Transcription, replication, and repair involve interactions of specific genomic loci with many different proteins. How these interactions are orchestrated at any given location and under changing ...cellular conditions is largely unknown because systematically measuring protein-DNA interactions at a specific locus in the genome is challenging. To address this problem, we developed Epi-Decoder, a Tag-chromatin immunoprecipitation-Barcode-Sequencing (TAG-ChIP-Barcode-Seq) technology in budding yeast. Epi-Decoder is orthogonal to proteomics approaches because it does not rely on mass spectrometry (MS) but instead takes advantage of DNA sequencing. Analysis of the proteome of a transcribed locus proximal to an origin of replication revealed more than 400 interacting proteins. Moreover, replication stress induced changes in local chromatin proteome composition prior to local origin firing, affecting replication proteins as well as transcription proteins. Finally, we show that native genomic loci can be decoded by efficient construction of barcode libraries assisted by clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Thus, Epi-Decoder is an effective strategy to identify and quantify in an unbiased and systematic manner the proteome of an individual genomic locus by DNA sequencing.
Pralatrexate (Folotyn; PLX) and belinostat (Beleodaq; BLS) are registered for the treatment of patients with peripheral T-cell lymphoma (PTCL) and are being considered for other lymphomas. In this ...study we investigated whether BLS had the ability to potentiate the cytotoxicity of PLX. A panel of lymphoma cell lines was used for the combination studies: the B-cell SUDHL-4, SUDHL-5, HT, Jeko-1 and T-cell Karpas-299 and Hut-78. Uptake of PLX was mediated by the reduced folate carrier (RFC). PLX showed a 6-fold better RFC substrate affinity compared to methotrexate, and 2-fold better than levoleucovorin (l-LV). Sensitivity expressed as the concentration that resulted in 50% growth inhibition (IC50) after 72 hr exposure to PLX varied from 2.8 to 20 nM and for BLS from 72 to 233 nM, independent of the background of the cell lines. The interaction between BLS and PLX was studied using the median-drug effect analysis. At a fixed molar ratio between the drugs based on the IC50 concentration the average combination index (CI) for all cell lines showed additivity (CI: around 1.0). In three selected cell lines (SUDHL-4, SUDHL-5, and HT) sequential exposure (24 h pretreatment with BLS, followed by 48 h to PLX + BLS), did not improve interaction (CI: 0.9–1.4). As an alternative approach a non-fixed ratio was used by exposing SUDHL-4, SUDHL-5, and HT cells to IC25 concentrations of either BLS or PLX in combination with the other drug. Exposure to IC25 of PLX did not decrease the IC50 for BLS (CI from 0.6–1.2), but exposure to IC25 of BLS markedly increased PLX sensitivity (low CIs from 0.40 to 0.66). Mechanistic studies focused on induction of apoptosis, and showed cleavage of predominantly caspase-9 in HT and SUDHL-4 cells for both drugs at their IC50s, being similar in the combination setting. Moreover, at these concentrations, the drugs were shown to confer an S-phase arrest. In conclusion, the combination of PLX and BLS showed additivity in various lymphoma cell lines, with a schedule-dependent synergism in B-cell lymphoma. Based on these data, proficient inhibition of HDAC activity by BLS holds promise in sensitization of tumor cells to PLX.
Most tumors lack the G1/S phase checkpoint and are insensitive to antigrowth signals. Loss of G1/S control can severely perturb DNA replication as revealed by slow replication fork progression and ...frequent replication fork stalling. Cancer cells may thus rely on specific pathways that mitigate the deleterious consequences of replication stress. To identify vulnerabilities of cells suffering from replication stress, we performed an shRNA-based genetic screen. We report that the RECQL helicase is specifically essential in replication stress conditions and protects stalled replication forks against MRE11-dependent double strand break (DSB) formation. In line with these findings, knockdown of RECQL in different cancer cells increased the level of DNA DSBs. Thus, RECQL plays a critical role in sustaining DNA synthesis under conditions of replication stress and as such may represent a target for cancer therapy.
This book is the third publication from the Eurogang Network, a cross-national collaboration of researchers (from both North America and Europe) devoted to comparative and multi-national research on ...youth gangs. It provides a unique insight into the influence of migration on local gang formation and development, paying particular attention to the importance of ethnicity. The book also explores the challenges that migration and ethnicity pose for responding effectively to the growth of such gangs, particularly in areas where public discourse on such issues is restricted. Chapters in the book are concerned to address both situations where there have been longstanding problems with street gangs as well as areas where such issues have just started to emerge. A variety of different research traditions and approaches are represented, including ethnographic methods, self-report surveys and interviews, official records data and victim interviews. It will be essential reading for anybody interested in the phenomenon of street and youth gangs.
Liver cancer is one of the most commonly diagnosed cancers and the fourth leading cause of cancer-related death worldwide. Broad-spectrum kinase inhibitors like sorafenib and lenvatinib provide only ...modest survival benefit to patients with hepatocellular carcinoma (HCC). This study aims to identify novel therapeutic strategies for HCC patients.
Integrated bioinformatics analyses and a non-biased CRISPR loss of function genetic screen were performed to identify potential therapeutic targets for HCC cells. Whole-transcriptome sequencing (RNA-Seq) and time-lapse live imaging were performed to explore the mechanisms of the synergy between CDC7 inhibition and ATR or CHK1 inhibitors in HCC cells. Multiple in vitro and in vivo assays were used to validate the synergistic effects.
Through integrated bioinformatics analyses using the Cancer Dependency Map and the TCGA database, we identified ATR-CHK1 signaling as a therapeutic target for liver cancer. Pharmacological inhibition of ATR or CHK1 leads to robust proliferation inhibition in liver cancer cells having a high basal level of replication stress. For liver cancer cells that are resistant to ATR or CHK1 inhibition, treatment with CDC7 inhibitors induces strong DNA replication stress and consequently such drugs show striking synergy with ATR or CHK1 inhibitors. The synergy between ATR-CHK1 inhibition and CDC7 inhibition probably derives from abnormalities in mitosis inducing mitotic catastrophe.
Our data highlights the potential of targeting ATR-CHK1 signaling, either alone or in combination with CDC7 inhibition, for the treatment of liver cancer.
Kickboksen 2.0 van Gemert, Frank
Justitiële verkenningen,
05/2023, Volume:
49, Issue:
1
Journal Article
Kickboxing 2.0. On the positive relaunch of a martial art Kickboxing has undergone a remarkable transformation in the Netherlands in recent decades. The maligned full-contact combat sport with links ...to the criminal underworld changed into a mainstream activity serving a broad market. This article outlines the background against which this change took place and mentions three levels: national and international society (macro), the gym and the criminal scene (meso), and the fighter and the consumer (micro). In addition to being a martial art, kickboxing is looked upon as a commodity that is put on the market and consumed.
Research Summary
To reduce individual and social harms, most nations prohibit certain psychoactive drugs. Yet, prior scholarship has suggested that prohibition reduces illicit drug sellers’ access to ...law and thereby increases predation against and retaliation by them. No prior study, however, has directly tested that theory by comparing drug sellers of different legal statuses operating in a single place and time. This study analyzes rates of victimization, legal mobilization, and violent retaliation in three retail drug markets in Amsterdam, the Netherlands: the legally regulated alcohol trade of cafés, the decriminalized cannabis market of “coffeeshops,” and the illegal street drug market. Results from interviews conducted with 50 sellers in each market indicate, as expected, that illicit drug dealers have the highest rates of victimization and violent retaliation and the lowest rates of legal mobilization. Contrary to expectations, we find coffeeshops experience less victimization than cafés and have similar rates of violent retaliation and legal mobilization.
Policy Implications
Our findings suggest that state regulation of drug markets affects victimization and conflict management of sellers, but the relationship does not seem to be linear. Prohibition undercuts the state's regulatory capacity by producing zones of virtual statelessness in which formal means of dispute resolution are unavailable, and thus, victimization and retaliation are more common. At the other extreme is laissez faire regulation, which may make sellers more likely to address problems only after they occur (instead of preventing their occurrence). The Dutch government originally instituted coffeeshops as a harm‐reduction method meant to separate the market for cannabis from that of hard drugs. The policy also seems to work well when it comes to reducing victimization, perhaps by encouraging the use of preventive measures by coffeeshop owners and employees. The Dutch experience offers lessons for drug policy reforms elsewhere.
Abstract
Standard induction chemotherapy, consisting of an anthracycline and cytarabine, has been the first-line therapeutic to treat acute myeloid leukemia (AML). Although this treatment induces ...complete remissions (CR) in the majority of patients, many face a relapse. Relapse is caused by the survival of chemotherapy-resistant leukemia cells (minimal residual disease, MRD), that may have acquired drug resistance by non-genetic mechanisms. Persistence of MRD is driven by intra-leukemic heterogeneity and plasticity in response to therapy. Leukemia cells within MRD capable of initiating relapse are thought to have stem cell features and are therefore named “leukemic stem cells” (LSCs). In this study, we demonstrated that the anthracycline doxorubicin epigenetically reprograms leukemia cells by inducing tri-methylation of histone 3 lysine 27 (H3K27) and H3K4. Moreover, to understand chemotherapy resistance at the single-cell level and to study intra-leukemia heterogeneity with respect to anthracycline sensitivity, we seeded K562 myeloid leukemia cells in 480 wells, each containing 10.000 cells, and treated the cells with increasing concentrations of doxorubicin. Using this approach, a subpopulation of reversible anthracycline-tolerant cells (ATCs) was identified within the doxorubicin-sensitive leukemia cell population, which lacked upregulation of H3K27me3 or H3K4me3 and exhibited epigenetically regulated expression of stem cell features similar to leukemic stem cells (LSCs). These ATCs have a distinct transcriptional landscape from the leukemia bulk and could be eradicated by the KDM6 inhibitor GSK-J4. In primary human AML, GSK-J4 treatment could eliminate AML blasts and LSCs at diagnosis but importantly it could significantly reduce chemotherapy resistant AML patient cell load (MRD) and survival of LSCs residing within MRD, ex vivo and in vivo. An efficient response to GSK-J4 was associated with upregulation of H3K27 and/or H3K4 methylation, and downregulation of STAT5B. Together, our results reveal plasticity of anthracycline resistance in AML cells and highlight the potential of epigenetic-based therapeutics to target chemotherapy-resistant AML cells.
Citation Format: Noortje van Gils, Han J.M.P. Verhagen, Tània Martiáñez, Fedor Denkers, Eline Vermue, Arjo Rutten, Tamás Csikós, Meyrem Çil, Marjon Al, Frank van Gemert, Jeroen J.W.M. Janssen, Gert J. Ossenkoppele, Renee X. Menezes, Linda Smit. Targeting histone methylation to overrule transcriptionally driven drug resistance in myeloid leukemia abstract. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-125.
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