Long Interspersed Nuclear Element-1 Analytes in Extracellular Vesicles as Tools for Molecular Diagnostics of Non-Small Cell Lung Cancer.

Aberrant expression of the oncogenic retrotransposon LINE-1 is a hallmark of various cancer types, including non-small cell lung cancers (NSCLCs). Here, we present proof-of-principle evidence that LINE-1 analytes in extracellular vesicles (EVs) serve as tools for molecular diagnostics of NSCLC, with LINE-1 status in tumor cells and tissues mirroring the LINE-1 mRNA and ORF1p cargos of EVs from lung cancer cell culture conditioned media or human plasma. The levels of LINE-1 analytes in plasma EVs from ostensibly healthy individuals were higher in females than males. While the profiles of LINE-1 mRNA and ORF1p in African Americans compared to Hispanics were not significantly different, African Americans showed slightly higher ORF1p content, and 2-3 times greater ranges of LINE-1 values compared to Hispanics. Whole plasma ORF1p levels correlated with EV ORF1p levels, indicating that most of the circulating LINE-1 protein is contained within EVs. EV LINE-1 mRNA levels were elevated in patients with advanced cancer stages and in select patients with squamous cell carcinoma and metastatic tumors compared to adenocarcinomas. The observed EV LINE-1 mRNA profiles paralleled the patterns of ORF1p expression in NSCLC tissue sections suggesting that LINE-1 analytes in plasma EVs may serve to monitor the activity of LINE-1 retroelements in lung cancer.

Role of the Histone Acetyl Transferase MOF and the Histone Deacetylase Sirtuins in Regulation of H4K16ac During DNA Damage Repair and Metabolic Programming: Implications in Cancer and Aging.

The accurate repair of genomic damage mediated by ionizing radiation (IR), chemo- or radiomimetic drugs, or other exogenous agents, is necessary for maintenance of genome integrity, preservation of cellular viability and prevention of oncogenic transformation. Eukaryotes have conserved mechanisms designed to perceive and repair the damaged DNA quite efficiently. Among the different types of DNA damage, double strand breaks (DSB) are the most detrimental. The cellular DNA DSB response is a hierarchical signaling network that integrates damage sensing and repair with chromatin structural changes that involve a range of pre-existing and induced covalent modifications. Recent studies have revealed that pre-existing histone modifications are important contributors within this signaling/repair network. This chapter discusses the role of a critical histone acetyl transferase (HAT) known as MOF (males absent on the first) and the histone deacetylases (HDACs) Sirtuins on histone H4K16 acetylation (H4K16ac) and DNA damage repair. We also discuss the role of this important histone modification in light of metabolic rewiring and its role in regulating human pathophysiologic states.

Pattern matching for high precision detection of LINE-1s in human genomes.

BACKGROUND: Long interspersed element 1 (LINE-1 or L1) retrotransposons are mobile elements that constitute 17-20% of the human genome. Strong correlations between abnormal L1 expression and several human diseases have been reported. This has motivated increasing interest in accurate quantification of the number of L1 copies present in any given biologic specimen. A main obstacle toward this aim is that L1s are relatively long DNA segments with regions of high variability, or largely present in the human genome as truncated fragments. These particularities render traditional alignment strategies, such as seed-and-extend inefficient, as the number of segments that are similar to L1s explodes exponentially. This study uses the pattern matching methodology for more accurate identification of L1s. We validate experimentally the superiority of pattern matching for L1 detection over alternative methods and discuss some of its potential applications. RESULTS: Pattern matching detected full-length L1 copies with high precision, reasonable computational time, and no prior input information. It also detected truncated and significantly altered copies of L1 with relatively high precision. The method was effectively used to annotate L1s in a target genome and to calculate copy number variation with respect to a reference genome. Crucial to the success of implementation was the selection of a small set of k-mer probes from a set of sequences presenting a stable pattern of distribution in the genome. As in seed-and-extend methods, the pattern matching algorithm sowed these k-mer probes, but instead of using heuristic extensions around the seeds, the analysis was based on distribution patterns within the genome. The desired level of precision could be adjusted, with some loss of recall. CONCLUSION: Pattern matching is more efficient than seed-and-extend methods for the detection of L1 segments whose characterization depends on a finite set of sequences with common areas of low variability. We propose that pattern matching may help establish correlations between L1 copy number and disease states associated with L1 mobilization and evolution.

LINE-1 Cargo and Reverse Transcriptase Activity Profiles in Extracellular Vesicles from Lung Cancer Cells and Human Plasma.

Long Interspersed Element-1 (LINE-1) is an oncogenic human retrotransposon that 'copies and pastes' DNA into new locations via reverse transcription. Given that enzymatically active LINE-1 can be exported in extracellular vesicles (EVs), and that LINE-1 mRNA and its two encoded proteins, ORF1p and ORF2p, are required for retrotransposition, the present study examined LINE-1 EV loading patterns relative to reverse transcriptase (RT) activity in vivo and in vitro. Density gradient ultracentrifugation identified conserved patterns of LINE-1 mRNA and protein distribution in EVs, with RT activity readily detected in EV fractions containing both LINE-1 mRNA and protein. Unlike whole cell and tissue lysates, the ORF1p in EVs was detected as a dimer. EVs from ostensibly healthy plasma donors showed variable but consistent ORF1p profiles, with residual levels of LINE-1 mRNA measured in some but not all samples. EVs from cancer cell lines had elevated mean LINE-1 levels and 5-85 times greater RT activity than EVs from normal cells or healthy plasma. EV RT activity was associated with EV LINE-1 mRNA content and was highest in cell lines that also expressed an elevated expression of ORF1p and ORF2p. Given that LINE-1 activation is a hallmark of many cancer types, our findings suggest that an EV LINE-1 'liquid biopsy' may be developed to monitor LINE-1 activity during the course of malignant progression.

Role of Transposable Elements in Genome Stability: Implications for Health and Disease.

Most living organisms have in their genome a sizable proportion of DNA sequences capable of mobilization; these sequences are commonly referred to as transposons, transposable elements (TEs), or jumping genes. Although long thought to have no biological significance, advances in DNA sequencing and analytical technologies have enabled precise characterization of TEs and confirmed their ubiquitous presence across all forms of life. These findings have ignited intense debates over their biological significance. The available evidence now supports the notion that TEs exert major influence over many biological aspects of organismal life. Transposable elements contribute significantly to the evolution of the genome by giving rise to genetic variations in both active and passive modes. Due to their intrinsic nature of mobility within the genome, TEs primarily cause gene disruption and large-scale genomic alterations including inversions, deletions, and duplications. Besides genomic instability, growing evidence also points to many physiologically important functions of TEs, such as gene regulation through cis-acting control elements and modulation of the transcriptome through epigenetic control. In this review, we discuss the latest evidence demonstrating the impact of TEs on genome stability and the underling mechanisms, including those developed to mitigate the deleterious impact of TEs on genomic stability and human health. We have also highlighted the potential therapeutic application of TEs.

Pediatric pharmacogenomics: challenges and opportunities: on behalf of the Sanford Children's Genomic Medicine Consortium.

The advent of digital, electronic, and molecular technologies has allowed the study of complete genomes. Integrating this information into drug development has opened the door for pharmacogenomic (PGx) interventions in direct patient care. PGx allows clinicians to better identify drug of choice and optimize dosing regimens based on an individual's genetic characteristics. Integrating PGx into pediatric care is a priority for the Sanford Children's Genomic Medicine Consortium, a partnership of ten children's hospitals across the US committed to the innovation and advancement of genomics in pediatric care. In this white paper, we review the current state of PGx research and its clinical utility in pediatrics, a largely understudied population, and make recommendations for advancing cutting-edge practice in pediatrics.

Pharmacogenomics insights into precision pediatric oncology.

PURPOSE OF REVIEW: Pharmacogenomic insights provide an opportunity to optimize medication dosing regimens and patient outcomes. However, the potential for interindividual genomic variability to guide medication dosing and toxicity monitoring is not yet standard of care. In this review, we present advances for the thiopurines, anthracyclines and vincristine and provide perspectives on the actionability of pharmacogenomic guidance in the future. RECENT FINDINGS: The current guideline on thiopurines recommends that those with normal predicted thiopurine methyltransferase and NUDT15 expression receive standard-of-care dosing, while 'poor metabolizer' haplotypes receive a decreased 6-mercaptopurine starting dose to avoid bone marrow toxicity. Emerging evidence established significant polygenic contributions that predispose to anthracycline-induced cardiotoxicity and suggest this knowledge be used to identify those at higher risk of complications. In the case of vincristine, children who express CYP3A5 have a significantly reduced risk of peripheral neuropathy compared with those expressing an inactive form or the CYP3A4 isoform. SUMMARY: The need for adequately powered pediatric clinical trials, coupled with the study of epigenetic mechanisms and their influence on phenotypic variation and the integration of precision survivorship into precision approaches are featured as important areas for focused investments in the future.

Driving the precision medicine highway: community health workers and patient navigators.

The general public is currently bombarded with direct-to-consumer advertising, real time "medical" guidance through the internet, access to digital devices that capture health information, and science-based adds that promote foods, cosmetics, and dietary supplements. Unfortunately, much of this information relies on terminology and concepts not well-understood by consumers, particularly those with lower levels of health and genomic literacy. Such constraints align with the limitations of the American public to obtain and process the basic medical information needed to make appropriate healthcare decisions. Low levels of health and genomic literacy render the American public ill-equipped to make informed decisions, use and interpret genomic information, or appreciate the benefits afforded by genomics-based technologies. We propose that coordinated expansion of the roles of community health workers and patient navigators within the precision medicine space can be effectively used to disseminate the knowledge required for the public to benefit from precision medicine advances in healthcare. A well-organized and trained community health worker and patient navigator workforce will provide a voice for the disadvantaged, especially among recent immigrants likely to be experiencing social isolation, language barriers, and economic deprivation. Armed with this knowledge, community health workers and patient navigators can advance the precision medicine agenda and empower disadvantaged communities to take advantage of major advances in the precision medicine era.

Academic medical centers as innovation ecosystems to address population -omics challenges in precision medicine.

While the promise of the Human Genome Project provided significant insights into the structure of the human genome, the complexities of disease at the individual level have made it difficult to utilize -omic information in clinical decision making. Some of the existing constraints have been minimized by technological advancements that have reduced the cost of sequencing to a rate far in excess of Moore's Law (a halving in cost per unit output every 18 months). The reduction in sequencing costs has made it economically feasible to create large data commons capturing the diversity of disease across populations. Until recently, these data have primarily been consumed in clinical research, but now increasingly being considered in clinical decision- making. Such advances are disrupting common diagnostic business models around which academic medical centers (AMCs) and molecular diagnostic companies have collaborated over the last decade. Proprietary biomarkers and patents on proprietary diagnostic content are no longer driving biomarker collaborations between industry and AMCs. Increasingly the scope of the data commons and biorepositories that AMCs can assemble through a nexus of academic and pharma collaborations is driving a virtuous cycle of precision medicine capabilities that make an AMC relevant and highly competitive. A rebalancing of proprietary strategies and open innovation strategies is warranted to enable institutional precision medicine asset portfolios. The scope of the AMC's clinical trial and research collaboration portfolios with industry are increasingly dependent on the currency of data, and less on patents. Intrapeneurial support of internal service offerings, clinical trials and clinical laboratory services for example, will be important new points of emphasis at the academic-industry interface. Streamlining these new models of industry collaboration for AMCs are a new area for technology transfer offices to offer partnerships and to add value beyond the traditional intellectual property offering.

LINE-1 silencing by retinoblastoma proteins is effected through the nucleosomal and remodeling deacetylase multiprotein complex.

BACKGROUND: Long Interspersed Nuclear Element-1 (L1) is an oncogenic mammalian retroelement silenced early in development via tightly controlled epigenetic mechanisms. We have previously shown that the regulatory region of human and murine L1s interact with retinoblastoma (RB) proteins to effect retroelement silencing. The present studies were conducted to identify the corepressor complex responsible for RB-mediated silencing of L1. METHODS: Chromatin immunoprecipitation and silencing RNA technology were used to identify the repressor complex that silences L1 in human and murine cells. RESULTS: Components of the Nucleosomal and Remodeling Deacetylase (NuRD) multiprotein complex specifically enriched the L1 5'-untranslated DNA sequence in human and murine cells. Genetic ablation of RB proteins in murine cells destabilized interactions within the NuRD macromolecular complex and mediated nuclear rearrangement of Mi2-ß, an ATP-dependent helicase subunit with nucleosome remodeling activity. Depletion of Mi2-ß, RbAP46 and HDAC2 reduced the repressor activity of the NuRD complex and reactivated a synthetic L1 reporter in human cells. Epigenetic reactivation of L1 in RB-null cells by DNA damage was markedly enhanced compared to wild type cells. CONCLUSIONS: RB proteins stabilize interactions of the NuRD corepressor complex within the L1 promoter to effect L1 silencing. L1 retroelements may serve as a scaffold on which RB builds heterochromatic regions that regulate chromatin function.

Residential Proximity to Major Roadways Is Associated With Increased Levels of AC133+ Circulating Angiogenic Cells.

OBJECTIVES: Previous studies have shown that residential proximity to a roadway is associated with increased cardiovascular disease risk. Yet, the nature of this association remains unclear, and its effect on individual cardiovascular disease risk factors has not been assessed. The objective of this study was to determine whether residential proximity to roadways influences systemic inflammation and the levels of circulating angiogenic cells. APPROACH AND RESULTS: In a cross-sectional study, cardiovascular disease risk factors, blood levels of C-reactive protein, and 15 antigenically defined circulating angiogenic cell populations were measured in participants (n=316) with moderate-to-high cardiovascular disease risk. Attributes of roadways surrounding residential locations were assessed using geographic information systems. Associations between road proximity and cardiovascular indices were analyzed using generalized linear models. Close proximity (<50 m) to a major roadway was associated with lower income and higher rates of smoking but not C-reactive protein levels. After adjustment for potential confounders, the levels of circulating angiogenic cells in peripheral blood were significantly elevated in people living in close proximity to a major roadway (CD31(+)/AC133(+), AC133(+), CD34(+)/AC133(+), and CD34(+)/45(dim)/AC133(+) cells) and positively associated with road segment distance (CD31(+)/AC133(+), AC133(+), and CD34(+)/AC133(+) cells), traffic intensity (CD31(+)/AC133(+) and AC133(+) cells), and distance-weighted traffic intensity (CD31(+)/34(+)/45(+)/AC133(+) cells). CONCLUSIONS: Living close to a major roadway is associated with elevated levels of circulating cells positive for the early stem marker AC133(+). This may reflect an increased need for vascular repair. Levels of these cells in peripheral blood may be a sensitive index of cardiovascular injury because of residential proximity to roadways.

Regulation of p53-targeting microRNAs by polycyclic aromatic hydrocarbons: Implications in the etiology of multiple myeloma.

Multiple myeloma (MM) is a common and deadly cancer of blood plasma cells. A unique feature of MM is the extremely low somatic mutation rate of the p53 tumor suppressor gene, in sharp contrast with about half of all human cancers where this gene is frequently mutated. Eleven miRNAs have been reported to repress p53 through direct interaction with the 3' untranslated region. The expression of nine of them is higher in MM plasma cells than in healthy donor counterparts, suggesting that miRNA overexpression is responsible for p53 inactivation in MM. Here, we report that the environmental carcinogen benzo[a]pyrene (BaP) upregulated the expression of seven p53-targeting miRNAs (miR-25, miR-15a, miR-16, miR-92, miR-125b, miR-141, and miR-200a), while 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) upregulated two of them (miR-25 and miR-92) in MM cells. The miR-25 promoter was activated by both BaP and TCDD, and this response was mediated by the aryl hydrocarbon receptor (AhR). We screened 727 compounds that inhibit MM cell survival and down-regulate the expression of p53-targeting miRNAs. We found that (-)-epigallocatechin-3-gallate (EGCG), a constituent of green tea and a major component of the botanical drug Polyphenon® E, reduced the expression of four p53-targeting miRNAs, including miR-25, miR-92, miR-141, and miR-200a. Collectively, these data implicate polycyclic aromatic hydrocarbons and AhR in the regulation of p53-targeting miRNAs in MM and identify a potential therapeutic and preventive agent to combat this deadly disease.

Single nucleotide variation in the TP53 3' untranslated region in diffuse large B-cell lymphoma treated with rituximab-CHOP: a report from the International DLBCL Rituximab-CHOP Consortium Program.

We identified multiple single nucleotide variants (SNVs) in the TP53 3' untranslated region (3'UTR) in tumor specimens from 244 patients with diffuse large B-cell lymphoma (DLBCL). Patients carrying a wild-type TP53 coding sequence (CDS) and 1 or more 3'UTR SNVs had a better 5-year survival rate than patients carrying a wild-type CDS and the reference 3'UTR, yet there is no statistically significance difference in overall survival (OS). In contrast, 3'UTR variation predicted poorer OS for patients with a mutant TP53 CDS. We then sequenced TP53 3'UTR in 247 additional DLBCL patients as a validation set. Altogether, we identified 187 novel SNVs; 36 occurred at least twice. Most of the newly identified 3'UTR SNVs were located at sites that are complementary to seed sequences of microRNAs (miRNAs) that are predicted or experimentally known to target TP53. Three SNVs disrupt the seed match between miR-125b and the TP53 3'UTR, thereby impeding suppression of p53 by this miRNA. In addition, a germline SNV (rs78378222) located in the TP53 polyadenylation signal resulted in downregulation of both p53 messenger RNA and protein levels and reduction of cellular apoptosis. This study is the first to demonstrate the prognostic value of the TP53 3'UTR in cancer.

De novo LINE-1 retrotransposition in HepG2 cells preferentially targets gene poor regions of chromosome 13.

Long interspersed nuclear elements (Line-1 or L1s) account for ~17% of the human genome. While the majority of human L1s are inactive, ~80-100 elements remain retrotransposition competent and mobilize through RNA intermediates to different locations within the genome. De novo insertions of L1s account for polymorphic variation of the human genome and disruption of target loci at their new location. In the present study, fluorescence in situ hybridization and DNA sequencing were used to characterize retrotransposition profiles of L1(RP) in cultured human HepG2 cells. While expression of synthetic L1(RP) was associated with full-length and truncated insertions throughout the entire genome, a strong preference for gene-poor regions, such as those found in chromosome 13 was observed for full-length insertions. These findings shed light into L1 targeting mechanisms within the human genome and question the putative randomness of L1 retrotransposition.

Case Report: Life-Threatening Fluoxetine-Linked Postoperative Bleeding Informed by Pharmacogenetic Evaluation.

BACKGROUND: Selective serotonin reuptake inhibitors (SSRI) are commonly used for the treatment of depression and anxiety. Inhibition of serotonin reuptake in platelets increases bleeding risk in patients taking SSRIs. CASE: Here, we present the case of a 52-year-old patient who developed severe postsurgical bleeding requiring blood transfusion following panniculectomy. CONCLUSION: SSRI-induced bleeding is dose-related and strongly influenced by individual variations in drug metabolizing enzymes and transporters. Supplementary file1 (MP4 8441 KB).

A prismatic view of the epigenetic-metabolic regulatory axis in breast cancer therapy resistance.

Epigenetic regulation established during development to maintain patterns of transcriptional expression and silencing for metabolism and other fundamental cell processes can be reprogrammed in cancer, providing a molecular mechanism for persistent alterations in phenotype. Metabolic deregulation and reprogramming are thus an emerging hallmark of cancer with opportunities for molecular classification as a critical preliminary step for precision therapeutic intervention. Yet, acquisition of therapy resistance against most conventional treatment regimens coupled with tumor relapse, continue to pose unsolved problems for precision healthcare, as exemplified in breast cancer where existing data informs both cancer genotype and phenotype. Furthermore, epigenetic reprograming of the metabolic milieu of cancer cells is among the most crucial determinants of therapeutic resistance and cancer relapse. Importantly, subtype-specific epigenetic-metabolic interplay profoundly affects malignant transformation, resistance to chemotherapy, and response to targeted therapies. In this review, we therefore prismatically dissect interconnected epigenetic and metabolic regulatory pathways and then integrate them into an observable cancer metabolism-therapy-resistance axis that may inform clinical intervention. Optimally coupling genome-wide analysis with an understanding of metabolic elements, epigenetic reprogramming, and their integration by metabolic profiling may decode missing molecular mechanisms at the level of individual tumors. The proposed approach of linking metabolic biochemistry back to genotype, epigenetics, and phenotype for specific tumors and their microenvironment may thus enable successful mechanistic targeting of epigenetic modifiers and oncometabolites despite tumor metabolic heterogeneity.

Recommendations for the Clinician Role in Reducing Gun Violence.

There is a growing need for clear and definitive guidelines to prevent firearm violence in communities across the United States. Recommendations explore the utility and feasibility of universal screenings and recommend utilizing universal screening due to a lack of a clear risk to it. Providers should also work to create risk reduction plans with patients as well. Furthermore, recommendations for mental health care, counseling, and bystander training are made for institutions and their providers.

Scope and financial impact of unpublished data and unused samples among U.S. academic and government researchers.

Unpublished data and unused samples are common byproducts of research activity, but little is known about the scope and economic impact of their disuse. To fill this knowledge gap, we collected self-reported anonymous survey responses from 301 academic and government scientists from randomly selected institutions. Respondents estimated that they published ∼60% of their data and 95% had unpublished data. Of those collecting specimens, 60% stored unused samples. Systemic and logistical issues were identified as major contributory factors. The median cumulative self-reported estimated value of unused resources per researcher was $28,857, with life science ($36k) and government ($109k) researchers reporting the costliest assets. Using NSF headcounts, we estimated that the current cumulative value of unused resources at universities is approximately $6.2 billion, about 7% of the current annual R&D budget. These findings provide actionable information that can be used by decision makers to reduce obstacles that undermine scientific progress and productivity.

External control arms: COVID-19 reveals the merits of using real world evidence in real-time for clinical and public health investigations.

Randomized controlled trials are considered the 'gold standard' to reduce bias by randomizing patients to an experimental intervention, versus placebo or standard of care cohort. There are inherent challenges to enrolling a standard of care or cohorts: costs, site engagement logistics, socioeconomic variability, patient willingness, ethics of placebo interventions, cannibalizing the treatment arm population, and extending study duration. The COVID-19 pandemic has magnified aspects of constraints in trial recruitment and logistics, spurring innovative approaches to reducing trial sizes, accelerating trial accrual while preserving statistical rigor. Using data from medical records and databases allows for construction of external control arms that reduce the costs of an external control arm (ECA) randomized to standard of care. Simultaneously examining covariates of the clinical outcomes in ECAs that are being measured in the interventional arm can be particularly useful in phase 2 trials to better understand social and genetic determinants of clinical outcomes that might inform pivotal trial design. The FDA and EMA have promulgated a number of publicly available guidance documents and qualification reports that inform the use of this regulatory science tool to streamline clinical development, of phase 4 surveillance, and policy aspects of clinical outcomes research. Availability and quality of real-world data (RWD) are a prevalent impediment to the use of ECAs given such data is not collected with the rigor and deliberateness that characterizes prospective interventional control arm data. Conversely, in the case of contemporary control arms, a clinical trial outcome can be compared to a contemporary standard of care in cases where the standard of care is evolving at a fast pace, such as the use of checkpoint inhibitors in cancer care. Innovative statistical methods are an essential aspect of an ECA strategy and regulatory paths for these innovative approaches have been navigated, qualified, and in some cases published.