Browsing by Subject "Epigenetics"
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Item A phase 1 study of combined guadecitabine and cisplatin in platinum refractory germ cell cancer(Wiley, 2021) Albany, Costantine; Fazal, Zeeshan; Singh, Ratnakar; Bikorimana, Emmanuel; Adra, Nabil; Hanna, Nasser H.; Einhorn, Lawrence H.; Perkins, Susan M.; Sandusky, George E.; Christensen, Brock C.; Keer, Harold; Fang, Fang; Nephew, Kenneth P.; Spinella, Michael J.; Medicine, School of MedicinePurpose: Germ cell tumors (GCTs) are cured with therapy based on cisplatin, although a clinically significant number of patients are refractory and die of progressive disease. Based on preclinical studies indicating that refractory testicular GCTs are hypersensitive to hypomethylating agents (HMAs), we conducted a phase I trial combining the next-generation HMA guadecitabine (SGI-110) with cisplatin in recurrent, cisplatin-resistant GCT patients. Methods: Patients with metastatic GCTs were treated for five consecutive days with guadecitabine followed by cisplatin on day 8, for a 28-day cycle for up to six cycles. The primary endpoint was safety and toxicity including dose-limiting toxicity (DLT) and maximum tolerated dose (MTD). Results: The number of patients enrolled was 14. The majority of patients were heavily pretreated. MTD was determined to be 30 mg/m2 guadecitabine followed by 100 mg/m2 cisplatin. The major DLTs were neutropenia and thrombocytopenia. Three patients had partial responses by RECIST criteria, two of these patients, including one with primary mediastinal disease, completed the study and qualified as complete responses by serum tumor marker criteria with sustained remissions of 5 and 13 months and survival of 16 and 26 months, respectively. The overall response rate was 23%. Three patients also had stable disease indicating a clinical benefit rate of 46%. Conclusions: The combination of guadecitabine and cisplatin was tolerable and demonstrated activity in patients with platinum refractory germ cell cancer.Item Activation of AKT induces EZH2-mediated β-catenin trimethylation in colorectal cancer(Elsevier, 2023-08-16) Ghobashi, Ahmed H.; Vuong, Truc T.; Kimani, Jane W.; Ladaika, Christopher A.; Hollenhorst, Peter C.; O’Hagan, Heather M.; Biochemistry and Molecular Biology, School of MedicineColorectal cancer (CRC) develops in part through the deregulation of different signaling pathways, including activation of the WNT/β-catenin and PI3K/AKT pathways. Additionally, the lysine methyltransferase enhancer of zeste homologue 2 (EZH2) is commonly overexpressed in CRC. EZH2 canonically represses gene transcription by trimethylating lysine 27 of histone H3, but also has non-histone substrates. Here, we demonstrated that in CRC, active AKT phosphorylated EZH2 on serine 21. Phosphorylation of EZH2 by AKT induced EZH2 to interact with and methylate β-catenin at lysine 49, which increased β-catenin’s binding to the chromatin. Additionally, EZH2-mediated β-catenin trimethylation induced β-catenin to interact with TCF1 and RNA polymerase II and resulted in dramatic gains in genomic regions with β-catenin occupancy. EZH2 catalytic inhibition decreased stemness but increased migratory phenotypes of CRC cells with active AKT. Overall, we demonstrated that EZH2 modulates AKT-induced changes in gene expression through the AKT/EZH2/β-catenin axis in CRC.Item Analysis of differentiation capacity of Cfp1 null embyronic stem cells(2014) Bowen, Tamara R.; Skalnik, David Gordon; Marrs, James; Chang, Hua-ChenEpigenetics is defined as “the study of stable, often heritable, changes that influence gene expression that are not mediated by DNA sequence” (Fingerman et al., 2013). Epigenetic marks such as covalent histone modifications and DNA methylation are important for maintaining chromatin structure and epigenetic inheritance. Several proteins have been found to bind and/ or regulate epigenetic marks. One such protein, CXXC finger protein 1 (Cfp1) is an important chromatin regulator that binds to unmethylated CpG islands. It has been found to be essential for mammalian development. Mice lacking Cfp1 exhibit an embryonic- lethal phenotype. However, the function of Cfp1 can be studied using Cfp1 Null mouse ES cells, which are viable. Thus far, Cfp1 has been shown to be important for cell growth, cytosine methylation, histone modifications, subnuclear localization of Set1A histone H3K4 methyltransferase, and cellular differentiation. When Cfp1 Null ES cells are induced to differentiate by removal of Leukemia Inhibitory Factor (LIF), the cells are not able to turn off pluripotency markers such as Oct4 and alkaline phosphatase and fail to express differentiation markers such as Gata4 and Brachyury. In this study, we used established protocols to further examine the differentiation capacity of Cfp1 Null cells. Specifically, we tested the ability of Cfp1 Null ES cells to retain stem cell properties in the absence of LIF, differentiate into cardiomyocytes in the presence of TGF-β2 and differentiate into neuron precursors in the presence of retinoic acid (RA). While the differentiation effects of RA were inconclusive, Null cells were able to start differentiating in the absence of LIF, either as individual cells or EBs, and the presence of TGF-β2 when seeded on gelatin coated tissue culture dishes. However, no difference was seen between cells treated without LIF and those treated with TGF-β2. In both conditions, only a small portion of cells were able to differentiate, while the majority of the cell population retained stem cell characteristics. Cell growth and the differentiation capacity of Cfp1 Null cells were also compromised in comparison to WT cells. Thus, further supporting the need for the correct epigenetic patterns maintained by Cfp1 during cellular differentiation.Item Asparagine starvation suppresses histone demethylation through iron depletion(Elsevier, 2023-03-16) Jiang, Jie; Srivastava, Sankalp; Liu, Sheng; Seim, Gretchen; Claude, Rodney; Zhong, Minghua; Cao, Sha; Davé, Utpal; Kapur, Reuben; Mosley, Amber L.; Zhang, Chi; Wan, Jun; Fan, Jing; Zhang, Ji; Pediatrics, School of MedicineIntracellular α-ketoglutarate is an indispensable substrate for the Jumonji family of histone demethylases (JHDMs) mediating most of the histone demethylation reactions. Since α-ketoglutarate is an intermediate of the tricarboxylic acid cycle and a product of transamination, its availability is governed by the metabolism of several amino acids. Here, we show that asparagine starvation suppresses global histone demethylation. This process is neither due to the change of expression of histone-modifying enzymes nor due to the change of intracellular levels of α-ketoglutarate. Rather, asparagine starvation reduces the intracellular pool of labile iron, a key co-factor for the JHDMs to function. Mechanistically, asparagine starvation suppresses the expression of the transferrin receptor to limit iron uptake. Furthermore, iron supplementation to the culture medium restores histone demethylation and alters gene expression to accelerate cell death upon asparagine depletion. These results suggest that suppressing iron-dependent histone demethylation is part of the cellular adaptive response to asparagine starvation.Item Association of peripheral blood DNA methylation level with Alzheimer’s disease progression(BMC, 2021-10-15) Li, Qingqin S.; Vasanthakumar, Aparna; Davis, Justin W.; Idler, Kenneth B.; Nho, Kwangsik; Waring, Jeffrey F.; Saykin, Andrew J.; Radiology and Imaging Sciences, School of MedicineBackground: Identifying biomarkers associated with Alzheimer's disease (AD) progression may enable patient enrichment and improve clinical trial designs. Epigenome-wide association studies have revealed correlations between DNA methylation at cytosine-phosphate-guanine (CpG) sites and AD pathology and diagnosis. Here, we report relationships between peripheral blood DNA methylation profiles measured using Infinium® MethylationEPIC BeadChip and AD progression in participants from the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort. Results: The rate of cognitive decline from initial DNA sampling visit to subsequent visits was estimated by the slopes of the modified Preclinical Alzheimer Cognitive Composite (mPACC; mPACCdigit and mPACCtrailsB) and Clinical Dementia Rating Scale Sum of Boxes (CDR-SB) plots using robust linear regression in cognitively normal (CN) participants and patients with mild cognitive impairment (MCI), respectively. In addition, diagnosis conversion status was assessed using a dichotomized endpoint. Two CpG sites were significantly associated with the slope of mPACC in CN participants (P < 5.79 × 10-8 [Bonferroni correction threshold]); cg00386386 was associated with the slope of mPACCdigit, and cg09422696 annotated to RP11-661A12.5 was associated with the slope of CDR-SB. No significant CpG sites associated with diagnosis conversion status were identified. Genes involved in cognition and learning were enriched. A total of 19, 13, and 5 differentially methylated regions (DMRs) associated with the slopes of mPACCtrailsB, mPACCdigit, and CDR-SB, respectively, were identified by both comb-p and DMRcate algorithms; these included DMRs annotated to HOXA4. Furthermore, 5 and 19 DMRs were associated with conversion status in CN and MCI participants, respectively. The most significant DMR was annotated to the AD-associated gene PM20D1 (chr1: 205,818,956 to 205,820,014 [13 probes], Sidak-corrected P = 7.74 × 10-24), which was associated with both the slope of CDR-SB and the MCI conversion status. Conclusion: Candidate CpG sites and regions in peripheral blood were identified as associated with the rate of cognitive decline in participants in the ADNI cohort. While we did not identify a single CpG site with sufficient clinical utility to be used by itself due to the observed effect size, a biosignature composed of DNA methylation changes may have utility as a prognostic biomarker for AD progression.Item Bromodomains in Protozoan Parasites: Evolution, Function, and Opportunities for Drug Development(American Society for Microbiology, 2017-01-11) Jeffers, Victoria; Yang, Chunlin; Huang, Sherri; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of MedicineParasitic infections remain one of the most pressing global health concerns of our day, affecting billions of people and producing unsustainable economic burdens. The rise of drug-resistant parasites has created an urgent need to study their biology in hopes of uncovering new potential drug targets. It has been established that disrupting gene expression by interfering with lysine acetylation is detrimental to survival of apicomplexan (Toxoplasma gondii and Plasmodium spp.) and kinetoplastid (Leishmania spp. and Trypanosoma spp.) parasites. As "readers" of lysine acetylation, bromodomain proteins have emerged as key gene expression regulators and a promising new class of drug target. Here we review recent studies that demonstrate the essential roles played by bromodomain-containing proteins in parasite viability, invasion, and stage switching and present work showing the efficacy of bromodomain inhibitors as novel antiparasitic agents. In addition, we performed a phylogenetic analysis of bromodomain proteins in representative pathogens, some of which possess unique features that may be specific to parasite processes and useful in future drug development.Item Canonical histone H2Ba and H2A.X dimerize in an opposite genomic localization to H2A.Z/H2B.Z dimers in Toxoplasma gondii(Elsevier, 2014-10) Bogado, Silvina S.; Dalmasso, Carolina; Ganuza, Agustina; Kim, Kami; Sullivan, William J., Jr.; Angel, Sergio O.; Vanagas, Laura; Department of Pharmacology and Toxicology, IU School of MedicineHistone H2Ba of Toxoplasma gondii was expressed as recombinant protein (rH2Ba) and used to generate antibody in mouse that is highly specific. Antibody recognizing rH2Ba detects a single band in tachyzoite lysate of the expected molecular weight (12kDa). By indirect immunofluorescence (IFA) in in vitro grown tachyzoites and bradyzoites, the signal was detected only in the parasite nucleus. The nucleosome composition of H2Ba was determined through co-immunoprecipitation assays. H2Ba was detected in the same immunocomplex as H2A.X, but not with H2A.Z. Through chromatin immunoprecipitation (ChIP) assays and qPCR, it was observed that H2Ba is preferentially located at promoters of inactive genes and silent regions, accompanying H2A.X and opposed to H2A.Z/H2B.Z dimers.Item CFP1 governs uterine epigenetic landscapes to intervene in progesterone responses for uterine physiology and suppression of endometriosis(Springer Nature, 2023-06-03) Yang, Seung Chel; Park, Mira; Hong, Kwon-Ho; La, Hyeonwoo; Park, Chanhyeok; Wang, Peike; Li, Gaizhen; Chen, Qionghua; Choi, Youngsok; DeMayo, Francesco J.; Lydon, John P.; Skalnik, David G.; Lim, Hyunjung J.; Hong, Seok-Ho; Park, So Hee; Kim, Yeon Sun; Kim, Hye-Ryun; Song, Haengseok; Biology, School of ScienceProgesterone (P4) is required for the preparation of the endometrium for a successful pregnancy. P4 resistance is a leading cause of the pathogenesis of endometrial disorders like endometriosis, often leading to infertility; however, the underlying epigenetic cause remains unclear. Here we demonstrate that CFP1, a regulator of H3K4me3, is required for maintaining epigenetic landscapes of P4-progesterone receptor (PGR) signaling networks in the mouse uterus. Cfp1f/f;Pgr-Cre (Cfp1d/d) mice showed impaired P4 responses, leading to complete failure of embryo implantation. mRNA and chromatin immunoprecipitation sequencing analyses showed that CFP1 regulates uterine mRNA profiles not only in H3K4me3-dependent but also in H3K4me3-independent manners. CFP1 directly regulates important P4 response genes, including Gata2, Sox17, and Ihh, which activate smoothened signaling pathway in the uterus. In a mouse model of endometriosis, Cfp1d/d ectopic lesions showed P4 resistance, which was rescued by a smoothened agonist. In human endometriosis, CFP1 was significantly downregulated, and expression levels between CFP1 and these P4 targets are positively related regardless of PGR levels. In brief, our study provides that CFP1 intervenes in the P4-epigenome-transcriptome networks for uterine receptivity for embryo implantation and the pathogenesis of endometriosis.Item Characterization of protein arginine methyltransferase of TgPRMT5 in Toxoplasma gondii(Springer Nature, 2019-05-08) Liu, Min; Li, Fen-Xiang; Li, Chun-Yuan; Li, Xiao-Cong; Chen, Long-Fei; Wu, Kun; Yang, Pei-Liang; Lai, Zhi-Fa; Liu, Ting-kai; Sullivan, William J., Jr.; Cui, Liwang; Chen, Xiao-Guang; Pharmacology and Toxicology, School of MedicineBACKGROUND: Protein arginine methylation is a prevalent post-translational modification. The protein arginine methyltransferase family (PRMT) is involved in many cellular processes in eukaryotes, including transcriptional regulation, epigenetic regulation, RNA metabolism, and DNA damage repair. Toxoplasma gondii, an opportunistic protozoan parasite, encodes five conserved PRMTs. PRMT5 is thought to be responsible for substantial PRMT activity in T. gondii; however, it has not yet been characterized. METHODS: We tagged the 3' end of the endogenous TgPRMT5 genomic locus with sequence encoding a 3X hemagglutinin (HA) epitope. IFA and WB were performed to check the expression and subcellular localization of TgPRMT5 in tachyzoites and bradyzoites. In vitro methylation assays were performed to determine whether endogenous TgPRMT5 has arginine methyltransferase activity. RESULTS: IFA and WB results showed that T. gondii PRMT5 (TgPRMT5) was localized in the cytoplasm in the tachyzoite stage; however, it shifts largely to the nuclear compartment in the bradyzoite stage. The in vitro methylation showed that TgPRMT5 has authentic type II PRMT activity and forms monomethylarginines and symmetric dimethylarginines. CONCLUSIONS: We determined the expression and cellular localization of TgPRMT5 in tachyzoites and bradyzoites and confirmed its type II PRMT activity. We demonstrated the major changes in expression and cellular localization of TgPRMT5 during the tachyzoite and bradyzoite stages in T. gondii. Our findings suggest that TgPRMT5 protein may be involved in tachyzoite-bradyzoite transformation.Item Combinatorial Inhibition of Epigenetic Regulators to Treat Glioblastoma(2022-07-29) Burket, Noah; Koenig, Jenna; Saratsis, AmandaGlioblastoma (GBM) is a deadly primary brain cancer that affects 12,000 patients in the US annually with a median survival time of 15 months. Temozolomide is the standard-of-care chemotherapy for GBM; however, many tumors are resistant, necessitating the expansion of therapeutic options. EZH2 and JMJD3 are two proteins responsible for epigenetic regulation of the genome via histone methylation, with EZH2 also affecting non-histone targets. Prior studies showed that inhibition of these proteins decreased cell counts and induced radiosensitivity in GBM cells. Thus, we investigated combined use of EZH2 inhibitor, EPZ6438, and JMJD3 inhibitor, GSK-J4, in the treatment of temozolomide-resistant GBM10 cells. Non-irradiated cells were treated with both drugs singly and combined, and counted at 24-, 48-, and 72-hour intervals. Irradiated cells were pre-treated with each drug and combination therapy for three days, irradiated, and then counted at 24-, 48-, and 72-hour intervals. Western blot was used to investigate dsDNA damage biomarker y-H2AX, gene-silencing modification H3K27me3, tumor suppressor p53, EZH2, and JMJD3 expression in non-irradiated and irradiated cells following drug treatment. Single EPZ-6438 and GSK-J4 treatments reduced cell counts with increasing concentration and time. GSK-J4 appears to reduce cell counts more than EPZ-6438 alone, and combinatorial use reduces this further. Western blot reveals increased H3K27me3 expression with GSK-J4 treatment following radiation, but not with EPZ-6438. y-H2AX expression is increased after EPZ-6438 treatment but is not further increased with radiation. Meanwhile, GSK-J4 increased y-H2AX, but only after irradiation. Reduced cell counts following treatment with GSK-J4 may be due to its effects on gene silencing from inhibition of H3K27 demethylation. Additionally, increased dsDNA breaks seen in EPZ-6438 and GSK-J4 supports their roles in radiosensitizing GBM cells. This study highlights the importance of further investigation into GSK-J4 and EPZ-6438 combination therapy in temozolomide-resistant GBM tumors.