Cross-talk between Lysine-Modifying Enzymes Controls Site-Specific DNA Amplifications.

Acquired chromosomal DNA amplifications are features of many tumors. Although overexpression and stabilization of the histone H3 lysine 9/36 (H3K9/36) tri-demethylase KDM4A generates transient site-specific copy number gains (TSSGs), additional mechanisms directly controlling site-specific DNA copy gains are not well defined. In this study, we uncover a collection of H3K4-modifying chromatin regulators that function with H3K9 and H3K36 regulators to orchestrate TSSGs. Specifically, the H3K4 tri-demethylase KDM5A and specific COMPASS/KMT2 H3K4 methyltransferases modulate different TSSG loci through H3K4 methylation states and KDM4A recruitment. Furthermore, a distinct chromatin modifier network, MLL1-KDM4B-KDM5B, controls copy number regulation at a specific genomic locus in a KDM4A-independent manner. These pathways comprise an epigenetic addressing system for defining site-specific DNA rereplication and amplifications.

KDM4A lysine demethylase induces site-specific copy gain and rereplication of regions amplified in tumors.

Acquired chromosomal instability and copy number alterations are hallmarks of cancer. Enzymes capable of promoting site-specific copy number changes have yet to be identified. Here, we demonstrate that H3K9/36me3 lysine demethylase KDM4A/JMJD2A overexpression leads to localized copy gain of 1q12, 1q21, and Xq13.1 without global chromosome instability. KDM4A-amplified tumors have increased copy gains for these same regions. 1q12h copy gain occurs within a single cell cycle, requires S phase, and is not stable but is regenerated each cell division. Sites with increased copy number are rereplicated and have increased KDM4A, MCM, and DNA polymerase occupancy. Suv39h1/KMT1A or HP1γ overexpression suppresses the copy gain, whereas H3K9/K36 methylation interference promotes gain. Our results demonstrate that overexpression of a chromatin modifier results in site-specific copy gains. This begins to establish how copy number changes could originate during tumorigenesis and demonstrates that transient overexpression of specific chromatin modulators could promote these events.

Atypical histone targets of PHD fingers.

Plant homeodomain (PHD) fingers are structurally conserved zinc fingers that selectively bind unmodified or methylated at lysine 4 histone H3 tails. This binding stabilizes transcription factors and chromatin-modifying proteins at specific genomic sites, which is required for vital cellular processes, including gene expression and DNA repair. Several PHD fingers have recently been shown to recognize other regions of H3 or histone H4. In this review, we detail molecular mechanisms and structural features of the noncanonical histone recognition, discuss biological implications of the atypical interactions, highlight therapeutic potential of PHD fingers, and compare inhibition strategies.

A unifying model for the selective regulation of inducible transcription by CpG islands and nucleosome remodeling.

We describe a broad mechanistic framework for the transcriptional induction of mammalian primary response genes by Toll-like receptors and other stimuli. One major class of primary response genes is characterized by CpG-island promoters, which facilitate promiscuous induction from constitutively active chromatin without a requirement for SWI/SNF nucleosome remodeling complexes. The low nucleosome occupancy at promoters in this class can be attributed to the assembly of CpG islands into unstable nucleosomes, which may lead to SWI/SNF independence. Another major class consists of non-CpG-island promoters that assemble into stable nucleosomes, resulting in SWI/SNF dependence and a requirement for transcription factors that promote selective nucleosome remodeling. Some stimuli, including serum and tumor necrosis factor-alpha, exhibit a strong bias toward activation of SWI/SNF-independent CpG-island genes. In contrast, interferon-beta is strongly biased toward SWI/SNF-dependent non-CpG-island genes. By activating a diverse set of transcription factors, Toll-like receptors induce both classes and others for an optimal response to microbial pathogens.

Initiation Patterns and Transitions Among Adults Using Stimulant Drugs: Latent Transition Analysis.

BACKGROUND: The fourth wave of the drug overdose epidemic in the United States includes increasing rates of stimulant-involved overdose. Recent studies of transitions leading to stimulant misuse have shown complex patterns that are not universally applicable because they have isolated individual populations or individual behaviors. A comprehensive analysis of transitions between behaviors and the associations with present-day problematic drug use has not been conducted. OBJECTIVE: This study aims to determine whether adults from the general population who use stimulants initiate use through a heterogeneous combination of behaviors and quantify the association between these typologies with present-day problematic drug use. METHODS: Individuals who have reported use of any stimulant in their lifetime were recruited from the 2021 Survey of Nonmedical Use of Prescription Drugs Program, a nationally representative web-based survey on drug use, to participate in a rapid follow-up survey about their past stimulant use. Individuals were asked which stimulants they used, the reasons for use, the routes of administration, and the sources of the stimulant. For each stimulant-related behavior, they were asked at what age, between 6 and 30 years, they initiated each behavior in a 6-year time window. A latent transition analysis was used to characterize heterogeneity in initiation typologies. Mutually exclusive pathways of initiation were identified manually by the researchers. The association of these pathways with present-day problematic drug use was calculated using logistic regression adjusted by the current age of the respondent. RESULTS: From a total of 1329 participants, 740 (55.7%) reported lifetime prescription stimulant use and 1077 (81%) reported lifetime illicit stimulant use. Three typologies were identified. The first typology was characterized by illicit stimulant initiation to get high, usually via oral or snorting routes and acquisition from friends or family or a dealer (illicit experimentation). The second typology was characterized by low, but approximately equal probabilities of initiating 1-2 new behaviors in a time window, but no singular set of behaviors characterized the typology (conservative initiation). The third was characterized by a high probability of initiating many diverse combinations of behaviors (nondiscriminatory experimentation). The choice of drug initiated was not a strong differentiator. Categorization of pathways showed those who were only in an illicit experimentation status (reference) had the lowest odds of having severe present-day problematic drug use. Odds were higher for a conservative initiation-only status (odds ratio [OR] 1.84, 95% CI 1.14-2.94), which is higher still for those moving from illicit experimentation to conservative initiation (OR 3.50, 95% CI 2.13-5.74), and highest for a nondiscriminatory experimentation status (OR 5.45, 95% CI 3.39-8.77). CONCLUSIONS: Initiation of stimulant-related use behaviors occurred across many time windows, indicating that multiple intervention opportunities are presented. Screening should be continued throughout adulthood to address unhealthy drug use before developing into full substance use disorders.

Differing Behaviors Around Adult Nonmedical Use of Prescription Stimulants and Opioids: Latent Class Analysis.

BACKGROUND: The availability of central nervous system stimulants has risen in recent years, along with increased dispensing of stimulants for treatment of, for example, parent-reported attention-deficit/hyperactivity disorder in children and new diagnoses during adulthood. Typologies of drug use, as has been done with opioids, fail to include a sufficient range of behavioral factors to contextualize person-centric circumstances surrounding drug use. Understanding these patterns across drug classes would bring public health and regulatory practices toward precision public health. OBJECTIVE: The objective of this study was to quantitatively delineate the unique behavioral profiles of adults who currently nonmedically use stimulants and opioids using a latent class analysis and to contrast the differences in findings by class. We further evaluated whether the subgroups identified were associated with an increased Drug Abuse Screening Test-10 (DAST-10) score, which is an indicator of average problematic drug use. METHODS: This study used a national cross-sectional web-based survey, using 3 survey launches from 2019 to 2020 (before the COVID-19 pandemic). Data from adults who reported nonmedical use of prescription stimulants (n=2083) or prescription opioids (n=6127) in the last 12 months were analyzed. A weighted latent class analysis was used to identify the patterns of use. Drug types, motivations, and behaviors were factors in the model, which characterized unique classes of behavior. RESULTS: Five stimulant nonmedical use classes were identified: amphetamine self-medication, network-sourced stimulant for alertness, nonamphetamine performance use, recreational use, and nondiscriminatory behaviors. The drug used nonmedically, acquisition through a friend or family member, and use to get high were strong differentiators among the stimulant classes. The latter 4 classes had significantly higher DAST-10 scores than amphetamine self-medication (P<.001). In addition, 4 opioid nonmedical use classes were identified: moderate pain with low mental health burden, high pain with higher mental health burden, risky behaviors with diverse motivations, and nondiscriminatory behaviors. There was a progressive and significant increase in DAST-10 scores across classes (P<.001). The potency of the opioid, pain history, the routes of administration, and psychoactive effect behaviors were strong differentiators among the opioid classes. CONCLUSIONS: A more precise understanding of how behaviors tend to co-occur would improve efficacy and efficiency in developing interventions and supporting the overall health of those who use drugs, and it would improve communication with, and connection to, those at risk for severe drug outcomes.

Hypoxia drives transient site-specific copy gain and drug-resistant gene expression.

Copy number heterogeneity is a prominent feature within tumors. The molecular basis for this heterogeneity remains poorly characterized. Here, we demonstrate that hypoxia induces transient site-specific copy gains (TSSGs) in primary, nontransformed, and transformed human cells. Hypoxia-driven copy gains are not dependent on HIF1α or HIF2α; however, they are dependent on the KDM4A histone demethylase and are blocked by inhibition of KDM4A with a small molecule or the natural metabolite succinate. Furthermore, this response is conserved at a syntenic region in zebrafish cells. Regions with site-specific copy gain are also enriched for amplifications in hypoxic primary tumors. These tumors exhibited amplification and overexpression of the drug resistance gene CKS1B, which we recapitulated in hypoxic breast cancer cells. Our results demonstrate that hypoxia provides a biological stimulus to create transient site-specific copy alterations that could result in heterogeneity within tumors and cell populations. These findings have major implications in our understanding of copy number heterogeneity and the emergence of drug resistance genes in cancer.

Isolation and analysis of rereplicated DNA by Rerep-Seq.

Changes in gene copy number contribute to genomic instability, the onset and progression of cancer, developmental abnormalities and adaptive potential. The origins of gene amplifications have remained elusive; however, DNA rereplication has been implicated as a source of gene amplifications. The inability to determine which sequences are rereplicated and under what conditions have made it difficult to determine the validity of the proposed models. Here we present Rerep-Seq, a technique that selectively enriches for rereplicated DNA in preparation for analysis by DNA sequencing that can be applied to any species. We validated Rerep-Seq by simulating DNA rereplication in yeast and human cells. Using Rerep-Seq, we demonstrate that rereplication induced in Saccharomyces cerevisiae by deregulated origin licensing is non-random and defined by broad domains that span multiple replication origins and topological boundaries.

Measuring prescription opioid misuse and its consequences.

AIMS: Prescription drug misuse in the USA increased during the 1990s to 2010. The epidemic stimulated the need new analytical strategies and techniques to understand the medications involved, user characteristics and other factors needed to address the epidemic. METHODS: A strategy of mosaic surveillance has evolved. Using real world evidence, the goal is to paint a more complete profile of a drug's real world misuse using triangulation-integrating results from multiple sources, where each approach has unrelated sources of bias. RESULTS: Research findings have been remarkably consistent across multiple data sources. The most commonly misused opioid medications: hydrocodone = oxycodone > methadone = buprenorphine = tramadol = fentanyl (prescription form) > morphine > hydromorphone = oxymorphone > tapentadol. This rank order is similar to the number of prescriptions dispensed for each product in the USA. In the USA, prescription opioid misuse started to decrease about 2011. Typically, multiple drugs are misused together, particularly in lethal cases. Immediate release formulations are more commonly misused than extended release formulations. The introduction of tamper resistant formulations to resist crushing were followed by a decrease in misuse of those products. CONCLUSIONS: The rapid expansion of opioid prescribing was accompanied by increasing misuse and mortality. Interventions such as prescription drug monitoring programmes, increased law enforcement and abuse deterrent formulations have been followed by decreases in misuse of most opioid analgesics.

An evaluation of online discussion relating to nonmedical use of prescription opioids within the UK.

AIM: To identify and describe the nature of online discussion relating to prescription opioids within the UK. METHODS: We performed analysis of posts originating in the UK related to buprenorphine, hydrocodone, oxycodone and tramadol using Social Studio, a web-monitoring platform. The study included posts published between January 2014 and December 2016. The data were cleaned to produce a final dataset consisting only of substantive mentions, which were then categorised by defined themes. RESULTS: The final dataset included a total of 17 361 substantive mentions (2936 buprenorphine, 2894 hydrocodone, 3826 oxycodone and 7705 tramadol). The most common theme for all 4 drugs was sharing experience or opinion comprising over 90% of mentions for each drug, while discussion related to polysubstance use was present in >1/4 of mentions across drug substances. Mentions related to diversion were more common for hydrocodone and oxycodone (8.1% [6.3-10.1 95% confidence interval] and 7.8% [6.5-9.2], respectively) than buprenorphine or tramadol (4.1 and 3.9% [3.5-4.3], respectively). CONCLUSION: This investigation shows that there is substantial online discussion relating to a variety of nonmedical use (NMU) behaviours of prescription opioids within the UK, including for hydrocodone, which is not medically available. Web monitoring provides useful data and merits future investigation; this could include expansion to other categories of drugs and a more in-depth analysis of motivations behind NMU, both of which could add timely evidence regarding the current situation in the UK and help inform public health interventions for NMU of prescription drugs.

Nonmedical use of benzodiazepines and Z-drugs in the UK.

AIMS: To estimate prevalence of last 12-month nonmedical use (NMU) of benzodiazepines and Z-drugs (the nonbenzodiazepine hypnotics zaleplon, zolpidem and zopiclone) in the UK. METHODS: Data were collected using the Non-Medical Use of Prescription Drugs survey with poststratification weighting applied to be representative of the UK population (≥16 years). Participants were questioned about whether they had nonmedically used benzodiazepines and/or Z-drugs in the last 12-months and from where they had obtained the drug (including via a prescription, or illicitly from a friend/family member, a dealer or via the internet). Additional questions were asked about last 12-month use of illicit drugs (cannabis, cocaine, 3,4-methylenedioxymethylamphetamine [MDMA], non-pharmaceutical amphetamine, crack cocaine and/or heroin). RESULTS: The study included 10 006 eligible participants representing approximately 52 927 000 UK adults. The estimated prevalence of past 12-month NMU of any benzodiazepine and/or Z-drug was 1.2% (95% confidence interval: 1.0-1.5) corresponding to approximately 635 000 adults; amongst this group only an estimated 4.6% (1.2-8.0) had NMU of both a benzodiazepine and a Z-drug. The highest prevalence of NMU for only Z-drugs was among those who had used heroin in the last 12-months (5.4%, 2.7-10.5), whilst the highest prevalence of NMU for only benzodiazepines was among those who had used illicit stimulants in the last 12-months: cocaine (5.9%, 3.8-8.9), amphetamine (5.6%, 3.1-10.0) and MDMA (5.2%, 3.1-8.8). The drug non-medically used was more commonly acquired without than with a prescription for both only benzodiazepines (70.2%, 59.4-81.1 compared to 51.3%, 41.5-64.6) and only Z-drugs (75.6%, 61.6-89.7 compared to 33.9%, 16.9-51.0). CONCLUSION: There is little overlap between benzodiazepine and Z-drug NMU suggesting distinct nonmedical use of the drugs; future studies need to explore whether this relates to personal preference, drug availability or other factors. A significant proportion are acquiring these drugs for NMU without a prescription, so without guidance and monitoring from a medical practitioner. While the dangers of mixing benzodiazepines and heroin/other opioids are well documented, there is a paucity of data regarding concomitant NMU of benzodiazepines and stimulant drugs, or NMU of Z-drugs and opioids, and, given the prevalence of these combinations, this requires further investigation.

Non-medical use of benzodiazepines and GABA analogues in Europe.

AIMS: We investigated the prevalence of non-medical use (NMU) of benzodiazepines and GABA analogues in Europe. METHODS: Data were collected using the online Non-Medical Use of Prescription Drugs (NMURx) survey from France, Germany, Italy, Spain and the UK. RESULTS: The study included 55 223 eligible surveys which, after post-stratification weights were applied, represented approximately 260 million European adults. Lifetime NMU of benzodiazepines was highest in Spain (6.5%, 95% CI: 6.0-7.0) and lowest in Germany (1.7%, 1.5-2.0). Lifetime NMU of GABA analogues was highest in Germany (5.4%, 5.0-5.7) and lowest in France (2.2%, 1.9-2.5) and the UK (2.2%, 1.9-2.6) While no notable difference was observed for France or the UK, there was a higher prevalence of last 12-month NMU of benzodiazepines compared to GABA analogues in Italy (2.4 times higher) and Spain (3.0 times higher) and a higher prevalence of NMU of GABA analogues compared to benzodiazepines in Germany (2.6 times higher). CONCLUSION: This study shows that there is variation in NMU of benzodiazepines and GABA analogues among countries. Of particular interest is the high incidence of GABA analogue NMU in Germany and benzodiazepine NMU in Spain. Further research to identify factors and motivations responsible for the higher prevalence observed are essential to inform public health policies in those countries.

Drug product dispensing and estimates of use in a general population survey as a signal detection problem.

PURPOSE: Understanding potential bias due to rarity of the outcome is important when monitoring newly approved drugs and drugs with low availability to the general public. Although there is an increasing use of online surveys to investigate health outcomes, the limits of inference due to drug availability have not been studied. The goal of this study was to quantify the relationship between dispensing of prescription drugs and estimates of use in an online general population survey. METHODS: An online repeated, cross-sectional survey from 2018 to 2020 was used to estimate the number of adults in the United States who used prescription drugs in the general population and compared to estimated number of prescriptions dispensed over an equivalent time period. Joinpoint regression was used to quantify thresholds. A sample of respondents was retested to estimate reliability statistics. RESULTS: A model with a single threshold was the best fit, with the estimated threshold of 565 000 (95% CI: 9500-11 600 000) prescriptions dispensed per year. Above the threshold, there was a significant association between dispensing and estimates (p < 0.001); below the threshold, the relationship was not significant (p = 0.912). Above the threshold, responses were more reliable than random chance, and reliability steadily increased with increased dispensing. CONCLUSIONS: These results suggest the threshold demarcates two distinct pharmacoepidemiological paradigms when investigating drug use in general population surveys. Dispensing can be used as a guide to determine the epidemiological paradigm that is best suited.

Changes in Mortality Involving Extended-Release and Long-Acting Opioids After Implementation of a Risk Evaluation and Mitigation Strategy.

OBJECTIVE: To assess changes in mortality rates in extended-release and long-acting (ER/LA) opioid analgesics after the implementation of the Risk Evaluation and Mitigation Strategy (REMS). SETTING: All drug poisoning deaths in three states: Florida, Oregon, and Washington. Data were obtained through state vital records offices and the Researched Abuse, Diversion and Addiction-Related Surveillance System Medical Examiner Program. METHODS: Using cause-of-death literal text from death certificates, individual opioid active pharmaceutical ingredients (APIs) involved in each death were identified using rules-based natural language processing. Population-adjusted and prescriptions dispensed-adjusted mortality rates were calculated for all ER/LA opioid analgesic and individual opioid APIs. Rates before and after implementation of the REMS were compared. Rate changes were compared with rates from two APIs with little or no inclusion in the REMS: benzodiazepines and hydrocodone. RESULTS: The mean ER/LA opioid analgesic population-adjusted mortality rate significantly decreased in all three states (FL: P = 0.003; OR: P = 0.003; WA: P < 0.001). Mortality rates for benzodiazepines and hydrocodone also decreased and were not statistically different. Significant heterogeneity in mortality rates of individual opioids was observed between the three states. When adjusted for prescription volume, the ER/LA opioid analgesic mortality rate decreased in all three states, but was significant only for Washington (P < 0.001). CONCLUSIONS: The population-adjusted mortality rate of ER/LA opioid analgesics has decreased in three states. Notably, the contributions to mortality rates by individual opioid analgesics were not uniform across the three states in this study. However, these changes were not generally distinct from changes in mortality rates where comparator substances were involved.

LOX out, histones: a new enzyme is nipping at your tails.

In the current issue of Molecular Cell, Herranz et al. (2012) demonstrate that LOXL2 deaminates trimethylated histone 3 lysine 4 (H3K4me3), which uncovers a new chromatin modification and a new enzymatic mechanism with the potential to regulate additional lysine residues.

Histone lysine methylation dynamics: establishment, regulation, and biological impact.

Histone lysine methylation has emerged as a critical player in the regulation of gene expression, cell cycle, genome stability, and nuclear architecture. Over the past decade, a tremendous amount of progress has led to the characterization of methyl modifications and the lysine methyltransferases (KMTs) and lysine demethylases (KDMs) that regulate them. Here, we review the discovery and characterization of the KMTs and KDMs and the methyl modifications they regulate. We discuss the localization of the KMTs and KDMs as well as the distribution of lysine methylation throughout the genome. We highlight how these data have shaped our view of lysine methylation as a key determinant of complex chromatin states. Finally, we discuss the regulation of KMTs and KDMs by proteasomal degradation, posttranscriptional mechanisms, and metabolic status. We propose key questions for the field and highlight areas that we predict will yield exciting discoveries in the years to come.

Nonmedical use of alprazolam in the UK: Results from a nationally representative survey.

There is concern in the UK about nonmedical use (NMU) of alprazolam (Xanax). We investigated the epidemiology of alprazolam NMU compared with diazepam using data from the Survey of Non-Medical Use of Prescription Drugs (NMURx) programme (collected 28 September-1 December 2017). The survey included 10 019 respondents and was weighted by age, sex and region to represent 52 927 659 UK adults. The estimated national prevalence of lifetime NMU of alprazolam was 0.32% (95% confidence interval: 0.19-0.46), and 1.30% (1.06-1.54) for diazepam. The prevalence of NMU in the last 90 days was significantly different when split by age category for alprazolam (P < .001), but not for diazepam (P = .262) with alprazolam NMU being more common among younger adults (age 16-24 years: 0.37%; age 25-34 years: 0.14%; 35 years or older: 0.01%). Further research is needed to fully understand the motivations of alprazolam NMU and to monitor whether the popularity of alprazolam will rise.

Conserved antagonism between JMJD2A/KDM4A and HP1γ during cell cycle progression.

The KDM4/JMJD2 family of histone demethylases is amplified in human cancers. However, little is known about their physiologic or tumorigenic roles. We have identified a conserved and unappreciated role for the JMJD2A/KDM4A H3K9/36 tridemethylase in cell cycle progression. We demonstrate that JMJD2A protein levels are regulated in a cell cycle-dependent manner and that JMJD2A overexpression increased chromatin accessibility, S phase progression, and altered replication timing of specific genomic loci. These phenotypes depended on JMJD2A enzymatic activity. Strikingly, depletion of the only C. elegans homolog, JMJD-2, slowed DNA replication and increased ATR/p53-dependent apoptosis. Importantly, overexpression of HP1γ antagonized JMJD2A-dependent progression through S phase, and depletion of HPL-2 rescued the DNA replication-related phenotypes in jmjd-2(-/-) animals. Our findings describe a highly conserved model whereby JMJD2A regulates DNA replication by antagonizing HP1γ and controlling chromatin accessibility.