Systemic Toxicity Reported for CDK8/19 Inhibitors CCT251921 and MSC2530818 Is Not Due to Target Inhibition

Abstract

CDK8/19 kinases, which mediate transcriptional reprogramming, have become an active target for cancer drug discovery. Several small-molecule CDK8/19 inhibitors showed in vivo efficacy and two have entered clinical trials, with no significant toxicities reported. However, Clarke et al. (eLife 2016; 5; e20722) found severe systemic toxicity associated with two potent CDK8/19 inhibitors, Cmpd3 (CCT251921) and Cmpd4 (MSC2530818), and suggested that their toxicity was due to on-target effects. Here, we compared five CDK8/19 inhibitors: Cmpd3, Cmpd4, Senexin B, 16-didehydro-cortistatin A (dCA) and 15w, in different assays. Only Cmpd4 showed striking toxicity in developing zebrafish. In cell-based assays for CDK8 and CDK19 inhibition, Cmpd3, Cmpd4, dCA and 15w showed similar low-nanomolar potency and efficacy against CDK8 and CDK19, while Senexin B was less potent. Only dCA produced sustained inhibition of CDK8/19-dependent gene expression. While toxicity of different compounds did not correlate with their effects on CDK8 and CDK19, kinome profiling identified several off-target kinases for both Cmpd3 and Cmpd4, which could be responsible for their toxicity. Off-target activities could have been achieved in the study of Clarke et al. due to high in vivo doses of Cmpd3 and Cmpd4, chosen for the ability to inhibit STAT1 S727 phosphorylation in tumor xenografts. We show here that STAT1 S727 phosphorylation is induced by various cytokines and stress stimuli in CDK8/19-independent manner, indicating that it is not a reliable pharmacodynamic marker of CDK8/19 activity. These results illustrate the need for careful off-target analysis and dose selection in the development of CDK8/19 inhibitors.

The CDK8 inhibitor DCA promotes a tolerogenic chemical immunophenotype in CD4+ T cells via a novel CDK8-GATA3-FOXP3 pathway

“…Recent studies identify AS2863619 and CCT251921 as CDK8/19 inhibitors that enhance Treg differentiation (22,23). Our findings that DCA and BRD-6989 enhance Treg differentiation are important not only because they identify additional CDK8/19 inhibitors with similar effects, but also because recent studies point to DCA as having higher specificity and lower toxicity (24). Our studies here show important novel aspects.…”

“…These findings highlight the importance of better understanding which immune processes are regulated by CDK8 inhibition, and how. In particular, recent studies pointing to DCA as the CDK8 inhibitor with highest specificity and lowest toxicity highlight DCA as a particularly important compound to investigate (24).…”

The Inhibition of CDK8/19 Mediator Kinases Prevents the Development of Resistance to EGFR-Targeting Drugs

“…To test the effect of CDK8/19 inhibition on the outcome of selection, we have used the compound senexin B (4-((2-(6-(4-methylpiperazine-1-carbonyl)naphthalen-2-yl)ethyl)amino)quinazoline-6-carbonitrile), which is highly selective for CDK8/19 based on the lack of off-target inhibition in extensive kinome profiling [ 45 , 46 ] and lack of phenotypic effects in CDK8/19 knockout cells [ 38 , 47 ]. In contrast, when selection was carried out in the presence of 1 μM senexin B (concentration sufficient for near-maximum CDK8/19 kinase inhibition in cell-based assays [ 33 , 46 ]), cells did not grow out even after 8 weeks and were undetectable by crystal violet staining ( Figure 1 A) or showed minimal numbers by phase contrast microscopy ( Figure 1 B,C).…”

“…To test the effect of CDK8/19 inhibition on the outcome of selection, we have used the compound senexin B (4-((2-(6-(4-methylpiperazine-1-carbonyl)naphthalen-2-yl)ethyl)amino)quinazoline-6-carbonitrile), which is highly selective for CDK8/19 based on the lack of off-target inhibition in extensive kinome profiling [ 45 , 46 ] and lack of phenotypic effects in CDK8/19 knockout cells [ 38 , 47 ]. In contrast, when selection was carried out in the presence of 1 μM senexin B (concentration sufficient for near-maximum CDK8/19 kinase inhibition in cell-based assays [ 33 , 46 ]), cells did not grow out even after 8 weeks and were undetectable by crystal violet staining ( Figure 1 A) or showed minimal numbers by phase contrast microscopy ( Figure 1 B,C). To confirm the effects of CDK8/19 inhibition on the development of EGFR inhibitor resistance, we employed a chemically unrelated CDK8/19 inhibitor, 15w (3-amino-4-(4-(4-(2-(dimethylamino)-2-oxoethyl)phenyl)-1,4-diazepan-1-yl)thieno [2,3-b]pyridine-2-carboxamide), which is also highly selective for CDK8/19 based on kinome profiling [ 36 ] and phenotypic analysis [ 37 , 46 ].…”

CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

“…Previous studies have described CDKs as regulators of STAT1 Ser phosphorylation in different systems ( Bancerek et al., 2013 ; Chen et al., 2019 ; Kosciuczuk et al., 2019 ; Putz et al., 2013 ). Thus, we next tested whether the STAT3 Ser phosphorylation induced by HyIL-6 was mediated by CDKs.…”