in gene length-dependent elongation defects, leading to premature cleavage and polyadenylation (PCPA) as well as loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlated with an increased proportion of intronic polyadenylation sites, a feature that was especially prominent among DDR genes. Finally, phosphoproteomic analysis indicated that pre-mRNA processing factors, including those involved in PCPA, are direct phosphotargets of CDKs 12 and 13. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition due primarily to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites. 5 CDK12 inhibition preferentially affects DDR genes. CDK12 inhibition has been shown to affect the expression of genes involved in the DDR 5,6,10 . To determine whether similar effects are produced by our selective inhibitor in NB cells, we analyzed the gene expression profiles of cells treated with and without THZ531 for 6 hr., a time point at which there were little or no confounding effects due to cell cycle changes ( Supplementary Fig. 1e).Unlike the effects seen with THZ1 17 , predominantly an inhibitor of CDK7 with some activity against CDK12/13 28 , we failed to observe a complete and global transcriptional shutdown in THZ531-treated NB cells; instead, only 57.4% of the transcripts were downregulated (n=10,707), with 0.35% (n=66) upregulated [false discovery rate (FDR) <0.05] (Supplementary Fig. 2a; Supplementary Table 1). Consistent with earlier studies 15,16 , THZ531 led to significant downregulation of both transcription-associated and DDR genes ( Fig. 1c, Supplementary Fig. 2b, c), the latter of which were primarily associated with homologous recombination (HR) repair and are crucial for the maintenance of genome stability, including BRCA1, BARD1 and RAD51 29 (Fig. 1c, Supplementary Fig. 2c, d). To determine whether these effects were due to inhibition of CDK12 or 13, we depleted the expression of each kinase individually in NB cells, and in keeping with prior studies 6,10,30 , observed selective downregulation of DDR genes with CDK12 and not CDK13 knockdown (KD) (Supplementary Fig. 2e). Additionally, the expression of DDR genes was not affected in Kelly E9R THZ531-resistant cells, further implicating the selective role of CDK12 in regulating the DDR (Fig. 1d). Consistent with these observations, THZ531 also led to increased DNA damage with elevated γ-H2AX levels (Fig. 1e) and decreased radiationinduced RAD51 foci, indicating defects in DNA repair (Fig. 1f). Thus, our findings indicate that DDR genes are selectively affected by THZ531 and that such regulation is driven predominantly by CDK12.CDK12/13 inhibition with TH...

Connections between 3′ end processing and DNA damage response: Ten years later

“…It remains to be determined whether this CDK12‐mediated effect on ALE splicing in breast cancer is dependent on RNAP II CTD or another undescribed pathway of CDK12. Recent studies showed that CDK12 not only play a role in ALE regulation but also in protecting the cells from premature CpA and allowing expression of long genes, a sizable portion of which include DDR genes (Krajewska et al, ). p38 kinase (Danckwardt et al, ) and protein kinase C (PKC)δ (W. Li et al, ) are involved in the modulation of poly(A) signal usage of prothrombin pre‐mRNA in response to the stress‐inducing agent anisomycin and BIK pre‐mRNA in response to DNA damage, respectively.…”

Emerging approaches to CDK inhibitor development, a structural perspective

“…2A). 34,35,63,68 However, while the binding of p27 to A and D-cyclins is conserved, significant differences are found when comparing inhibitor binding to the kinase subunit in the CDK2-cyclin A-p27 and CDK4-cyclin D1-p27 structures (Fig. 2B).…”

“…38,67 p21 and p27 are classed as intrinsically disordered proteins and fold upon binding to CDK-cyclin complexes. 37,64,65 To act as an inhibitor, exposure of the p27 D1 region (residues [27][28][29][30][31][32][33][34][35][36][37] which contains an RXL motif that binds to the cyclin recruitment site (Fig. 2A) primes binding of the p27 kinase inhibitory domain (KID, residues 28-89) via conformational sampling.…”

“…However, though challenging to develop, there is mounting evidence to support the therapeutic value of transcriptional inhibitors particularly in cancer. [33][34][35] This review discusses recent CDK structural studies (in particular those published since 2018) that have advanced our understanding of CDK-protein interactions. 9 We explore the contributions of structural flexibility, allostery and short linear motifs (SLiMs) to the regulation of CDK activity and the use of small molecules and chemically-induced targeted CDK degradation to perturb CDK signalling.…”

Research progress of anticancer drugs targeting CDK12

“…4 CDK12 plays a vital biological role in transcription, mRNA splicing, the cell cycle and DNA damage response (DDR) repair. [5][6][7][8] CDK12 overexpression increases DNAJB6-L through ALE splicing which increases the aggressiveness of breast cancer cells. 5 High CDK12 expression promotes IRS1-p85-ErbB receptor binding and activates the ErbB-PI3K-AKT signaling cascade for HER2-positive breast cancer development.…”