Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay

Cell-based assays for CDK8/19 inhibition are not easily defined, since there are no known cellular functions unique to these kinases. To solve this problem, we generated derivatives of 293 cells with CRISPR knockout of one or both of CDK8 and CDK19. Double knockout (dKO) of CDK8 and CDK19 together (but not individually) decreased the induction of transcription by NFκB (a CDK8/19-potentiated transcription factor) and abrogated the effect of CDK8/19 inhibitors on such induction. We generated wild type (WT) and dKO cell lines expressing luciferase from an NFκB-dependent promoter. Inhibitors selective for CDK8/19 over other CDKs decreased TNFα-induced luciferase expression in WT cells by ~80% with no effect on luciferase induction in dKO cells. In contrast, non-selective CDK inhibitors flavopiridol and dinaciclib and a CDK7/12/13 inhibitor THZ1 (but not CDK4/6 inhibitor palbociclib) suppressed luciferase induction in both WT and dKO cells, indicating a distinct role for other CDKs in the NFκB pathway. We used this assay to characterize a series of thienopyridines with in vitro bone anabolic activity, one of which was identified as a selective CDK8/19 inhibitor. Thienopyridines inhibited luciferase induction in the WT but not dKO cells and their IC50 values in the WT reporter assay showed near-perfect correlation (R2 = 0.98) with their reported activities in a bone anabolic activity assay, confirming that the latter function is mediated by CDK8/19 and validating our assay as a robust and quantitative method for CDK8/19 inhibition

Regulation of Sp1 by cell cycle related proteins

Sp1 transcription factor regulates the expression of multiple genes, including the Sp1 gene itself. We analyzed the ability of different cell cycle regulatory proteins to interact with Sp1 and to affect Sp1 promoter activity. Using an antibody array, we observed that CDK4, SKP2, Rad51, BRCA2 and p21 could interact with Sp1 and we confirmed these interactions by co-immunoprecipitation. CDK4, SKP2, Rad51, BRCA2 and p21 also activated the Sp1 promoter. Among the known Sp1-interacting proteins, E2F-DP1, Cyclin D1, Stat3 and Rb activated the Sp1 promoter, whereas p53 and NFκB inhibited it. The proteins that regulated Sp1 gene expression were shown by positive chromatin immunoprecipitation to be bound to the Sp1 promoter. Moreover, SKP2, BRCA2, p21, E2F-DP1, Stat3, Rb, p53 and NFκB had similar effects on an artificial promoter containing only Sp1 binding sites. Transient transfections of CDK4, Rad51, E2F-DP1, p21 and Stat3 increased mRNA expression from the endogenous Sp1 gene in HeLa cells whereas overexpression of NFκB, and p53 decreased Sp1 mRNA levels. p21 expression from a stably integrated inducible promoter in HT1080 cells activated Sp1 expression at the promoter and mRNA levels, but at the same time it decreased Sp1 protein levels due to the activation of Sp1 degradation. The observed multiple effects of cell cycle regulators on Sp1 suggest that Sp1 may be a key mediator of cell cycle associated changes in gene expression

Identification of single nucleotide polymorphisms in the p21 (CDKN1A) gene and correlations with longevity in the Italian population

Longevity in humans is determined by multiple environmental and genetic factors. We have investigated possible associations between longevity and Single Nucleotide Polymorphisms (SNPs) in the p21 (CDKN1A) gene, a stress-inducible senescence-associated cell cycle inhibitor, expression of which upregulates genes implicated in several age-related diseases. By sequencing the promoter and exons of p21 in genomic DNA of ten individuals over 90 years old, we have identified 30 SNPs, many of which had not been previously characterized. A cluster of minor alleles within the -4547/-3489 bp region did not alter the basal activity or p53 responsiveness of the p21 promoter. We then compared the frequency of 41 p21 SNPs between 184 centenarians and 184 younger subjects in the Italian population. Rare alleles of two exon-derived SNPs, rs1801270 and rs1059234, were significantly under-represented among the centenarians; no significant differences were found for 39 non-exonic SNPs. SNP rs1801270 causes Ser to Arg substitution at amino acid 31 and SNP rs1059234 leads to a nucleotide change in the 3'-untranslated region. Previous studies showed that the rare alleles of these two SNPs may play a role in cancer. These p21 alleles may be potentially detrimental to longevity and therefore are rare in centenarians

A structural interpretation of the effect of GC-content on efficiency of RNA interference

BACKGROUND: RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful technique for eukaryotic gene knockdown. siRNA GC-content negatively correlates with RNAi efficiency, and it is of interest to have a convincing mechanistic interpretation of this observation. We here examine this issue by considering the secondary structures for both the target messenger RNA (mRNA) and the siRNA guide strand. RESULTS: By analyzing a unique homogeneous data set of 101 shRNAs targeted to 100 endogenous human genes, we find that: 1) target site accessibility is more important than GC-content for efficient RNAi; 2) there is an appreciable negative correlation between GC-content and RNAi activity; 3) for the predicted structure of the siRNA guide strand, there is a lack of correlation between RNAi activity and either the stability or the number of free dangling nucleotides at an end of the structure; 4) there is a high correlation between target site accessibility and GC-content. For a set of representative structural RNAs, the GC content of 62.6% for paired bases is significantly higher than the GC content of 38.7% for unpaired bases. Thus, for a structured RNA, a region with higher GC content is likely to have more stable secondary structure. Furthermore, by partial correlation analysis, the correlation for GC-content is almost completely diminished, when the effect of target accessibility is controlled. CONCLUSION: These findings provide a target-structure-based interpretation and mechanistic insight for the effect of GC-content on RNAi efficiency

Senescence and cancer : a review of clinical implications of senescence and senotherapies

Cellular senescence is a key component of human aging that can be induced by a range of stimuli, including DNA damage, cellular stress, telomere shortening, and the activation of oncogenes. Senescence is generally regarded as a tumour suppressive process, both by preventing cancer cell proliferation and suppressing malignant progression from pre-malignant to malignant disease. It may also be a key effector mechanism of many types of anticancer therapies, such as chemotherapy, radiotherapy, and endocrine therapies, both directly and via bioactive molecules released by senescent cells that may stimulate an immune response. However, senescence may contribute to reduced patient resilience to cancer therapies and may provide a pathway for disease recurrence after cancer therapy. A new group of drugs, senotherapies, (drugs which interact with senescent cells to interfere with their pro-aging impacts by either selectively destroying senescent cells (senolytic drugs) or inhibiting their function (senostatic drugs)) are under active investigation to determine whether they can enhance the efficacy of cancer therapies and improve resilience to cancer treatments. Senolytic drugs include quercetin, navitoclax, and fisetin and preclinical and early phase clinical data are emerging of their potential role in cancer treatments, although none are yet in routine use clinically. This article provides a review of these issues

Repression of the Sumo-Specific Protease SENP1 Induces P53-Dependent Premature Senescence in Normal Human Fibroblasts

The proliferative lifespan of normal somatic human cells in culture terminates in a permanent growth-arrested state known as replicative senescence. In this study, we show that RNA interference-mediated repression of the genes encoding the small ubiquitin-related modifier (SUMO)-specific proteases, Senp1, Senp2, and Senp7, induced low passage primary human fibroblasts to senesce rapidly. Following Senp1 repression, we observed a global increase in sumoylated proteins and in the number and size of nuclear SUMO-containing promyelocytic leukemia (PML) bodies. SUMO/PML bodies also increased during replicative senescence. p53 transcriptional activity was enhanced towards known p53 target genes following repression of Senp1, and inhibition of p53 function prevented senescence after Senp1 repression. These data indicate that Senp1 repression induces p53-mediated premature senescence and that SUMO proteases may thus be required for proliferation of normal human cells

TP53 codon 72 polymorphism affects accumulation of mtDNA damage in human cells

Human TP53 gene is characterised by a polymorphism at codon 72 leading to an Arginine-to-Proline (R/P) substitution. The two resulting p53 isoforms have a different subcellular localisation after stress (more nuclear or more mitochondrial for the P or R isoform, respectively). p53P72 variant is more efficient than p53R72 in inducing the expression of genes involved in nuclear DNA repair. Since p53 is involved also in mitochondrial DNA (mtDNA) maintenance, we wondered whether these p53 isoforms are associated with different accumulation of mtDNA damage. We observed that cells bearing p53R72 accumulate lower amount of mtDNA damage upon rotenone stress with respect to cells bearing p53P72, and that p53R72 co-localises with polymerase gamma more than p53P72. We also analysed the in vivo accumulation of heteroplasmy in a 300 bp fragment of mtDNA D-loop of 425 aged subjects. We observed that subjects with heteroplasmy higher than 5% are significantly less than expected in the p53R72/R72 group. On the whole, these data suggest that the polymorphism of TP53 at codon 72 affects the accumulation of mtDNA mutations, likely through the different ability of the two p53 isoforms to bind to polymerase gamma, and may contribute to in vivo accumulation of mtDNA mutations

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

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