Jan Eickhoff

The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a ‘pill’ that awakens the innate immune system to kill cancer metastases.

Proteomic characterization of the angiogenesis inhibitor SU6668 reveals multiple impacts on cellular kinase signaling

Knowledge about molecular drug action is critical for the development of protein kinase inhibitors for cancer therapy. Here, we establish a chemical proteomic approach to profile the anticancer drug SU6668, which was originally designed as a selective inhibitor of receptor tyrosine kinases involved in tumor vascularization. By employing immobilized SU6668 for the affinity capture of cellular drug targets in combination with mass spectrometry, we identified previously unknown targets of SU6668 including Aurora kinases and TANK-binding kinase 1. Importantly, a cell cycle block induced by SU6668 could be attributed to inhibition of Aurora kinase activity. Moreover, SU6668 potently suppressed antiviral and inflammatory responses by interfering with TANK-binding kinase 1-mediated signal transmission. These results show the potential of chemical proteomics to provide rationales for the development of potent kinase inhibitors, which combine rather unexpected biological modes of action by simultaneously targeting defined sets of both serine/threonine and tyrosine kinases involved in cancer progression.

Protein kinase inhibitors of the quinazoline class exert anti-cytomegaloviral activity in vitro and in vivo

Cytomegalovirus infection is associated with severe disease in immunocompromised individuals. Current antiviral therapy faces several limitations. In a search of novel drug candidates, we describe here the anti-cytomegaloviral properties of two compounds of the chemical class of quinazolines, gefitinib (Iressa) and Ax7396 (RGB-315389). Both compounds showed strong inhibitory effects in vitro against human and animal cytomegaloviruses with IC(50)s in a low micromolar range. Cytotoxicity did not occur at these effective concentrations. The antiviral mode of action was based on the inhibition of protein kinase activity, mainly directed to a viral target kinase (UL97/M97) in addition to cellular target candidates. This was demonstrated by a high sensitivity of the respective protein kinases in vitro and by infection experiments with viral mutants carrying genomic alterations in the ORF UL97/M97 modulating viral drug sensitivity. In a guinea pig model, gefitinib showed inhibition of cytomegaloviral loads in blood and lung tissue. Importantly, the rate of mortality of infected animals was reduced by gefitinib treatment. In contrast to the in vitro data, Ax7396 showed no significant antiviral activity in a mouse model. Further in vivo analyses have to assess the potential use of gefitinib in the treatment of cytomegalovirus disease.

Cyclin-dependent Kinases Phosphorylate the Cytomegalovirus RNA Export Protein pUL69 and Modulate Its Nuclear Localization and Activity.

Replication of human cytomegalovirus (HCMV) is subject to regulation by cellular protein kinases. Recently, we and others reported that inhibition of cyclin-dependent protein kinases (CDKs) or the viral CDK ortholog pUL97 can induce intranuclear speckled aggregation of the viral mRNA export factor, pUL69. Here we provide the first evidence for a direct regulatory role of CDKs on pUL69 functionality. Although replication of all HCMV strains was dependent on CDK activity, we found strain-specific differences in the amount of CDK inhibitor-induced pUL69 aggregate formation. In all cases analyzed, the inhibitor-induced pUL69 aggregates were clearly localized within viral replication centers but not subnuclear splicing, pore complex, or aggresome structures. The CDK9 and cyclin T1 proteins colocalized with these pUL69 aggregates, whereas other CDKs behaved differently. Phosphorylation analyses in vivo and in vitro demonstrated pUL69 was strongly phosphorylated in HCMV-infected fibroblasts and that CDKs represent a novel class of pUL69-phosphorylating kinases. Moreover, the analysis of CDK inhibitors in a pUL69-dependent nuclear mRNA export assay provided evidence for functional impairment of pUL69 under suppression of CDK activity. Thus, our data underline the crucial importance of CDKs for HCMV replication, and indicate a direct impact of CDK9-cyclin T1 on the nuclear localization and activity of the viral regulator pUL69.

Phosphotyrosine-specific phosphatase PTP-SL regulates the ERK5 signaling pathway

The duration and the magnitude of mitogen-activated protein kinase (MAPK) activation specifies signal identity and thus allows the regulation of diverse cellular functions by the same kinase cascade. A tight and finely tuned regulation of MAPK activity is therefore critical for the definition of a specific cellular response. We investigated the role of tyrosine-specific phosphatases (PTPs) in the regulation of ERK5. Although unique in its structure, ERK5 is activated in analogy to other MAPKs by dual phosphorylation of threonine and tyrosine residues in its activation motif. In this study we concentrated on whether and how PTP-SL, a kinase-interacting motif-containing PTP, might be involved in the down-regulation of the ERK5 signal. We found that both proteins interact directly with each other in vitro and in intact cells, resulting in mutual modulation of their enzymatic activities. PTP-SL is a substrate of ERK5 and independent of phosphorylation binding to the kinase enhances its catalytic phosphatase activity. On the other hand, interaction with PTP-SL not only down-regulates endogenous ERK5 activity but also effectively impedes the translocation of ERK5 to the nucleus. These findings indicate a direct regulatory influence of PTP-SL on the ERK5 pathway and corresponding downstream responses of the cell.

Characterization of molecular and cellular functions of the cyclin-dependent kinase CDK9 using a novel specific inhibitor

The cyclin‐dependent kinase CDK9 is an important therapeutic target but currently available inhibitors exhibit low specificity and/or narrow therapeutic windows. Here we have used a new highly specific CDK9 inhibitor, LDC000067 to interrogate gene control mechanisms mediated by CDK9.

RICK Activates a NF-κB-dependent Anti-human Cytomegalovirus Response

The adapter kinase receptor interacting protein-like interacting caspase-like apoptosis regulatory protein kinase (RICK, also called RIP2 and CARDIAK) was found to be elevated at both the protein and RNA levels during human cytomegalovirus (HCMV) replication, suggesting either that the virus may require RICK for replication or that RICK is part of an unsuccessful host attempt to inhibit HCMV replication. It is demonstrated here that forced expression of RICK in either a kinase active or inactive form activates nuclear factor (NF)-κB by means of its intermediate domain and potently blocks HCMV replication in human fibroblasts. Importantly, NF-κB activation, which exerted a modestly positive effect on the early phase of infection, clearly had a strongly negative impact during later viral steps. A stable inhibitor of NF-κB (IκB) reverses the RICK inhibitory effect, and activation of NF-κB by IκB kinase β expression is inhibitory to HCMV, demonstrating that NF-κB activation is part of a potent anti-HCMV response. Supernatant transfer experiments identified interferon-β as a downstream component of the RICK inhibitory pathway. RICK expression was found to synergize with HCMV infection in the induction of interferon-β expression. This study identifies an endogenous RICK-activated, NF-κB- and interferon-β-dependent antiviral pathway that is either inhibited or faulty under normal HCMV replication conditions; efforts to bolster this pathway may lead to novel anti-viral approaches.

A Novel CDK7 Inhibitor of the Pyrazolotriazine Class Exerts Broad-Spectrum Antiviral Activity at Nanomolar Concentrations

ABSTRACT Protein kinases represent central and multifunctional regulators of a balanced virus-host interaction. Cyclin-dependent protein kinase 7 (CDK7) plays crucial regulatory roles in cell cycle and transcription, both connected with the replication of many viruses. Previously, we developed a CDK7 inhibitor, LDC4297, that inhibits CDK7 in vitro in the nano-picomolar range. Novel data from a kinome-wide evaluation (>330 kinases profiled in vitro) demonstrate a kinase selectivity. Importantly, we provide first evidence for the antiviral potential of the CDK7 inhibitor LDC4297, i.e., in exerting a block of the replication of human cytomegalovirus (HCMV) in primary human fibroblasts at nanomolar concentrations (50% effective concentration, 24.5 ± 1.3 nM). As a unique feature compared to approved antiherpesviral drugs, inhibition occurred already at the immediate-early level of HCMV gene expression. The mode of antiviral action was considered multifaceted since CDK7-regulated cellular factors that are supportive of HCMV replication were substantially affected by the inhibitors. An effect of LDC4297 was identified in the interference with HCMV-driven inactivation of retinoblastoma protein (Rb), a regulatory step generally considered a hallmark of herpesviral replication. In line with this finding, a broad inhibitory activity of the drug could be demonstrated against a selection of human and animal herpesviruses and adenoviruses, whereas other viruses only showed intermediate drug sensitivity. Summarized, the CDK7 inhibitor LDC4297 is a promising candidate for further antiviral drug development, possibly offering new options for a comprehensive approach to antiviral therapy.

Cyclin-Dependent Kinase 7 Controls mRNA Synthesis by Affecting Stability of Preinitiation Complexes, Leading to Altered Gene Expression, Cell Cycle Progression, and Survival of Tumor Cells

ABSTRACT Cyclin-dependent kinase 7 (CDK7) activates cell cycle CDKs and is a member of the general transcription factor TFIIH. Although there is substantial evidence for an active role of CDK7 in mRNA synthesis and associated processes, the degree of its influence on global and gene-specific transcription in mammalian species is unclear. In the current study, we utilize two novel inhibitors with high specificity for CDK7 to demonstrate a restricted but robust impact of CDK7 on gene transcription in vivo and in in vitro-reconstituted reactions. We distinguish between relative low- and high-dose responses and relate them to distinct molecular mechanisms and altered physiological responses. Low inhibitor doses cause rapid clearance of paused RNA polymerase II (RNAPII) molecules and sufficed to cause genome-wide alterations in gene expression, delays in cell cycle progression at both the G1/S and G2/M checkpoints, and diminished survival of human tumor cells. Higher doses and prolonged inhibition led to strong reductions in RNAPII carboxyl-terminal domain (CTD) phosphorylation, eventual activation of the p53 program, and increased cell death. Together, our data reason for a quantitative contribution of CDK7 to mRNA synthesis, which is critical for cellular homeostasis.

The Role of RIP2 in p38 MAPK Activation in the Stressed Heart

The activation of p38 MAPK by dual phosphorylation aggravates myocardial ischemic injury and depresses cardiac contractile function. SB203580, an ATP-competitive inhibitor of p38 MAPK and other kinases, prevents this dual phosphorylation during ischemia. Studies in non-cardiac tissue have shown receptor-interacting protein 2 (RIP2) lies upstream of p38 MAPK, is SB203580-sensitive and ischemia-responsive, and aggravates ischemic injury. We therefore examined the RIP2-p38 MAPK signaling axis in the heart. Adenovirus-driven expression of wild-type RIP2 in adult rat ventricular myocytes caused robust, SB203580-sensitive dual phosphorylation of p38 MAPK associated with activation of p38 MAPK kinases MKK3, MKK4, and MKK6. The effect of SB203580 was recapitulated by unrelated inhibitors of RIP2 or the downstream MAPK kinase kinase, TAK1. However, overexpression of wild-type, kinase-dead, caspase recruitment domain-deleted, or kinase-dead and caspase recruitment domain-deleted forms of RIP2 had no effect on the activating dual phosphorylation of p38 MAPK during simulated ischemia. Similarly, p38 MAPK activation and myocardial infarction size in response to true ischemia did not differ between hearts from wild-type and RIP2 null mice. However, both p38 MAPK activation and the contractile depression caused by the endotoxin component muramyl dipeptide were attenuated by SB203580 and in RIP2 null hearts. Although RIP2 can cause myocardial p38 MAPK dual phosphorylation in the heart under some circumstances, it is not responsible for the SB203580-sensitive pattern of activation during ischemia.