Chromatin compaction upon CDK12 inhibition drives long gene silencing and a combinatorial lethal dependency on PAF1

Loss-of-function mutations in cyclin-dependent kinase 12 (CDK12) define an aggressive subtype of metastatic castration-resistant prostate cancer (mCRPC) characterized by genomic instability, and depletion of CDK12 activity downregulates the expression of long DNA repair genes. While these transcriptional defects are well-documented, potential alterations in chromatin that may topologically affect this silencing and the subsequent adaptive survival mechanisms of tumor cells remain poorly understood. Here, we employ a dynamic multi-omics strategy to map the remodeling of the RNA Polymerase II (RNA Pol II) complex and chromatin landscape. We utilized 5-ethynyl uridine (5-EU) metabolic labeling combined with mass spectrometry (MS) to specifically capture the transcriptionally active RNA Pol II complex, distinguishing active elongation complexes from static background, and map how its interactome changes when the activity of the major transcriptional kinases CDK7, CDK9 or CDK12/13 is inhibited. These data are then integrated with ATAC-seq, transcriptomics, and targeted synthetic lethality screen to validate findings from the RNA Pol II capture. We show that, unlike CDK9 inhibition, CDK12/13 inhibition triggers a progressive chromatin compaction. This is characterized by the decrease in the binding activity of CTCF to chromosomes closing, which acts as a physical blockade specifically silencing long genes when CDK12 activity is lost. In response to this topological remodeling, cells launch an adaptive survival program by recruiting the transcription elongation factor PAF1 and the DNA damage sensor DDB1 to the stalled RNA Pol II. We identify PAF1 as a critical vulnerability in this context. Collectively, we identify CDK12 as a regulator of chromatin topology rather than merely an elongation kinase. The chromatin compaction upon CDK12/13 inhibition creates a unique dependency on the PAF1-mediated adaptive remodeling. Specifically, targeting PAF1 converts this stress response into a lethal trap, establishing a potent, kinase-selective combination lethal strategy for CDK12-deficient prostate cancer.