Grzegorz Nalepa

Drug discovery in the ubiquitin- proteasome system

Regulated protein turnover via the ubiquitin–proteasome system (UPS) underlies a wide variety of signalling pathways, from cell-cycle control and transcription to development. Recent evidence that pharmacological inhibition of the proteasome can be efficacious in the treatment of human cancers has set the stage for attempts to selectively inhibit the activities of disease-specific components of the UPS. Here, we review recent advances linking UPS components with specific human diseases, most prominently cancer and neurodegenerative disorders, and emphasize potential sites of therapeutic intervention along the regulated protein-degradation pathway.

Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities

The spindle checkpoint prevents chromosome mis-segregation by delaying sister chromatid separation until all chromosomes have achieved bipolar attachment to the mitotic spindle. Its operation is essential for accurate chromosome segregation, whereas its dysregulation can contribute to birth defects and tumorigenesis. The target of the spindle checkpoint is the anaphase-promoting complex (APC), a ubiquitin ligase that promotes sister chromatid separation and progression to anaphase. Using a short hairpin RNA screen targeting components of the ubiquitin-proteasome pathway in human cells, we identified the deubiquitinating enzyme USP44 (ubiquitin-specific protease 44) as a critical regulator of the spindle checkpoint. USP44 is not required for the initial recognition of unattached kinetochores and the subsequent recruitment of checkpoint components. Instead, it prevents the premature activation of the APC by stabilizing the APC-inhibitory Mad2–Cdc20 complex. USP44 deubiquitinates the APC coactivator Cdc20 both in vitro and in vivo, and thereby directly counteracts the APC-driven disassembly of Mad2–Cdc20 complexes (discussed in an accompanying paper). Our findings suggest that a dynamic balance of ubiquitination by the APC and deubiquitination by USP44 contributes to the generation of the switch-like transition controlling anaphase entry, analogous to the way that phosphorylation and dephosphorylation of Cdk1 by Wee1 and Cdc25 controls entry into mitosis.

Identification and Functional Evaluation of Cellular and Viral Factors Involved in the Alteration of Nuclear Architecture during Herpes Simplex Virus 1 Infection

ABSTRACT Herpes simplex virus 1 (HSV-1) replicates in the nucleus of host cells and radically alters nuclear architecture as part of its replication process. Replication compartments (RCs) form, and host chromatin is marginalized. Chromatin is later dispersed, and RCs spread past it to reach the nuclear edge. Using a lamin A-green fluorescent protein fusion, we provide direct evidence that the nuclear lamina is disrupted during HSV-1 infection and that the UL31 and UL34 proteins are required for this. We show nuclear expansion from 8 h to 24 h postinfection and place chromatin rearrangement and disruption of the lamina in the context of this global change in nuclear architecture. We show HSV-1-induced disruption of the localization of Cdc14B, a cellular protein and component of a putative nucleoskeleton. We also show that UL31 and UL34 are required for nuclear expansion. Studies with inhibitors of globular actin (G-actin) indicate that G-actin plays an essential role in nuclear expansion and chromatin dispersal but not in lamina alterations induced by HSV-1 infection. From analyses of HSV infections under various conditions, we conclude that nuclear expansion and chromatin dispersal are dispensable for optimal replication, while lamina rearrangement is associated with efficient replication.

Recognition of phosphodegron motifs in human cyclin E by the SCF Fbw7 ubiquitin ligase

Turnover of cyclin E is controlled by SCFFbw7. Three isoforms of Fbw7 are produced by alternative splicing. Whereas Fbw7α and -γ are nuclear and the β-isoform is cytoplasmic in 293T cells, all three isoforms induce cyclin E destruction in an in vivo degradation assay. Cyclin E is phosphorylated on Thr62, Ser88, Ser372, Thr380, and Ser384 in vivo. To examine the roles of phosphorylation in cyclin E turnover, a series of alanine point mutations in each of these sites were analyzed for Fbw7-driven degradation. As expected, mutation of the previously characterized residue Thr380 to alanine led to profound defects of cyclin E turnover, and largely abolished association with Fbw7. Mutation of Thr62 to alanine led to a dramatic reduction in the extent of Thr380 phosphorylation, suggesting an indirect effect of this mutation on cyclin E turnover. Nevertheless, phosphopeptides centered at Thr62 associated with Fbw7, and residual binding of cyclin ET380A to Fbw7 was abolished upon mutation of Thr62, suggesting a minor role for this residue in direct association with Fbw7. Mutation of Ser384 to alanine also rendered cyclin E resistant to degradation by Fbw7, with the largest effects being observed with Fbw7β. Cyclin ES384A associated more weakly with Fbw7α and -β isoforms but was not defective in Thr380 phosphorylation. Analysis of the localization of cyclin E mutant proteins indicated selective accumulation of cyclin ES384A in the nucleus, which may contribute to the inability of cytoplasmic Fbw7β to promote turnover of this cyclin E mutant protein.

The Cyclin E/Cdk2 Substrate p220NPAT Is Required for S-Phase Entry, Histone Gene Expression, and Cajal Body Maintenance in Human Somatic Cells

ABSTRACT Cyclin E/Cdk2, a central regulator of the G1/S transition, coordinates multiple cell cycle events, including DNA replication, centrosome duplication, and activation of the E2F transcriptional program. Recent studies suggest a role for cyclin E/Cdk2 in activation of histone transcription during S phase via the Cajal body-associated protein p220NPAT, and in addition, p220 can promote S-phase entry independently of histone transcriptional activation when overexpressed. Here we have examined the requirement for p220 in histone transcription, cell cycle progression, and Cajal body function through analysis of human somatic HCT116 cells engineered to contain a conditional p220 allele. p220 is required for proliferation of HCT116 cells, as assessed after expression of Cre recombinase in p220flox/− cells. This defect was due to an inability of these cells to transit from G0/G1 into S phase, and cell cycle arrest occurred in the presence of elevated Cdk2 kinase activity. Expression of human papillomavirus E7, but not E6, eliminated cell cycle arrest in response to p220 depletion. Optimal expression of all four core histone genes required p220, as did optimal transcription of a histone H4 promoter-luciferase construct. Basal histone H4 expression in G0/G1, although p220 dependent, occurs in the absence of detectable phosphorylation of p220 on Cdk2 sites. Cells lacking p220 displayed defects in the localization of the Cajal body component p80coilin as cells progressed from G0 to S phase in response to mitogenic signals. These finding indicate that p220 is an essential downstream component of the cyclin E/Cdk2 signaling pathway and functions to coordinate multiple elements of the G1/S transition.

Imatinib mesylate for plexiform neurofibromas in patients with neurofibromatosis type 1: a phase 2 trial

BACKGROUND Plexiform neurofibromas are slow-growing chemoradiotherapy-resistant tumours arising in patients with neurofibromatosis type 1 (NF1). Currently, there are no viable therapeutic options for patients with plexiform neurofibromas that cannot be surgically removed because of their proximity to vital body structures. We undertook an open-label phase 2 trial to test whether treatment with imatinib mesylate can decrease the volume burden of clinically significant plexiform neurofibromas in patients with NF1. METHODS Eligible patients had to be aged 3-65 years, and to have NF1 and a clinically significant plexiform neurofibroma. Patients were treated with daily oral imatinib mesylate at 220 mg/m(2) twice a day for children and 400 mg twice a day for adults for 6 months. The primary endpoint was a 20% or more reduction in plexiform size by sequential volumetric MRI imaging. Clinical data were analysed on an intention-to-treat basis; a secondary analysis was also done for those patients able to take imatinib mesylate for 6 months. This trial is registered with ClinicalTrials.gov, number NCT01673009. FINDINGS Six of 36 patients (17%, 95% CI 6-33), enrolled on an intention-to-treat basis, had an objective response to imatinib mesylate, with a 20% or more decrease in tumour volume. Of the 23 patients who received imatinib mesylate for at least 6 months, six (26%, 95% CI 10-48) had a 20% or more decrease in volume of one or more plexiform tumours. The most common adverse events were skin rash (five patients) and oedema with weight gain (six). More serious adverse events included reversible grade 3 neutropenia (two), grade 4 hyperglycaemia (one), and grade 4 increases in aminotransferase concentrations (one). INTERPRETATION Imatinib mesylate could be used to treat plexiform neurofibromas in patients with NF1. A multi-institutional clinical trial is warranted to confirm these results. FUNDING Novartis Pharmaceuticals, the Indiana University Simon Cancer Centre, and the Indiana University Herman B Wells Center for Pediatric Research.

The tumor suppressor CYLD regulates entry into mitosis

Mutations in the cylindromatosis (CYLD) gene cause benign tumors of skin appendages, referred to as cylindromas. The CYLD gene encodes a deubiquitinating enzyme that removes Lys-63-linked ubiquitin chains from IκB kinase signaling components and thereby inhibits NF-κB pathway activation. The dysregulation of NF-κB activity has been proposed to promote cell transformation in part by increasing apoptosis resistance, but it is not clear whether this is CYLDs only or predominant tumor-suppressing function. Here, we show that CYLD is also required for timely entry into mitosis. Consistent with a cell-cycle regulatory function, CYLD localizes to microtubules in interphase and the midbody during telophase, and its protein levels decrease as cells exit from mitosis. We identified the protein kinase Plk1 as a potential target of CYLD in the regulation of mitotic entry, based on their physical interaction and similar loss-of-function and overexpression phenotypes. Our findings raise the possibility that, as with other genes regulating tumorigenesis, CYLD has not only tumor-suppressing (apoptosis regulation) but also tumor-promoting activities (enhancer of mitotic entry). We propose that this additional function of CYLD could provide an explanation for the benign nature of most cylindroma lesions.

A functional genomic screen identifies a role for TAO1 kinase in spindle-checkpoint signalling

Defects in chromosome–microtubule attachment trigger spindle-checkpoint activation and delay mitotic progression. How microtubule attachment is sensed and integrated into the steps of checkpoint-signal amplification is poorly understood. In a functional genomic screen targeting human kinases and phosphatases, we identified a microtubule affinity-regulating kinase kinase, TAO1 (also known as MARKK) as an important regulator of mitotic progression, required for both chromosome congression and checkpoint-induced anaphase delay. TAO1 interacts with the checkpoint kinase BubR1 and promotes enrichment of the checkpoint protein Mad2 at sites of defective attachment, providing evidence for a regulatory step that precedes the proposed Mad2–Mad1 dependent checkpoint-signal amplification step. We propose that the dual functions of TAO1 in regulating microtubule dynamics and checkpoint signalling may help to coordinate the establishment and monitoring of correct congression of chromosomes, thereby protecting genomic stability in human cells.

Therapeutic anti-cancer targets upstream of the proteasome

Polyubiquitination of a protein is generally the first step in its degradation. This article discusses how altered protein destruction pathways impact the cell cycle and allow for abnormal cell proliferation, and explores how this process can be utilized in anticancer therapy. There are several levels of possible therapeutic intervention in ubiquitin-dependent proteolysis pathways upstream of the proteasome. In principle, targeting specific components of the ubiquitin system may offer an opportunity to develop selective drugs. However, the fact that general proteasome inhibitors have been demonstrated to be effective in cancer therapy suggests that other ubiquitin components that are common to many destruction pathways may also be clinically useful. We will, therefore, evaluate both the specific, rate-limiting enzymes and a number of general, nonselective enzymes as targets for anticancer therapy. Potential nonselective therapeutic strategies that are under investigation in a variety of human cancers include the identification and inhibition of individual F-box proteins, such as Skp2, and the inhibition of the ubiquitin ligases such as the SCF family, Mdm2, and Efp. A general pathway under investigation is the cullin neddylation and deneddylation system, with promising enzymatic targets such as csn5 and Rpn11.

Genetic disruption of both Fancc and Fancg in mice recapitulates the hematopoietic manifestations of Fanconi anemia.

Fanconi anemia (FA) is an inherited chromosomal instability syndrome characterized by bone marrow failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). Eight FA proteins associate in a nuclear core complex to monoubiquitinate FANCD2/FANCI in response to DNA damage. Additional functions have been described for some of the core complex proteins; however, in vivo genetic proof has been lacking. Here we show that double-mutant Fancc(-/-);Fancg(-/-) mice develop spontaneous hematologic sequelae including bone marrow failure, AML, MDS and complex random chromosomal abnormalities that the single-mutant mice do not. This genetic model provides evidence for unique core complex protein function independent of their ability to monoubiquitinate FANCD2/FANCI. Importantly, this model closely recapitulates the phenotypes found in FA patients and may be useful as a preclinical platform to evaluate the molecular pathogenesis of spontaneous bone marrow failure, MDS and AML in FA.