ABSTRACT: Acute myeloid leukemia (AML) with leukemic stem cell (LSC) characteristics represents a therapeutic challenge due to inherent resistance to differentiation. CDK8, a kinase component of the Mediator complex, has emerged as a regulator of oncogenic transcription, yet its role in LSC maintenance and potential as a combination therapy target remains incompletely defined. RVU120 (Romaciclib) and CCT251921 are selective, orally bioavailable small-molecule inhibitors of CDK8. Of them, RVU120 is currently tested as monotherapy in stage II clinical trials for Myelodysplastic Syndrome. Here, we demonstrate that pharmacologic CDK8 inhibition rapidly suppresses STAT5 Ser726 phosphorylation in LSC-like TEX cells, reduces CD34+ populations, and drives erythroid/megakaryocytic differentiation. Integrated time-resolved transcriptomic, proteomic, and phosphoproteomic analyses of TEX cells, revealed highly concordant responses to both inhibitors, with progressive activation of cholesterol biosynthesis, inflammatory signaling, and erythroid-like differentiation. Phosphoproteomic profiling identified a restricted set of altered phosphosites enriched in nucleic acid-binding proteins. Co-immunoprecipitation mass spectrometry mapping of the CDK8 interactome in TEX and AML models identified core proteins, including Mediator components, chromatin remodelers (INO80), and BRD3, a BET family bromodomain protein. CUT&Tag genomic profiling demonstrated that CDK8 inhibition triggers widespread enhancer activation (H3K27ac, H3K4me1) with coordinated redistribution of Polymerase II RNA, BRD3, and MLL4, while uncoupling proliferative from immune-regulatory chromatin programs. Functional screening revealed context-dependent synergy between CDK8 and BET inhibition in AML cell lines and patient-derived xenograft models. 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.
