Date of Award
Summer 2023
Document Type
Open Access Dissertation
Department
College of Pharmacy
First Advisor
Igor Roninson
Abstract
Following androgen deprivation therapy (ADT), prostate cancers (PCa) progress to castration resistant prostate cancer (CRPC) status, where their growth becomes independent of androgen signaling, through altered androgen receptor (AR) signaling or other genetic or transcriptional changes. Metastatic CRPC remains incurable, and the most potent new drugs prolong survival by only a few months. There is an urgent need for novel agents against advanced CRPC. Here we have investigated the roles of CDK8 and CDK19, two isoforms of Mediator kinase, which regulates transcriptional reprogramming, in regard to their biological activities and as potential novel targets for the treatment of CRPC.
To evaluate the activity and target selectivity of different Mediator kinase inhibitors, we have developed a robust cell-based assay for CDK8/19-dependent inhibition of the induction of NFκB transcriptional activity. Using this and other cell-based and cell-free assays, we have investigated whether systemic toxicity that was reported for two CDK8/19 inhibitors but not for three other chemically distinct inhibitors was due to the toxic inhibitors’ interactions with CDK8/19 or with other, off-target kinases. This analysis revealed that the reported toxicities were due to the off-target effects of the toxic inhibitors, whereas selective CDK8/19 inhibitors were well-tolerated.
We have conducted a detailed transcriptomic, proteomic and phosphoproteomic analysis of Mediator kinase activities, using genetic modifications of CDK8 and CDK19, selective CDK8/19 inhibitors and a potent CDK8/19 PROTAC degrader. CDK8/19 inhibition in human embryonic kidney 293 cells exposed to serum or to agonists of NFκB or protein kinase C (PKC) reduced the induction of signal-responsive genes, indicating a pleiotropic role of Mediator kinases in signal-induced transcriptional reprogramming. CDK8/19 inhibition under basal conditions initially downregulated a small group of genes, most of which were inducible by serum or PKC stimulation. Prolonged CDK8/19 inhibition or mutagenesis upregulated a larger gene set, and this transcriptomic effect was accompanied by a post-transcriptional increase in the proteins comprising the core Mediator complex and its kinase module. Regulation of both RNA and protein expression required CDK8/19 kinase activities. Analysis of isogenic cell populations expressing CDK8, CDK19 or their kinase-inactive mutants revealed that CDK8 and CDK19 have the same qualitative effects on protein phosphorylation and gene expression at the RNA and protein levels, whereas differential effects of CDK8 versus CDK19 knockouts were attributable to quantitative differences in their expression and activity rather than different functions.
To study the role of Mediator kinases in PCa, we have used bioinformatic analysis of gene expression in clinical cancers, selective Mediator kinase inhibitors and genetic modification of CDK19 and CDK8. These tools were used to investigate the effects of Mediator kinase inhibition on in vitro and in vivo growth of different PCa cell lines and patient-derived xenografts (PDX). Expression of CDK8 and CDK19 in androgen-responsive PCa cells was found to be responsive to androgen, which downregulated CDK8 but upregulated CDK19. In agreement with these results, CDK8 is decreased but CDK19 is increased in primary PCa (where AR signaling is upregulated) but both CDK19, CDK8 and their interacting proteins of the Mediator-associated CDK module become upregulated in metastatic CRPC (where AR-mediated transcription is drastically altered). Pharmacological Mediator kinase inhibitors did not suppress androgen-dependent PCa proliferation but selectively inhibited the in vivo growth of CRPC cell line-based and patient-derived xenografts, especially under the conditions of androgen deprivation. Genetic inactivation of CDK8 and CDK19 suppressed in vivo growth of 22Rv1 CRPC cells, which express the most common clinical variant of androgen-independent AR. CRPC tumor growth was selectively inhibited in surgically or chemically castrated (but not in intact) male mice, whereas the expression of either CDK19 or CDK8 reversed this phenotype. RNA-Seq analysis revealed that Mediator kinase inhibition preferentially affected tumor gene expression in castrated relative to intact mice and potentiated the tumor-restraining effects of castration on tumor and stromal transcriptomes. These results offer a mechanistic explanation for the selective CRPC suppression under the conditions of ADT in vivo. Remarkably, prolonged CDK8/19 inhibition (up to 300 days of continuous inhibitor treatment) was well tolerated and not only inhibited tumor growth but also induced shrinkage of CRPC tumors and even induced cures in 18- 27% of animals (unprecedented in CRPC models). These results warrant the exploration of Mediator kinase inhibitors for the treatment of the presently incurable CRPC.
Rights
© 2023, Jing Li
Recommended Citation
Li, J.(2023). CDK8 AND CDK19 Mediator Kinases as Novel Targets in Advanced Prostate Cancer. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/7388