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FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL., Competing Interests: The authors declare no conflicts of interest.

Muscle acetylcholine receptor ion channels mediate neurotransmission by depolarizing the postsynaptic membrane at the neuromuscular junction. Inherited disorders of neuromuscular transmission, termed congenital myasthenic syndromes, are commonly caused by mutations in genes encoding the five subunits of the acetylcholine receptor that severely reduce endplate acetylcholine receptor numbers and/or cause kinetic abnormalities of acetylcholine receptor function. We tracked the cause of the myasthenic disorder in a female with onset of first symptoms at birth, who displayed mildly progressive bulbar, respiratory and generalized limb weakness with ptosis and ophthalmoplegia. Direct DNA sequencing revealed heteroallelic mutations in exon 8 of the acetylcholine receptor ε-subunit gene. Two alleles were identified: one with the missense substitution p.εP282R, and the second with a deletion, c.798&#95;800delCTT, which result in the loss of a single amino acid, residue F266, within the M2 transmembrane domain. When these acetylcholine receptor mutations were expressed in HEK 293 cells, the p.εP282R mutation caused severely reduced expression on the cell surface, whereas p.εΔF266 gave robust surface expression. Single-channel analysis for p.εΔF266 acetylcholine receptor channels showed the longest burst duration population was not different from wild-type acetylcholine receptor (4.39 ± 0.6 ms versus 4.68 ± 0.7 ms, n = 5 each) but that the amplitude of channel openings was reduced. Channel amplitudes at different holding potentials showed that single-channel conductance was significantly reduced in p.εΔF266 acetylcholine receptor channels (42.7 ± 1.4 pS, n = 8, compared with 70.9 ± 1.6 pS for wild-type, n = 6). Although a phenylalanine residue at this position within M2 is conserved throughout ligand-gated excitatory cys-loop channel subunits, deletion of equivalent residues in the other subunits of muscle acetylcholine receptor did not have equivalent effects. Modelling the impact of p.εΔF266 revealed only a minor alteration to channel structure. In this study we uncover the novel mechanism of reduced acetylcholine receptor channel conductance as an underlying cause of congenital myasthenic syndrome, with the 'low conductance' phenotype that results from the p.εΔF266 deletion mutation revealed by the coinheritance of the low-expressor mutation p.εP282R.

Focal adhesion assembly and disassembly are essential for cell migration and cancer invasion, but the detailed molecular mechanisms regulating these processes remain to be elucidated. Phosphatidylinositol phosphate kinase type Iγ (PIPKIγ) binds talin and is required for focal adhesion formation in EGF-stimulated cells, but its role in regulating focal adhesion dynamics and cancer invasion is poorly understood. We show here that overexpression of PIPKIγ promoted focal adhesion formation, whereas cells expressing either PIPKIγ(K188,200R) or PIPKIγ(D316K), two kinase-dead mutants, had much fewer focal adhesions than those expressing WT PIPKIγ in CHO-K1 cells and HCT116 colon cancer cells. Furthermore, overexpression of PIPKIγ, but not PIPKIγ(K188,200R), resulted in an increase in both focal adhesion assembly and disassembly rates. Depletion of PIPKIγ by using shRNA strongly inhibited formation of focal adhesions in HCT116 cells. Overexpression of PIPKIγ(K188,200R) or depletion of PIPKIγ reduced the strength of HCT116 cell adhesion to fibronection and inhibited the invasive capacities of HCT116 cells. PIPKIγ depletion reduced PIP₂ levels to ∼40% of control and PIP₃ to undetectable levels, and inhibited vinculin localizing to focal adhesions. Taken together, PIPKIγ positively regulates focal adhesion dynamics and cancer invasion, most probably through PIP₂-mediated vinculin activation.

Dok-7 is a cytoplasmic activator of muscle-specific receptor-tyrosine kinase (MuSK). Both Dok-7 and MuSK are required for neuromuscular synaptogenesis. Mutations in DOK7 underlie a congenital myasthenic syndrome (CMS) associated with small and simplified neuromuscular synapses likely due to impaired Dok-7/MuSK signaling. The overwhelming majority of patients with DOK7 CMS have at least one allele with a frameshift mutation that causes a truncation in the COOH-terminal region of Dok-7 and affects MuSK activation. Dok-7 has pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains in the NH2-terminal moiety, both of which are indispensable for MuSK activation in myotubes, but little is known about additional functional elements. Here, we identify a chromosome region maintenance 1-dependent nuclear export signal (NES) in the COOH-terminal moiety and demonstrate that the NES-mediated cytoplasmic location of Dok-7 is essential for regulating the interaction with MuSK in myotubes. The NH2-terminal PH domain is responsible for the nuclear import of Dok-7. We also show that the Src homology 2 target motifs in the COOH-terminal moiety of Dok-7 are active and crucial for MuSK activation in myotubes. In addition, CMS-associated missense mutations found in the PH or PTB domain inactivate Dok-7. Together, these findings demonstrate that, in addition to the NH2-terminal PH and PTB domains, the COOH-terminal NES and Src homology 2 target motifs play key roles in Dok-7/MuSK signaling for neuromuscular synaptogenesis. Ablation or disruption of these functional elements in Dok-7 probably underlies the neuromuscular junction synaptopathy observed in DOK7 CMS.

The congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders affecting neuromuscular transmission. Underlying mutations have been identified in at least 11 different genes. The majority of CMS patients have disorders due to mutations in postsynaptic proteins. Initial studies focused on dysfunction of the acetylcholine receptor (AChR) itself as the major cause of CMS. However, it is becoming apparent that mutations of proteins involved in clustering the AChR and maintaining neuromuscular junction structure form important subgroups. Analysis of the mutations in the AChR-clustering protein, rapsyn, show diverse causes for defective AChR localization and suggest that the common mutation rapsyn-N88K results in AChR clusters that are less stable than those generated by wild-type rapsyn. More recently, mutations in the newly identified endplate protein Dok-7 have been shown to affect AChR clustering and the generation and maintenance of specialized structures at the endplate. Dok-7 binds MuSK and many of the mutations of DOK7 impair the MuSK signaling pathway. Components of this pathway will provide attractive gene candidates for additional forms of CMS. The phenotypic characteristics of the different CMS in which muscle groups may be differentially affected not only provide clues for targeted genetic screening, but also pose further intriguing questions about underlying molecular mechanisms.

Multiple pterygium syndromes (MPS) comprise a group of multiple congenital anomaly disorders characterized by webbing (pterygia) of the neck, elbows, and/or knees and joint contractures (arthrogryposis). MPS are phenotypically and genetically heterogeneous but are traditionally divided into prenatally lethal and nonlethal (Escobar) types. Previously, we and others reported that recessive mutations in the embryonal acetylcholine receptor g subunit (CHRNG) can cause both lethal and nonlethal MPS, thus demonstrating that pterygia resulted from fetal akinesia. We hypothesized that mutations in acetylcholine receptor-related genes might also result in a MPS/fetal akinesia phenotype and so we analyzed 15 cases of lethal MPS/fetal akinesia without CHRNG mutations for mutations in the CHRNA1, CHRNB1, CHRND, and rapsyn (RAPSN) genes. No CHRNA1, CHRNB1, or CHRND mutations were detected, but a homozygous RAPSN frameshift mutation, c.1177-1178delAA, was identified in a family with three children affected with lethal fetal akinesia sequence. Previously, RAPSN mutations have been reported in congenital myasthenia. Functional studies were consistent with the hypothesis that whereas incomplete loss of rapsyn function may cause congenital myasthenia, more severe loss of function can result in a lethal fetal akinesia phenotype.

Congenital myasthenic syndromes are inherited disorders of neuromuscular transmission characterized by fatigable muscle weakness. Autosomal recessive acetylcholine receptor (AChR) deficiency syndromes, in which levels of this receptor at the neuromuscular junction are severely reduced, may be caused by mutations within genes encoding the AChR or the AChR-clustering protein, rapsyn. Most patients have mutations within the rapsyn coding region and are either homozygous for N88K or heteroallelic for N88K and a second mutation. In some cases the second allele carries a null mutation but in many the mutations are missense, and are located in different functional domains. Little is known about the functional effects of these mutations, but we hypothesize that they would have an effect on AChR clustering by a variety of mechanisms that might correlate with disease severity. Here we expressed RAPSN mutations A25V, N88K, R91L, L361R and K373del in TE671 cells and in rapsyn-/- myotubes to determine their pathogenic mechanisms. The A25Vmutation impaired colocalization of rapsyn with AChR and prevented agrin-induced AChR clusters in rapsyn-/- myotubes. In TE671 cells, R91L reduced the ability of rapsyn to self-associate, and K373del-rapsyn was significantly less stable than wild-type. The effects of mutations L361R and N88K were more subtle: in TE671 cells, in comparison with wild-type rapsyn, L361R-rapsyn showed reduced expression/stability, and both N88K-rapsyn and L361R-rapsyn showed significantly reduced co-localization with AChR. N88K-rapsyn and L361R-rapsyn could effectively mediate agrin-induced AChR clusters, but these were reduced in number and were less stable than with wild-type rapsyn. The disease severity of patients harbouring the compound allelic mutations was greater than that of patients with homozygous rapsyn mutation N88K, suggesting that the second mutant allele may largely determine severity.

Congenital myasthenic syndromes (CMSs) are a group of inherited disorders of neuromuscular transmission characterized by fatigable muscle weakness. One major subgroup of patients shows a characteristic "limb girdle" pattern of muscle weakness, in which the muscles have small, simplified neuromuscular junctions but normal acetylcholine receptor and acetylcholinesterase function. We showed that recessive inheritance of mutations in Dok-7, which result in a defective structure of the neuromuscular junction, is a cause of CMS with proximal muscle weakness.

Anemia is a common clinical problem, and there is much interest in its role in promoting left ventricular hypertrophy through increasing cardiac workload. Normally, red blood cell production is adjusted through the regulation of erythropoietin (Epo) production by the kidney. One important cause of anemia is relative deficiency of Epo, which occurs in most types of renal disease. Clinically, this can be corrected by supplementation with recombinant Epo. Here we describe an oxygen-regulated gene therapy approach to treating homozygous erythropoietin-SV40 T antigen (Epo-TAg(h)) mice with relative erythropoietin deficiency. We used vectors in which murine Epo expression was directed by an Oxford Biomedica hypoxia response element (OBHRE) or a constitutive cytomegalovirus (CMV) promoter. Both corrected anemia, but CMV-Epo-treated mice acquired fatal polycythemia. In contrast, OBHRE-Epo corrected the hematocrit level in anemic mice to a normal physiologic level that stabilized without resulting in polycythemia. Importantly, the OBHRE-Epo vector had no significant effect on the hematocrit of control mice. Homozygous Epo-TAg(h) mice display cardiac hypertrophy, a common adaptive response in patients with chronic anemia. In the OBHRE-Epo-treated Epo-TAg(h) mice, we observed a significant reversal of cardiac hypertrophy. We conclude that the OBHRE promoter gives rise to physiologically regulated Epo secretion such that the hematocrit level is corrected to healthy in anemic Epo-TAg(h) mice. This establishes that a hypoxia regulatory mechanism similar to the natural mechanism can be achieved, and it makes EPO gene therapy more attractive and safer in clinical settings. We envisage that this control system will allow regulated delivery of therapeutic gene products in other ischemic settings.

Human cytochrome P450 2B6 (CYP2B6) metabolizes the prodrug cyclophosphamide (CPA) to produce phosphoramide mustard that cross-links DNA leading to cell death. We have constructed a novel retroviral vector encoding CYP2B6 (designated "MetXia-P450") and used it to transduce the human tumor cell lines HT29 and T47D. MetXia-P450 transduction sensitised these cells to the cytotoxic effects of the prodrug CPA. Results from in vitro experiments demonstrated adverse effects on the clonogenic survival of cyclophosphamide-treated cells transduced with MetXia-P450. Cytotoxic activity accompanied by bystander effect was particularly evident in 3-D multicellular spheroid models suggesting that this in vitro system may be a more appropriate model for assessing the efficacy of gene directed-enzyme prodrug therapy (GDEPT). We have applied this approach in a clinically relevant gene therapy protocol on established subcutaneous tumor xenografts. These studies show for the first time the efficacy of a P450-based GDEPT strategy mediated by a direct retroviral gene transfer in vivo.

A high therapeutic index is as important for gene-based therapies as it is for chemotherapy or radiotherapy. One approach has been transcriptional targeting through the use of tissue-specific regulatory elements. A more versatile approach would be to use a regulatory element that is controlled via a parameter common to a broad range of diseases. Ischemia is characteristic of a number of pathologies that range from vascular occlusion through to cancer. The state of low oxygen, hypoxia, triggers a transcriptional signaling pathway that is mediated by transcription factors binding to a specific enhancer, the hypoxia response element (HRE). These observations have therefore led to the use of HREs to drive gene expression in a number of target tissues from tumors to cardiac muscle. To translate these observations into a clinically useful vector system we have now assessed the potency of a number of naturally derived HREs in various configurations combined with minimal promoters. The optimal HRE has been introduced into a single transcription unit retroviral vector that can deliver regulated gene expression in response to hypoxia. An important feature of this new physiologically regulated vector is the combination of low basal expression and high-level activated expression that is on a par with that obtained with the cytomegalovirus immediate-early (CMV IE) promoter. The role of elements that stabilize mRNA in the presence of hypoxia has also been assessed. These hypoxia-regulated vectors may have utility for restricting the delivery of therapeutic proteins to tumors and ischemic sites.