Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

CDK9 inhibition strategy defines distinct sets of target genes.

BACKGROUND: CDK9 is the catalytic subunit of the Positive Transcription Elongation Factor b (P-TEFb), which phosphorylates the CTD of RNAPII and negative elongation factors enabling for productive elongation after initiation. CDK9 associates with T-type cyclins and cyclin K and its activity is tightly regulated in cells at different levels. CDK9 is also the catalytic subunit of TAK (Tat activating Kinase), essential for HIV1 replication. Because of CDK9's potential as a therapeutic target in AIDS, cancer, inflammation, and cardiomyophathy it is important to understand the consequences of CDK9 inhibition. A previous gene expression profiling study performed with human glioblastoma T98G cells in which CDK9 activity was inhibited either with a dominant negative mutant form of CDK9 (dnCDK9) or the pharmacological inhibitor Flavopiridol unveiled striking differences in gene expression effects. In the present report we extended these studies by (1) using both immortalized normal human fibroblasts and primary human astrocytes, (2) eliminating potential experimental variability due to transduction methodology and (3) also modulating CDK9 activity with siRNA. FINDINGS: Striking differences in the effects on gene expression resulting from the strategy used to inhibit CDK9 activity (dnCDK9 or FVP) remain even when potential variability due to viral transduction is eliminated. siRNA mediated CDK9 knockdown in human fibroblasts and astrocytes efficiently reduced CDK9 expression and led to potent changes in gene expression that exhibit little correlation with the effects of dnCDK9 or FVP. Interestingly, HEXIM1 a validated CDK9 target gene, was found to be potently downregulated by dnCDK9, FVP and siCDK9, but the cluster of genes with expression profiles similar to HEXIM1 was small. Finally, cluster analysis of all treatments revealed higher correlation between treatments than cell type origin. CONCLUSION: The nature of the strategy used to inhibit CDK9 profoundly affects the patterns of gene expression resulting from CDK9 inhibition. These results suggest multiple variables that affect outcome, including kinetics of inhibition, potency, off-target effects, and selectivity issues. This is particularly important when considering CDK9 as a potential target for therapeutic intervention.

Activation of p107 by fibroblast growth factor, which is essential for chondrocyte cell cycle exit, is mediated by the protein phosphatase 2A/B55α holoenzyme.

The phosphorylation state of pocket proteins during the cell cycle is determined at least in part by an equilibrium between inducible cyclin-dependent kinases (CDKs) and serine/threonine protein phosphatase 2A (PP2A). Two trimeric holoenzymes consisting of the core PP2A catalytic/scaffold dimer and either the B55α or PR70 regulatory subunit have been implicated in the activation of p107/p130 and pRB, respectively. While the phosphorylation state of p107 is very sensitive to forced changes of B55α levels in human cell lines, regulation of p107 in response to physiological modulation of PP2A/B55α has not been elucidated. Here we show that fibroblast growth factor 1 (FGF1), which induces maturation and cell cycle exit in chondrocytes, triggers rapid accumulation of p107-PP2A/B55α complexes coinciding with p107 dephosphorylation. Reciprocal solution-based mass spectrometric analysis identified the PP2A/B55α complex as a major component in p107 complexes, which also contain E2F/DPs, DREAM subunits, and/or cyclin/CDK complexes. Of note, p107 is one of the preferred partners of B55α, which also associates with pRB in RCS cells. FGF1-induced dephosphorylation of p107 results in its rapid accumulation in the nucleus and formation of larger complexes containing p107 and enhances its interaction with E2F4 and other p107 partners. Consistent with a key role of B55α in the rapid activation of p107 in chondrocytes, limited ectopic expression of B55α results in marked dephosphorylation of p107 while B55α knockdown results in hyperphosphorylation. More importantly, knockdown of B55α dramatically delays FGF1-induced dephosphorylation of p107 and slows down cell cycle exit. Moreover, dephosphorylation of p107 in response to FGF1 treatment results in early recruitment of p107 to the MYC promoter, an FGF1/E2F-regulated gene. Our results suggest a model in which FGF1 mediates rapid dephosphorylation and activation of p107 independently of the CDK activities that maintain p130 and pRB hyperphosphorylation for several hours after p107 dephosphorylation in maturing chondrocytes.

B55alpha PP2A holoenzymes modulate the phosphorylation status of the retinoblastoma-related protein p107 and its activation.

Pocket proteins negatively regulate transcription of E2F-dependent genes and progression through the G(0)/G(1) transition and the cell cycle restriction point in G(1). Pocket protein repressor activities are inactivated via phosphorylation at multiple Pro-directed Ser/Thr sites by the coordinated action of G(1) and G(1)/S cyclin-dependent kinases. These phosphorylations are reversed by the action of two families of Ser/Thr phosphatases: PP1, which has been implicated in abrupt dephosphorylation of retinoblastoma protein (pRB) in mitosis, and PP2A, which plays a role in an equilibrium that counteracts cyclin-dependent kinase (CDK) action throughout the cell cycle. However, the identity of the trimeric PP2A holoenzyme(s) functioning in this process is unknown. Here we report the identification of a PP2A trimeric holoenzyme containing B55α, which plays a major role in restricting the phosphorylation state of p107 and inducing its activation in human cells. Our data also suggest targeted selectivity in the interaction of pocket proteins with distinct PP2A holoenzymes, which is likely necessary for simultaneous pocket protein activation.

Coordinated activation of the origin licensing factor CDC6 and CDK2 in resting human fibroblasts expressing SV40 small T antigen and cyclin E.

We have previously shown that SV40 small t antigen (st) cooperates with deregulated cyclin E to activate CDK2 and bypass quiescence in normal human fibroblasts (NHF). Here we show that st expression in serum-starved and density-arrested NHF specifically induces up-regulation and loading of CDC6 onto chromatin. Coexpression of cyclin E results in further accumulation of CDC6 onto chromatin concomitantly with phosphorylation of CDK2 on Thr-160 and CDC6 on Ser-54. Investigation of the mechanism leading to CDC6 accumulation and chromatin loading indicates that st is a potent inducer of cdc6 mRNA expression and increases CDC6 protein stability. We also show that CDC6 expression in quiescent NHF efficiently promotes cyclin E loading onto chromatin, but it is not sufficient to activate CDK2. Moreover, we show that CDC6 expression is linked to phosphorylation of the activating T loop of CDK2 in serum-starved NHF stimulated with mitogens or ectopically expressing cyclin E and st. Our data suggest a model where the combination of st and deregulated cyclin E result in cooperative and coordinated activation of both an essential origin licensing factor, CDC6, and an activity required for origin firing, CDK2, resulting in progression from quiescence to S phase.

Cyclin E and SV40 small T antigen cooperate to bypass quiescence and contribute to transformation by activating CDK2 in human fibroblasts.

Cyclin E overexpression is observed in multiple human tumors and linked to poor prognosis. We have previously shown that ectopic expression of cyclin E is sufficient to induce mitogen-independent cell cycle entry in a variety of tumor/immortal cell lines. Here we have investigated the rate-limiting step leading to cell cycle entry in quiescent normal human fibroblasts (NHF) ectopically expressing cyclin E. We found that in serum-starved NHF, cyclin E forms inactive complexes with CDK2 and fails to induce DNA synthesis. Coexpression of SV40 small t antigen (st), but not other tested oncogenes, efficiently induces mitogen-independent CDK2 phosphorylation on Thr-160, CDK2 activation, and DNA synthesis. Additionally, in contact-inhibited NHF ectopically expressing cyclin E, st induces cell cycle entry, continued proliferation, and foci formation. Coexpression of cyclin E and st also bypasses G(0)/G(1) arrests induced by CDK inhibitors. Although CDK2 is dispensable for G(0)/G(1) cell cycle entry and normal proliferation in mammals, CDK2 activity is an essential rate-limiting step in NHF with deregulated cyclin E expression and altered PP2A activity, which endows primary cells with transformed features. Consequently, CDK2 could be targeted therapeutically in tumors that involve these alterations. These data also suggest that alterations prior to cyclin E deregulation facilitate proliferation of tumor cells by bypassing mitogenic requirements and negative regulation by adjacent cells.

Metabolic effects of ethanol on primary cell cultures of rat skeletal muscle.

Individuals who have consumed alcohol chronically accumulate glycogen in their skeletal muscles. Changes in the energy balance caused by alcohol consumption might lead to alcoholic myopathy. Experimental models used in the past, such as with skeletal muscle biopsy samples of alcohol-dependent individuals or in animal models, do not distinguish between direct effects and indirect effects (i.e., alterations to the nervous or endocrine system) of alcohol. In the current study, we evaluated the direct effect of ethanol on skeletal muscle glycogen concentrations and related glycolytic pathways. We measured the changes in metabolite concentrations and enzyme activities of carbohydrate metabolism in primary cell cultures of rat skeletal muscle exposed to ethanol for two periods. The concentrations of glycolytic metabolites and the activities of several enzymes that regulate glucose and glycogen metabolism were measured. After a short exposure to ethanol (6 h), glucose metabolism slowed. After 48 h of exposure, glycogen accumulation was observed.

A dynamic equilibrium between CDKs and PP2A modulates phosphorylation of pRB, p107 and p130.

It is thought that G(1) cyclin/CDK mediated phosphorylation of pocket proteins from mid G(1) to mitosis is reversed via dephosphorylation in mitosis. We examined the mechanisms involved in the unexpectedly rapid dephosphorylation of the pocket proteins induced via inhibition of cellular protein synthesis by cycloheximide (CHX) as well as direct inhibition of CDKs by flavopiridol. CHX and flavopiridol-induced dephosphorylation of pocket proteins is attributable to inactivation of D-type cyclin/CDKs and G(1)/S CDKs, respectively, which unmasks a phosphatase activity that targets the three pocket proteins apparently throughout the cell cycle. Treatment of cells with phosphatase inhibitors at concentrations selective for PP2A inhibition prevents CHX and flavopiridol-mediated dephosphorylation of pocket proteins in vivo. Also, ectopic expression of SV40 small t antigen, which inhibits PP2A via disruption of trimeric PP2A holoenzymes, delays CHX-induced pocket protein dephosphorylation. Moreover, dephosphorylation of p130 and p107 in cell extracts is inhibited by concentrations of okadaic acid known to inhibit PP2A, but not PP1. Finally, the PP2A catalytic subunit (PP2A/C) specifically interacts with both p130 and p107 in quiescent cells as well as cells progressing throughout the cell cycle. Together, these results demonstrate that the overall phosphorylation state of pocket proteins is determined, at least in part, by a dynamic equilibrium between CDKs and PP2A, or a closely related PP2A-like enzyme. These findings have important implications, as cell cycle or checkpoint-dependent inhibition of CDK activities counteracted by an active PP2A should have imminent effects on the phosphorylation state and activities of pocket proteins.

CDK9 is constitutively expressed throughout the cell cycle, and its steady-state expression is independent of SKP2.

CDK9 is a CDC2-related kinase and the catalytic subunit of the positive-transcription elongation factor b and the Tat-activating kinase. It has recently been reported that CDK9 is a short-lived protein whose levels are regulated during the cell cycle by the SCF(SKP2) ubiquitin ligase complex (R. E. Kiernan et al., Mol. Cell. Biol. 21:7956-7970, 2001). The results presented here are in contrast to those observations. CDK9 protein levels remained unchanged in human cells entering and progressing through the cell cycle from G(0), despite dramatic changes in SKP2 expression. CDK9 levels also remained unchanged in cells exiting from mitosis and progressing through the next cell cycle. Similarly, the levels of CDK9 protein did not change as cells exited the cell cycle and differentiated along various lineages. In keeping with these observations, the kinase activity associated with CDK9 was found to not be regulated during the cell cycle. We have also found that endogenous CDK9 is a very stable protein with a half-life (t(1/2)) of 4 to 7 h, depending on the cell type. In contrast, when CDK9 is overexpressed, it is not stabilized and is rapidly degraded, with a t(1/2) of less than 1 h, depending on the level of expression. Treatment of cells with proteasome inhibitors blocked the degradation of short-lived proteins, such as p27, but did not affect the expression of endogenous CDK9. Ectopic overexpression of SKP2 led to reduction of p27 protein levels but had no effect on the expression of endogenous CDK9. Finally, downregulation of endogenous SKP2 gene expression by interfering RNA had no effect on CDK9 protein levels, whereas p27 protein levels increased dramatically. Therefore, the SCF(SKP2) ubiquitin ligase does not regulate CDK9 expression in a cell cycle-dependent manner.

SKP2 associates with p130 and accelerates p130 ubiquitylation and degradation in human cells.

p130 is a member of the retinoblastoma family of pocket proteins, which includes pRB and p107. Unlike pRB and p107, p130 protein levels decrease dramatically following its hyperphosphorylation starting in the mid-G1 phase of the cell cycle. However, the mechanism leading to p130 downregulation is unknown. We have found that the proteasome inhibitor, lactacystin, inhibited p130 downregulation in T98G cells progressing through the G1/S transition and S phase and that p130 is multiubiquitylated in 293 cells. We have previously shown that ectopic expression of both cyclin D and E induces phosphorylation and downregulation of p130. Since the SKP1/Cul1/SKP2 E3 ubiquitin ligase complex mediates ubiquitylation of substrates previously phosphorylated by cyclin-dependent kinases, we investigated the potential role of this ubiquitin ligase in mediating p130 downregulation. We found that p130 interacts with SKP1, Cul-1 and SKP2 in human 293 cells. We also found that ectopic coexpression of SKP2 and p130 leads to dose-dependent downregulation of p130, reduces p130 protein half-life and induces p130 ubiquitylation in these cells. Moreover, adenoviral-mediated expression of SKP2 accelerates downregulation of endogenous hyperphosphorylated p130 in mitogen-stimulated T98G cells and primary WI38 fibroblasts. We conclude that p130 is a substrate of the SCF(SKP2) ubiquitin ligase and this E3 ligase regulates p130 abundance during the cell cycle.

G1 cyclin/cyclin-dependent kinase-coordinated phosphorylation of endogenous pocket proteins differentially regulates their interactions with E2F4 and E2F1 and gene expression.

Mitogenic stimulation leads to activation of G(1) cyclin-dependent kinases (CDKs), which phosphorylate pocket proteins and trigger progression through the G(0)/G(1) and G(1)/S transitions of the cell cycle. However, the individual role of G(1) cyclin-CDK complexes in the coordinated regulation of pocket proteins and their interaction with E2F family members is not fully understood. Here we report that individually or in concert cyclin D1-CDK and cyclin E-CDK complexes induce distinct and coordinated phosphorylation of endogenous pocket proteins, which also has distinct consequences in the regulation of pocket protein interactions with E2F4 and the expression of p107 and E2F1, both E2F-regulated genes. The up-regulation of these two proteins and the release of p130 and pRB from E2F4 complexes allows formation of E2F1 complexes not only with pRB but also with p130 and p107 as well as the formation of p107-E2F4 complexes. The formation of these complexes occurs in the presence of active cyclin D1-CDK and cyclin E-CDK complexes, indicating that whereas phosphorylation plays a role in the abrogation of certain pocket protein/E2F interactions, these same activities induce the formation of other complexes in the context of a cell expressing endogenous levels of pocket and E2F proteins. Of note, phosphorylated p130 "form 3," which does not interact with E2F4, readily interacts with E2F1. Our data also demonstrate that ectopic overexpression of either cyclin is sufficient to induce mitogen-independent growth in human T98G and Rat-1 cells, although the effects of cyclin D1 require downstream activation of cyclin E-CDK2 activity. Interestingly, in T98G cells, cyclin D1 induces cell cycle progression more potently than cyclin E. This suggests that cyclin D1 activates pathways independently of cyclin E that ensure timely progression through the cell cycle.