DNA methylation can mediate epigenetic silencing of tumor suppressor and cancer protective genes. The protein ubiquitin-like containing PHD and ring finger domains 1 (UHRF1) is an essential component in cells for DNA methylation maintenance. The SET- and RING-associated (SRA) domain of UHRF1 can bind hemimethylated DNA, and mediate recruitment of DNA methyltransferases to copy the methylation pattern to the newly synthesized daughter strand. Loss of UHRF1 function can lead to demethylation and re-expression of epigenetically silenced tumor suppressor genes and can reduce cancer cell growth and survival. We created a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay to screen for inhibitors capable of disrupting the interaction between the UHRF1-SRA domain and hemimethylated DNA. Using this assay (Z’ factor of 0.74 in 384-well format) we screened the Library of Pharmacologically Active Compounds (LOPAC) for UHRF1-SRA inhibitors, and validated 7 hit compounds. These compounds included the anthracycline derivatives idarubicin and mitoxantrone, which are commonly used chemotherapeutic drugs known to mediate cytotoxicity by acting as class II topoisomerase (TOP2) poisons. In a panel of additional known topoisomerase poisons, only the anthracycline derivatives showed dose responsive inhibition of UHRF1-SRA. Additionally, mitoxantrone and doxorubicin showed dose-responsive global DNA demethylation and demonstrated a synergistic growth inhibition of multiple cancer cell lines when combined with the DNA methyltransferase (DNMT) inhibitor decitabine. These data validate a novel TR-FRET assay for identification of UHRF1 inhibitors, and revealed unexpected epigenetic properties of commonly used chemotherapeutic drugs that showed synergistic cytotoxicity of cancer cells when combined with a demethylating agent.
Background Resistance to androgen deprivation therapies is a major driver of mortality in advanced prostate cancer. Therefore, there is a need to develop new preclinical models that allow the investigation of resistance mechanisms and the assessment of drugs for the treatment of castration‐resistant prostate cancer. Methods We generated two novel cell line models (LAPC4‐CR and VCaP‐CR) which were derived by passaging LAPC4 and VCaP cells in vivo and in vitro under castrate conditions. We performed detailed transcriptomic (RNA‐seq) and proteomic analyses (SWATH‐MS) to delineate expression differences between castration‐sensitive and castration‐resistant cell lines. Furthermore, we characterized the in vivo and in vitro growth characteristics of these novel cell line models. Results The two cell line derivatives LAPC4‐CR and VCaP‐CR showed castration‐resistant growth in vitro and in vivo which was only minimally inhibited by AR antagonists, enzalutamide, and bicalutamide. High‐dose androgen treatment resulted in significant growth arrest of VCaP‐CR but not in LAPC4‐CR cells. Both cell lines maintained AR expression, but exhibited distinct expression changes on the mRNA and protein level. Integrated analyses including data from LNCaP and the previously described castration‐resistant LNCaP‐abl cells revealed an expression signature of castration resistance. Conclusions The two novel cell line models LAPC4‐CR and VCaP‐CR and their comprehensive characterization on the RNA and protein level represent important resources to study the molecular mechanisms of castration resistance.
In order to prescribe an antibiotic, a physician must go through a series of decision-making processes that involve both the drug and the host. In this review article, we outline exactly what those decision-making processes are and some of their limitations. Before a medication can be prescribed, a physician has to determine if the antibiotic works against the host pathogen. To do this, basic science techniques are employed including phenotypic methods such as broth dilution methods, Kirby-Bauer susceptibility testing, Epsilometer test (E-test), and genotypic methods such as the new and upcoming automated tests. After determining if a drug has potential to work, the physician must consider the drug’s mechanism of action in order to determine a dosing regimen. Some groups of drugs should be administered at high concentrations infrequently, others should be given more frequently in smaller doses, and others lie somewhere between this spectrum. Finally, external factors such as the patient's age, especially for pediatrics and geriatrics patients, need to be considered, as these groups have the highest health care burden but are among the most vulnerable when it comes to the side effects of drugs.
BACKGROUND: Frequent neurological examinations in patients with traumatic brain injury (TBI) disrupt sleep-wake cycles and potentially contribute to the development of delirium. OBJECTIVE: To evaluate the risk of delirium among patients with TBI with respect to their neuro-check frequencies. METHODS: A retrospective study of patients presenting with TBI at a single level I trauma center between January 2018 and December 2019. The primary exposure was the frequency of neurological examinations (neuro-checks) assigned at the time of admission. Patients admitted with hourly (Q1) neuro-check frequencies were compared with those who received examinations every 2 (Q2) or 4 (Q4) hours. The primary outcomes were delirium and time-to-delirium. The onset of delirium was defined as the first documented positive Confusion Assessment Method for the Intensive Care Unit score. RESULTS: Of 1552 patients with TBI, 458 (29.5%) patients experienced delirium during their hospital stay. The median time-to-delirium was 1.8 days (IQR: 1.1, 2.9). Kaplan-Meier analysis demonstrated that patients assigned Q1 neuro-checks had the greatest rate of delirium compared with the patients with Q2 and Q4 neuro-checks (P < .001). Multivariable Cox regression modeling demonstrated that Q2 neuro-checks (hazard ratio: 0.439, 95% CI: 0.33-0.58) and Q4 neuro-checks (hazard ratio: 0.48, 95% CI: 0.34-0.68) were protective against the development of delirium compared with Q1. Other risk factors for developing delirium included pre-existing dementia, tobacco use, lower Glasgow Coma Scale score, higher injury severity score, and certain hemorrhage patterns. CONCLUSION: Patients with more frequent neuro-checks had a higher risk of developing delirium compared with those with less frequent neuro-checks.
DNA hypermethylation can trigger silencing of tumor suppressor genes during cancer development and progression, partly through binding by methylated-DNA binding proteins (MBD), such as MBD2, that function as “epigenetic readers” and recruit co-repressor complexes to promote gene repression. Inhibiting MBD2-mediated repression represents an attractive cancer therapeutic strategy. Here, we used a cell-based screen to identify small molecules capable of reactivating hypermethylated promoter sequences. We used biochemical, molecular biologic, and pharmacologic approaches to characterize mechanism of action of identified MBD2 inhibitors. A subset of these compounds represent a new class of inhibitors capable of selectively antagonizing interactions between MBD2 and methylated DNA, leading to reactivation of the hypermethylated gene GSTP1 and the epigenetically silenced retinoic acid signaling pathway. Combinations of one of the newly identified MBD2 inhibitors, KCC-08, with the retinoic acid receptor agonist, isotretinoin, significantly reduced cancer cell growth/survival in vitro and in vivo. These novel MBD2 inhibitors are thus positioned for further pharmacologic lead development for use as probes to interrogate epigenetic gene silencing mechanisms and as cancer therapeutics. Citation Format: Hugh Giovinazzo, Zachary R. Reichert, Andries Bergman, Xiaohui Lin, Nicolas Wyhs, David Esopi, Ajay Vaghasia, Jianyong Liu, Yash Jain, Akshay Bhamidipati, Ruchama Steinberg, Traci Speed, Matthew Vaughn, Yonggang Zhang, Nate Brennen, Theodore Deweese, Srinivasan Yegnasubramanian, William G. Nelson. Novel inhibitors of the epigenetic reader protein MBD2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5881.
e17037 Background: The poly(ADP-ribose) polymerase inhibitors (PARPi) olaparib and rucaparib have been approved for the treatment of metastatic castration resistant prostate cancer (mCRPC) in the setting of homologous recombination deficiency (HRD). Additionally, PARPi have been shown to modulate androgen receptor (AR) signaling, with a recent report demonstrating mono-ADP-ribosylation of cysteine residues in AR by Parp7. Here, we further evaluated the effect of PARPi on AR activity and localization. Methods: The effect of PARPi on cell growth and survival of CRPC cell lines was evaluated in vitro and in vivo. AR ribosylation was assessed in CRPC cell lines by immunoprecipitation (IP) assays and proximity ligation assays (PLA). The subcellular localization of AR was determined by quantitative immunofluorescence microscopy in CRPC cell lines and xenograft models. Changes in AR activity with PARPi treatment were evaluated by a luciferase reporter assay and AR target gene expression in a PDX model. Finally, PARPi mediated alteration in the AR protein interactome was evaluated by liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics. Results: The PARPi olaparib and talazoparib, and to a lesser extent veliparib, inhibited CRPC cell growth. Evidence of AR ribosylation was seen by IP and PLA. PARPi treatment of multiple in vitro and in vivo prostate cancer models resulted in a shift of AR subcellular localization, from predominantly nuclear to cytoplasmic compartments. In luciferase reporter assays, AR transactivation activity was decreased after PARPi treatment in a dose dependent manner. In vivo, in prostate cancer xenograft models, decreased AR target gene expression was seen upon PARPi treatment. LC-MS/MS proteomic studies revealed that PARP inhibition resulted in significant changes in the composition of AR interaction partners, in particular of proteins related to intracellular trafficking and nuclear transport. This suggests a potential link between altered AR complex assembly and the observed changes in AR subcellular localization in the context of PARPi treatment. Conclusions: We describe a novel sequela of PARPi therapy to alter AR localization and activity in CRPC. Single agent PARP inhibition can alter prostate cancer cell growth in vitro and in vivo. With PARPi treatment, AR localization is shifted to the cytoplasm, the AR interactome is altered, and AR transcriptional activity decreased. These findings implicate a collateral mechanism of PARPi in preventing prostate cancer cell growth/survival that may augment previously described mechanisms related to HDR and synthetic lethality. It is essential to understand the role of PARPi beyond synthetic lethality in the context of HRD in order to better define the spectrum of response to PARPi across patients, and for development of biology-informed combination therapies.
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