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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 anemia is a complex heterogeneous genetic disorder with a high incidence of bone marrow failure, clonal evolution to acute myeloid leukemia and mesenchymal-derived congenital anomalies. Increasing evidence in Fanconi anemia and other genetic disorders points towards an interdependence of skeletal and hematopoietic development, yet the impact of the marrow microenvironment in the pathogenesis of the bone marrow failure in Fanconi anemia remains unclear. Here we demonstrated that mice with double knockout of both Fancc and Fancg genes had decreased bone formation at least partially due to impaired osteoblast differentiation from mesenchymal stem/progenitor cells. Mesenchymal stem/progenitor cells from the double knockout mice showed impaired hematopoietic supportive activity. Mesenchymal stem/progenitor cells of patients with Fanconi anemia exhibited similar cellular deficits, including increased senescence, reduced proliferation, impaired osteoblast differentiation and defective hematopoietic stem/progenitor cell supportive activity. Collectively, these studies provide unique insights into the physiological significance of mesenchymal stem/progenitor cells in supporting the marrow microenvironment, which is potentially of broad relevance in hematopoietic stem cell transplantation.

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.

Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a complex genetic disorder with a high predisposition of numerous skeletal dysplasias including short stature, osteoporosis, kyphoscoliosis, and fracture non-union (pseudoarthrosis). We have developed murine models that phenocopy many of the skeletal dysplasias observed in NF1 patients, including reduced bone mass and fracture non-union. We also show that the development of these skeletal manifestations requires an Nf1 haploinsufficient background in addition to nullizygous loss of Nf1 in mesenchymal stem/progenitor cells (MSCs) and/or their progenies. This is replicated in two animal models of NF1, PeriCre(+);Nf1(flox/-) and Col2.3Cre(+);Nf1(flox/-) mice. Adoptive transfer experiments demonstrate a critical role of the Nf1+/- marrow microenvironment in the impaired fracture healing in both models and adoptive transfer of WT bone marrow cells improves fracture healing in these mice. To our knowledge, this is the first demonstration of a non-cell autonomous mechanism in non-malignant NF1 manifestations. Collectively, these data provide evidence of a combinatory effect between nullizygous loss of Nf1 in osteoblast progenitors and haploinsufficiency in hematopoietic cells in the development of non-malignant NF1 manifestations.

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.

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.