Forward genetic screen of human transposase genomic rearrangements

BACKGROUND: Numerous human genes encode potentially active DNA transposases or recombinases, but our understanding of their functions remains limited due to shortage of methods to profile their activities on endogenous genomic substrates. RESULTS: To enable functional analysis of human transposase-derived genes, we combined forward chemical genetic hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) screening with massively parallel paired-end DNA sequencing and structural variant genome assembly and analysis. Here, we report the HPRT1 mutational spectrum induced by the human transposase PGBD5, including PGBD5-specific signal sequences (PSS) that serve as potential genomic rearrangement substrates. CONCLUSIONS: The discovered PSS motifs and high-throughput forward chemical genomic screening approach should prove useful for the elucidation of endogenous genome remodeling activities of PGBD5 and other domesticated human DNA transposases and recombinases

Passive water control at the surface of a superhydrophobic lichen

Some lichens have a super-hydrophobic upper surface, which repels water drops, keeping the surface dry but probably preventing water uptake. Spore ejection requires water and is most efficient just after rainfall. This study was carried out to investigate how super-hydrophobic lichens manage water uptake and repellence at their fruiting bodies, or podetia. Drops of water were placed onto separate podetia of Cladonia chlorophaea and observed using optical microscopy and cryo-scanning-electron microscopy (cryo-SEM) techniques to determine the structure of podetia and to visualise their interaction with water droplets. SEM and optical microscopy studies revealed that the surface of the podetia was constructed in a three-level structural hierarchy. By cryo-SEM of water-glycerol droplets placed on the upper part of the podetium, pinning of the droplet to specific, hydrophilic spots (pycnidia/apothecia) was observed. The results suggest a mechanism for water uptake, which is highly sophisticated, using surface wettability to generate a passive response to different types of precipitation in a manner similar to the Namib Desert beetle. This mechanism is likely to be found in other organisms as it offers passive but selective water control

Genomic DNA transposition induced by human PGBD5

Transposons are mobile genetic elements that are found in nearly all organisms, including humans. Mobilization of DNA transposons by transposase enzymes can cause genomic rearrangements, but our knowledge of human genes derived from transposases is limited. In this study, we find that the protein encoded by human PGBD5, the most evolutionarily conserved transposable element-derived gene in vertebrates, can induce stereotypical cut-and-paste DNA transposition in human cells. Genomic integration activity of PGBD5 requires distinct aspartic acid residues in its transposase domain, and specific DNA sequences containing inverted terminal repeats with similarity to piggyBac transposons. DNA transposition catalyzed by PGBD5 in human cells occurs genome-wide, with precise transposon excision and preference for insertion at TTAA sites. The apparent conservation of DNA transposition activity by PGBD5 suggests that genomic remodeling contributes to its biological function

Small-molecule dual PLK1 and BRD4 inhibitors are active against preclinical models of pediatric solid tumors

Simultaneous inhibition of multiple molecular targets is an established strategy to improve the continuance of clinical response to therapy. Here, we screened 49 molecules with dual nanomolar inhibitory activity against BRD4 and PLK1, best classified as dual kinase-bromodomain inhibitors, in pediatric tumor cell lines for their antitumor activity. We identified two candidate dual kinase-bromodomain inhibitors with strong and tumor-specific activity against neuroblastoma, medulloblastoma, and rhabdomyosarcoma tumor cells. Dual PLK1 and BRD4 inhibitor treatment suppressed proliferation and induced apoptosis in pediatric tumor cell lines at low nanomolar concentrations. This was associated with reduced MYCN-driven gene expression as assessed by RNA sequencing. Treatment of patient-derived xenografts with dual inhibitor UMB103 led to significant tumor regression. We demonstrate that concurrent inhibition of two central regulators of MYC protein family of protooncogenes, BRD4, and PLK1, with single small molecules has strong and specific antitumor effects in preclinical pediatric cancer models

GW25-e0848 The effects of anticoagulant therapy on coagulant state and platelet function following transcatheter closure of atrial septal defect

BACKGROUND: Motor cortex stimulation (MCS) was introduced in the early 1990s by Tsubokawa and his group for patients diagnosed with drug-resistant, central neuropathic pain. Inconsistencies concerning the details of this therapy and its outcomes and poor methodology of most clinical essays divide the neuromodulation society worldwide into "believers" and "nonbelievers." A European expert meeting was organized in Brussels, Belgium by the Benelux Neuromodulation Society in order to develop uniform MCS protocols in the preoperative, intraoperative, and postoperative courses. METHODS: An expert meeting was organized, and a questionnaire was sent out to all the invited participants before this expert meeting. An extensive literature research was conducted in order to enrich the results. RESULTS: Topics that were addressed during the expert meeting were 1) inclusion and exclusion criteria, 2) targeting and methods of stimulation, 3) effects of MCS, and 4) results from the questionnaire. CONCLUSIONS: Substantial commonalities but also important methodologic divergencies emerged from the discussion of MCS experts from 7 European Centers. From this meeting and questionnaire, all participants concluded that there is a need for more homogenous standardized protocols for MCS regarding patient selection, implantation procedure, stimulation parameters, and follow-up-course

Reflection of neuroblastoma intratumor heterogeneity in the new OHC-NB1 disease model

Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC-NB1, from a bone marrow metastasis from a patient diagnosed with MYCN-amplified neuroblastoma and performed whole-exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC-NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80-540 single-nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with 4 clones being reflected in the model systems. The set of OHC-NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system

Detection and validation of circular DNA fragments using nanopore sequencing

Occurrence of extra-chromosomal circular DNA is a phenomenon frequently observed in tumor cells, and the presence of such DNA has been recognized as a marker of adverse outcome across cancer types. We here describe a computational workflow for identification of DNA circles from long-read sequencing data. The workflow is implemented based on the Snakemake workflow management system. Its key step uses a graph-theoretic approach to identify putative circular fragments validated on simulated reads. We then demonstrate robustness of our approach using nanopore sequencing of selectively enriched circular DNA by highly sensitive and specific recovery of plasmids and the mitochondrial genome, which is the only circular DNA in normal human cells. Finally, we show that the workflow facilitates detection of larger circular DNA fragments containing extrachromosomal copies of the MYCN oncogene and the respective breakpoints, which is a potentially useful application in disease monitoring of several cancer types

Combined inhibition of Aurora-A and ATR kinase results in regression of MYCN-amplified neuroblastoma

Amplification of MYCN is the driving oncogene in a subset of high-risk neuroblastoma. The MYCN protein and the Aurora-A kinase form a complex during S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription-replication conflicts and activates the Ataxia telangiectasia and Rad3 related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription-replication conflicts is an effective therapy for MYCN-driven neuroblastoma (141 words)

CD28 co-stimulus achieves superior CAR T cell effector function against solid tumors than 4-1BB co-stimulus

Spacer or co-stimulatory components in chimeric antigen receptor (CAR) design influence CAR T cell effector function. Few preclinical mouse models optimally support CAR candidate pre-selection for clinical development. Here we use a model in which murine CAR T cells can be exploited with human tumor xenografts. This mouse-in-mouse approach avoids limitations caused by species-specific factors crucial for CAR T cell survival, trafficking and function. We compared trafficking, expansion and tumor control for T cells expressing different CAR construct designs targeting two antigens (L1CAM or HER2), structurally identical except for spacer (long or short) or co-stimulatory (4-1BB or CD28) domains to be evaluated. Using monoclonal, murine-derived L1CAM-specific CAR T cells in Rag-/- mice harboring established xenografted tumors from a human neuroblastoma cell line revealed a clear superiority in CAR T cell trafficking using CD28 co-stimulation. L1CAM-targeting short spacer-CD28/ζ CAR T cells expanded the most at the tumor site and induced initial tumor regression. Treating patient-derived neuroblastoma xenografts with human L1CAM-targeting CAR T cells confirmed the superiority of CD28 co-stimulus. CD28 superiority was also demonstrated with HER2-specific CAR T cells (targeting ovarian carcinoma xenografts). Our findings encourage incorporating CD28 signaling into CAR design for adoptive T cell treatment of solid tumors

Long-read sequencing of diagnosis and post-therapy medulloblastoma reveals complex rearrangement patterns and epigenetic signatures

Cancer genomes harbor a broad spectrum of structural variants (SVs) driving tumorigenesis, a relevant subset of which escape discovery using short-read sequencing. We employed Oxford Nanopore Technologies (ONT) long-read sequencing in a paired diagnostic and post-therapy medulloblastoma to unravel the haplotype-resolved somatic genetic and epigenetic landscape. We assembled complex rearrangements, including a 1.55-Mbp chromothripsis event, and we uncover a complex SV pattern termed templated insertion (TI) thread, characterized by short (mostly <1 kb) insertions showing prevalent self-concatenation into highly amplified structures of up to 50 kbp in size. TI threads occur in 3% of cancers, with a prevalence up to 74% in liposarcoma, and frequent colocalization with chromothripsis. We also perform long-read-based methylome profiling and discover allele-specific methylation (ASM) effects, complex rearrangements exhibiting differential methylation, and differential promoter methylation in cancer-driver genes. Our study shows the advantage of long-read sequencing in the discovery and characterization of complex somatic rearrangements