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Mediator-associated protein kinases CDK8 and CDK19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We identified two structurally differentiated chemical series, suitable for exploring their function. In addition to tools that fulfil the criteria set out for chemical probes, the lead compounds from each series, CCT251921 and MSC2530818, had optimal pharmacological and pharmaceutical properties making them suitable for preclinical studies. Having potent, highly selective, orally bioavailable exemplar compounds from these series in hand, we were well positioned to investigate the therapeutic potential of dual CDK8/19 inhibition. The compounds exhibited modest anti-tumor activity in colorectal cancer cell line xenograft models with modulation of p-STAT1SER727, a target engagement biomarker, and altered gene expression profiles, including super-enhancer regulated gene expression, consistent with the inhibition of CDK8/19. In PDX-derived cell cultures we observed inhibition of soft-agar growth in cells derived from different tumor types. However, we only detected significant antitumour activity in 1 of 6 colorectal PDX models tested in vivo, and one example of sensitization to standard of care chemotherapy, despite showing inhibition of p-STAT1SER727. Acute myeloid leukemia cells were the most sensitive cancer type in the PDX panel with therapeutic potency seen in systemic and sub-cutaneous models. Significantly, the compounds impacted on stem cell biology. In a bone progenitor model we saw dose-responsive activation and inhibition of markers of bone matrix and bone deposition that was distinct from WNT blockade. Treatment of a diverse collection of normal cell co-culture models detected a unique response profile consistent with stimulation of an immune/inflammatory response. In vivo treatment of a genetically engineered mouse model expressing oncogenic beta-catenin shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. Finally, in pre-clinical tolerability studies we observed a similar, widespread adverse safety profile at therapeutically relevant exposures for both CCT251921 and MSC2530818. At the concentrations tested we detected >80% inhibition of p-STAT1SER727 and increased IL-12 plasma levels. Since the observed pathological effects were generated with two potent, highly selective, but structurally distinct compounds, we conclude that the adverse consequences of treatment are the direct result of inhibition of CDK8 and/or CDK19. The serious and complex nature of the toxicity observed indicates that the clinical development of either series of CDK8/19 modulators, or other chemotypes with similar profiles, will be extremely challenging. Citation Format: Paul A. Clarke, Maria-Jesus Ortiz-Ruiz, Robert Te Poele, Olajumoke Adeniji-Popoola, Gary Box, Christina Esdar, Kenneth Ewan, Sharon Gowan, Alexis De Haven Brandon, Phllip Hewitt, Wolfgang Kaufmann, Aurelie Mallinger, Florence Raynaud, Felix Rohdich, Kai Schiemann, Stephanie Simon, Richard Schneider, Melanie Valenti, Julian Blagg, Trevor Dale, Suzanne Eccles, Paul Workman, Dirk Wienke Dirk Wienke. Assessing the mechanism and therapeutic potential of modulators of the human mediator complex-associated protein kinases CDK8 and CDK19 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 129. doi:10.1158/1538-7445.AM2017-129

Background The Mediator complex-associated kinases CDK8 and CDK19 are cyclin C-dependent enzymes that, with MED12 and MED13, form the kinase module of the Mediator complex. CDK8 expression correlates with activation of β-catenin in colon and gastric cancers and has also been associated with increased mortality in colorectal, breast and ovarian cancers. CDK8 is located in a region of chromosome 13 known to undergo copy number gain in ∼60% of colorectal cancers and inducible shRNA-mediated knockdown of CDK8 protein reduces the growth of colorectal cancer human tumor xenograft animal models harboring CDK8 gene amplification. Results Here we report the discovery and evaluation of CCT251545, a potent, selective and orally bioavailable small molecule chemical probe for CDK8 and CDK19 that we identified from a cell-based WNT pathway screen [1]. We also report a structure-based design approach to the discovery of CCT251921, a potent, selective and orally bioavailable inhibitor of CDK8, with equipotent affinity for CDK19, that has optimised pharmacokinetic and pharmaceutical properties suitable for preclinical development. Furthermore, we describe the discovery of MSC2530818, a structurally differentiated back-up candidate with equivalent pharmacological profile to CCT251921, from a high throughput screen versus CDK8 and subsequent structure-based design. Taking advantage of these two structurally distinct and highly selective dual CDK8/19 modulators we were able to reliably define on-target effects of targeting both CDK8 and CDK19 in the cellular context and in in vivo animal models. We describe gene expression profiles resulting from dual inhibition of CDK8 and CDK19 to demonstrate robust modulation of WNT signalling and additional pathways, including stress and immune response, consistent with the multiple contexts in which Mediator complex is known to regulate gene transcription. We show that both CCT251921 and MSC2530818 exhibit potent cell-based and in vivo inhibition of STAT1SER727 phosphorylation, a target engagement biomarker of CDK8 inhibition, and further demonstrate in vivo antiproliferative activity in human tumour xenograft animal models of colorectal cancer and acute myeloid leukaemia at exposures where pharmacodynamics biomarker modulation is evident. Recent observations suggest CDK8 as a novel anticancer therapeutic target; here we will disclose, for the first time, comprehensive preclinical efficacy, toleration and safety findings for both CCT251921 and MSC2530818 which will inform on the potential for dual CDK8/19 inhibition in the clinical setting. References 1. Dale, T. et. al. Identification of a potent and selective chemical probe for exploring the role of Mediator complex-associated protein kinases CDK8 and CDK19 in human disease. 2015, Nat. Chem. Biol., 11, 973-980. Citation Format: Paul Clarke, Christina Esdar, Aurelie Mallinger, Kai Schiemann, Dennis Waalboer, Simon Crumpler, Christian Rink, Frank Stieber, Michel Calderini, Olajumoke Adeniji-Popoola, Maria-Jesus Ortiz-Ruiz, Rahul S. Samant, Paul Czodrowski, Djordje Musil, Daniel Schwarz, Klaus Schneider, Michael Busch, Mark Stubbs, Rosemary Burke, Robert TePoele, Sharon Gowan, Felix Rohdich, Florence Raynaud, Richard Schneider, Oliver Poeschke, Andree Blaukat, Klaus Urbahns, Paul Workman, Wolfgang Kaufmann, Stephanie Simon, Suzanne A. Eccles, Trevor Dale, Dirk Wienke, Julian Blagg. Discovery of preclinical development candidate inhibitors of the mediator complex-associated kinases CDK8 and CDK19 and evaluation of their therapeutic potential. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3025.