Activation of signaling pathways in models of t(6;9)-acute myeloid leukemia.

Patients within the WHO-subgroup of t(6;9)-positive acute myeloid leukemia (AML) differ from other AML subgroups as they are characterised by younger age and a grim prognosis. Leukemic transformation can often be attributed to single chromosomal aberrations encoding oncogenes, in the case of t(6;9)-AML to the fusion protein DEK-CAN (also called DEK-NUP214). As being a rare disease there is the urgent need for models of t(6;9)-AML. The only cell line derived from a t(6;9)-AML patient currently available is FKH1. By using phospho-proteomics on FKH1 cells, we found a strongly activated ABL1 kinase. Further investigation revealed the presence of ETV6-ABL1. This finding renders necessary to determine DEK-CAN- and ETV6-ABL1-related features when using FKH1. This can be done as ETV6-ABL1 activity in FKH1 is responsive to imatinib. Nevertheless, we provided evidence that both SFK and mTOR activation in FKH1 are DEK-CAN-related features as they were activated also in other t(6;9) and DEK-CAN-positive models. The activation of STAT5 previously shown to be strong in t(6;9)-AML and activated by DEK-CAN is regulated in FKH1 by both DEK-CAN and ETV6-ABL1. In conclusion, FKH1 cells still represent a model for t(6;9)-AML and could serve as model for ETV6-ABL1-positive AML if the presence of these leukemia-inducing oncogenes is adequately considered.Taken together, all our results provide clear evidence of novel and specific interdependencies between leukemia-inducing oncogenes and cancer signaling pathways which will influence the design of therapeutic strategies to better address the complexity of cancer signaling.

Genome-Wide Association Analysis for Hybrid Breeding in Wheat.

Disclosure of markers that are significantly associated with plant traits can help develop new varieties with desirable properties. This study determined the genome-wide associations based on DArTseq markers for six agronomic traits assessed in eight environments for wheat. Moreover, the association study for heterosis and analysis of the effects of markers grouped by linkage disequilibrium were performed based on mean values over all experiments. All results were validated using data from post-registration trials. GWAS revealed 1273 single nucleotide polymorphisms with biologically significant effects. Most polymorphisms were predicted to be modifiers of protein translation, with only two having a more pronounced effect. Markers significantly associated with the considered set of features were clustered within chromosomes based on linkage disequilibrium in 327 LD blocks. A GWAS for heterosis revealed 1261 markers with significant effects.

Evaluation of genetic structure in European wheat cultivars and advanced breeding lines using high-density genotyping-by-sequencing approach.

BACKGROUND: The genetic diversity and gene pool characteristics must be clarified for efficient genome-wide association studies, genomic selection, and hybrid breeding. The aim of this study was to evaluate the genetic structure of 509 wheat accessions representing registered varieties and advanced breeding lines via the high-density genotyping-by-sequencing approach. RESULTS: More than 30% of 13,499 SNP markers representing 2162 clusters were mapped to genes, whereas 22.50% of 26,369 silicoDArT markers overlapped with coding sequences and were linked in 3527 blocks. Regarding hexaploidy, perfect sequence matches following BLAST searches were not sufficient for the unequivocal mapping to unique loci. Moreover, allelic variations in homeologous loci interfered with heterozygosity calculations for some markers. Analyses of the major genetic changes over the last 27 years revealed the selection pressure on orthologs of the gibberellin biosynthesis-related GA2 gene and the senescence-associated SAG12 gene. A core collection representing the wheat population was generated for preserving germplasm and optimizing breeding programs. CONCLUSIONS: Our results confirmed considerable differences among wheat subgenomes A, B and D, with D characterized by the lowest diversity but the highest LD. They revealed genomic regions that have been targeted by breeding.

The effect of RO3201195 and a pyrazolyl ketone P38 MAPK inhibitor library on the proliferation of Werner syndrome cells.

Microwave-assisted synthesis of the pyrazolyl ketone p38 MAPK inhibitor RO3201195 in 7 steps and 15% overall yield, and the comparison of its effect upon the proliferation of Werner Syndrome cells with a library of pyrazolyl ketones, strengthens the evidence that p38 MAPK inhibition plays a critical role in modulating premature cellular senescence in this progeroid syndrome and the reversal of accelerated ageing observed in vitro on treatment with SB203580.

A single nuclear transcriptomic characterisation of mechanisms responsible for impaired angiogenesis and blood-brain barrier function in Alzheimer's disease.

Brain perfusion and blood-brain barrier (BBB) integrity are reduced early in Alzheimer's disease (AD). We performed single nucleus RNA sequencing of vascular cells isolated from AD and non-diseased control brains to characterise pathological transcriptional signatures responsible for this. We show that endothelial cells (EC) are enriched for expression of genes associated with susceptibility to AD. Increased ß-amyloid is associated with BBB impairment and a dysfunctional angiogenic response related to a failure of increased pro-angiogenic HIF1A to increased VEGFA signalling to EC. This is associated with vascular inflammatory activation, EC senescence and apoptosis. Our genomic dissection of vascular cell risk gene enrichment provides evidence for a role of EC pathology in AD and suggests that reducing vascular inflammatory activation and restoring effective angiogenesis could reduce vascular dysfunction contributing to the genesis or progression of early AD.

p38 (MAPK) stress signalling in replicative senescence in fibroblasts from progeroid and genomic instability syndromes.

Werner Syndrome (WS) is a human segmental progeria resulting from mutations in a DNA helicase. WS fibroblasts have a shortened replicative capacity, an aged appearance, and activated p38 MAPK, features that can be modulated by inhibition of the p38 pathway. Loss of the WRNp RecQ helicase has been shown to result in replicative stress, suggesting that a link between faulty DNA repair and stress-induced premature cellular senescence may lead to premature ageing in WS. Other progeroid syndromes that share overlapping pathophysiological features with WS also show defects in DNA processing, raising the possibility that faulty DNA repair, leading to replicative stress and premature cellular senescence, might be a more widespread feature of premature ageing syndromes. We therefore analysed replicative capacity, cellular morphology and p38 activation, and the effects of p38 inhibition, in fibroblasts from a range of progeroid syndromes. In general, populations of young fibroblasts from non-WS progeroid syndromes do not have a high level of cells with an enlarged morphology and F-actin stress fibres, unlike young WS cells, although this varies between strains. p38 activation and phosphorylated HSP27 levels generally correlate well with cellular morphology, and treatment with the p38 inhibitor SB203580 effects cellular morphology only in strains with enlarged cells and phosphorylated HSP27. For some syndromes fibroblast replicative capacity was within the normal range, whereas for others it was significantly shorter (e.g. HGPS and DKC). However, although in most cases SB203580 extended replicative capacity, with the exception of WS and DKC the magnitude of the effect was not significantly different from normal dermal fibroblasts. This suggests that stress-induced premature cellular senescence via p38 activation is restricted to a small subset of progeroid syndromes.

Reactivation of hepatitis B virus infection in a seafarer: an omitted problem of maritime medicine.

Infection with hepatitis B virus (HBV), despite the implementation of extensive preventive measures, has remained one of the biggest health problems worldwide. There are still people not covered by the compulsory vaccination programme and carriers of an actively replicating virus among professionally active seafarers. The article is based on a case report of a seafarer with life-threatening reactivation of long-term uncontrolled HBV infection that resulted in decompensated cirrhosis and liver transplant. The case shows clinical aspects of chronic hepatitis B and contributes to discussion about HBV infection with regard to seafarers. The article also analyses the current legal regulations and guidelines in terms of preventing new infections and detecting people already infected with HBV. Considering the specific nature of work on seagoing ships, it is justified to recognise the seafaring as a profession with a high risk of HBV infection. Monitoring the course of the disease can prevent reactivation of inflammatory process and serious consequences of chronic hepatitis B during a cruise. The elementary issue is specific prophylaxis, that is, covering the unvaccinated persons with the vaccination programme. The prevalence of HBV infection and the specifics of the seafarer labour market require development of new international regulations, which will unify Pre-Employment Medical Examination (PEME) protocols and take into consideration compulsory vaccination.

Mapping of quantitative trait loci for traits linked to fusarium head blight in barley.

Fusarium head blight (FHB) is a devastating disease occurring in small grain cereals worldwide. The disease results in the reduction of grain yield, and mycotoxins accumulated in grain are also harmful to both humans and animals. It has been reported that response to pathogen infection may be associated with the morphological and developmental traits of the host plant, e.g. earliness and plant height. Despite many studies, effective markers for selection of barley genotypes with increased resistance to FHB have not been developed. In the present study, we investigated 100 recombinant inbred lines (RIL) of spring barley. Plants were examined in field conditions (three locations) in a completely randomized design with three replications. Barley genotypes were artificially infected with spores of Fusarium culmorum before heading. Apart from the main phenotypic traits (plant height, spike characteristic, grain yield), infected kernels were visually scored and the content of deoxynivalenol (DON) mycotoxin was investigated. A set of 70 Quantitative Trait Loci (QTLs) were detected through phenotyping of the mapping population in field conditions and genotyping using a barley Ilumina 9K iSelect platform. Six loci were detected for the FHB index on chromosomes 2H, 3H, 5H, and 7H. A region on the short arm of chromosome 2H was detected in which many QTLs associated with FHB- and yield-related traits were found. This study confirms that agromorphological traits are tightly related to FHB and should be taken into consideration when breeding barley plants for FHB resistance.

Microwave-assisted Ullmann C-S bond formation: synthesis of the P38alpha MAPK clinical candidate VX-745.

Microwave irradiation promotes the rapid and efficient reaction of a thiophenol and aryl or heteroaryl halide using a copper or palladium catalyst and a range of ligands, depending upon substrate. Of particular utility is the use of copper(I) iodide (5 mol %) and trans-cyclohexane-1,2-diol as ligand under basic conditions and microwave irradiation to give the corresponding sulfide in high yield. This method for C-S bond formation is applied in the four-step synthesis of the clinical candidate VX-745 in 38% overall yield. The inhibitory activity of VX-745 against p38alpha MAPK is confirmed in Werner syndrome dermal fibroblasts at 1.0 microM concentration by immunoblot assay.

Microwave-assisted synthesis of 5-aminopyrazol-4-yl ketones and the p38(MAPK) inhibitor RO3201195 for study in Werner syndrome cells.

5-Aminopyrazol-4-yl ketones are prepared rapidly and efficiently using microwave dielectric heating from beta-ketonitriles by treatment with N,N'-diphenylformamidine followed by heterocyclocondensation by irradiation with a hydrazine. The inhibitory activity of RO3201195 prepared by this methodology was confirmed in hTERT-immortalized HCA2 and WS dermal fibroblasts at 200nM concentration, both by ELISA and immunoblot assay, and displays excellent kinase selectivity for p38alpha MAPK over the related stress-activated kinase JNK.

The role of cellular senescence in Werner syndrome: toward therapeutic intervention in human premature aging.

Werner syndrome (WS) is a premature aging disorder used as a model of normal human aging. WS individuals have several characteristics of normal aging, such as cataracts, hair graying, and skin aging, but manifest these at an early age. Additionally, WS individuals have high levels of inflammatory diseases, such as atherosclerosis and type 2 diabetes. The in vivo aging in WS is associated with accelerated aging of fibroblasts in culture. The cause of the accelerated senescence is not understood, but may be due to the genomic instability that is a hallmark of WS. Genome instability results in activation of stress kinases, such as p38, and the p38-specific inhibitor SB203580, prevents the accelerated senescence seen in WS fibroblasts. However, oxidative damage plays a role, as low oxygen conditions and antioxidant treatment revert some of the accelerated senescence phenotype. The effects of oxidative stress appear to be suppressible by SB203580; however, it does not appear to be transduced by p38. As SB203580 is known to inhibit other kinases in addition to p38, this suggests that more than one kinase pathway is involved. The recent development of p38 inhibitors with different binding properties, specificities, and oral bioavailability, and of new potent and selective inhibitors of JNK and MK2, will make it possible to dissect the roles of various kinase pathways in the accelerated senescence of WS cells. If this accelerated senescence is reflective of WS aging in vivo, these kinase inhibitors may well form the basis of antiaging therapies for individuals with WS.

Synthesis and in vivo activity of MK2 and MK2 substrate-selective p38alpha(MAPK) inhibitors in Werner syndrome cells.

A benzopyranopyridine inhibitor of mitogen-activated protein kinase-activated protein kinase 2 (MK2) is prepared rapidly and efficiently in one step using microwave dielectric heating, whereas a substrate-selective p38 MAPK inhibitor was prepared using conventional heating techniques. The former had MK2 inhibitory activity above 2.5 microM concentration, whereas the latter showed no MK2 inhibition at 10 microM. However, rather than rescuing the reduced cellular growth rate and aged morphology of hTERT-immortalised WS dermal fibroblasts, both induce a state resembling stress-induced cellular senescence, suggesting that these inhibitors may have limited therapeutic use.

Evaluating the Role of p38 MAPK in the Accelerated Cell Senescence of Werner Syndrome Fibroblasts.

Progeroid syndromes show features of accelerated ageing and are used as models for human ageing, of which Werner syndrome (WS) is one of the most widely studied. WS fibroblasts show accelerated senescence that may result from p38 MAP kinase activation since it is prevented by the p38 inhibitor SB203580. Thus, small molecule inhibition of p38-signalling may be a therapeutic strategy for WS. To develop this approach issues such as the in vivo toxicity and kinase selectivity of existing p38 inhibitors need to be addressed, so as to strengthen the evidence that p38 itself plays a critical role in mediating the effect of SB203580, and to find an inhibitor suitable for in vivo use. In this work we used a panel of different p38 inhibitors selected for: (1) having been used successfully in vivo in either animal models or human clinical trials; (2) different modes of binding to p38; and (3) different off-target kinase specificity profiles, in order to critically address the role of p38 in the premature senescence seen in WS cells. Our findings confirmed the involvement of p38 in accelerated cell senescence and identified p38 inhibitors suitable for in vivo use in WS, with BIRB 796 the most effective.

The effect of small-molecule inhibition of MAPKAPK2 on cell ageing phenotypes of fibroblasts from human Werner syndrome.

Fibroblasts derived from the progeroid Werner syndrome (WS) show reduced replicative lifespan and a "stressed" morphology, both phenotypes being alleviated by using the p38 MAP kinase inhibitor SB203580. Because p38 is a major hub for the control of stress-signalling pathways we were interested in examining the possible role for downstream kinases in order to refine our understanding of the role of p38 signalling in regulation of WS cell growth. To this end we treated WS and normal fibroblasts with MK2 inhibitors to determine whether MK2 inhibition would affect either the growth or morphology of WS cells. The first inhibitor, 7,8-dihydroxy-2,4-diamino-3-cyanobenzopyranopyridine (inhibitor 2), resulted in inhibition of WS cell growth and had no effect on morphology, effects that occurred below the level needed to inhibit MK2 and thus suggestive of inhibitor toxicity. The second inhibitor, 2-(2-quinolin-3-ylpyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo-[3,2-c]pyridin-4-one (CMPD16), resulted in a significant extension of WS fibroblast replicative capacity compared to normal cells. In addition, CMPD16 reverted the WS cellular morphology to that seen in normal dermal fibroblasts. These data suggest that MK2 activity plays a substantial role in proliferation control in WS cells. CMPD16 was not as effective in cellular lifespan extension as SB203580, however, suggesting that, although MK2 is a downstream kinase involved in cell cycle arrest, other p38 targets may play a role. Alternatively, as CMPD16 is toxic to cell growth at levels just above those that extend lifespan, it is possible that the therapeutic window is too small. However, as CMPD16 does show significant effects in WS fibroblasts, this acts as proof-of-principle for the efforts to design and synthesise improved MK2 inhibitors. As MK2 is involved in inflammatory processes and inflammation plays a major role in WS phenotypes, these data suggest MK2 as a potential therapeutic target for the treatment of Werner syndrome.

Activation of p38 MAP kinase and stress signalling in fibroblasts from the progeroid Rothmund-Thomson syndrome.

Rothmund-Thomson fibroblasts had replicative lifespans and growth rates within the range for normal fibroblasts; however, they show elevated levels of the stress-associated p38 MAP kinase, suggestive of stress during growth. Treatment with the p38 MAP kinase inhibitor SB203580 increased both lifespan and growth rate, as did reduction of oxidative stress using low oxygen in some strains. At replicative senescence p53, p21(WAF1) and p16(INK4A) levels were elevated, and abrogation of p53 using shRNA knockdown allowed the cells to bypass senescence. Ectopic expression of human telomerase allowed Rothmund-Thomson fibroblasts to bypass senescence. However, activated p38 was still present, and continuous growth for some telomerised clones required either a reduction in oxidative stress or SB203580 treatment. Overall, the evidence suggests that replicative senescence in Rothmund-Thomson cells resembles normal senescence in that it is telomere driven and p53 dependent. However, the lack of RECQL4 leads to enhanced levels of stress during cell growth that may lead to moderate levels of stress-induced premature senescence. As replicative senescence is believed to underlie human ageing, a moderate level of stress-induced premature senescence and p38 activity may play a role in the relatively mild ageing phenotype seen in Rothmund-Thomson.

Small molecule inhibition of p38 MAP kinase extends the replicative life span of human ATR-Seckel syndrome fibroblasts.

Ataxia-telangiectasia and rad3 (ATR)-related Seckel syndrome is associated with growth retardation and premature aging features. ATR-Seckel fibroblasts have a reduced replicative capacity in vitro and an aged morphology that is associated with activation of stress-associated p38 mitogen-activated protein kinase and phosphorylated HSP27. These phenotypes are prevented using p38 inhibitors, with replicative capacity restored to the normal range. However, this stressed phenotype is retained in telomerase-immortalized ATR-Seckel fibroblasts, indicating that it is independent of telomere erosion. As with normal fibroblasts, senescence in ATR-Seckel is bypassed by p53 abrogation. Young ATR-Seckel fibroblasts show elevated levels of p21(WAF1), p16(INK4A), phosphorylated actin-binding protein cofilin, and phosphorylated caveolin-1, with small molecule drug inhibition of p38 reducing p16(INK4A) and caveolin-1 phosphorylation. In conclusion, ATR-Seckel fibroblasts undergo accelerated aging via stress-induced premature senescence and p38 activation that may underlie certain clinical features of Seckel syndrome, and our data suggest a novel target for pharmacological intervention in this human syndrome.

Investigating the role of c-Jun N-terminal kinases in the proliferation of Werner syndrome fibroblasts using diaminopyridine inhibitors.

Fibroblasts derived from the progeroid Werner syndrome show reduced replicative lifespan and a "stressed" morphology, both alleviated using the MAP kinase inhibitor SB203580. However, interpretation of these data is problematical because although SB203580 has the stress-activated kinases p38 and JNK1/2 as its preferred targets, it does show relatively low overall kinase selectivity. Several lines of data support a role for both p38 and JNK1/2 activation in the control of cellular proliferation and also the pathology of diseases of ageing, including type II diabetes, diseases to which Werner Syndrome individuals are prone, thus making the use of JNK inhibitors attractive as possible therapeutics. We have thus tested the effects of the widely used JNK inhibitor SP600125 on the proliferation and morphology of WS cells. In addition we synthesised and tested two recently described aminopyridine based inhibitors. SP600125 treatment resulted in the cessation of proliferation of WS cells and resulted in a senescent-like cellular phenotype that does not appear to be related to the inhibition of JNK1/2. In contrast, use of the more selective aminopyridine CMPD 6o at concentrations that fully inhibit JNK1/2 had a positive effect on cellular proliferation of immortalised WS cells, but no effect on the replicative lifespan of primary WS fibroblasts. In addition, CMPD 6o corrected the stressed WS cellular morphology. The aminopyridine CMPD 6r, however, had little effect on WS cells. CMDP 6o was also found to be a weak inhibitor of MK2, which may partially explain its effects on WS cells, since MK2 is known to be involved in regulating cellular morphology via HSP27 phosphorylation, and is thought to play a role in cell cycle arrest. These data suggest that total JNK1/2 activity does not play a substantial role in the proliferation control in WS cells.

Gram-scale synthesis of the p38α MAPK-inhibitor VX-745 for preclinical studies into Werner syndrome.

BACKGROUND: The ATP-competitive p38α MAPK inhibitor VX-745 exhibits an exquisite kinase selectivity profile, is effective in blocking p38 stress signaling in Werner syndrome dermal fibroblasts, has efficacy in clinical trials and may have therapeutic value against Werner syndrome. Previous synthetic routes, however, have only resulted in milligram quantities suitable for cell-based studies, whereas gram quantities would be required for in vivo use. RESULTS & DISCUSSION: Microwave irradiation using a stop-flow monomodal microwave reactor has been found to facilitate scale-up of the synthesis of VX-745. Ullmann-type C-S bond formation using thiophenol, chloropyridazine, copper(I) catalyst and diol ligand proceeds rapidly and efficiently in this apparatus for elaboration to the pyrimido[1,6-b]pyridazinone core of VX-745 on gram scale and with good overall yield. CONCLUSION: This method delivers the p38 inhibitor VX-745 in sufficient quantities for preclinical studies to rescue the aging phenotype in Werner syndrome.

Synthesis of the highly selective p38 MAPK inhibitor UR-13756 for possible therapeutic use in Werner syndrome.

BACKGROUND: UR-13756 is a potent and selective p38 mitogen-activated protein kinase (MAPK) inhibitor, reported to have good bioavailability and pharmacokinetic properties and, thus, is of potential use in the treatment of accelerated aging in Werner syndrome. RESULTS AND DISCUSSION: Irradiation of 2-chloroacrylonitrile and methylhydrazine in ethanol at 100 °C gives 1-methyl-3-aminopyrazole, which reacts with 4-fluorobenzaldehyde and a ketone, obtained by Claisen condensation of 4-picoline, in a Hantzsch-type 3-component hereocyclocondensation, to give the pyrazolopyridine UR-13756. UR-13756 shows p38 MAPK inhibitory activity in human telomerase reverse transcriptase-immortalized HCA2 dermal fibroblasts, with an IC(50) of 80 nm, as shown by ELISA, is 100% efficacious for up to 24 h at 1.0 µm and displays excellent kinase selectivity over the related stress-activated c-Jun kinases. In addition, UR-13756 is an effective p38 inhibitor at 1.0 µm in Werner syndrome cells, as shown by immunoblot. CONCLUSION: The convergent synthesis of UR-13756 is realized using microwave dielectric heating and provides a highly selective inhibitor that shows excellent selectivity for p38 MAPK over c-Jun N-terminal kinase.

Microwave-assisted synthesis of a pyrazolyl ketone library for evaluation as p38 MAPK inhibitors in Werner syndrome cells.

BACKGROUND: The pyrazolyl ketone motif of RO3201195, which exhibits good oral bioavailability and high selectivity for p38 MAPK over other kinases, is a key pharmacophore that could find application in the treatment of Werner syndrome. RESULTS AND DISCUSSION: Microwave irradiation promotes Knoevenagel condensation of a ß-ketonitrile and formamidine, to give ß-aminovinyl ketones, and their subsequent cyclocondensation with a subset of hydrazines to provide rapid access to a 24-membered library of pyrazolyl ketones. The library was evaluated in human hTERT-immortalized HCA2 dermal cells and Werner syndrome cells. CONCLUSION: Four compounds display comparable, if not slightly improved, potency over RO3201195.