Background Transcription, metabolism and DNA damage response are tightly regulated to preserve the genomic integrity, and O-GlcNAc transferase (OGT) is positioned to connect the three. Prostate cancer is the most common cancer in men, and androgen-ablation therapy halts disease progression. However, a significant number of prostate cancer patients develop resistance against anti-androgens, and this incurable disease is termed castration-resistant prostate cancer (CRPC). We have shown that combined inhibition of OGT and the transcription elongation kinase CDK9 induce CRPC-selective anti-proliferative effects. Here, we explain the functional basis for these combinatorial effects. Methods We used comprehensive mass spectrometry profiling of short-term CDK9 inhibitor effects on O-GlcNAcylated proteins in an isogenic cell line system that models transition from PC to CRPC. In addition, we used both ChIP-seq and RNA-seq profiling, and pulldown experiments in multiple CRPC models. Finally, we validated our findings in prostate cancer patient samples. Results Inhibition of CDK9 results in an OGT-dependent remodeling of the proteome in prostate cancer cells. More specifically, the activity of the DNA damage repair protein MRE11 is regulated in response to CDK9 inhibition in an OGT-dependent manner. MRE11 is enriched at the O-GlcNAc-marked loci. CDK9 inhibition does not decrease the expression of mRNAs whose genes are bound by both O-GlcNAc and MRE11. Combined inhibition of CDK9 and OGT or MRE11 further decreases RNA polymerase II activity, induces DNA damage signaling, and blocks the survival of prostate cancer cells. These effects are seen in CRPC cells but not in normal prostate cells. Mechanistically, OGT activity is required for MRE11 chromatin-loading in cells treated with CDK9 inhibitor. Finally, we show that MRE11 and O-GlcNAc are enriched at the prostate cancer-specific small nucleotide polymorphic sites, and the loss of MRE11 activity results in a hyper-mutator phenotype in patient tumors. Conclusions Both OGT and MRE11 are essential for the repair of CDK9 inhibitor-induced DNA damage. Our study raises the possibility of targeting CDK9 to elicit DNA damage in CRPC setting as an adjuvant to other treatments. Graphical Abstract

Purpose Prostate cancer (PC) is successfully treated with anti-androgens; however, a significant proportion of patients develop resistance against this therapy. Anti-androgen-resistant disease (castration-resistant prostate cancer; CRPC) is currently incurable. Cyclin-dependent kinase 7 (CDK7) is positioned to positively regulate both cell cycle and transcription, the two features critical for the rapid proliferation of the CRPC cells. Here, we assess if CDK7 is a viable target to halt the proliferation of CRPC cells. Methods We use recently developed clinically relevant compounds targeting CDK7 and multiple cell proliferation assays to probe the importance of this kinase for the proliferation of normal, androgen-dependent, and CRPC cells. PC patient data were used to evaluate expression of CDK7 at different disease-stages. Finally, comprehensive glycoproteome-profiling was performed to evaluate CDK7 inhibitor effects on androgen-dependent and CRPC cells. Results We show that CDK7 is overexpressed in PC patients with poor prognosis, and that CRPC cells are highly sensitive to compounds targeting CDK7. Inhibition of O-GlcNAc transferase sensitizes the CRPC, but not androgen-dependent PC cells, to CDK7 inhibitors. Glycoproteome-profiling revealed that CDK7 inhibition induces hyper-O-GlcNAcylation of the positive transcription elongation complex (pTEFB: CDK9 and CCNT1) in the CRPC cells. Accordingly, co-targeting of CDK7 and CDK9 synergistically blocks the proliferation of the CRPC cells but does not have anti-proliferative effects in the normal prostate cells. Conclusion We show that CRPC cells, but not normal prostate cells, are addicted on the high activity of the key transcriptional kinases, CDK7 and CDK9.

Prostate cancer (PC) is the most common cancer in men and after development of the castration-resistant PC (CRPC), there are no curative treatment options. Inactivating mutations in cyclin-dependent kinase 12 (CDK12) define an aggressive sub-type of CRPC. We hypothesized that compromised CDK12 activity leads to a significant rewiring of the CRPC cells, and that this rewiring results in actionable synthetic lethal interactions. Methods: We used combinatorial lethal screening, ChIP-seq data, RNA-seq data, global alternative splicing analysis, and comprehensive mass spectrometry (MS) profiling to understand how the compromised CDK12 activity rewires the CRPC cells. In addition, we used DepMap-, PC- and CRPC-datasets as a strategy to identify factors that are selectively required by the CDK12-mutant cells. Results: We show that inhibition of O-GlcNAc transferase (OGT) and CDK12 induces cancer cell-selective growth-defect. OGT catalyzes all nucleocytoplasmic O-GlcNAcylation, and we use unbiased MS-profiling to show that the short-term CDK12 inhibition induces hyper-O-GlcNAcylation of the spliceosome-machinery in PC and CRPC cells. Integration of DepMap- and a small scale-drug screen data reveled that depletion of CDK12 activity causes addiction to non-essential spliceosome components (CLK1/4 and SRPK1). CDK12-mutant tumors overexpress CLK1/4 and SRPK1. Finally, we show that the genomes of the CDK12-mutant tumors have lowered DNA methylation, and that CDK12 inhibition induces the expression of the genes marked by DNA methylation. Conclusions: Compromised CDK12 activity rewires DNA methylation, transcription and splicing, and this rewiring renders the affected cells addicted on the non-essential spliceosome components. We propose that inactivation of CDK12 is a biomarker for sensitivity against inhibitors of the non-essential spliceosome components just entering the clinical trials.