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1. EZH2 is a key component of hepatoblastoma tumor cell growth

   https://doi.org/10.1002/pbc.30774  

   Glaser, Kathryn ; Schepers, Emily J. ; Zwolshen, Harrison M. ; Lake, Charissa M. ; Timchenko, Nikolai A. ; Karns, Rebekah A. ; Cairo, Stefano ; Geller, James I. ; Tiao, Gregory M. ; Bondoc, Alexander J. ( November 2023 , Pediatric Blood & Cancer)

   Enhancer of zeste homolog 2 (EZH2) catalyzes the trimethylation of histone H3 at lysine 27 via the polycomb recessive complex 2 (PRC2) and plays a time‐specific role in normal fetal liver development. EZH2 is overexpressed in hepatoblastoma (HB), an embryonal tumor. EZH2 can also promote tumorigenesis via a noncanonical, PRC2‐independent mechanism via proto‐oncogenic, direct protein interaction, including β‐catenin. We hypothesize that the pathological activation of EZH2 contributes to HB propagation in a PRC2‐independent manner.

   We demonstrate that EZH2 promotes proliferation in HB tumor‐derived cell lines through interaction with β‐catenin. Although aberrant EZH2 expression occurs, we determine that both canonical and noncanonical EZH2 signaling occurs based on specific gene‐expression patterns and interaction with SUZ12, a PRC2 component, and β‐catenin. Silencing and inhibition of EZH2 reduce primary HB cell proliferation.

   EZH2 overexpression promotes HB cell proliferation, with both canonical and noncanonical function detected. However, because EZH2 directly interacts with β‐catenin in human tumors and EZH2 overexpression is not equal to SUZ12, it seems that a noncanonical mechanism is contributing to HB pathogenesis. Further mechanistic studies are necessary to elucidate potential pathogenic downstream mechanisms and translational potential of EZH2 inhibitors for the treatment of HB.

    

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2. Ref-1 redox activity alters cancer cell metabolism in pancreatic cancer: exploiting this novel finding as a potential target

   https://doi.org/10.1186/s13046-021-02046-x  

   Gampala, Silpa ; Shah, Fenil ; Lu, Xiaoyu ; Moon, Hye-ran ; Babb, Olivia ; Umesh Ganesh, Nikkitha ; Sandusky, George ; Hulsey, Emily ; Armstrong, Lee ; Mosely, Amber L. ; et al ( December 2021 , Journal of Experimental & Clinical Cancer Research)

   Abstract Background Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy. Redox factor-1 (Ref-1), a redox signaling protein, regulates the conversion of several transcription factors (TFs), including HIF-1α, STAT3 and NFκB from an oxidized to reduced state leading to enhancement of their DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia. Methods scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model and validated using proteomics and qRT-PCR. The identified Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays, qRT-PCR, western blotting and NADP assay. Further, the effect of Ref-1 redox function inhibition against pancreatic cancer metabolism was assayed using 3D co-culture in vitro and xenograft studies in vivo. Results Distinct transcriptional variation in central metabolism, cell cycle, apoptosis, immune response, and genes downstream of a series of signaling pathways and transcriptional regulatory factors were identified in Ref-1 knockdown vs Scrambled control from the scRNA-seq data. Mitochondrial DEG subsets downregulated with Ref-1 knockdown were significantly reduced following Ref-1 redox inhibition and more dramatically in combination with Devimistat in vitro. Mitochondrial function assays demonstrated that Ref-1 knockdown and Ref-1 redox signaling inhibition decreased utilization of TCA cycle substrates and slowed the growth of pancreatic cancer co-culture spheroids. In Ref-1 knockdown cells, a higher flux rate of NADP + consuming reactions was observed suggesting the less availability of NADP + and a higher level of oxidative stress in these cells. In vivo xenograft studies demonstrated that tumor reduction was potent with Ref-1 redox inhibitor similar to Devimistat. Conclusion Ref-1 redox signaling inhibition conclusively alters cancer cell metabolism by causing TCA cycle dysfunction while also reducing the pancreatic tumor growth in vitro as well as in vivo. 

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3. Artesunate inhibits myofibroblast formation via induction of apoptosis and antagonism of pro‐fibrotic gene expression in human dermal fibroblasts

   https://doi.org/10.1002/cbin.11220  

   Larson, Sara A. ; Dolivo, David M. ; Dominko, Tanja ( August 2019 , Cell Biology International)

   The anti‐malaria drug artesunate and other chemical analogs of artemisinin have demonstrated cytostatic and cytotoxic effects in bacterial and cancer cells. Artemisinin‐derived compounds have also been demonstrated to attenuate fibrosis in preclinical animal models, but the mechanisms by which this inhibition occurs are not well‐understood. We investigated the effects of artesunate on the emergence of the myofibroblast, which is causally implicated in pro‐fibrotic pathologies. CRL‐2097 human dermal fibroblasts were analyzed for protein and transcript expression after treatment with artesunate to analyze fibroblast activation. Proliferation and apoptosis were also evaluated following treatment with artesunate in this cell line. Treatment of human dermal fibroblasts with artesunate antagonized fibroblast activation and pro‐fibrotic extracellular matrix (ECM) deposition, both at basal culture conditions and when cultured in the presence of exogenous transforming growth factor‐β1 (TGF‐β1), a major pro‐fibrotic cytokine. Artesunate‐treated fibroblasts also demonstrated decreased proliferation and increased apoptosis. Transcript analysis by quantitative real‐time polymerase chain reaction demonstrated that artesunate downregulated expression of pro‐fibrotic genes including canonical myofibroblast markers, ECM genes, and several TGF‐β receptors and ligands, and upregulated expression of cell cycle inhibitors and matrix‐metalloproteinases. Together, these data demonstrate that artesunate antagonizes fibroblast activation and decreases expression of pro‐fibrotic genes, while also promoting myofibroblast apoptosis, suggesting that these mechanisms may be responsible in part for the anti‐fibrotic effects of artesunate described previously.

    

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4. Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy

   Ramamurthy, R ; Zheng, WT ; George, S ; Wan, M ; Zhou, Y ; Lu, B ; Bishop, C ; Atala, A ; Porada, C ; Almeida-Porada, G ( December 2021 , ASH)

   2938 Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy 63rd ASH Annual Meeting and Exposition, December 11-14, 2021, Georgia World Congress Center, Atlanta, GA Program: Oral and Poster Abstracts Session: 801. Gene Therapies: Poster II Hematology Disease Topics & Pathways: Bleeding and Clotting, Biological, Translational Research, Hemophilia, Genetic Disorders, Clinically Relevant, Diseases, Gene Therapy, Therapies Sunday, December 12, 2021, 6:00 PM-8:00 PM Ritu M Ramamurthy1*, Wen Ting Zheng2*, Sunil George, PhD1*, Meimei Wan1*, Yu Zhou, PhD1*, Baisong Lu, PhD1*, Colin E Bishop, PhD1*, Anthony Atala, M.D.1*, Christopher D Porada, PhD1* and M. Graca Almeida-Porada, MD3 1Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC 2Massachusetts Institute of Technology, Cambridge, MA 3Fetal Research and Therapy Program, Wake Forest Institute For Regenerative Medicine, Winston-Salem, NC Clinical trials employing AAV vectors for hemophilia A have been hindered by unanticipated immunological and/or inflammatory responses in some of the patients. Also, these trials have often yielded lower levels of transgene expression than were expected based upon preclinical studies, highlighting the poor correlation between the transduction efficiency observed in traditional 2D cultures of primary cells in vitro, and that observed in those same cell types in vivo. It has been also recognized that there are marked species-specific differences in AAV-vector tropism, raising the critical question of the accuracy with which various animal models will likely predict tropism/vector transduction efficiency, and eventual treatment success in humans. Human liver tissue equivalents (hLTEs) are comprised of major cell types in the liver in physiologically relevant frequencies and possess the ability to recapitulate the biology and function of native human liver. Here, we hypothesize that hLTEs can be used as a better model to predict the efficacy and safety of AAV gene therapy in humans. We fabricated hLTEs using 75% hepatocytes, 10% stellate cells, 10% Kupffer cells, and 5% liver sinusoid-derived endothelial cells in 96-well Elplasia plates with 79 microwells per well. hLTEs were transduced at an MOI of 105vg/cell, on the day of fabrication, with the clinically relevant serotypes AAV5 (hLTE-5) or AAV3b (hLTE-3b), both encoding a GFP reporter. After 4 days of self-aggregation, live/dead assay was performed to confirm viability. Non-transduced hLTEs served as negative controls (hLTE(-)), and hLTEs exposed to 20 mM acetaminophen were used as positive controls for liver inflammation/damage. Incucyte® Live-Cell Imaging system was used to track the aggregation and GFP expression of hLTEs. Over the course of the next 5 days, media was collected to determine hepatic functionality, RNA was isolated to assess dysregulation of genes involved in inflammation and fibrosis, DNA was isolated to determine whether AAV vectors integrate into the genome of human hepatocytes and, if so, to define the frequency at which this occurs and the genomic loci of integration, and hLTEs were fixed and processed at appropriate times for histological analyses and transmission electron microscopy (TEM). TEM analysis revealed that all groups exhibited microvilli and bile-canaliculus-like structures, demonstrating the formation of a rudimentary biliary system and, more importantly, proving that hLTEs resemble native liver structure. Incucyte® imaging showed that AAV5 and AAV3b transduction impaired formation of hLTEs (57.57 ± 2.42 and 24.57 ± 4.01 spheroids/well, respectively) in comparison with hLTE(-) (74.86 ± 3.8 spheroids/well). Quantification of GFP expression demonstrated that AAV5 yielded the most efficient transduction of hLTEs (fold change in GFP expression compared to control: 2.73 ± 0.09 and 1.19 ± 0.03 for hLTE-5 and hLTE-3b, respectively). Chromogenic assays showed decreased urea production in cell culture supernatants of AAV transduced groups compared to the non-transduced hLTEs on days 6 and 10 of culture, demonstrating decreased hepatocyte functionality. However, ALT and AST levels were similar in all groups. On day 10, hLTEs were either used for RNA isolation or fixed in 4% PFA and processed for histology. Masson’s Trichrome and Alcian Blue/Sirius Red staining was performed to detect fibrosis, which was then quantified using ImageJ. These analyses showed no significant increase in fibrosis in either hLTE-5 or hLTE-3b compared to hLTE(-). Nevertheless, RT2 PCR Array for Human Fibrosis detected dysregulation of several genes involved in fibrosis/inflammation in both hLTE-5 and hLTE-3b (16/84 and 26/84, respectively). In conclusion, data collected thus far show successful recapitulation of native liver biology and demonstrate that AAV5 transduces hLTEs more efficiently than AAV3b. However, impaired self-aggregation and decreased hepatocyte functionality was observed in both AAV-transduced groups. Studies to address the incidence and location(s) of AAV integration are ongoing. We have thus shown that the hLTE system can provide critical new knowledge regarding the efficacy and safety of AAV gene therapy in the human liver. Disclosures: No relevant conflicts of interest to declare. 

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5. A Polycomb repressive complex is required for RNAi-mediated heterochromatin formation and dynamic distribution of nuclear bodies

   https://doi.org/10.1093/nar/gkaa1262  

   Xu, Jing ; Zhao, Xiaolu ; Mao, Fengbiao ; Basrur, Venkatesha ; Ueberheide, Beatrix ; Chait, Brian T ; Allis, C David ; Taverna, Sean D ; Gao, Shan ; Wang, Wei ; et al ( January 2021 , Nucleic Acids Research)

   Abstract Polycomb group (PcG) proteins are widely utilized for transcriptional repression in eukaryotes. Here, we characterize, in the protist Tetrahymena thermophila, the EZL1 (E(z)-like 1) complex, with components conserved in metazoan Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2). The EZL1 complex is required for histone H3 K27 and K9 methylation, heterochromatin formation, transposable element control, and programmed genome rearrangement. The EZL1 complex interacts with EMA1, a helicase required for RNA interference (RNAi). This interaction is implicated in co-transcriptional recruitment of the EZL1 complex. Binding of H3K27 and H3K9 methylation by PDD1—another PcG protein interacting with the EZL1 complex—reinforces its chromatin association. The EZL1 complex is an integral part of Polycomb bodies, which exhibit dynamic distribution in Tetrahymena development: Their dispersion is driven by chromatin association, while their coalescence by PDD1, likely via phase separation. Our results provide a molecular mechanism connecting RNAi and Polycomb repression, which coordinately regulate nuclear bodies and reorganize the genome. 

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