Targeting of Mediator Kinases for Cancer Therapy and Resistance Prevention

Estrogen receptor (ER)-positive breast cancers make up 70% of all breast cancer incidences in the US. Cell cycle progression of ER-positive breast cancer is highly dependent on cyclin-dependent kinases (CDK) 4 and 6, the G1/S checkpoint kinases. Inhibitors of CDK4/6 have become a major addition to the clinical arsenal against ER-positive breast cancers. CDK4/6 inhibitor palbociclib (IBRANCETM) has been approved for the treatment of ER-positive breast cancers. However, palbociclib treatment eventually fails due to the development of resistance. Laboratory studies revealed several diverse mechanisms of palbociclib resistance, making universal re-sensitization difficult to achieve. However, prevention of the adaptive process that precedes resistance development, such as transcriptional changes, might be a more promising synergistic approach. Here we discuss the role of CDK8/19 Mediator kinase, a broad-spectrum regulator of transcriptional reprogramming, in adaptation to the CDK4/6 inhibitor palbociclib. To investigate pre-resistant adaptation to CDK4/6 inhibition, we have characterized the dynamics of cell growth of MCF-7 ER+ breast cancer cells in the presence of different concentrations of palbociclib. Cells undergo rapid adaptation to the drug and resume cell growth within 2-3 weeks of treatment. Similar results were obtained with other cell lines, including ZR-75-1 ER+ breast cancer and SW-620 colon cancer cells, where adaptation took longer (3-4 weeks). Although the adapted cells proliferated in the presence of palbociclib, their insensitivity was unstable, indicating its non-genetic nature. Remarkably, palbociclib adaptation in all the cell lines was prevented by the addition of CDK8/19 inhibitors (which by themselves had only a moderate antiproliferative effect). To delineate transcriptional features of CDK8/19-dependent adaptation to palbociclib, we carried out RNA-Seq analysis of MCF-7 cells after 28-day adaptation and after short-term (3 days) exposure to palbociclib, with or without CDK8/19 inhibitor SNX631. Remarkably, all 10 of the 28-day palbociclib-adapted cell populations showed very similar changes in gene expression, including prominent upregulation of interferon-γ and interferon-α pathways. Notable effects of CDK8/19 inhibition on genes affected by 3-day treatment with palbociclib include downregulation of palbociclib-inducible genes and upregulation of palbociclib-inhibited genes. Our analysis identified several changes in gene expression that were associated with both 3-day and 28-day palbociclib treatment and counteracted by CDK8/19 inhibition, as potential mediators of CDK8/19-dependent transcriptional adaptation. One such change is the upregulation of CDK6, which has been previously linked to palbociclib resistance. We have also characterized the heterogeneity of MCF-7 cells regarding their inherent sensitivity to palbociclib, SNX631, and their combination. 44 single-cell clones were isolated and characterized by 7-day growth inhibition assays. This set of clones showed a wide range of palbociclib sensitivity, but the inherent resistance was unstable. All the clones were strongly inhibited when palbociclib was combined with SNX631. These results suggest that palbociclib adaptation is a robust and rapid process driven by phenotypic heterogeneity and by transcriptional reprogramming mediated by CDK8/19. To identify other cancers where these novel CDK8/19 inhibitors may provide benefit, we queried genomic and transcriptomic databases for the potential impact of CDK8, CDK19, or their binding partner CCNC. Among the candidates revealed by this analysis was ovarian cancer, where CDK8 correlated with shorter survival in samples with a below-median tumor mutation burden. These results suggest that Mediator kinases are especially important in ovarian cancers driven primarily by transcriptional, rather than mutational, changes and warrant an investigation of their role in ovarian cancer. To elucidate the dependence on CDK8/19 in ovarian cancer, we characterized several immortalized ovarian cancer cell lines (ES-2, OVCAR-8, P76, and SKOV-3) in response to selective CDK8/19 inhibitors (Senexin B and SNX631). These experiments also aid in establishing the newer SNX631 compound as a more stable inhibitor of CDK8/19 than Senexin B, as all 7-day SNX631 IC50s were found to be lower. Results were diverse, as P76 showed considerable CDK8/19i sensitivity, ES-2 and SKOV-3 showed moderate CDK8/19i sensitivity, and OVCAR-8 cells were resistant. To test whether the degree of malignancy of an ovarian tumor sample correlates with the amount of CDK8 present, tissue microarrays (TMAs) of primary and metastatic growths from 4 subtypes of ovarian cancer were fluorescently probed for CDK8. Benign tissue samples were used to establish a baseline average CDK8 expression. While all malignant subtypes exhibited CDK8 expression above this baseline, the difference was only significant in the mucinous and clear cell carcinoma subtypes. Only ovarian clear cell carcinoma had significantly higher CDK8 expression in the metastatic tumors relative to the primary growth of the same subtype. This subtype was selected for further studies