Genetic alteration of SJ293TS cells and modification of serum-free media enhances lentiviral vector production.

Successful cell and gene therapy clinical trials have resulted in the US Food and Drug Administration and European Medicines Agency approving their use for treatment of patients with certain types of cancers and monogenetic diseases. These novel therapies, which rely heavily on lentiviral vectors to deliver therapeutic transgenes to patient cells, have driven additional investigations, increasing demand for both pre-clinical and current Good Manufacturing Practices-grade viral vectors. To better support novel studies by improving current production methods, we report the development of a genetically modified HEK293T-based cell line that is null for expression of both Protein Kinase R and Beta-2 microglobulin and grows in suspension using serum-free media, SJ293TS-DPB. Absence of Protein Kinase R increased anti-sense lentiviral vector titers by more than 7-fold, while absence of Beta-2 microglobulin, a key component of major histocompatibility complex class I molecules, has been reported to reduce the immunogenicity of lentiviral particles. Furthermore, we describe an improved methodology for culturing SJ293TS-DPB that facilitates expansion, reduces handling, and increases titers by 2-fold compared with previous methods. SJ293TS-DPB stably produced lentiviral vectors for over 4 months and generated lentiviral vectors that efficiently transduce healthy human donor T cells and CD34+ hematopoietic stem cells.

Tetrazole and acylsulfonamide bioisosteric replacements of the carboxylic acid in a dual MCL-1/BCL-xL inhibitor are tolerated.

Overexpression of the anti-apoptotic protein MCL-1 is associated with a plethora of human cancers, and it reduces the sensitivity of cancer cells to approved chemotherapies. Accordingly, the discovery of MCL-1 inhibitors is an active area of interest. Many inhibitors of the anti-apoptotic MCL-1 protein bear a crucial carboxylic acid that may engage Arg263 in the BH3-binding groove. We previously described the salicylic acid-based dual MCL-1/BCL-xL inhibitor 17cd, which is currently undergoing lead optimization. As part of that process, we wished to investigate bioisosteric replacement of 17cd's key carboxylic acid. Herein we describe the synthesis of a variety of analogues of a simpler analogue of 17cd presenting carboxylic acid surrogates. The acylsulfonamide and tetrazole motifs, which exhibit comparable pKas to the carboxylic acid function, displayed similar, or better, binding affinities to MCL-1 and BCL-xL as the corresponding carboxylic acid-containing lead. Our best compound was acylsulfonamide 7d with a Ki of 800 nM against MCL-1 and 1.82 mM against BCL-xL, and demonstrated an improved effect on the viability of the HL60 acute myeloid leukemia cell line relative to the parent carboxylic acid-containing dual inhibitor from which it was derived.

Expert Algorithm for Substance Identification Using Mass Spectrometry: Application to the Identification of Cocaine on Different Instruments Using Binary Classification Models.

This is the second of two manuscripts describing how general linear modeling (GLM) of a selection of the most abundant normalized fragment ion abundances of replicate mass spectra from one laboratory can be used in conjunction with binary classifiers to enable specific and selective identifications with reportable error rates of spectra from other laboratories. Here, the proof-of-concept uses a training set of 128 replicate cocaine spectra from one crime laboratory as the basis of GLM modeling. GLM models for the 20 most abundant fragments of cocaine were then applied to 175 additional test/validation cocaine spectra collected in more than a dozen crime laboratories and 716 known negative spectra, which included 10 spectra of three diastereomers of cocaine. Spectral similarity and dissimilarity between the measured and predicted abundances were assessed using a variety of conventional measures, including the mean absolute residual and NIST's spectral similarity score. For each spectral measure, GLM predictions were compared to the traditional exemplar approach, which used the average of the cocaine training set as the consensus spectrum for comparisons. In unsupervised models, EASI provided better than a 95% true positive rate for cocaine with a 0% false positive rate. A supervised binary logistic regression model provided 100% accuracy and no errors using EASI-predicted abundances of only four peaks at m/z 152, 198, 272, and 303. Regardless of the measure of spectral similarity, error rates for identifications using EASI were superior to the traditional exemplar/consensus approach. As a supervised binary classifier, EASI was more reliable than using Mahalanobis distances.

Expert Algorithm for Substance Identification Using Mass Spectrometry: Statistical Foundations in Unimolecular Reaction Rate Theory.

This study aims to resolve one of the longest-standing problems in mass spectrometry, which is how to accurately identify an organic substance from its mass spectrum when a spectrum of the suspected substance has not been analyzed contemporaneously on the same instrument. Part one of this two-part report describes how Rice-Ramsperger-Kassel-Marcus (RRKM) theory predicts that many branching ratios in replicate electron-ionization mass spectra will provide approximately linear correlations when analysis conditions change within or between instruments. Here, proof-of-concept general linear modeling is based on the 20 most abundant fragments in a database of 128 training spectra of cocaine collected over 6 months in an operational crime laboratory. The statistical validity of the approach is confirmed through both analysis of variance (ANOVA) of the regression models and assessment of the distributions of the residuals of the models. General linear modeling models typically explain more than 90% of the variance in normalized abundances. When the linear models from the training set are applied to 175 additional known positive cocaine spectra from more than 20 different laboratories, the linear models enabled ion abundances to be predicted with an accuracy of <2% relative to the base peak, even though the measured abundances vary by more than 30%. The same models were also applied to 716 known negative spectra, including the diastereomers of cocaine: allococaine, pseudococaine, and pseudoallococaine, and the residual errors were larger for the known negatives than for known positives. The second part of the manuscript describes how general linear regression modeling can serve as the basis for binary classification and reliable identification of cocaine from its diastereomers and all other known negatives.

Subtelomeric Chromatin in the Fission Yeast S. pombe.

Telomeres play important roles in safeguarding the genome. The specialized repressive chromatin that assembles at telomeres and subtelomeric domains is key to this protective role. However, in many organisms, the repetitive nature of telomeric and subtelomeric sequences has hindered research efforts. The fission yeast S. pombe has provided an important model system for dissection of chromatin biology due to the relative ease of genetic manipulation and strong conservation of important regulatory proteins with higher eukaryotes. Telomeres and the telomere-binding shelterin complex are highly conserved with mammals, as is the assembly of constitutive heterochromatin at subtelomeres. In this review, we seek to summarize recent work detailing the assembly of distinct chromatin structures within subtelomeric domains in fission yeast. These include the heterochromatic SH subtelomeric domains, the telomere-associated sequences (TAS), and ST chromatin domains that assemble highly condensed chromatin clusters called knobs. Specifically, we review new insights into the sequence of subtelomeric domains, the distinct types of chromatin that assemble on these sequences and how histone H3 K36 modifications influence these chromatin structures. We address the interplay between the subdomains of chromatin structure and how subtelomeric chromatin is influenced by both the telomere-bound shelterin complexes and by euchromatic chromatin regulators internal to the subtelomeric domain. Finally, we demonstrate that telomere clustering, which is mediated via the condensed ST chromatin knob domains, does not depend on knob assembly within these domains but on Set2, which mediates H3K36 methylation.

Submonomer synthesis of peptoids containing trans-inducing N-imino- and N-alkylamino-glycines.

The use of hydrazones as a new type of submonomer in peptoid synthesis is described, giving access to peptoid monomers that are structure-inducing. A wide range of hydrazones were found to readily react with α-bromoamides in routine solid phase peptoid submonomer synthesis. Conditions to promote a one-pot cleavage of the peptoid from the resin and reduction to the corresponding N-alkylamino side chains were also identified, and both the N-imino- and N-alkylamino glycine residues were found to favor the trans-amide bond geometry by NMR, X-ray crystallography, and computational analyses.

Surprising phenotypic diversity of cancer-associated mutations of Gly 34 in the histone H3 tail.

Sequencing of cancer genomes has identified recurrent somatic mutations in histones, termed oncohistones, which are frequently poorly understood. Previously we showed that fission yeast expressing only the H3.3G34R mutant identified in aggressive pediatric glioma had reduced H3K36 trimethylation and acetylation, increased genomic instability and replicative stress, and defective homology-dependent DNA damage repair. Here we show that surprisingly distinct phenotypes result from G34V (also in glioma) and G34W (giant cell tumors of bone) mutations, differentially affecting H3K36 modifications, subtelomeric silencing, genomic stability; sensitivity to irradiation, alkylating agents, and hydroxyurea; and influencing DNA repair. In cancer, only 1 of 30 alleles encoding H3 is mutated. Whilst co-expression of wild-type H3 rescues most G34 mutant phenotypes, G34R causes dominant hydroxyurea sensitivity, homologous recombination defects, and dominant subtelomeric silencing. Together, these studies demonstrate the complexity associated with different substitutions at even a single residue in H3 and highlight the utility of genetically tractable systems for their analysis.

Histone H3 Mutations: An Updated View of Their Role in Chromatin Deregulation and Cancer.

In this review, we describe the attributes of histone H3 mutants identified in cancer. H3 mutants were first identified in genes encoding H3.3, in pediatric high-grade glioma, and subsequently in chondrosarcomas and giant cell tumors of bone (GCTB) in adolescents. The most heavily studied are the lysine to methionine mutants K27M and K36M, which perturb the target site for specific lysine methyltransferases and dominantly perturb methylation of corresponding lysines in other histone H3 proteins. We discuss recent progress in defining the consequences of these mutations on chromatin, including a newly emerging view of the central importance of the disruption of H3K36 modification in many distinct K to M histone mutant cancers. We also review new work exploring the role of H3.3 G34 mutants identified in pediatric glioma and GCTB. G34 is not itself post-translationally modified, but G34 mutation impinges on the modification of H3K36. Here, we ask if G34R mutation generates a new site for methylation on the histone tail. Finally, we consider evidence indicating that histone mutations might be more widespread in cancer than previously thought, and if the perceived bias towards mutation of H3.3 is real or reflects the biology of tumors in which the histone mutants were first identified.

Histone H3G34R mutation causes replication stress, homologous recombination defects and genomic instability in S. pombe.

Recurrent somatic mutations of H3F3A in aggressive pediatric high-grade gliomas generate K27M or G34R/V mutant histone H3.3. H3.3-G34R/V mutants are common in tumors with mutations in p53 and ATRX, an H3.3-specific chromatin remodeler. To gain insight into the role of H3-G34R, we generated fission yeast that express only the mutant histone H3. H3-G34R specifically reduces H3K36 tri-methylation and H3K36 acetylation, and mutants show partial transcriptional overlap with set2 deletions. H3-G34R mutants exhibit genomic instability and increased replication stress, including slowed replication fork restart, although DNA replication checkpoints are functional. H3-G34R mutants are defective for DNA damage repair by homologous recombination (HR), and have altered HR protein dynamics in both damaged and untreated cells. These data suggest H3-G34R slows resolution of HR-mediated repair and that unresolved replication intermediates impair chromosome segregation. This analysis of H3-G34R mutant fission yeast provides mechanistic insight into how G34R mutation may promote genomic instability in glioma.

SHREC Silences Heterochromatin via Distinct Remodeling and Deacetylation Modules.

Nucleosome remodeling and deacetylation (NuRD) complexes are co-transcriptional regulators implicated in differentiation, development, and diseases. Methyl-CpG binding domain (MBD) proteins play an essential role in recruitment of NuRD complexes to their target sites in chromatin. The related SHREC complex in fission yeast drives transcriptional gene silencing in heterochromatin through cooperation with HP1 proteins. How remodeler and histone deacetylase (HDAC) cooperate within NuRD complexes remains unresolved. We determined that in SHREC the two modules occupy distant sites on the scaffold protein Clr1 and that repressive activity of SHREC can be modulated by the expression level of the HDAC-associated Clr1 domain alone. Moreover, the crystal structure of Clr2 reveals an MBD-like domain mediating recruitment of the HDAC module to heterochromatin. Thus, SHREC bi-functionality is organized in two separate modules with separate recruitment mechanisms, which work together to elicit transcriptional silencing at heterochromatic loci.

Cancer-associated mutants of RNA helicase DDX3X are defective in RNA-stimulated ATP hydrolysis.

The DEAD-box RNA helicase DDX3X is frequently mutated in pediatric medulloblastoma. We dissect how these mutants affect DDX3X function with structural, biochemical, and genetic experiments. We identify an N-terminal extension ("ATP-binding loop", ABL) that is critical for the stimulation of ATP hydrolysis by RNA. We present crystal structures suggesting that the ABL interacts dynamically with ATP and confirming that the interaction occurs in solution by NMR chemical shift perturbation and isothermal titration calorimetry. DEAD-box helicases require interaction between two conserved RecA-like helicase domains, D1 and D2 for function. We use NMR chemical shift perturbation to show that DDX3X interacts specifically with double-stranded RNA through its D1 domain, with contact mediated by residues G302 and G325. Mutants of these residues, G302V and G325E, are associated with pediatric medulloblastoma. These mutants are defective in RNA-stimulated ATP hydrolysis. We show that DDX3X complements the growth defect in a ded1 temperature-sensitive strain of Schizosaccharomyces pombe, but the cancer-associated mutants G302V and G325E do not complement and exhibit protein expression defects. Taken together, our results suggest that impaired translation of important mRNA targets by mutant DDX3X represents a key step in the development of medulloblastoma.

Histone H3 mutations--a special role for H3.3 in tumorigenesis?

Brain tumors are the most common solid tumors in children. Pediatric high-grade glioma (HGG) accounts for ∼8-12 % of these brain tumors and is a devastating disease as 70-90 % of patients die within 2 years of diagnosis. The failure to advance therapy for these children over the last 30 years is largely due to limited knowledge of the molecular basis for these tumors and a lack of disease models. Recently, sequencing of tumor cells revealed that histone H3 is frequently mutated in pediatric HGG, with up to 78 % of diffuse intrinsic pontine gliomas (DIPGs) carrying K27M and 36 % of non-brainstem gliomas carrying either K27M or G34R/V mutations. Although mutations in many chromatin modifiers have been identified in cancer, this was the first demonstration that histone mutations may be drivers of disease. Subsequent studies have identified high-frequency mutation of histone H3 to K36M in chondroblastomas and to G34W/L in giant cell tumors of bone, which are diseases of adolescents and young adults. Interestingly, the G34 mutations, the K36M mutations, and the majority of K27M mutations occur in genes encoding the replacement histone H3.3. Here, we review the peculiar characteristics of histone H3.3 and use this information as a backdrop to highlight current thinking about how the identified mutations may contribute to disease development.

Sir2 is required for Clr4 to initiate centromeric heterochromatin assembly in fission yeast.

Heterochromatin assembly in fission yeast depends on the Clr4 histone methyltransferase, which targets H3K9. We show that the histone deacetylase Sir2 is required for Clr4 activity at telomeres, but acts redundantly with Clr3 histone deacetylase to maintain centromeric heterochromatin. However, Sir2 is critical for Clr4 function during de novo centromeric heterochromatin assembly. We identified new targets of Sir2 and tested if their deacetylation is necessary for Clr4-mediated heterochromatin establishment. Sir2 preferentially deacetylates H4K16Ac and H3K4Ac, but mutation of these residues to mimic acetylation did not prevent Clr4-mediated heterochromatin establishment. Sir2 also deacetylates H3K9Ac and H3K14Ac. Strains bearing H3K9 or H3K14 mutations exhibit heterochromatin defects. H3K9 mutation blocks Clr4 function, but why H3K14 mutation impacts heterochromatin was not known. Here, we demonstrate that recruitment of Clr4 to centromeres is blocked by mutation of H3K14. We suggest that Sir2 deacetylates H3K14 to target Clr4 to centromeres. Further, we demonstrate that Sir2 is critical for de novo accumulation of H3K9me2 in RNAi-deficient cells. These analyses place Sir2 and H3K14 deacetylation upstream of Clr4 recruitment during heterochromatin assembly.

Centromeric heterochromatin assembly in fission yeast--balancing transcription, RNA interference and chromatin modification.

Distinct regions of the eukaryotic genome are packaged into different types of chromatin, with euchromatin representing gene rich, transcriptionally active regions and heterochromatin more condensed and gene poor. The assembly and maintenance of heterochromatin is important for many aspects of genome control, including silencing of gene transcription, suppression of recombination, and to ensure proper chromosome segregation. The precise mechanisms underlying heterochromatin establishment and maintenance are still unclear, but much progress has been made towards understanding this process during the last few years, particularly from studies performed in fission yeast. In this review, we hope to provide a conceptual model of centromeric heterochromatin in fission yeast that integrates our current understanding of the competing forces of transcription, replication, and RNA decay that influence its assembly and propagation.