Multiple Hits Research Articles

Hit-to-Lead Medicinal Chemistry

Hit-to-Lead medicinal chemistry is the process through which “hits” are converted into “leads.” Fundamental to the process is to gain confidence, first that a hit compound can be developed into a robust (lead) series, and second that the leads have the potential to be converted into drug candidates. Such confidence is gained in a stepwise fashion: initially, hit compounds must have their activity confirmed, and second it must be demonstrated that structural modification of the hit can result in changes in biological activity, thus generating a series. Third, members of the series must pass a series of so-called “developability filters,” thus ensuring that there is a high chance of success in the subsequent phase of lead optimization. The hit-to-lead phase is highly important, as chemists are frequently presented with multiple hits from which to identify a small number of series to take forward. Selection at this point will have a direct impact on the success of lead optimization. Keywords: hit; lead; lead optimization; fragment; ligand efficiency

Projectile Penetrating Multilayer Composite Armor

Objectives: The objective of this paper is to study the influence of interface between multi layered composite structures in penetration problems, using finite element solver RADIOSS. Methods/Statistical Analysis: Finite element modeling of the penetrating projectile, multilayer composite and analysis using RADIOSS is done. The analysis is confined to Johnson Cook material model for plasticity and failure. One of the important aspects of material model is the representation of the plasticity and failure region. The modified interface creates a realistic environment with combination of TYPE 2 and TYPE 7 influences multiple hit of projectiles and does not influence the depth of penetration when compared to the TYPE 7 interface. Findings: Hyper Works software tool is used to simulate the projectile penetrating armor plate and Hyper Mesh used for preprocessing and RADIOSS as a solver. The depth of penetration is not significantly affected due to the TYPE 2 interface. However, the delamination behavior of the layers is captured in the TYPE 2 interface when compared to TYPE7. The modified interface with combination of TYPE 2 and TYPE 7 does not influence the depth of penetration when compared to the TYPE 7 interface. TYPE 7 formulations gives considerably acceptable results for capturing the depth of penetration in case of multilayer composite structures. TYPE 2 interfaces has to be studied in cases where delamination has influence, for example in multiple hits of projectiles etc. Application/Improvements: Development of alternative material or sandwich structure is required for the future war scenario to prevent the main threats for Armored Fighting Vehicles (AFVs), shock and penetration as future war scenario insists on light tanks that can withstand penetration with higher order of destruction.

Abstract A27: European Network NGS-PTL preliminary data: Whole exome sequencing identifies mutations of ALDH2, RETSAT, HSPG2, CHPF and other metabolic genes as a novel functional category in acute myeloid leukemia

Abstract Next Generation Sequencing (NGS) studies identified 9 functional categories of mutations in acute myeloid leukemia (AML), with >99% of cases having at least one of those mutations (Ley et al. NEJM 2013). However, multiple genetic hits participate to AML pathogenesis, and metabolic dysregulations, as the one induced by IDH1/2 mutations, play oncogenic functions (Ward et al. Cancer Cell 2010). Aim of the study was to define novel functional categories of AML mutations affecting relevant and druggable biological processes, with focus on genetic determinants of metabolic plasticity. Out of 455 whole exome sequencing (WES) cases from onco-hematological patients collected in the NGS-PTL project, we analyzed 37 AML cases, belonging to our cohort of 239 FLT3-WT samples (886 AML total). We performed 100 bp paired-end WES (HiSeq2000, Illumina) and mapped the sequenced reads with Burrows-Wheeler Aligner. Variants where called with MuTect or GATK for single nucleotide variant (SNV) and indels detection, respectively (>90% confidence). Gene expression profiling was performed using HTA2.0 microarray (Affymetrix) on 56 bone marrow samples, including AML (≥80% blasts) and healthy controls. By WES analysis, we detected an average of 26 somatic variants per patient (range, 7 to 65). Gene ontology annotation identified 8 novel relevant functional categories of mutated genes: transcription, translation and post-translational modifications, protein degradation, cytoskeleton, cell cycle, DNA damage, cell survival and metabolism. Since metabolic pathways are promising targets for tailored therapies (e.g. IDH1/2 and glutaminase inhibitors), we focused our analysis on them. We identified 82 variants (74 SNVs, 2 frameshift and 4 nonframeshift deletions, 2 stopgains) targeting 70 genes involved in metabolism, with 78% of patients carrying at least one mutation in a metabolic gene and 35 variants rated as damaging by CONDEL algorithm. Among mutations in metabolic genes, the most represented pathways according to Recon X database were amino acids, lipids, CoA and nucleotides metabolism, transport and bioenergetics pathways. Notably, IMPDH2, a mediator of MYC-induced proliferation involved in nucleotide interconversion, was mutated and overexpressed in our AML cohort (p=0.01), suggesting a potential oncogenic function. Moreover, ALDH2, a regulator of hematopoietic stem cell functions which is involved in multiple metabolic pathways and associates with metabolic remodeling, was mutated and 2-fold downregulated in AML blasts. Seven genes were mutated in 5-8% of samples: RETSAT, HSPG2, CHPF, ABCA2, ND1, APOBR, NAAA. Among them, RETSAT, HSPG2, CHPF mutations were also predicted as “drivers” by DOTS-Finder tool. Bioenergetics pathways were affected by mutations in glycolysis and gluconeogenesis (GPI, ITPA), oxidative phosphorylation (ND1, ND4, ND5, CYTB), pentose phosphate pathway (H6PD, PGLS). Patients carrying mutations in the bioenergetics pathway showed a strong trend towards reduced overall survival, which did not associate with unfavorable molecular mutations. In conclusion, metabolism is the most represented class of mutated genes (8.6% of variants) in our FLT3-WT AML cohort after signaling, leading us to propose a novel functional category. Our data suggest that, along with mutations in established oncogenes and tumor suppressors involved in metabolic control (KRAS, TP53, MYC pathway), a number of genetic determinants participate to leukemia metabolic plasticity and oncogenic mutations of metabolic enzymes may drive leukemogenesis, impact on patient's survival and become novel targets for personalized therapies. Acknowledgements: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. GS and AP equally contributed to this work. Citation Format: Giorgia Simonetti, Antonella Padella, Ilaria Iacobucci, Italo Do Valle, Gabriele Fontanarosa, Elisa Zago, Francesca Griggio, Marianna Garonzi, Simona Bernardi, Cristina Papayannidis, Maria Chiara Abbenante, Giovanni Marconi, Giorgio Melloni, Laura Riva, Viviana Guadagnuolo, Mariachiara Fontana, Samantha Bruno, Elisa Zuffa, Eugenia Franchini, Annalisa Astolfi, Carmen Baldazzi, Elisa Dan, Barbara Sinigaglia, Michele Cavo, Nicoletta Testoni, Emanuela Ottaviani, Pier Giuseppe Pelicci, Marco Sazzini, Alberto Ferrarini, Massimo Delledonne, Daniel Remondini, Giovanni Martinelli. European Network NGS-PTL preliminary data: Whole exome sequencing identifies mutations of ALDH2, RETSAT, HSPG2, CHPF and other metabolic genes as a novel functional category in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A27.

Identification and Mechanistic Characterization of CMPD1 As a Selective Sensitizer of Histone Deacetylase Inhibitors in Myeloid Malignancies

Background: Histone deacetylase inhibitors (HDIs) have shown clinical activity in myeloid malignancies, albeit insufficient to justify single-agent therapy. Thus, we have sought targets/therapies that synergistically enhance the activity of HDIs for combination therapy. We conducted RNA-interference (RNAi) drug modifier screens with the HDI SAHA, and have previously reported convergence of multiple screen hits on different points of the p38-SAPK/JNK signaling pathway. However, in subsequent pharmacological studies, well-characterized p38α/β inhibitors SB2012190 and LY2228820, and JNK inhibitor SP600125 did not modulate SAHA anti-leukemic activity. Nonetheless, a putative MK2 substrate-selective p38 inhibitor known as CMPD1 (Biochemistry 2004 Sep 21; 43(37):11658-71) was found to potently synergize with SAHA in all AML cell lines tested (N=8) in a dose-dependent manner. Further, using ex vivo cultures of primary myeloid malignancies (N=14), CMPD1 showed strong, selective synergy with SAHA in malignant CD34+ isolated cells, as compared to little or no synergy in CD34-depleted cells. Furthermore, CMPD1 specifically synergized with SAHA and a similar HDI panobinostat, but was found to interact antagonistically with cytarabine, and only additively with azacitidine. Herein, we present additional data from our investigation to better understand the mechanism of CMPD1 synergy with HDIs for future clinical translation.Results: Concurrent cell cycle and apoptosis measurements by flow cytometry show that single-agent CMPD1 results in a potent G2/M arrest that is resolved over time without a significant induction of apoptosis, whereas the combination of SAHA + CMPD1 results in a similar or increased level of G2/M arrest, which conversely culminates in a significant induction of apoptosis. The synergistic dose of single-agent SAHA used in these flow cytometry studies did not cause any significant cell cycle changes, and did not induce significant apoptosis alone in the AML cell lines studied. CMPD1 is known to be a MK2 substrate-selective p38 inhibitor; however, we find that CMPD1 potently increases the phosphorylation of MK2 at doses strongly synergistic with SAHA. This is not a universal activity of p38 inhibitors in our in vitro AML models, as the p38 inhibitor SB202190 does not increase MK2 phosphorylation alone or in combination with SAHA. We further hypothesized that CMPD1 synergizes with SAHA through a transcription-based mechanism, thus we measured mRNA expression by next-generation sequencing after SAHA and CMPD1 treatment alone, and in combination, in an AML cell line TF-1. We find that SAHA + CMPD1 treatment induces a synergistic increase in transcript levels of: i) several chemokines such as IL-8, CXCL1, CXCL2, and CXCL3, ii) inhibitor of DNA binding proteins ID2 and ID3, and iii) cell cycle regulator p21. Transcript levels of the Hippo pathway transcription factor YAP1 were also found to be increased by CMPD1 treatment in TF-1. We confirm an increase of total YAP1 protein levels by western blot. We also find an increase of YAP1 phosphorylation at serine 127 in CMPD1 or CMPD1 + SAHA treated samples in several AML cell lines, yet we find that Ser-127 phosphorylated YAP1 is located in the nucleus. Consistent with YAP1 Ser-127 phosphorylation, we find that upstream signaling kinase LATS1 is activated/phosphorylated at serine 909 in AML cell lines after treatment with CMPD1 or SAHA + CMPD1. We also confirm a synergistic increase in p21 protein levels upon SAHA + CMPD1 treatment by western blot in several AML cell lines. Changes in the protein levels of ID2 and ID3 were confirmed in TF-1 by western blot; however, ID protein levels appear to be uniquely affected by CMPD1 or the combination in a cell line-dependent manner.Conclusion: CMPD1 selectively synergizes with histone deacetylases inhibitors in myeloid malignancies. The synergistic activity of this combination highlights the potential for selectively targeting malignant CD34+ cells. The phosphorylation of LATS1 observed is consistent with i) the known roles for LATS1 in cell cycle regulation, ii) the G2/M arrest observed with CMPD1, and iii) the downstream phosphorylation of YAP1 observed. These findings linking CMPD1 with LATS1/YAP1 await further study and characterization. Although CMPD1 is not a clinically-relevant compound, CMPD1 may be used as a scaffold or tool compound to enable future development and research. DisclosuresMesa:Pfizer: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Genentech: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; Gilead: Research Funding; CTI Biopharma: Research Funding; Incyte Corporation: Research Funding.

From Memory Impairment to Posttraumatic Stress Disorder-Like Phenotypes: The Critical Role of an Unpredictable Second Traumatic Experience

Arousal and stress critically regulate memory formation and retention. Increasing levels of stress produce an inverted U-shaped effect on cognitive performance, including the retention of explicit memories, and experiencing a severe stress during a traumatic event may lead to posttraumatic stress disorder (PTSD). The molecular mechanisms underlying the impairing effect of a severe stress on memory and the key contribution of traumatic experiences toward the development of PTSD are still unknown. Here, using increasing footshock intensities in an inhibitory avoidance paradigm, we reproduced the inverted U-shaped curve of memory performance in rats. We then show that the inverted U profile of memory performance correlates with an inverted U profile of corticosterone level in the circulation and of brain-derived neurotrophic factor, phosphorylated tropomyosin-receptor kinase B, and methyl CpG binding protein in the dorsal hippocampus. Furthermore, training with the highest footshock intensity (traumatic experience) led to a significant elevation of hippocampal glucocorticoid receptors. Exposure to an unpredictable, but not to a predictable, highly stressful reminder shock after a first traumatic experience resulted in PTSD-like phenotypes, including increased memory of the trauma, high anxiety, threat generalization, and resistance to extinction. Systemic corticosterone injection immediately after the traumatic experience, but not 3 d later, was sufficient to produce PTSD-like phenotypes. We suggest that, although after a first traumatic experience a suppression of the corticosterone-dependent response protects against the development of an anxiety disorder, experiencing more than one trauma (multiple hits) is a critical contributor to the etiology of PTSD.

Juvenile myelomonocytic leukemia displays mutations in components of the RAS pathway and the PRC2 network.

Juvenile myelomonocytic leukemia (JMML) is a rare and severe myelodysplastic and myeloproliferative neoplasm of early childhood initiated by germline or somatic RAS-activating mutations. Genetic profiling and whole-exome sequencing of a large JMML cohort (118 and 30 cases, respectively) uncovered additional genetic abnormalities in 56 cases (47%). Somatic events were rare (0.38 events/Mb/case) and restricted to sporadic (49/78; 63%) or neurofibromatosis type 1 (NF1)-associated (8/8; 100%) JMML cases. Multiple concomitant genetic hits targeting the RAS pathway were identified in 13 of 78 cases (17%), disproving the concept of mutually exclusive RAS pathway mutations and defining new pathways activated in JMML involving phosphoinositide 3-kinase (PI3K) and the mTORC2 complex through RAC2 mutation. Furthermore, this study highlights PRC2 loss (26/78; 33% of sporadic JMML cases) that switches the methylation/acetylation status of lysine 27 of histone H3 in JMML cases with altered RAS and PRC2 pathways. Finally, the association between JMML outcome and mutational profile suggests a dose-dependent effect for RAS pathway activation, distinguishing very aggressive JMML rapidly progressing to acute myeloid leukemia.

Exploring causal networks underlying fat deposition and muscularity in pigs through the integration of phenotypic, genotypic and transcriptomic data.

BackgroundJoint modeling and analysis of phenotypic, genotypic and transcriptomic data have the potential to uncover the genetic control of gene activity and phenotypic variation, as well as shed light on the manner and extent of connectedness among these variables. Current studies mainly report associations, i.e. undirected connections among variables without causal interpretation. Knowledge regarding causal relationships among genes and phenotypes can be used to predict the behavior of complex systems, as well as to optimize management practices and selection strategies. Here, we performed a multistep procedure for inferring causal networks underlying carcass fat deposition and muscularity in pigs using multi-omics data obtained from an F2 Duroc x Pietrain resource pig population.ResultsWe initially explored marginal associations between genotypes and phenotypic and expression traits through whole-genome scans, and then, in genomic regions with multiple significant hits, we assessed gene-phenotype network reconstruction using causal structural learning algorithms. One genomic region on SSC6 showed significant associations with three relevant phenotypes, off-midline10th-rib backfat thickness, loin muscle weight, and average intramuscular fat percentage, and also with the expression of seven genes, including ZNF24, SSX2IP, and AKR7A2. The inferred network indicated that the genotype affects the three phenotypes mainly through the expression of several genes. Among the phenotypes, fat deposition traits negatively affected loin muscle weight.ConclusionsOur findings shed light on the antagonist relationship between carcass fat deposition and lean meat content in pigs. In addition, the procedure described in this study has the potential to unravel gene-phenotype networks underlying complex phenotypes.

Evidence for the multiple hits genetic theory for inherited language impairment: a case study.

Communication disorders have complex genetic origins, with constellations of relevant gene markers that vary across individuals. Some genetic variants are present in healthy individuals as well as those affected by developmental disorders. Growing evidence suggests that some variants may increase susceptibility to these disorders in the presence of other pathogenic gene mutations. In the current study, we describe eight children with specific language impairment and four of these children had a copy number variant in one of these potential susceptibility regions on chromosome 15. Three of these four children also had variants in other genes previously associated with language impairment. Our data support the theory that 15q11.2 is a susceptibility region for developmental disorders, specifically language impairment.

Genetic polymorphisms and oxidative stress in non-alcoholic steatohepatitis (NASH): A mini review

Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease, thus becoming an epidemic in the Western world with a major impact on public health. NAFLD encompasses a large spectrum of disease ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and may progress to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). The role of genetic polymorphisms is not clear. Evidence supports the hypothesis that genetic factors are involved in the predisposition to NAFLD, and thus should emphasize the polygenic nature of the disease as a limiting factor in these studies. However, the polymorphic allele associated with increased hepatic steatosis appears to be associated with various different combinations of phenotypes, including increase or decrease of the biochemical and clinical parameters. It is possible that SNPs in genes involved in excessive fatty acid oxidation would predispose to NASH. On the other hand, the SNPs could determine the inadequate mitochondrial overload during times of excessive FFA supply. However due to the multiple hits involving some pathways, a brief review of genetic variants on mediators of oxidative stress, inflammation and lipid metabolism pathways is presented. It is clear that the discovery of genetic and environmental associations, robust enough to direct the treatment and to trace specific prevention strategies would only be possible with studies examining the susceptibility of NAFLD in a number of individuals considerably higher than assessed so far. These studies need a large number of well phenotyped cases and controls and certainly require national and international collaboration.

The effect of age at exposure on the inactivating mechanisms and relative contributions of key tumor suppressor genes in radiation-induced mouse T-cell lymphomas

Children are considered more sensitive to radiation-induced cancer than adults, yet any differences in genomic alterations associated with age-at-exposure and their underlying mechanisms remain unclear. We assessed genome-wide DNA copy number and mutation of key tumor suppressor genes in T-cell lymphomas arising after weekly irradiation of female B6C3F1 mice with 1.2Gy X-rays for 4 consecutive weeks starting during infancy (1 week old), adolescence (4 weeks old) or as young adults (8 weeks old). Although T-cell lymphoma incidence was similar, loss of heterozygosity at Cdkn2a on chromosome 4 and at Ikaros on chromosome 11 was more frequent in the two older groups, while loss at the Pten locus on chromosome 19 was more frequent in the infant-irradiated group. Cdkn2a and Ikaros mutation/loss was a common feature of the young adult-irradiation group, with Ikaros frequently (50%) incurring multiple independent hits (including deletions and mutations) or suffering a single hit predicted to result in a dominant negative protein (such as those lacking exon 4, an isoform we have designated Ik12, which lacks two DNA binding zinc-finger domains). Conversely, Pten mutations were more frequent after early irradiation (60%) than after young adult-irradiation (30%). Homozygous Pten mutations occurred without DNA copy number change after irradiation starting in infancy, suggesting duplication of the mutated allele by chromosome mis-segregation or mitotic recombination. Our findings demonstrate that while deletions on chromosomes 4 and 11 affecting Cdkn2a and Ikaros are a prominent feature of young adult irradiation-induced T-cell lymphoma, tumors arising after irradiation from infancy suffer a second hit in Pten by mis-segregation or recombination. This is the first report showing an influence of age-at-exposure on genomic alterations of tumor suppressor genes and their relative involvement in radiation-induced T-cell lymphoma. These data are important for considering the risks associated with childhood exposure to radiation.

Preliminary analysis of the contributions of major histocompatibility complex-H2 to autoantibody production in a murine model of systemic lupus erythematosus. (BA3P.116)

Abstract The stochastic and heterogeneous nature of Systemic Lupus Erythematosus (SLE) supports a model whereby multiple genetic and/or environmental hits culminate in loss of tolerance and autoantibody production. Consistent with this model, the phenotype of CD45E613R mice that contain a single point mutation in the juxtamembrane wedge of CD45 depends on genetic context. Despite similar dysregulated phosphatase activity in all immune cells, CD45E613R mice on a C57Bl/6 (B6) background have no overt phenotype while BALB/c (BA) mice develop anti-double stranded (ds) DNA antibodies. An unbiased genome-wide screen for modifiers of autoantibody production between CD45E613R B6 and BA mice identified two candidate loci: Wedge Associated Modifier (Wam) 1 on Chromosome (Chr) 9 encompassing tlr9 and Wam2 on Chr 17 encompassing MHC H2. Previous work has shown that the hyporesponsive BA TLR9 allele permits anti-ds DNA antibodies while the B6 TLR9 allele is resistant. Here, we analyze the contribution of the MHC to this phenotype. CD45E613R BALB/c mice congenic for the B6 H2 locus develop anti-Sm antibodies while B6 mice congenic for the BALB/c H2 locus develop anti-Sm and anti-dsDNA antibodies. H2 congenic CD45E613R BALB/c mice have increased transitional B cells and a trend in increased B1 cells compared to congenic B6 mice. Thus, we hypothesize that MHC influences autoantibody specificity.

Integrated analysis of whole-exome sequencing and transcriptome profiling in males with autism spectrum disorders.

BackgroundAutism spectrum disorders (ASD) are a group of neurodevelopmental disorders with high heritability. Recent findings support a highly heterogeneous and complex genetic etiology including rare de novo and inherited mutations or chromosomal rearrangements as well as double or multiple hits.MethodsWe performed whole-exome sequencing (WES) and blood cell transcriptome by RNAseq in a subset of male patients with idiopathic ASD (n = 36) in order to identify causative genes, transcriptomic alterations, and susceptibility variants.ResultsWe detected likely monogenic causes in seven cases: five de novo (SCN2A, MED13L, KCNV1, CUL3, and PTEN) and two inherited X-linked variants (MAOA and CDKL5). Transcriptomic analyses allowed the identification of intronic causative mutations missed by the usual filtering of WES and revealed functional consequences of some rare mutations. These included aberrant transcripts (PTEN, POLR3C), deregulated expression in 1.7% of mutated genes (that is, SEMA6B, MECP2, ANK3, CREBBP), allele-specific expression (FUS, MTOR, TAF1C), and non-sense-mediated decay (RIT1, ALG9). The analysis of rare inherited variants showed enrichment in relevant pathways such as the PI3K-Akt signaling and the axon guidance.ConclusionsIntegrative analysis of WES and blood RNAseq data has proven to be an efficient strategy to identify likely monogenic forms of ASD (19% in our cohort), as well as additional rare inherited mutations that can contribute to ASD risk in a multifactorial manner. Blood transcriptomic data, besides validating 88% of expressed variants, allowed the identification of missed intronic mutations and revealed functional correlations of genetic variants, including changes in splicing, expression levels, and allelic expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13229-015-0017-0) contains supplementary material, which is available to authorized users.

Nonalcoholic fatty liver disease: update on pathogenesis, diagnosis, treatment and the role of S-adenosylmethionine.

Nonalcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide affecting over one-third of the population in the U.S. It has been associated with obesity, type 2 diabetes, hyperlipidemia, and insulin resistance and is initiated by the accumulation of triglycerides in hepatocytes. Isolated hepatic steatosis (IHS) remains a benign process, while a subset develops superimposed inflammatory activity and progression to nonalcoholic steatohepatitis (NASH) with or without fibrosis. However, the molecular mechanisms underlying NAFLD progression are not completely understood. Liver biopsy is still required to differentiate IHS from NASH as easily accessible noninvasive biomarkers are lacking. In terms of treatments for NASH, pioglitazone, vitamin E, and obeticholic acid have shown some benefit. All of these agents have potential complications associated with long-term use. Nowadays, a complex hypothesis suggests that multiple parallel hits are involved in NASH development. However, the 'key switch' between IHS and NASH remains to be discovered. We have recently shown that knocking out enzymes involved in S-adenosylmethionine (SAMe) metabolism, the main biological methyl donor in humans that is abundant in the liver, will lead to NASH development in mice. This could be due to the fact that a normal SAMe level is required to establish the proper ratio of phosphatidylethanolamine to phosphatidylcholine that has been found to be important in NAFLD progression. New data from humans have also suggested that these enzymes play a role in the pathogenesis of NAFLD and that some of SAMe cycle metabolites may serve as noninvasive biomarkers of NASH. In this review, we discuss the evidence of the role of SAMe in animal models and humans with NAFLD and how studying this area may lead to the discovery of new noninvasive biomarkers and possibly personalized treatment for NASH.

Flexible, fast and accurate sequence alignment profiling on GPGPU with PaSWAS.

MotivationTo obtain large-scale sequence alignments in a fast and flexible way is an important step in the analyses of next generation sequencing data. Applications based on the Smith-Waterman (SW) algorithm are often either not fast enough, limited to dedicated tasks or not sufficiently accurate due to statistical issues. Current SW implementations that run on graphics hardware do not report the alignment details necessary for further analysis.ResultsWith the Parallel SW Alignment Software (PaSWAS) it is possible (a) to have easy access to the computational power of NVIDIA-based general purpose graphics processing units (GPGPUs) to perform high-speed sequence alignments, and (b) retrieve relevant information such as score, number of gaps and mismatches. The software reports multiple hits per alignment. The added value of the new SW implementation is demonstrated with two test cases: (1) tag recovery in next generation sequence data and (2) isotype assignment within an immunoglobulin 454 sequence data set. Both cases show the usability and versatility of the new parallel Smith-Waterman implementation.

Inferring the demographic history of Drosophila subobscura from nucleotide variation at regions not affected by chromosomal inversions.

Drosophila subobscura presents a rich and complex chromosomal inversion polymorphism. It can thus be considered a model system (i) to study the mechanisms originating inversions and how inversions affect the levels and patterns of variation in the inverted regions and (ii) to study adaptation at both the single-gene and chromosomal inversion levels. It is therefore important to infer its demographic history as previous information indicated that its nucleotide variation is not at mutation-drift equilibrium. For that purpose, we sequenced 16 noncoding regions distributed across those parts of the J chromosome not affected by inversions in the studied population and possibly either by other selective events. The pattern of variation detected in these 16 regions is similar to that previously reported within different chromosomal arrangements, suggesting that the latter results would, thus, mainly reflect recent demographic events rather than the partial selective sweep imposed by the origin and frequency increase of inversions. Among the simple demographic models considered in our Approximate Bayesian Computation analysis of variation at the 16 regions, the model best supported by the data implies a population size expansion soon after the penultimate glacial period. This model constitutes a better null model, and it is therefore an important resource for subsequent studies aiming among others to uncover selective events across the species genome. Our results also highlight the importance of introducing the possibility of multiple hits in the coalescent simulations with an outgroup.

Antimicrobial peptides in 2014.

This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms.

Global spectral deconvolution based on non-negative matrix factorization in GC × GC-HRTOFMS.

A global spectral deconvolution, based on non-negative matrix factorization (NMF) in comprehensive two-dimensional gas chromatography high-resolution time-of-flight mass spectrometry, was developed. We evaluated the ability of various instrumental parameters and NMF settings to derive high-performance detection in nontarget screening using a sediment sample. To evaluate the performance of the process, a NIST library search was used to identify the deconvoluted spectra. Differences of the instrumental scan rates (25 and 50 Hz) in deconvolution were evaluated and results show that a high scan rate enhanced the number of compounds detected in the sediment sample. A higher mass resolution in the range of 1,000 to 10,000 and a higher m/z precision in the deconvolution were needed to obtain an accurate mass database. After removal of multiple duplicate hits, which occurred in batch processes of NIST library search on the deconvolution result, 62 unique assignable spectra with a match factor ≥900 were obtained in the deconvoluted chromatogram from the sediment sample, including 54 spectra that were refined by the deconvolution. This method will help to detect and build up well-resolved reference spectra from various complex mixtures and will accelerate nontarget screening.

Identification of a First-in-Class PRMT5 Inhibitor with Potent in Vitro and in Vivo Activity in Preclinical Models of Mantle Cell Lymphoma

Protein Arginine Methyltransferase-5 (PRMT5) has been reported to play a role in multiple diverse cellular processes including tumorigenesis. Overexpression of PRMT5 has been demonstrated in cell lines and primary patient samples derived from lymphomas, particularly Mantle Cell Lymphoma (MCL). Furthermore, knockdown of PRMT5 expression inhibits the proliferation of MCL cell lines. The mechanisms behind the oncogenic potential of PRMT5 are unclear, but the protein has been postulated to regulate processes such as cell death, cell cycle progression, and RNA processing through the dimethylation of arginine residues within a variety of cytoplasmic and nuclear target proteins. Epizyme developed small molecule inhibitors of PRMT5 enzyme activity in order to understand the role of PRMT5-mediated arginine methylation in tumorigenesis and to develop PRMT5-targeted cancer therapeutics. Here, we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with anti-proliferative effects in both in vivo and in vitro models of MCL. A diverse compound library was screened for inhibitors of arginine methylation by purified recombinant PRMT5:MEP50 complex and multiple hits were identified. The inhibitors are SAM uncompetitive, peptide competitive and bind with the PRMT5:MEP50 complex in a unique binding mode not previously observed. Further optimization yielded YQ36286, an orally available inhibitor of PRMT5 with enzymatic activity in biochemical assays with an IC50 in the low nM range and broad selectivity against a panel of other histone methyltransferases. YQ36286 demonstrated potent cellular activity as measured by its ability to inhibit symmetric dimethylation of SmD3, a cytoplasmic PRMT5 substrate in a time- and concentration-dependent manner. Treatment of MCL cell lines with YQ36286 led to inhibition of SmD3 methylation and cell killing, with IC50s in the nM range. Oral dosing of YQ36286 demonstrated dose-dependent anti-tumor activity in multiple MCL xenograft models. In xenograft studies with the Z138 MCL cell line, near 95% tumor growth inhibition was observed after 21 days of dosing with a corresponding decrease in symmetrically dimethylated levels of PRMT5 substrates. In summary, we have developed the first potent and selective small molecule inhibitor of PRMT5 that has cellular activity and in vivo efficacy. MCL cells are dependent on PRMT5 activity for their survival as demonstrated with YQ36286. This small molecule represents a starting point for the development of PRMT5 inhibitors as potential cancer therapeutics. DisclosuresPenebre:Company stock options: Equity Ownership; Epizyme Inc.: Employment; GSK Research Funding: Research Funding. Kuplast:GSK research funding: Research Funding; Company Stock options: Equity Ownership; Epizyme Inc.: Employment. Majer:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Johnston:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Rioux:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Munchhof:Epizyme Inc.: Employment; GSK research funding: Research Funding. Jin:Epizyme Inc.: Employment; GSK research funding: Research Funding; Company stock options: Equity Ownership. Boriak-Sjodin:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Wigle:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Jacques:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. West:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Lingaraj:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Stickland:GSK research funding: Research Funding; Company Stock options: Equity Ownership; Epizyme Inc.: Employment. Ribich:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Raimondi:Epizyme: Employment, Equity Ownership; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Porter-Scott:Company stock options: Equity Ownership; GSK research funding: Research Funding; Epizyme Inc.: Employment. Waters:Epizyme, Inc: Employment, Equity Ownership; GSK research funding: Research Funding. Pollock:Epizyme: Employment, Equity Ownership; GSK research funding: Research Funding. Smith:GSK research funding: Research Funding; Epizyme: Employment, Equity Ownership. Barbash:GlaxoSmithKline Pharmaceuticals: Employment. Kruger:GlaxoSmithKline Pharmaceuticals: Employment, Equity Ownership. Copeland:Mersana: Membership on an entity’s Board of Directors or advisory committees; Epizyme, Inc: Employment, Equity Ownership; Celgene, Inc: Research Funding; Eisai Inc: Research Funding; Glaxo Smith Kline, Inc: Research Funding; Multiple Myeloma Research Foundation: Research Funding; Leukemia and Lymphoma Society: Research Funding; New Enterprise Associates: Ad hoc consultant, Ad hoc consultant Other. Moyer:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Chesworth:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Duncan:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding.

Chronic Lymphocytic Leukemia Patients Harbor Very Low Frequency Stereotypic Immunoglobulin CDR3 Sequences

Introduction : Recent studies have shown that leukemic clones in approximately 10-20% of CLL patients have stereotypic IgH gene sequences (Stamatopoulos et al., Blood 2007). These stereotypic IgH sequences are characterized by closely homologous complementarity-determining regions 3 (CDR3) sequences. The presence of these sequences supports the model of antigen-mediated disease pathogenesis in CLL. While previous studies have focused on the dominant CLL clone present at diagnosis, recent advances in next-generation sequencing enable analysis of the full IgH repertoire. Here we assessed whether stereotypic IgH sequences are present at very low frequency among the total IgH repertoire in diagnostic and follow-up samples from 36 CLL patients.Methods:We utilized the LymphoSIGHT method to sequence the IgH repertoire in 36 CLL patients and 185 patients with non-CLL lymphoid neoplasms. Using universal primer sets, we amplified IGH variable (V), diversity (D), and joining (J) gene segments in diagnostic and follow-up samples. Amplified products were sequenced, and CLL-specific clonotypes were identified in the diagnostic sample of each patient based on high-frequency within the B-cell repertoire. We selected a set of 289 stereotypic IgH CDR3 sequences that were previously curated (Stamatopoulos et al., Blood 2007; Messmer et al., Leukemia Res 2008). We assessed whether the stereotypic CDR3 regions were present in the IgH repertoire of both CLL and non-CLL patients using sequence alignment tools.Results: 154,318 IgH clonotypes were observed in the 36 CLL patients, while we identified 8,248,032 IgH clonotypes from 185 patients with non-CLL lymphoid neoplasms. Specifically, we tested 14 mantle cell lymphoma (MCL), 19 hodgkin disease (HD), and 28 acute lymphoblastic leukemia (ALL) patients as well as three cohorts of multiple myeloma (MM) with 26, 30, and 68 patients. We assessed whether each of the 289 CDR3 stereotypes were present in the IgH repertoire of the CLL or non-CLL patients. Twenty two of the stereotypic IgH sequences were observed in any patient: 2 in CLL patients and 20 in the non-CLL patients. This is not surprising given that non-CLL patients have over 50 times the total number of clones compared to the CLL group. One of the stereotypic IgH clonotypes was detected in one CLL patient (p-value= 0.018; not significant with Bonferroni correction). The other stereotypic IgH clonotype was present in two CLL patients but not in any of the non-CLL lymphoid neoplasm cohorts (p=0.00034; 0.0074 Bonferroni corrected). In contrast, we tested for association between each of the 20 stereotypic sequences and each of the 6 control cohorts (3 MM, 1 MCL, 1 HD, and 1 ALL), and only two significant associations were observed (p=0.045 and p=0.01; neither were significant with Bonferroni correction). This underscores that the only significant association was seen in the tested CLL cohort. Of note, both of these stereotypic IgH clonotypes were present at very low frequency in the IgH repertoire (<10-4) (Figure 1). The two stereotypic IgH sequences identified in CLL patients were unmutated and match the previously published V, D, and J segments. Specifically, the stereotypic sequence ARHRLGYCSSTSCYYYYYGMDVWGQGTT present in one CLL patient has IGHV4-39, IGHJ6, and IGHD2-2 and the stereotypic sequence CARDSPLVVPAAIFYYYYGMDVW present in two CLL patients has IGHV3-48, IGHJ6, IGHD2-2. In contrast the vast majority of the hits in the non-CLL samples have V, D, and J sequences that are different from the published segments for the specific stereotypic sequence.Conclusion : Our results demonstrate that CLL patients harbor stereotypic IgH sequences. These sequences were not the dominant, high-frequency clonotypes identified at diagnosis in the CLL patients and were absent in a much larger set of clones derived from a cohort of non-CLL cancer patients. These results suggest that a subset of CLL patients may possess an antigen environment that selects for multiple B cells with stereotypic IgH sequences at low frequencies. This selection process may predispose patients to CLL that develops later via accumulation of multiple genetic hits in a pre-leukemic cell containing the stereotypic IgH sequence driving it to high-frequency. [Display omitted] DisclosuresKlinger:Sequenta, Inc.: Employment, Equity Ownership. Hervold:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Novel Biological Effects and Distinct Patterns of Rhoa Mutations in Adult T-Cell Leukemia/Lymphoma and Angioimmunoblastic T Cell Lymphoma

Adult T-cell leukemia/lymphoma (ATL) is a distinct form of peripheral T-cell lymphoma (PTCL). ATL is initiated by immortalization of T-cells by human T-lymphotropic virus type I (HTLV-1) infection during early infancy, followed by an accumulation of multiple genetic hits to develop leukemia. However, little has been known about these genetic hits. Recently, frequent somatic mutations in RHOA, DNMT3A, IDH2 and TET2 have been reported in angioimmunoblastic T cell lymphoma (AITL) and PTCL not otherwise specified (PTCL-NOS), which are characterized by follicular helper T-cell phenotypes. Most of the RHOA mutations in AITL invariably cause an identical amino acid change (Gly17Val), which was shown to inhibit wild-type RHOA function in a dominant-negative manner. In addition, RHOA mutations have been also identified in gastric cancer and Burkitt lymphoma, with mutational hotspots including Tyr42 and Arg5. Interestingly, some of them were reported as working in a gain-of function manner.In the current study, we investigated the mutational status of RHOA, as well as TET2, IDH2, and DNMT3A in a total of 205 patients with ATL in order to reveal their role in ATL pathogenesis. In targeted deep sequencing, RHOA was mutated in 32 (15.6%) cases. In contrast to the exclusive G17V mutation found in AITL and PTCL-NOS, mutations in ATL was widely distributed within the regions responsible for GTP-binding with Cys16Arg being most frequently mutated (N = 11; 5.4%), although Gly17Val mutations were also found in 4 cases (1.9%). Being commonly located in the same amino acid with the GTP binding site, Ala161Pro and Ala161Glu mutations were exclusively found in ATL and AITL/PTCL-NOS, respectively. We found no significant differences in the RHOA mutational status among different ATL subtypes, and no significant prognostic impact of RHOA mutations on survival. TET2 mutations were very common and tightly associated with RHOA mutation in AITL, but much less common (N = 21; 10.2%) and may not associated with RHOA mutations in ATL. Mutations of IDH2 (N = 2; 1%) and DNMT3A (N = 2; 1%) were rare in ATL.RHOA encodes a ras-related GTP-binding protein that functions as a molecular switch in a variety of biological processes through cycling between an active (GTP-bound) state and an inactive (GDP-bound) state. Known biological functions of RHOA are related to actin organization, cell migration and transcriptional activation. Modeling of three-dimensional structures suggested that most of mutations identified in ATL located near nucleotide binding pocket, which affected the GTP-binding ability of RHOA protein. Indeed, we performed luciferase assay to measure transcriptional activities of wild-type and mutant RHOA. As previously reported, dominant-negative Gly17Val RHOA mutant suppressed transcriptional activity. In contrast, ATL specific RHOA mutant such as Cys16Arg or Ala161Pro enhanced transcriptional activity, similar to or even more than wild-type RHOA. In addition, when we assessed actin stress fiber formation in cells transduced with wild-type or mutant RHOA, Gly17Val or Ala161Glu RHOA attenuated actin stress fiber formations, whereas Cys16Arg or Ala161Pro RHOA induced actin stress fiber formation, similar to known gain-of-function Gly14Val RHOA mutant. These functional assays implicated Cys16Arg and Ala161Pro RHOA mutant showed different biological behaviors from Gly17Val and Ala161Glu RHOA mutant, indicating gain-of-function mechanisms of Cys16Arg or Ala161Pro mutants.In summary, RHOA mutations were also common in ATL as in AITL and PTCL-NOS. Nevertheless, mutational patterns and functional consequences of RHOA mutations identified in ATL were distinct from those in other PTCLs, suggesting differential driver roles of RHOA mutations in ATL compared to other PTCLs. DisclosuresNo relevant conflicts of interest to declare.