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The Fanconi anemia (FA) pathway safeguards genomic stability through cell cycle regulation and DNA damage repair. The canonical tumor suppressive role of FA proteins in the repair of DNA damage during interphase is well established, but their function in mitosis is incompletely understood. Here, we performed a kinome‐wide synthetic lethality screen in FANCA−/− fibroblasts, which revealed multiple mitotic kinases as necessary for survival of FANCA‐deficient cells. Among these kinases, we identified the depletion of the centrosome kinase SIK2 as synthetic lethal upon loss of FANCA. We found that FANCA colocalizes with SIK2 at multiple mitotic structures and regulates the activity of SIK2 at centrosomes. Furthermore, we found that loss of FANCA exacerbates cell cycle defects induced by pharmacological inhibition of SIK2, including impaired G2‐M transition, delayed mitotic progression, and cytokinesis failure. In addition, we showed that inhibition of SIK2 abrogates nocodazole‐induced prometaphase arrest, suggesting a novel role for SIK2 in the spindle assembly checkpoint. Together, these findings demonstrate that FANCA‐deficient cells are dependent upon SIK2 for survival, supporting a preclinical rationale for targeting of SIK2 in FA‐disrupted cancers.

Summary Background Data to inform surveillance and treatment for leukaemia predisposition syndromes are scarce and recommendations are largely based on expert opinion. This study aimed to investigate the clinical features and outcomes of patients with myelodysplastic syndrome or acute myeloid leukaemia and Shwachman-Diamond syndrome, an inherited bone marrow failure disorder with high risk of developing myeloid malignancies. Methods We did a multicentre, retrospective, cohort study in collaboration with the North American Shwachman-Diamond Syndrome Registry. We reviewed patient medical records from 17 centres in the USA and Canada. Patients with a genetic (biallelic mutations in the SBDS gene) or clinical diagnosis (cytopenias and pancreatic dysfunction) of Shwachman-Diamond syndrome who developed myelodysplastic syndrome or acute myeloid leukaemia were eligible without additional restriction. Medical records were reviewed between March 1, 2001, and Oct 5, 2017. Masked central review of bone marrow pathology was done if available to confirm leukaemia or myelodysplastic syndrome diagnosis. We describe the clinical features and overall survival of these patients. Findings We initially identified 37 patients with Shwachman-Diamond syndrome and myelodysplastic syndrome or acute myeloid leukaemia. 27 patients had samples available for central pathology review and were reclassified accordingly (central diagnosis concurred with local in 15 [56%] cases), 10 had no samples available and were classified based on the local review data, and 1 patient was excluded at this stage as not eligible. 36 patients were included in the analysis, of whom 10 (28%) initially presented with acute myeloid leukaemia and 26 (72%) initially presented with myelodysplastic syndrome. With a median follow-up of 4·9 years (IQR 3·9–8·4), median overall survival for patients with myelodysplastic syndrome was 7·7 years (95% CI 0·8–not reached) and 0·99 years (95% CI 0·2–2·4) for patients with acute myeloid leukaemia. Overall survival at 3 years was 11% (95% CI 1–39) for patients with leukaemia and 51% (29–68) for patients with myelodysplastic syndrome. Management and surveillance were variable. 18 (69%) of 26 patients with myelodysplastic syndrome received upfront therapy (14 haematopoietic stem cell transplantation and 4 chemotherapy), 4 (15%) patients received no treatment, 2 (8%) had unavailable data, and 2 (8%) progressed to acute myeloid leukaemia before receiving treatment. 12 patients received treatment for acute myeloid leukaemia—including the two patients initially diagnosed with myelodysplastic who progressed— two (16%) received HSCT as initial therapy and ten (83%) received chemotherapy with intent to proceed with HSCT. 33 (92%) of 36 patients (eight of ten with leukaemia and 25 of 26 with myelodysplastic syndrome) were known to have Shwachman-Diamond syndrome before development of a myeloid malignancy and could have been monitored with bone marrow surveillance. Bone marrow surveillance before myeloid malignancy diagnosis was done in three (33%) of nine patients with leukaemia for whom surveillance status was confirmed and 11 (46%) of 24 patients with myelodysplastic syndrome. Patients monitored had a 3-year overall survival of 62% (95% CI 32–82; n=14) compared with 28% (95% CI 10–50; n=19; p=0·13) without surveillance. Six (40%) of 15 patients with available longitudinal data developed myelodysplastic syndrome in the setting of stable blood counts. Interpretation Our results suggest that prognosis is poor for patients with Shwachman-Diamond syndrome and myelodysplastic syndrome or acute myeloid leukaemia owing to both therapy-resistant disease and treatment-related toxicities. Improved surveillance algorithms and risk stratification tools, studies of clonal evolution, and prospective trials are needed to inform effective prevention and treatment strategies for leukaemia predisposition in patients with Shwachman-Diamond syndrome. Funding National Institute of Health.

Background Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, short stature, congenital anomalies, and cancer predisposition. Most cases are due to mutations in genes encoding ribosomal proteins (RP) leading to RP haploinsufficiency. Effective treatments for the anemia of DBA include chronic red cell transfusions, long-term corticosteroid therapy, or hematopoietic stem cell transplantation. In a small patient series and in animal models, there have been hematologic responses to L-leucine with amelioration of anemia. The study objectives of this clinical trial were to determine feasibility, safety, and efficacy of L-leucine in transfusion-dependent patients with DBA. Procedure Patients ≥2 years of age received L-leucine 700 mg/m2 orally three times daily for nine months to determine a hematologic response and any improvement in growth (NCT01362595). Results This multicenter, phase I/II study enrolled 55 subjects; 43 were evaluable. There were 21 males; the median age at enrollment was 10.4 years (range, 2.5-46.1 years). No significant adverse events were attributable to L-leucine. Two subjects had a complete erythroid response and five had a partial response. Nine of 25, and 11 of 25, subjects experienced a positive weight and height percentile change, respectively, at the end of therapy. Conclusions L-leucine is safe, resulted in an erythroid response in 16% of subjects with DBA, and led to an increase in weight and linear growth velocity in 36% and 44% of evaluable subjects, respectively. Further studies will be critical to understand the role of L-leucine in the management of patients with DBA.

Fanconi anaemia (FA) is a genetic disorder that is characterized by bone marrow failure (BMF), developmental abnormalities and predisposition to cancer. Together with other proteins involved in DNA repair processes and cell division, the FA proteins maintain genome homeostasis, and germline mutation of any one of the genes that encode FA proteins causes FA. Monoallelic inactivation of some FA genes, such as FA complementation group D1 (FANCD1; also known as the breast and ovarian cancer susceptibility gene BRCA2), leads to adult-onset cancer predisposition but does not cause FA, and somatic mutations in FA genes occur in cancers in the general population. Carcinogenesis resulting from a dysregulated FA pathway is multifaceted, as FA proteins monitor multiple complementary genome-surveillance checkpoints throughout interphase, where monoubiquitylation of the FANCD2-FANCI heterodimer by the FA core complex promotes recruitment of DNA repair effectors to chromatin lesions to resolve DNA damage and mitosis. In this Review, we discuss how the FA pathway safeguards genome integrity throughout the cell cycle and show how studies of FA have revealed opportunities to develop rational therapeutics for this genetic disease and for malignancies that acquire somatic mutations within the FA pathway.

1. Richa Sharma, MD* 2. Grzegorz Nalepa, MD, PhD*,† 1. *Department of Pediatrics, Division of Pediatric Hematology-Oncology, Indiana University School of Medicine, Indianapolis, IN. 2. †Department of Medical and Molecular Genetics and Department of Biochemistry, Indiana University School of Medicine; Bone Marrow Failure Program, Riley Hospital for Children at IU Health, Indianapolis, IN. Diagnostic evaluation and management of children with chronic pancytopenia is highly complex. (1)(2)(3) Clinicians should be aware of the subtle signs and symptoms of acquired and congenital childhood pancytopenias to maintain a high index of suspicion, streamline the referral process, and function within their patients’ multidisciplinary medical homes. After completing this article, readers should be able to: 1. Identify signs and symptoms of pancytopenia. 2. Identify patients who require emergency referral due to a life-threatening illness. 3. Discuss the basics of clinical presentation, diagnostics, and management of childhood severe aplastic anemia. 4. Discuss the basics of clinical presentation, diagnostics, and management of common genetic syndromes associated with pancytopenia. 5. Understand the basic concepts behind laboratory tests and be aware of which tests are appropriate in specific clinical situations. Pancytopenia is characterized by a decreased number of at least two blood cell lines. Pancytopenia may progress acutely, such as with decreased blood cell counts in fulminant sepsis, disseminated intravascular coagulation, or rapid hemolysis. Alternatively, pancytopenia may evolve insidiously over weeks to months. In this review we focus on the latter group of acquired and congenital pancytopenias in children. Chronic pancytopenia is a diagnostic challenge. The range of potential causes is bewildering, signs and symptoms overlap substantially, and many diseases presenting with pancytopenia are life-threatening if not recognized and managed properly. Therefore, clinicians must be familiar with clinical scenarios that should prompt evaluation of blood counts and a pediatric hematology referral. ### Causes Pancytopenia may be …

Correct diagnosis of inherited bone marrow failure syndromes is a challenge because of the significant overlap in clinical presentation of these disorders. Establishing right genetic diagnosis is crucial for patients’ optimal clinical management and family counseling. A nondysmorphic infant reported here developed severe transfusion-dependent anemia and met clinical criteria for diagnosis of Diamond–Blackfan anemia (DBA). However, whole-exome sequencing demonstrated that the child was a compound heterozygote for a paternally inherited pathogenic truncating variant (SPTA1c.4975 C>T) and a novel maternally inherited missense variant of uncertain significance (SPTA1c.5029 G>A) within the spectrin gene, consistent with hereditary hemolytic anemia due to disruption of red blood cell (RBC) cytoskeleton. Ektacytometry demonstrated abnormal membrane flexibility of the child's RBCs. Scanning electron microscopy revealed morphological aberrations of the patient's RBCs. Both parents were found to have mild hereditary elliptocytosis. Importantly, patients with severe RBC membrane defects may be successfully managed with splenectomy to minimize peripheral destruction of misshapen RBCs, whereas patients with DBA require lifelong transfusions, steroid therapy, or hematopoietic stem cell transplantation. As suggested by the WES findings, splenectomy rendered our patient transfusion-independent, improving the family's quality of life and preventing transfusion-related iron overload. This case illustrates the utility of whole-exome sequencing in clinical care of children with genetic disorders of unclear presentation.

The partially understood phosphoinositide signaling cascade regulates multiple aspects of cellular metabolism. Previous studies revealed that INPP5E, the inositol polyphosphate-5-phosphatase that is mutated in the developmental disorders Joubert and MORM syndromes, is essential for the function of the primary cilium and maintenance of phosphoinositide balance in nondividing cells. Here, we report that INPP5E further contributes to cellular homeostasis by regulating cell division. We found that silencing or genetic knockout of

Fanconi anemia (FA) is a heterogenous genetic disease with a high risk of cancer. The FA proteins are essential for interphase DNA damage repair; however, it is incompletely understood why FA-deficient cells also develop gross aneuploidy, leading to cancer. Here, we systematically evaluated the role of the FA proteins in chromosome segregation through functional RNAi screens and analysis of primary cells from patients with FA. We found that FA signaling is essential for the spindle assembly checkpoint and is therefore required for high-fidelity chromosome segregation and prevention of aneuploidy. Furthermore, we discovered that FA proteins differentially localize to key structures of the mitotic apparatus in a cell cycle–dependent manner. The essential role of the FA pathway in mitosis offers a mechanistic explanation for the aneuploidy and malignant transformation known to occur after disruption of FA signaling. Collectively, our findings provide insight into the genetically unstable cancers resulting from inactivation of the FA/BRCA pathway.

Background Atopic dermatitis (AD) is characterized by intense pruritis and is a common childhood inflammatory disease. Many factors are known to affect AD development, including the pleiotropic cytokine IL-4. Yet little is known regarding the direct effects of IL-4 on keratinocyte function. Objective and Methods In this report RNA sequencing and functional assays were used to define the effect of the allergic environment on primary keratinocyte function and wound repair in mice. Results Acute or chronic stimulation by IL-4 modified expression of more than 1000 genes expressed in human keratinocytes that are involved in a broad spectrum of nonoverlapping functions. Among the IL-4–induced changes, repression of fibronectin critically impaired the human keratinocyte wound response. Moreover, in mouse models of spontaneous and induced AD-like lesions, there was delayed re-epithelialization. Importantly, topical treatment with fibronectin restored the epidermal repair response. Conclusion Keratinocyte gene expression is critically shaped by IL-4, altering cell fate decisions, which are likely important for the clinical manifestations and pathology of allergic skin disease.

Fanconi anemia (FA) is an inherited disorder of genomic instability associated with high risk of myelodysplasia and acute myeloid leukemia (AML). Young mice deficient in FA core complex genes do not naturally develop cancer, hampering preclinical studies on malignant hematopoiesis in FA. Here we describe that aging Fancc −/− mice are prone to genomically unstable AML and other hematologic neoplasms. We report that aneuploidy precedes malignant transformation during Fancc −/− hematopoiesis. Our observations reveal that Fancc −/− mice develop hematopoietic chromosomal instability followed by leukemia in an age-dependent manner, recapitulating the clinical phenotype of human FA and providing a proof of concept for future development of preclinical models of FA-associated leukemogenesis.

A 23 year old man with short stature, congenital malformation of the left thumb and both ears, and a single kidney presented with back pain, shortness of breath, fatigue, and easy bruising that had developed over several months. Physical examination revealed hepatosplenomegaly. His brother had died in early childhood, more than 20 years ago. The patient’s parents reported that his brother, “had similar but worse congenital malformations, including bad kidney problems.” Full blood count showed pancytopaenia, which prompted further laboratory investigations (table 1). View this table: Table 1 Initial laboratory investigations Radiography of the patient’s left hand (fig 1) revealed a single fused phalanx, anomalous carpal bones, and a hypoplastic radial styloid. Fig 1 (A) Dysmorphic left thumb. …

Defects in chromosome–microtubule attachment trigger spindle-checkpoint activation and delay mitotic progression1,2. 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 step3. 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.

We describe a child with dyserythropoietic anemia, thrombocytosis, functional platelet defect, and megakaryocyte dysplasia. We show that (i) this constellation of hematopoietic abnormalities was due to a germline mutation within the 5' untranslated region (5'UTR) of globin transcription factor 1 (GATA1); (ii) the mutation impaired a 5'UTR GATA1 splicing site, with promoted production of the shortened GATA1 isoform lacking the N-terminus; and (iii) expression of the GATA1 N-terminus is restricted to erythroblasts and megakaryocytes in normal marrow, consistent with the patient's abnormal erythropoiesis and megakaryopoiesis. Our findings provide insights into the clinically relevant in vivo function of the N-terminal domain of GATA1 in human hematopoiesis.

Dense fibrotic stroma associated with pancreatic ductal adenocarcinoma (PDAC) is a major obstacle for drug delivery to the tumor bed and plays a crucial role in pancreatic cancer progression. Current, anti-stromal therapies have failed to improve tumor response to chemotherapy and patient survival. Furthermore, recent studies show that stroma impedes tumor progression and its complete ablation accelerates PDAC progression. In an effort to understand the molecular mechanisms associated with tumor-stromal interactions, using in vitro and in vivo models and PDAC patient biopsies, we show that the loss of miR-29 is a common phenomenon of activated pancreatic stellate cells (PSCs)/fibroblasts, the major stromal cells responsible for fibrotic stromal reaction. Loss of miR-29 is correlated with a significant increase in extracellular matrix (ECM) deposition, a major component in PDAC stroma. Our in vitro miR-29 gain/loss-of-function studies document the role of miR-29 in PSC-mediated ECM stromal protein accumulation. Overexpression of miR-29 in activated stellate cells reduced stromal deposition, cancer cell viability and cancer growth in co-culture. Furthermore, the loss of miR-29 in TGF-β1 activated PSCs is SMAD3 dependent. These results provide insights into the mechanistic role of miR-29 in PDAC stroma and its potential use as a therapeutic agent to target PDAC.

For 30 years, the mammalian cell nucleus has been hypothesized to contain a filamentous framework, the nuclear matrix or karyoskeleton, which regulates nuclear structure and function. However, such an organized network of filaments has never been observed in living cells. Here we show that human Cdc14B phosphatase in living cells tightly associates with long filaments that begin at the nucleolar periphery and extend to the nuclear envelope, frequently making close connections with nuclear pore complexes. We demonstrate that Cdc14B contains a bipartite signal that directs it to the intranuclear filaments, and we also detect a small amount of Cdc14B on interphase and mitotic centrosomes. Furthermore, we show that Cdc14B is critical for the maintenance of proper nuclear structure together with polo-like kinase Plk1. This work provides the first direct evidence for the existence of an intranuclear filamentous framework in living mammalian cells and implicates Cdc14B in the control of mammalian nuclear architecture. Cell Motil. Cytoskeleton 59:94–108, 2004. © 2004 Wiley-Liss, Inc.

Diagnosis of bone marrow failure (BMF) disorders is challenging but essential for optimal patient management. Here, we report a young adult from nonconsanguineous parents with progressive pancytopenia since childhood, bone pain, increased bone density, and haphazard ossification replacing hematopoiesis within the bone marrow. Sequencing revealed two novel biallelic variants of unknown significance within the thromboxane A synthase gene, TBXAS1 (c.266T > C; c.989T > C), bioinformatically predicted to disrupt the protein. TBXAS1 mutations result in Ghosal hematodiaphyseal dysplasia (OMIM 231095), the autosomal recessive syndrome associated with abnormal bone structure and BMF. Identification of the genetic defect prompted steroid therapy leading to resolution of symptoms.

Cyclin E-CDK2 is a key regulator in G1/S transition. Previously, we identified a number of CDK2-interacting proteins, including PHF8 (plant homeodomain finger protein 8). In this report, we confirmed that PHF8 is a novel cyclin E-CDK2 substrate. By taking the approach of mass spectrometry, we identified that PHF8 Ser-844 is phosphorylated by cyclin E-CDK2. Immunoblotting analysis indicated that WT PHF8 demethylates histone H3K9me2 more efficiently than the cyclin E-CDK2 phosphorylation-deficient PHF8-S844A mutant. Furthermore, flow cytometry analysis showed that WT PHF8 promotes S phase progression more robustly than PHF8-S844A. Real-time PCR results demonstrated that PHF8 increases transcription of cyclin E, E2F3, and E2F7 to significantly higher levels compared with PHF8-S844A. Further analysis by ChIP assay indicated that PHF8 binds to the cyclin E promoter stronger than PHF8-S844A and reduces the H3K9me2 level at the cyclin E promoter more efficiently than PHF8-S844A. In addition, we found that cyclin E-CDK2-mediated phosphorylation of PHF8 Ser-844 promotes PHF8-dependent rRNA transcription in luciferase reporter assays and real-time PCR. Taken together, these results indicate that cyclin E-CDK2 phosphorylates PHF8 to stimulate its demethylase activity to promote rRNA transcription and cell cycle progression.

Neurofibromatosis type 2 (NF2) is an autosomal dominant genetic disorder resulting from germline mutations in the NF2 gene. Bilateral vestibular schwannomas, tumors on cranial nerve VIII, are pathognomonic for NF2 disease. Furthermore, schwannomas also commonly develop in other cranial nerves, dorsal root ganglia and peripheral nerves. These tumors are a major cause of morbidity and mortality, and medical therapies to treat them are limited. Animal models that accurately recapitulate the full anatomical spectrum of human NF2-related schwannomas, including the characteristic functional deficits in hearing and balance associated with cranial nerve VIII tumors, would allow systematic evaluation of experimental therapeutics prior to clinical use. Here, we present a genetically engineered NF2 mouse model generated through excision of the Nf2 gene driven by Cre expression under control of a tissue-restricted 3.9kbPeriostin promoter element. By 10 months of age, 100% of Postn-Cre; Nf2(flox/flox) mice develop spinal, peripheral and cranial nerve tumors histologically identical to human schwannomas. In addition, the development of cranial nerve VIII tumors correlates with functional impairments in hearing and balance, as measured by auditory brainstem response and vestibular testing. Overall, the Postn-Cre; Nf2(flox/flox) tumor model provides a novel tool for future mechanistic and therapeutic studies of NF2-associated schwannomas.

Summary 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 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. During cell division the spindle checkpoint ensures that chromosome segregation is delayed until all chromosomes are properly attached to the mitotic spindle. Two papers now identify a new regulatory mechanism that controls the spindle checkpoint. This involves the fine-tuned ubiquitination and de-ubiquitination of a coactivator of the anaphase promoting complex APC/C to regulate the timing of APC/C activation and thereby the onset of anaphase.

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.

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.

Turnover of cyclin E is controlled by SCF(Fbw7). Three isoforms of Fbw7 are produced by alternative splicing. Whereas Fbw7alpha and -gamma are nuclear and the beta-isoform is cytoplasmic in 293T cells, all three isoforms induce cyclin E destruction in an in vivo degradation assay. Cyclin E is phosphorylated on Thr(62), Ser(88), Ser(372), Thr(380), and Ser(384) 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 Thr(380) to alanine led to profound defects of cyclin E turnover, and largely abolished association with Fbw7. Mutation of Thr(62) to alanine led to a dramatic reduction in the extent of Thr(380) phosphorylation, suggesting an indirect effect of this mutation on cyclin E turnover. Nevertheless, phosphopeptides centered at Thr(62) associated with Fbw7, and residual binding of cyclin E(T380A) to Fbw7 was abolished upon mutation of Thr(62), suggesting a minor role for this residue in direct association with Fbw7. Mutation of Ser(384) to alanine also rendered cyclin E resistant to degradation by Fbw7, with the largest effects being observed with Fbw7beta. Cyclin E(S384A) associated more weakly with Fbw7alpha and -beta isoforms but was not defective in Thr(380) phosphorylation. Analysis of the localization of cyclin E mutant proteins indicated selective accumulation of cyclin E(S384A) in the nucleus, which may contribute to the inability of cytoplasmic Fbw7beta to promote turnover of this cyclin E mutant protein.

In response to DNA damage and DNA replication interference, cells activate an elaborate network of signaling pathways collectively called the DNA damage stress response pathway (Zhou and Elledge 2000). The central conduits of this network are two parallel but partially overlapping protein kinase cascades, the ATM-Chk2 and ATR-Chk1 kinase modules, that transduce the damage signal to downstream effectors involved in cell cycle control, DNA repair, and apoptosis (Zhou and Elledge 2000; Shiloh 2003). ATM primarily responds to DNA double-strand breaks, whereas ATR responds to agents that interfere with DNA replication in addition to double-strand breaks (Zou and Elledge 2003). The branches of these pathways used in arresting the cell cycle are called checkpoints. In many eukaryotes, the targets of checkpoint pathways are the cyclin-dependent kinases, Cdks, which control multiple cell cycle transitions including the G1/S transition, late origin firing during S phase, and the G2/M transition, each of which is inhibited in response to DNA damage. Cells have evolved multiple mechanisms to inhibit Cdk activity. In mammals, the Cdk inhibitor p21Cip1 is induced in response to DNA damage through activation of the p53 transcription factor, which is activated and stabilized in response to DNA damage (Giaccia and Kastan 1998; Zhou and Elledge 2000). In addition, levels of cyclin D1 rapidly decrease in response to DNA damage during G1, leading to redistribution of p21Cip1 from Cdk4 to Cdk2 (Agami and Bernards 2000). Cdks are also regulated by inhibitory phosphorylation on tyrosine, which is altered in response to DNA damage (Jin et al. 1997; Rhind et al. 1997; for review, see Takizawa and Morgan 2000; Donzelli and Draetta 2003). The regulatory mechanisms controlling Cdk phosphorylation have been extensively studied. Tyrosine phosphorylation is regulated by the opposing activities of the tyrosine kinases Wee1 and Myt1, and a group of tyrosine phosphatases known as Cdc25A, Cdc25B, Cdc25C (for review, see Morgan 1997). A direct connection between checkpoint signaling and Cdc25 was established when it was found that the checkpoint kinase Chk1, and later Chk2, could phosphorylate Cdc25C on a site relevant to its checkpoint function in vivo (Peng et al. 1996; Sanchez et al. 1996; Furnari et al. 1997; Matsuoka et al. 1998). Furthermore, these kinases were shown to phosphorylate all three Cdc25 family members, suggesting they were general targets of DNA damage stress response pathways (Sanchez et al. 1996; Matsuoka et al. 1998). Analysis of Chk regulation of Cdc25 from several systems showed that phosphorylation of Cdc25C both inhibited kinase activity (Blasina et al. 1999; Furnari et al. 1999) and maintained Cdc25C in the cytoplasm, where it cannot access Cdk/cyclin complexes efficiently (Zeng et al. 1998; Kumagai and Dunphy 1999; Lopez-Girona et al. 1999; Zeng and Piwnica-Worms 1999). However, mice lacking Cdc25C grow normally and have intact checkpoint responses (Chen et al. 2001), suggesting that other family members may play more prominent roles in Cdk regulation. A second family member implicated in the damage response is Cdc25A. Cdc25A is capable of removing inhibitory tyrosine phosphorylation from both Cdk1 and Cdk2 kinases to promote entry into and progression through S phase and mitosis (Hoffmann et al. 1994; Vigo et al. 1999; for review, see Donzelli and Draetta 2003). Cdc25A has also been shown to be a phosphorylation target of Chk kinases (Sanchez et al. 1996) and to be regulated by Chk kinases in response to DNA damage (for review, see Donzelli and Draetta 2003). In contrast to regulation of Cdc25C, Cdc25A is destroyed in response to ionizing radiation (IR) and ultraviolet (UV) light through a process involving ubiquitin-mediated proteolysis. During G1, UV treatment leads to Chk1-dependent elimination of Cdc25A (Mailand et al. 2000) and persistent Cdk2 Y15 phosphorylation. During an unperturbed S phase, Cdc25A is unstable and this instability requires Cdc25A phosphorylation by Chk1 (Falck et al. 2001; Sorensen et al. 2003). IR during S phase leads to accelerated Cdc25A phosphorylation by Chk1 with a concomitant increase in turnover. Defects in this intra-S-phase checkpoint lead to radio-resistant DNA synthesis (RDS; Falck et al. 2001; Xiao et al. 2003). Whereas depletion of Chk1 leads to an RDS phenotype, expression of a Cdk2 mutant that is resistant to inhibitory tyrosine phosphorylation overcomes IR-dependent S-phase arrest (Falck et al. 2001), implicating elimination of Cdc25A in the intra-S-phase checkpoint. Recent studies indicate that Cdc25A turnover through the ubiquitin pathway involves at least two temporally distinct components (Donzelli et al. 2002; Donzelli and Draetta 2003). During mitotic exit and early G1, Cdc25A stability is controlled by the anaphase-promoting complex in conjunction with Cdh1. During interphase, however, Cdc25A turnover is dependent on Cul1 (Donzelli et al. 2002), a central component of the SCF (Skp1/Cul1/F-box protein) ubiquitin ligase (Feldman et al. 1997; Skowyra et al. 1997). Precisely how Cul1 promotes turnover of Cdc25A is unknown. In SCF complexes, Cul1 together with the RING-H2 finger protein Rbx1 forms the core ubiquitin ligase that binds ubiquitin-conjugating enzymes (for review, see Deshaies 1999; Koepp et al. 1999). Specificity in these reactions is achieved by a substrate-binding module composed of Skp1 and a member of the F-box family of proteins. F-box proteins interact with Skp1 through the F-box motif (Bai et al. 1996) and with substrates through C-terminal protein interaction domains, including WD40 propellers (Skowyra et al. 1997; Wu et al. 2003; Orlicky et al. 2003). Frequently, association of SCF targets with the requisite F-box protein requires that the substrate be modified, typically by phosphorylation to produce a short peptide motif displaying properties of a phosphodegron (Winston et al. 1999a; Koepp et al. 2001; Nash et al. 2001; Wu et al. 2003; Orlicky et al. 2003). Here we report that constitutive and DNA-damage-induced turnover of Cdc25A occurs via the SCFβ-TRCP ubiquitin ligase. Depletion of β-TRCP by shRNA stabilizes Cdc25A, leading to inappropriately high levels of Cdk2 kinase activity characteristic of a checkpoint defect. Cdc25A ubiquitination by SCFβ-TRCP in vitro involves Chk1-dependent phosphorylation principally at S76, consistent with the requirement for Chk1 in vivo. However, Chk1-mediated phosphorylation of Cdc25A does not appear to be sufficient to generate the requisite phosphodegron for β-TRCP recruitment. We find that residues 79-84 of Cdc25A constitute a phosphodegron for recognition by β-TRCP and implicate S79 and S82 as phosphoacceptor sites in this motif. Indeed, S82 is in a sequence context (Asp-Ser-Gly-Phe) reminiscent of previously identified phosphodegrons in the β-TRCP substrates IκBα and β-catenin. We suggest that Chk1-mediated phosphorylation of S75 may promote Cdc25A turnover by facilitating the phosphorylation of the adjacent phosphodegron targeted by β-TRCP.

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 p220(flox/-) 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.

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.

Apoptosis (genetically programmed cell death) plays a key role in human physiology and pathogenesis of various diseases, including cancer. A suicide of cell can be initiated by many different factors, but activation of caspases, which are a special class of proteolytic enzymes, is always involved in this process. Activation of caspases may be achieved by several molecular pathways: the best known stimuli triggering caspase cascade are stimulation of Fas or TNF receptors, release of cytochrome c from the cellular mitochondria and exposure to granzymes, which are secreted by cytotoxic T cells. Activated caspases digest many cellular proteins responsible for cell cycle regulation (e.g. RB, MDM2), DNA damage recognition and repair (e.g. DNA-PK, P53, PARP), and regulation of the cellular structure (e.g. actin and lamins). All these functional and structural protein modifications lead directly to apoptosis. Further research on the mechanisms controlling caspase activity and the modes of action will provide better insight into pathogenesis of cancer and other disorders. It may be even the first step to design new and more efficient methods of conventional tumor treatment or gene therapy.

CDKN3 (cyclin-dependent kinase inhibitor 3), a dual specificity protein phosphatase, dephosphorylates cyclin-dependent kinases (CDKs) and thus functions as a key negative regulator of cell cycle progression. Deregulation or mutations of CDNK3 have been implicated in various cancers. However, the role of CDKN3 in Bcr-Abl-mediated chronic myelogenous leukemia (CML) remains unknown. Here we found that CDKN3 acts as a tumor suppressor in Bcr-Abl-mediated leukemogenesis. Overexpression of CDKN3 sensitized the K562 leukemic cells to imanitib-induced apoptosis and dramatically inhibited K562 xenografted tumor growth in nude mouse model. Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence. In contrast, depletion of CDKN3 expression conferred resistance to imatinib-induced apoptosis in the leukemic cells and accelerated the growth of xenograph leukemia in mice. In addition, we found that CDKN3 mutant (CDKN3-C140S) devoid of the phosphatase activity failed to affect the K562 leukemic cell survival and xenografted tumor growth, suggesting that the phosphatase of CDKN3 was required for its tumor suppressor function. Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression. Moreover, overexpression of CDKN3 delayed G1/S transition in K562 leukemic cells. Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.

A genome-wide screen of phosphatases that control mitosis identified CDKN3, which acts through the CDC2 signaling axis., Mitosis is controlled by a network of kinases and phosphatases. We screened a library of small interfering RNAs against a genome-wide set of phosphatases to comprehensively evaluate the role of human phosphatases in mitosis. We found four candidate spindle checkpoint phosphatases, including the tumor suppressor CDKN3. We show that CDKN3 is essential for normal mitosis and G1/S transition. We demonstrate that subcellular localization of CDKN3 changes throughout the cell cycle. We show that CDKN3 dephosphorylates threonine-161 of CDC2 during mitotic exit and we visualize CDC2pThr-161 at kinetochores and centrosomes in early mitosis. We performed a phosphokinome-wide mass spectrometry screen to find effectors of the CDKN3-CDC2 signaling axis. We found that one of the identified downstream phosphotargets, CKβ phosphorylated at serine 209, localizes to mitotic centrosomes and controls the spindle checkpoint. Finally, we show that CDKN3 protein is down-regulated in brain tumors. Our findings indicate that CDKN3 controls mitosis through the CDC2 signaling axis. These results have implications for targeted anticancer therapeutics.

Fanconi anemia (FA/BRCA) signaling network controls multiple genome-housekeeping checkpoints, from interphase DNA repair to mitosis. The in vivo role of abnormal cell division in FA remains unknown. Here, we quantified the origins of genomic instability in FA patients and mice in vivo and ex vivo. We found that both mitotic errors and interphase DNA damage significantly contribute to genomic instability during FA-deficient hematopoiesis and in non-hematopoietic human and murine FA primary cells. Super-resolution microscopy coupled with functional assays revealed that FANCA shuttles to the pericentriolar material (PCM) to regulate spindle assembly at mitotic entry. Loss of FA signaling rendered cells hypersensitive to spindle chemotherapeutics and allowed escape from the chemotherapy-induced spindle assembly checkpoint. In support of these findings, direct comparison of DNA cross-linking and antimitotic chemotherapeutics in primary FANCA−/− cells revealed genomic instability originating through divergent cell cycle checkpoint aberrations. Our data indicate that the FA/BRCA signaling functions as an in vivo gatekeeper of genomic integrity throughout interphase and mitosis, which may have implications for future targeted therapies in FA and FA-deficient cancers.