Integrated Multi-Omics and Interactome Analysis of CDK8 Inhibition Reveals Erythroid Differentiation Programs and BET Synergy in AML Stem-like Cells

Background. Acute myeloid leukemia (AML) remains among the most therapeutically challenging hematologic malignancies, driven largely by the self-renewal capacity, quiescence, and therapy resistance of leukemic stem cells (LSCs). CDK8, a kinase component of the Mediator complex, has emerged as a regulator of oncogenic transcription. We demonstrated that the selective CDK8/CDK19 inhibitor RVU120 (Romaciclib; SEL120-34A) potently targets AML cells with CD34⁺/pSTAT5-high LSC-like characteristics. However, the full spectrum of epigenetic and transcriptional events triggered by CDK8 blockade, and combinatorial strategies that may exploit them, remains incompletely defined. Methods. Using the TEX LSC-like cell line treated with CDK8 inhibitors, RVU120 and CCT251921, we performed time-resolved (3h, 24h, 72h) RNA-Seq, whole-proteome and phosphoproteomics mass spectrometry (MS). Chromatin occupancy of CDK8, Polymerase II RNA (RNAP2), BRD3, MLL-4, NFRKB and histone marks (H3K27ac, H3K4me1, H3K4me3) was profiled with CUT&Tag. CDK8 protein–protein interactions (PPIs) were surveyed by co-immunoprecipitation MS (co-IP-MS) and validated across five AML models. Synergy between CDK8 inhibitors and Pelabresib (BET inhibitor) or CB6644 (RUVBL1/2 inhibitor) was assessed by high-content screening microscopy in three cell lines and three patient-derived xenograft (PDX) models. Results. Both inhibitors suppressed STAT5 Ser726/731 phosphorylation; over subsequent days, TEX cells lost their CD34⁺/CD38⁻ phenotype and acquired surface markers of erythromegakaryocytic differentiation. Transcriptomics revealed a concordant response to both CDK8 inhibitors (r = 0.82–0.95), including early cholesterol biosynthesis, followed by inflammatory programs, and erythroid commitment with GATA1 upregulation. Whole-proteome changes significantly correlated with the transcriptomic response (r = 0.65–0.78). Phosphoproteomics identified 33 significantly different phosphosites with overrepresentation of nucleic acid-binding proteins. In the TEX cell line, co-IP-MS identified 125 CDK8-interacting proteins and a conserved PPI network across five AML models, including Mediator subunits, NFRKB, and BRD3. CUT&Tag genomic profiling demonstrated that CDK8 inhibition triggers widespread enhancer activation with coordinated redistribution of RNAP2, BRD3, and NFRKB, while uncoupling proliferative from immune-regulatory chromatin programs. CDK8 combined with Pelabresib acted synergistic in MOLM-16 cells and two of three PDX models. Conclusions. These findings position CDK8 as a central node in LSC transcriptional control and establish a mechanistic rationale for combined CDK8-BET inhibition as a precision therapeutic strategy in molecularly defined AML subsets.