This effect is mediated by RSPO2, a WNT/b-catenin agonist, and lymphoid enhancer binding factor 1 (LEF1), an essential nuclear effector of the WNT/b-catenin pathway, via conserved LEF1/T-cell factor binding sites in the Pitx3 promoter. LEF1 expression is restricted to a caudolateral mdDA progenitor subset that preferentially responds to WNT/b-catenin signaling and gives rise to a fraction of all mdDA neurons. Our data indicate that an attenuation of WNT/b-catenin signaling in mdDA progenitors is essential for their correct differentiation into specific mdDA neuron subsets. This is an important consideration for stem cell-based regenerative therapies and in vitro models of neuropsychiatric diseases.The mesodiencephalic dopaminergic (mdDA) neurons, including the nigrostriatal subset that preferentially degenerates in Parkinson’s Disease (PD), strongly depend on an accurately balanced Wingless-type MMTV integration site family member 1 (WNT1)/beta-catenin signaling pathway during their development. Loss of this pathway abolishes the generation of these neurons, whereas excessive WNT1/b-catenin signaling prevents their correct differentiation. The identity of the cells responding to this pathway in the developing mammalian ventral midbrain (VM) as well as the precise progression of WNT/b-catenin action in these cells are still unknown. We show that strong WNT/b-catenin signaling inhibits the differentiation of WNT/b-catenin-responding mdDA progenitors into PITX3 Ctnnb1) pathway  neurons in the mouse midbrain and from human and murine pluripotent stem cells (PSCs) crucially relies on signal transduction by the Wingless-type MMTV integration site family member 1 (WNT1)/beta-catenin  progenitors located in the ventricular/subventricular zone (VZ/SVZ) of the mesodiencephalic FP , of the developing midbrain and caudal diencephalon in mammals . This Wnhalic FP . Yet, WNhalic FP . Howeverhalic FP .Ctnnb1, like Wnt1, are necessary for the correct generation in particular of caudally positioned (VTA) mdDA neurons  domain  b-catenin upon binding of the WNT ligand to its receptor/co-receptor complex on the cell surface, and the subsequent translocation of stabilized b-catenin into the cell nucleus. In the nucleus, b-catenin activates the DNA-bound lymphoid enhancer binding factor 1 (LEF1)/T-cell factors (TCFs) and initiates the transcription of WNT target genes . Ctnnb1, neurons . Overexp) domain . These p) domain .BAT-gal) mouse of “canonical” WNT/b-catenin signaling. This analysis disclosed two components of this pathway, the potent agonist R-spondin 2 (RSPO2) and the nuclear effector LEF1 (WREs) in the murine Pitx3 promoter and WNT/b-catenin-mediated repression of this promoter. Accordingly, only a caudolateral subset of the mdDA progenitors and precursors responded to WNT1/b-catenin signaling throughout mouse embryonic development. Our results provide a mechanistic explanation in vitro for the subset-specific effects and the need of a tight regulation of WNT1/b-catenin signaling during mdDA neuron development. These findings might be explored in future applications of PSC-based protocols for the derivation of specific mdDA neuron subsets in vitro.We addressed these issues by a detailed transcriptome analysis of the WNT/b-catenin-responding domain in a transgenic reporter  was reported previously  or CD-1 (TOPGAL) genetic backgrounds. Generation and genotyping of Wnt1+ /− and En1Wnt1+/ mice were described by C57BL/6J genetic background for more than 10 generations. CD-1 and C57BL/6J mice were provided by the Transgenic Unit, Helmholtz Zentrum München. Heterozygote/hemizygote Wnt1+ /−; +BAT-gal and Lef1+ /−; +BAT-gal mice were generated by crossing Wnt1+ /− and Lef1+ /− mice with the transgenic BAT-gal mouse line. Homozygote/hemizygote Wnt1–/–; +BAT-gal and Lef1–/–; +BAT-gal embryos were obtained from intercrosses of heterozygote/hemizygote Wnt1+ /− or Lef1+ /−; +BAT-gal and Wnt1+ /− or Lef1+ /−; –BAT-gal mice. Pregnant dams were killed by CO2 asphyxiation or cervical dislocation. Collection of embryonic stages of either sex was done from timed-pregnant females; noon of the day of vaginal plug detection was designated as E0.5. This study was carried out in strict accordance with the recommendations in the European Union Directive 2010/63/EU and the Guide for the Care and Use of Laboratory Animals of the Federal Republic of Germany . The protocol was approved by the Institutional Animal Care and Use Committee  of the Helmholtz Zentrum München. All efforts were made to minimize suffering.Generation of eviously . These mBAT-gal embryo heads were dissected in ice-cold PBS, fixed for 2 h in 4% PFA at 4°C, incubated in 20% sucrose overnight, embedded in Neg-50 (Thermo Fisher Scientific), frozen in 2-methylbutane on dry ice and stored at −80°C. Consecutive coronal cryosections (20 μm) from the caudal forebrain  and midbrain were mounted on POL frame slides (Leica Microsystems) and stored at −80°C. Mounted sections were post-fixed in acetone at −20°C, dried at 40°C for 1 min, washed twice with cold 2 M NaCl PBS, permeabilized with 0.25% Triton X-100 in 2 M NaCl PBS for 2 h, incubated with primary antibodies  overnight and secondary antibodies  for 2 h, washed three times with 2 M NaCl PBS and stored in this solution at 4°C. Immediately before LMD, tissue sections were dehydrated in 70% and 100% ethanol (2 min each), the ROIs were outlined under visual inspection, and tissue fragments corresponding to the WNT/b-catenin-responding mdDA domain or the lateral non-WNT/b-catenin-responding, non-mdDA domain were isolated with an LMD6000 system (Leica Microsystems) and stored at −80°C.The RNA integrity-preserving tissue staining protocol by Pitx3GFP/+ mdDA neurons were reported by Total RNA was isolated from the laser-microdissected tissue fragments using the RNeasy FFPE kit (Qiagen), and RNA quality and quantity were determined with the 2100 Bioanalyzer and RNA 6000 Pico kit (Agilent Technologies) following the instructions of the manufacturers. Total RNA (40 ng) from each tissue fraction was amplified with the Ovation PicoSL WTA System V2 in combination with the Encore Biotin Module (Tecan). Amplified cDNAs were hybridized on Mouse Gene 1.0 ST arrays (Affymetrix), and staining and scanning were done according to the Affymetrix expression protocol including minor modifications as suggested in the Encore Biotin protocol. Expression console (Affymetrix) was used for quality control and to obtain annotated normalized RMA gene-level data . Fluorescence-activated cell sorting (FACS) and microarray analysis of E13.5/E14.5 35S]UTP, Perkin-Elmer) riboprobes as described previously (In Situ Hybridization (ISH) probes used were Wnt1  by PCR from E10.5 or E12.5 CD-1 whole embryo or head cDNA using the primer pairs listed in Serial paraffin sections (8 μm) were hybridized with radioactive  on serial 8 μm paraffin sections and Immunocytochemistry (ICC) on cultured cells were performed as reported previously . PolycloCD-1 mouse embryos as described by 5 cells/well were plated in a 24-well plate on poly-D-lysine (Merck)-coated coverslips in DMEM/F-12 medium supplemented with 1% penicillin/streptomycin (both from Thermo Fisher Scientific), 10% fetal bovine serum , and 100 μM ascorbic acid (Merck). 1 day (24 h) after plating in vitro (1 DIV), cells were incubated in expansion medium for 1 day (2 DIV) and then switched to differentiation medium  primary cultures were prepared from E11.5 n medium  for the BAT-gal VM, respectively, were counted bilaterally on every fourth , sixth (E14.5), or eighth (E17.5/E18.5) serial coronal midbrain section using Stereo Investigator 5.05.4 (MBF Bioscience) or LAS X 3.0.0 and ImageJ 1.52a (NIH/United States) software. Consecutive serial sections stained for b-GAL and TH were used to outline the mdDA domain in these cases. TH and PITX3 single-labeled cells in the wild-type (wt) and Lef1–/– VM were evaluated bilaterally by the optical fractionator method on every fourth (E12.5) or eighth (E17.5) serial coronal midbrain section using Stereo Investigator 5.05.4 software. Cell numbers were averaged for each genotype, stage, region or treatment and subjected to tests for the estimation of statistical significance.For ICC on VM primary cells, TH and PITX3 single- and double-labeled cells were counted on 1-2 coverslips/treatment/experiment. EdU- per DAPI-positive cells were determined using a CellInsight NXT high content screening platform (50 pictures at 10x magnification) and CellInsight NXT software (Thermo Fisher Scientific). For IHC, LMX1A, SOX6, PITX3, or OTX2 single- and b-GAL/LMX1A, b-GAL/SOX6, b-GAL/PITX3, or b-GAL/OTX2 double-positive cells in the E10.5, E12.5, E14.5, and E17.5 or E18.5 CD-1 mouse embryos were dissected in ice-cold PBS and homogenized in RIPA buffer . Total proteins  were separated by SDS-PAGE, blotted on Hybond-LFP membranes (Merck) and stripped as described by Ventral midbrain and dorsal midbrain (DM) tissues from E12.5 Pitx3 genes were derived from the ElDorado genome database . Orthologous promoter sequences from different mammalian species of the Genomatix homology group  were analyzed using the MatInspector program  to identify potential LEF1/TCF Binding Sites (BSs)/WREs and to extract matrix similarities. Conserved BSs/WREs were determined with the DiAlign TF program (Genomatix) and by searching for common LEF1/TCF BSs/WREs occurring at the same position in  orthologous Pitx3 promoter sequences. The Pitx3 promoter regions were defined as ∼2000 bp upstream, including the proximal region, and ∼500 bp downstream of the transcription start site (TSS).Promoter sequences for CD-1 embryos, and Chromatin Immunoprecipitation (ChIP) was done using the EZ-ChIP kit (Merck Millipore 17-371) according to the manufacturer’s instructions. Sheared genomic DNA fragments after sonication with a Sonopuls Sonicator  were enriched between 200–800 bp. Two different rabbit anti-LEF1 mAbs , anti-RNA Polymerase II antibody (Merck Millipore) or rabbit IgG  were used for ChIP. Purified DNA served as template for PCR amplification of Pitx3 genomic fragments with the six primer pairs listed in Ventral midbrain tissues were dissected from E11.5 L-glutamine; SH-SY5Y cells were kept in DMEM/F-12  and 15% FCS at 37°C and 5% CO2. HEK-293, COS-7 and SH-SY5Y cells (5 × 104 cells/well of a 24-well plate) were transfected with 200 ng/well BAT-gal plasmid  using LFOPFlash , or a wic vector ], 0.1 ngd 19286, ] with orEF1 cDNA , and vicRES-eGFP , or 353.NA #144, . The humTotal RNA was isolated from HEK-293, COS-7 and SH-SY5Y cells with Trizol Reagent (Thermo Fisher Scientific) or RNeasy Mini Kit (Qiagen), including a DNAse I treatment. 1 μg total RNA was reverse transcribed using Advantage RT-for-PCR Kit (Takara Bio) or Quantitect Reverse Transcription Kit (Qiagen) according to the instructions of the manufacturers. 1.0 or 2.5 μl cDNA were amplified by PCR using the intron-spanning primers and conditions listed in +BAT-gal embryos. For ICC on cultured cells, five or three independent experiments were evaluated in each case, as indicated in the figure legend. For ChIP-PCR experiments, data are from five independent ChIP experiments , and PCR assays were repeated three times for each experiment . The luciferase reporter assays were performed in triplicates , and data are derived from three to seven independent experiments , as indicated in the figure legends. For IHC and ISH, mutant [homozygote (-/-) or hemizygote +BAT-gal] embryos were compared to their wt [homozygote (+/+) and heterozygote (+/-) or hemizygote –BAT-gal] littermates and at least 3 embryos from at least 2 different litters were analyzed for each probe, genotype and stage, unless stated otherwise. For microarrays, statistical analyses were performed with the statistical programming environment R  < 10%]. Heat maps were generated with R. Genes associated with the WNT receptor signaling pathway were obtained from canonical pathways and pathway associations from GePS software (Genomatix). All other values are mean ± s.e.m. For comparison of two groups in the unbiased stereological quantifications, the Welsh t-test for unequal variances was applied using the R software with the nlme package  of laser-microdissected tissue pools from five E12.5 onment R  implemenonment R . Gene-wi package . All othBAT-gal mouse, in which nuclear b-GAL protein is expressed upon activation of a minimal promoter containing seven multimerized LEF1/TCF BSs by a “canonical” (b-catenin-mediated) WNT signal (+) mdDA domain was co-labeled with PITX3 for unambiguous identification and isolated from the VM of five E12.5 +BAT-gal embryos (–/PITX3–) domain at the alar-basal boundary (ABB) was laser-microdissected and profiled in the same experiment to enrich for specific transcripts of the WNT/b-catenin-responding mdDA domain (Pitx3GFP/+ mdDA precursors/neurons reported previously (Ddc (Aadc), Th, Nr4a2 (Nurr1), Foxa1/2 (Hnfa/b), En1/2, Slc18a2 (Vmat2), Lmx1a, Ret, Wnt5a, Dlk1, Lmx1b, Slc6a3 (Dat), Cxcr4, Shh, Pitx3, and Ferd3l (Nato3)  mouse embryo. At this embryonic stage, WNT/b-catenin signaling is fully active in the mouse VM . We usedT signal . The WNT embryos . A non-WA domain . This st (Nato3) ] were si (Nato3) . Notably (Nato3) .Fgf14  in the lateral domain at the ABB, thus confirming their selective enrichment in the mouse VM/mdDA domain (Cck (Smarca1 (Dlk1 [A/T]CAAAG-3′ in sense, 5′-CTTTG[T/A][T/A]-3′ in antisense orientation , indicating that this gene was being actively transcribed in the E11.5 mouse VM  cell line expressing detectable amounts of LEF1 mRNA and nuclear LMX1A protein  of S33Y-b-catenin tested in this context (LEF1 doses (12.5 and 6.25 ng) caused a 2.2 ± 0.15 to 2.5 ± 0.32-fold activation of the Pitx3 promoter/reporter construct, whereas high LEF1 amounts (200 and 100 ng) led to a 0.63 ± 0.088 to 0.93 ± 0.15-fold repression of this promoter after co-transfection of S33Y-b-catenin . This repression is turned into an activation of the Pitx3 promoter only when the levels of stabilized b-catenin or LEF1 protein are strongly decreased in the responding cell.We then determined the effect of WNT/b-catenin signaling on transcription from a previously characterized ∼2.5 kb BSs/WREs . We perf protein . As expeBSs/WREs . Decreasactivity . Transfereporter . These r93 cells . Surpris context . Consist-catenin . TogethePitx3 promoter is in fact mediated by the three conserved, LEF1-bound BSs/WREs in this promoter, through mutagenizing the WRE core sequence [5′-CAAAG-3′ or 5′-CTTTG-3′] in each of these three LEF1/TCF BSs  LEF1/TCF BS alone consistently resulted in a significant  1.27 ± 0.06 to 1.62 ± 0.15-fold de-repression (activation) of the mouse Pitx3 promoter, both in the complete absence and presence of high amounts (200 ng) of co-transfected S33Y-b-catenin or LEF1  LEF1/TCF BS also resulted in a significant (P = 0.016) 1.56 ± 0.11-fold de-repression (activation) of the mouse Pitx3 promoter in the presence of high amounts of LEF1 (WRE 1 and 3 represses the murine Pitx3 promoter. Mutation of WRE 3 together with WRE 1 and/or WRE 2 (in the intermediate LEF1/TCF BS) led to a small but significant (P = 0.039) 0.85 ± 0.09 to 0.88 ± 0.09-fold reduction of luciferase activity in the absence of co-transfected S33Y-b-catenin and LEF1 (S33Y-b-catenin or LEF1 (WRE 3 together with WRE 1 and/or WRE 2 directly or indirectly (via other TFs binding in the same region) contribute to the activation of the mouse Pitx3 promoter.Next, we determined whether the repressive effect of strong WNT/b-catenin/LEF1 signaling on the murine /TCF BSs . Mutatio or LEF1 . Mutatio of LEF1 , indicatand LEF1 , and a s or LEF1 . This rePitx3 promoter region in vivo  together resulted in a significant decrease of Pitx3 promoter activity relative to Lmx1a alone (P = 0.0123) or Lmx1a and LEF1 co-transfected (P = 0.0297) cells  ∼70% repression of the Pitx3 promoter in the presence of high amounts of S33Y-b-catenin and LEF1  cells . Repressls alone . We also93 cells . Reductiand LEF1 . These rTOPGAL  cells were detected in the DM (roof plate) of the E12.5 TOPGAL+ mouse embryo, whereas the remaining midbrain tissue was b-GAL–, which means non-WNT/b-catenin-responding  mdDA progenitors in the midgestational BAT-gal mouse embryo. At the beginning of mdDA neurogenesis [E10.5; (+ FP (mdDA) progenitors were also b-GAL+ and preferentially located in the medial and caudal VM , is responding to this pathway in the midgestational mouse VM.We then determined the location and overall proportion of WNT/b-catenin-responding  in the E12.5 +BAT-gal VM  and in the caudal linear nucleus of the raphe (CLi) at E18.5  in the midgestational +BAT-gal VM (+ SNc DA neurons (+ VTA DA neurons (+ SNc DA (26.57 ± 1.26%) and OTX2+ VTA DA (27.06 ± 0.94%) neurons were also b-GAL+ at this stage  mdDA neurons (see model below). Moreover, the almost exclusive detection of the LGR4/5 receptors for RSPO2 in progenitor cells located in the VZ/SVZ of the mouse VM  mdDA neurons while transiting toward the MZ. This part of our model is supported in vivo by the apparently slight de-repression of PITX3 expression in WNT/b-catenin-responding (b-GAL+) cells in the mediocaudal Lef1–/–; +BAT-gal VM . This seems rather unlikely, because the expression of Pbx1 is also restricted to postmitotic mdDA precursors in the IZ/MZ  in the adult mouse brain, but it remained unknown whether these cells were also responding to WNT1/b-catenin signaling (Wnt1-derived mdDA neurons in that study was lower (∼15% or less) than the proportion of WNT/b-catenin-responding mdDA precursors/neurons in our study (b-GAL+ cells: ≥32%). Thus, it is very likely that the subset of WNT/b-catenin-responding mdDA progenitors not only comprises Wnt1-expressing cells. Moreover, the Wnt1-lineage stopped to contribute to these neurons after E13.5  of the mediocaudal +BAT-gal VM. Nevertheless, the WNT/b-catenin-responding mdDA progenitors contributed to an almost equal proportion (∼27%) of SOX6+ and OTX2+ mdDA neurons at E18.5. This finding corresponds well with an equal contribution of Wnt1-expressing cells to postmitotic SNc and VTA DA neurons reported previously  is thus necessary for a precise fate-mapping of the mdDA progeny from WNT/b-catenin-responding cells. The conditional inactivation of the Ctnnb1 or Wnt1 genes in the developing mouse VM affects more pronouncedly the generation of mature TH+ mdDA neurons in the VTA at intermediate and caudal levels of the mutant midbrain  can be found below: The animal study was reviewed and approved by Institutional Animal Care and Use Committee , Helmholtz Zentrum München.PN, SG, BR, MI, CP, and CL performed experiments. PN, SG, BR, MI, DT, CKe, JB, CB, WW, and NP analyzed data. AB, AD, EE, and CKl contributed materials and reagents. PN, SG, BR, MI, and CP contributed to initial writing of the manuscript. NP designed research and wrote the manuscript. WW and NP provided funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Wnt/β-catenin signaling is implicated in many physiological processes, including development, tissue homeostasis, and tissue regeneration. In human cancers, Wnt/β-catenin signaling is highly activated, which has led to the development of various Wnt signaling inhibitors for cancer therapies. Nonetheless, the blockade of Wnt signaling causes side effects such as impairment of tissue homeostasis and regeneration. Recently, several studies have identified cancer-specific Wnt signaling regulators. In this review, we discuss the Wnt inhibitors currently being used in clinical trials and suggest how additional cancer-specific regulators could be utilized to treat Wnt signaling-associated cancer. More effective treatments for cancer could be developed by targeting signaling pathway regulators that are expressed solely in cancer cells. Disruption to a major signaling pathway known as Wnt, which is involved in processes including cell proliferation, tissue homeostasis and tissue regeneration, is now recognized as a significant contributor to the development of certain cancers. Jae-Il Park and Youn-Sang Jung at the University of Texas MD Anderson Cancer Center, Houston, USA, reviewed recent research into Wnt signaling in cancer and possible therapies. Scientists have developed Wnt inhibitors for cancer treatment, but these have detrimental side effects including skeletal degeneration and abdominal pain. New studies suggest there are Wnt signaling regulators that are specifically expressed in cancer cells, which may prove to be more effective drug targets than blocking Wnt signaling as a whole. Classically, Wnt signaling is divided into β-catenin-dependent  and β-catenin-independent  signaling7. Canonical Wnt signaling mainly regulates cell proliferation, and noncanonical Wnt signaling controls cell polarity and movement. However, this terminological distinction is unclear, and has been questions by studies proposing the involvement of both β-catenin-dependent and β-catenin-independent Wnt signaling in tumorigenesis8. For instance, APC and β-catenin are not only involved in cell proliferation but have also been linked to cell-to-cell adhesion9. In this review, we will discuss an ongoing effort to inhibit Wnt signaling and suggest potential approaches to target Wnt signaling for cancer therapies proposed from recent studies.Wnt signaling orchestrates various biological processes, such as cell proliferation, differentiation, organogenesis, tissue regeneration, and tumorigenesis11. The β-catenin protein destruction complex composed of adenomatous polyposis coli (APC), casein kinase 1 (CK1), glycogen synthase kinase 3α/β (GSK-3α/β), and AXIN1 tightly controls β-catenin via phosphorylation-mediated proteolysis16. In this section, we briefly describe how genetic alterations of Wnt signaling contribute to tumorigenesis and introduce recent clinical trials that have aimed to inhibit Wnt signaling for cancer treatment.β-Catenin is a crucial signaling transducer in Wnt signaling18. CRC displays a high mutation frequency in APC (~70%)21. In 1991, APC mutation was identified as the cause of hereditary colon cancer syndrome, also called familial adenomatous polyposis22. APC forms the β-catenin destruction complex in association with CK1, AXIN1, and GSK-3 and interacts with β-catenin24. This protein destruction complex downregulates β-catenin through phosphorylation and ubiquitin-mediated protein degradation16. Genetic mutations causing the loss of function of the destruction complex or gain of function of β-catenin lead to nuclear translocation of β-catenin, resulting in T-cell factor (TCF)4/β-catenin-mediated transactivation of Wnt target genes26. The Vogelstein group established a multistep tumorigenesis model of CRC. APC mutation is an early event that initiates CRC adenoma27. CRC progression also requires additional genetic alterations in KRAS, PI3K, TGF-β, SMAD4, and TP5327. Moreover, epigenetic silencing of negative regulators of Wnt signaling was also frequently found in the absence of APC mutations29. APC is a multifunctional protein. In addition to its role in β-catenin degradation, APC binds to actin and actin-regulating proteins33, which controls the interaction between E-cadherin and α-/β-catenin and various physiological processes, including migration and chromosomal fidelity38. Importantly, recent studies revealed that APC mutation is insufficient to fully activate Wnt signaling. Furthermore, even if APC is mutated, mutant APC still negatively regulates β-catenin to some extent40, which will be discussed later.Colorectal cancer (CRC) is the representative of human cancer caused by Wnt signaling hyperactivation42. In human cancer, AXIN1 mutations are scattered throughout the whole coding sequence of the AXIN1 gene44, which results in disassembly of the β-catenin destruction complex. As a priming kinase, CK1 initially phosphorylates β-catenin (Ser45), which induces the sequential phosphorylation of β-catenin by GSK3. Subsequently, phosphorylated β-catenin is recognized and degraded by E3 ubiquitin ligase (β-TrCP)16. GSK3 is a serine/threonine kinase that phosphorylates three serine/threonine residues of β-catenin 46. Since GSK3 does not bind to β-catenin directly, AXIN1 and APC facilitate the interaction of GSK3 with β-catenin48. Moreover, unphosphorylated AXIN1 shows a low binding affinity to β-catenin, which is increased by phosphorylation of AXIN1 via GSK3 kinase activity50. Low-density lipoprotein receptor-related protein 5/6 (LRP5/6) coreceptor is also phosphorylated by CK1 and GSK3, leading to the recruitment of AXIN1 to the membrane53.AXIN1 is a multidomain scaffolding protein that forms the β-catenin destruction complex in association with APC, CK1, and GSK354, which induces the recruitment of the protein destruction complex to the LRP receptors and the subsequent phosphorylation of the Ser/Pro-rich motif of the LRP cytoplasmic domain via GSK356. This event activates dishevelled (DVL) and inhibits GSK3, resulting in the inhibition of the phosphorylation-mediated β-catenin protein degradation and the stabilization/accumulation of β-catenin. Then, β-catenin undergoes nuclear translocation and transactivates Wnt target genes57. The secretion of WNT ligands mainly depends on acylation by Porcupine (PORCN)59. PORCN is a membrane-bound O-acyltransferase that mediates the palmitoylation of WNT ligands to induce their secretion. In line with this observation, PORCN shows increased genetic alterations in various human cancers, including esophageal, ovarian, uterine, lung, and cervical cancers60.Under physiological conditions, Wnt signaling is activated by the binding of secreted WNT ligands to LRP5/6 coreceptors and frizzled (FZD) receptorsAPC gene is frequently mutated, the CTNNB1 gene encoding β-catenin is predominantly mutated in hepatocellular carcinoma, endometrial cancer, and pancreatic cancer63. The CTNNB1/β-catenin gene harbors 16 exons. β-Catenin is mainly composed of three domains . The N-terminal domain contains the phosphorylation sites for GSK3 and CK146, which induces β-TrcP-mediated β-catenin degradation. The C-terminal domain is involved in transactivation of Wnt target genes via TCF/LEF interactions66. The armadillo repeat domain interacts with various proteins, including E-cadherin, APC, AXIN1, and PYGOs/Pygopus68. In human cancer, the phosphorylation sites (Ser/Thr) in the N-terminal domain of CTNNB1/β-catenin are mutational hotspots70, demonstrating that escape from destruction complex-mediated β-catenin protein degradation is a key process for Wnt signaling-induced tumorigenesis.Unlike CRC, in which the To suppress WNT ligands or receptors for cancer treatment, PORCN inhibitors, WNT ligand antagonists, and FZD antagonists/monoclonal antibodies have been examined in clinical trials of various Wnt signaling-associated human cancers Table .Table 1W(i) PORCN inhibitors74. Therefore, PORCN inhibitors block the secretion of WNT ligands through inhibition of posttranslational acylation of WNT ligands. However, similar to other cancer therapies targeting the Wnt pathway, skeletal side effects such as impairment of bone mass and strength and increase in bone resorption were caused by PORCN inhibitor administration75.WNT974 , ETC-1922159 , RXC004 (NCT03447470), and CGX1321 (NCT02675946 and NCT03507998) are orally administered PORCN inhibitors that commonly bind to PORCN in the endoplasmic reticulum(ii) SFRP and SFRP peptides78. Indeed, SFRPs or SFRP-derived peptides showed tumor suppressive activity in preclinical models80.SFRPs (secreted frizzled-related proteins) are soluble proteins. Given the structural homology of SFRPs with the WNT ligand-binding domain in the FZD receptors, SFRPs function as antagonists that bind to WNT ligands and prevent Wnt signaling activation(iii) FZD antagonist/monoclonal antibody81. Ipafricept  is a recombinant fusion protein that binds to a human IgG1 Fc fragment of FZD883. These reagents negatively regulate Wnt/β-catenin signaling through their direct binding to FZD, which thereby disrupts the function of LRPs/FZDs. Alternatively, a way of targeting and killing cancer cells that express high FZD receptors is also being examined. OTSA101 is a humanized monoclonal antibody against FZD10. OTSA101-DTPA-90Y  is labeled with a β-radiation delivering-yttrium Y90 for OSTA10184. OTSA101-DTPA-90Y selectively killed cancer cells highly expressing FZD10. The side effects of vanctumab include tiredness, diarrhea, vomiting, constipation, and abdominal pain. Vantictumab and ipafricept might also cause bone metabolism disorders82.Vantictumab  is a monoclonal antibody directly binding to FZD receptors, which blocks the binding of WNT ligands to FZD 1, 2, 5, 7, and 8(iv) Targeting of LRP degradation and FZD endocytosis87. In addition, niclosamide promotes FZD1 endocytosis, which downregulates WNT3A-stimulated β-catenin stabilization88. However, these reagents do not specifically target cancer-specific molecules, leading to side effects, including itchiness, abdominal pain, vomiting, dizziness, skin rash, and unpleasant taste89.Salinomycin, rottlerin, and monensin induce the phosphorylation of LRP6, resulting in the degradation of LRP692. Tankyrase inhibitors have been developed95. Indeed, Tankyrase inhibitors have been shown to negatively regulate Wnt signaling in APC-mutated cancer cells95.Given that the β-catenin protein destruction complex plays a crucial role in negatively regulating Wnt signaling, the restoration of this protein destruction complex may effectively inhibit Wnt/β-signaling. Tankyrase interacts with and degrades AXIN via ubiquitin-mediated proteasomal degradation(i) Tankyrase inhibitors92. However, currently, no clinical trials are being conducted with Tankyrase inhibitors.Tankyrase inhibitors downregulate β-catenin stabilization. In preclinical studies, Tankyrase inhibitors, including XAV939, JW-55, RK-287107, and G007-LK, stabilized AXIN by inhibiting the poly-ADP-ribosylating enzyme Tankyrase(ii) CK1 agonist96.Pyrvinium is an FDA-approved anti-helminthic drug. Pyrvinium binds to CK1 family members in vitro and promotes CK1 kinase activityCCND1, CD44, AXIN2, and MYC100. Thus, approaches inhibiting either β-catenin transcriptional activity or β-catenin target genes have been developed as potential therapeutic candidates for Wnt signaling-associated cancers (Table β-Catenin contributes to tumorigenesis via transactivation of Wnt target genes such as (i) Inhibitors of β-catenin transcriptional activity102. PRI-724  inhibits the interaction between CBP and β-catenin and prevents transcription of Wnt target genes103. Moreover, various inhibitors of TCF/LEF and β-catenin interactions have been identified and evaluated in preclinical settings104.β-Catenin/CBP binds to WRE  and activates target gene transcription106, which is inhibited by carnosic acid, compound 22, and SAH-BLC9108. In addition, Pyrvinium downregulates Wnt transcriptional activity through the degradation of PYGO96.To transactivate Wnt target genes, β-catenin forms a transcriptional complex with coactivators, including BCL9 and PYGO(ii) Inhibitor of Wnt target genesSM08502  is a small molecule that inhibits serine and arginine-rich splicing factor (SRSF) phosphorylation and disrupts spliceosome activity. Upon oral administration, SM08502 was shown to downregulate Wnt signaling-controlled gene expression.(iii) Proteasomal degradation of β-catenin109.MSAB (methyl 3-[(4-methylphenyl)sulfonyl]amino-benzoate) binds to β-catenin and facilitates the ubiquitination-mediated proteasomal degradation of β-catenin110.However, since β-catenin controls various physiological processes, downregulation of the transcriptional activity β-catenin was shown to induce diarrhea, hypophosphatemia, reversible elevated bilirubin, nausea, fatigue, anorexia, and thrombocytopeniaAPC mutations, β-catenin can be further activated by additional layers of regulation117, which demonstrated the complexity of Wnt signaling deregulation in cancer. Accumulating evidence supports the notion that additional regulatory processes contribute to Wnt signaling hyperactivation in cancer, as demonstrated in the following examples. (a) Mutant APC is still able to downregulate β-catenin40. (b) Even in the presence of APC mutations, blockade of WNT ligands triggers apoptosis or growth inhibition118. (c) β-Catenin fold induction is essential for the activation of β-catenin target genes121. (d) Increased AXIN1 by Tankyrase inhibitor suppresses cell proliferation of cancer cells where Wnt/β-catenin signaling is genetically hyperactive122. (e) Mutations in RNF43 and ZNRF3 E3 ligases that degrade Wnt receptors contribute to tumor development115. (f) Ras/MAPK signaling is also required for Wnt signaling activation123. These reports suggest that additional layers further enhance Wnt signaling activation in cancer.Wnt signaling hyperactivation by mutations in β-catenin destruction complex components or β-catenin itself contributes to tumorigenesis. In addition to 124. Lysosomal hydrolytic enzymes mediate the degradation of phagocytosed material and proteolysis of cytosolic proteins through fusion with the multivesicular body (MVB). Luminal acidification of the lysosome is required for lysosomal protein degradation, which is mainly controlled by vacuolar H+ transporters in the lysosomal membrane125.The lysosome contains 40 types of hydrolytic enzymes, including cathepsins, which become active under acidic conditions126. In human cancer, lysosomal dysfunction is involved in the generation of building blocks, cell proliferation, metastasis, angiogenesis, and tumor suppressor degradation127.Recently, this classical view of lysosomal functions has evolved into new perspectives highlighting the roles of lysosomes in transcriptional regulation and metabolic homeostasis128. GSK3 in the LRP signalosome is sequestered into the MVB, which leads to an increase in the level of cytosolic β-catenin and inhibition of Wnt signaling129. However, decreased GSK3 kinase activity by MVB sequestration lasts approximately 1 h130. Moreover, it is unclear how sequestrated APC, GSK3, AXIN, and CK1 in MVB are processed. A recent study showed that clathrin-mediated endocytosis is required for Wnt signaling activation, which is inhibited by APC131. These studies suggest that vesicular acidification and trafficking also play crucial roles in controlling Wnt/β-catenin signaling through modulation of the protein destruction complex. Next, we discuss how APC is deregulated for Wnt signaling hyperactivation in cancer cells.It has been reported that Wnt signaling is involved in the endocytosis-mediated formation of the LRP signalosome into the MVB+-ATPase; an electrogenic H+ transporter)133. Previous studies imply that in cancer cells, the upregulation of v-ATPase activity might trigger abnormal Wnt/β-catenin signaling and contribute to Wnt signaling-dependent tumorigenesis. Growing evidence has demonstrated the effect of v-ATPase on various oncogenic processes, including cellular signaling, survival, drug resistance, and metastasis134. Moreover, the v-ATPase subunits are highly expressed in colorectal, breast, prostate, liver, ovarian, and pancreatic cancer cells138. The v-ATPase complex is composed of the V1 domain (in the cytosol) and V0 domain (on the membrane)140. The V1 domain shows reversible disassociation from the V0 domain under physiological conditions, including glucose concentration, starvation of amino acids, and infection of cells by influenza virus144. Recently, TMEM9 (transmembrane protein 9) was identified as an activator of v-ATPase and is highly expressed in cancer39. TMEM9 amplifies Wnt signaling through the v-ATPase-mediated lysosomal protein degradation of APC39. Given that TMEM9 is highly expressed in CRC cells and that Tmem9 knockout mice are also viable39, molecular targeting of TMEM9 may selectively suppress Wnt signaling activity in cancer cells.Wnt signaling activation requires v-ATPase  and concanamycin (CON), directly bind to and inhibit v-ATPase147. Compared with CQ and HCQ, BAF and CON showed marked inhibition of Wnt/β-catenin signaling in CRC. In addition, BAF and CON displayed an antiproliferative effect in CRC patient-driven xenograft and animal models without toxicity to normal cells and animals39. In addition, BAF and CON also strongly inhibit Wnt signaling activity in CRC cells, regardless of APC mutations. Thus, further research may lead to the development of not only safer but also more potent anti-v-ATPase drugs as cancer-specific Wnt/β-catenin inhibitors  and hydroxychloroquine (HCQ), inhibitors of lysosomes and autophagy, are clinically used for the treatment of diseases such as malaria and rheumatoid arthritisors Fig. .Fig. 2InGenetic and epigenetic deregulation of Wnt/β-catenin signaling contributes to human cancer, which has led to the development of extensive approaches targeting Wnt/β-catenin signaling as cancer therapies. Nonetheless, the blockade of Wnt signaling impairs tissue homeostasis and regeneration, which needs to be resolved. Recent studies have identified several Wnt signaling regulators whose expression is specific to cancer cells. These cancer-specific regulatory processes of Wnt signaling may be druggable vulnerabilities of Wnt signaling-associated cancer.


Endoscopic procedures performed after-hours often require therapeutic interventions that are technically demanding for the endoscopist. The aim of this position paper is to provide guidance on the minimum standard of equipment that should be available on a mobile endoscopy cart for provision of a safe and effective after-hours emergency endoscopy service. The guidance is based on consensus among academic and community gastroenterologists in Canada.A modified Delphi process was used to establish consensus among 9 participants. A list of statements was prepared by an expert panel of endoscopists. The statements were divided into three broad sections for what should be on an after-hours endoscopy cart including medications, nonendoscopic tools and therapeutic/diagnostic equipment. Consensus for being on the endoscopy cart was achieved when 75% or more of voting members indicated ‘agree’.For nonendoscopic tools, there was agreement for having sterile saline, sterile water, endoscope lubricant, various syringes, bite blocks (paediatric and adult size), a water pump with foot peddle, formalin jars for biopsy specimens, digital photo and printing capability and an overtube. For medications, there was agreement for having hyoscine butylbromide and epinephrine on the cart. For therapeutic/diagnostic tools, there was agreement for having biopsy forceps (standard and jumbo), polypectomy snares, sclerotherapy needles and agent , band ligation kit, multipolar electrocautery probes, heater probe catheter, endoscopic clips, hemostatic powder and retrieval devices.This position paper provides guidance on the minimum standard of items that should be on an after-hours endoscopy cart. Standardization of equipment may help improve safety and quality of after-hours endoscopic procedures. It is not uncommon to need to perform therapeutic endoscopy after-hours. This requires a significant clinical and economic resource investment for the system. Acute upper gastrointestinal bleeds, foreign body ingestion and food bolus impaction are commonly encountered after hours. These cases can be technically challenging and require expertise and support. A survey of the practice of after-hours and emergency endoscopy in Canada revealed that 25% of adult gastroenterologists had no on-call endoscopy nurse . DespiteThere is literature to guide the design of endoscopy units adequately equipped to perform high quality diagnostic and therapeutic endoscopy services ,6. AfterA modified Delphi process was performed to establish the consensus for what should be on an after-hours endoscopy cart. This process was based on an anonymous single round voting system. A list of statements was initially prepared by an expert panel of endoscopists. This panel consisted of three Canadian academic therapeutic staff gastroenterologists with over 10 years of experience as staff. A total of nine endoscopists with at least 5 years of experience as a staff gastroenterologist in Canada who perform therapeutic endoscopy after hours were asked to complete the survey. This group of nine endoscopists included the expert panel. The statements were divided into three broad sections including medications, nontherapeutic and therapeutic/diagnostic equipment .The questionnaire was comprised of 31 questions. Participants were asked if they agreed or disagreed with each question. The data were summarized by calculating mean scores for each of the questions. Consensus for being on the endoscopy cart was achieved when 75% or above of voting members indicated ‘agree’. Consensus for not being on an endoscopy cart was achieved when 75% or above of voting members indicated ‘disagree’. Participants were also asked to provide comments for each question and to suggest additional supplies they felt were pivotal for an after-hours endoscopy cart, how their endoscopy carts are stocked and challenges they face with after-hours endoscopy.The expert panel reviewed the results and co-authors were involved in the final editing of the commentaries, consensus statements and the final manuscript.Nine endoscopists from different institutions completed the survey –4. Four There was at least 75% agreement for having sterile saline, sterile water, endoscope lubricant, various syringes, bite blocks (paediatric and adult size), a water pump with foot peddle, formalin jars for biopsy specimens, digital photo and printing capability and an overtube. Consensus was not achieved on having simethicone, a suction or vacuum pump and a large bore nasogastric tube.There was at least 75% agreement for having hyoscine butylbromide and epinephrine on the cart. At least 75% disagreed with having erythromycin and metoclopramide. Consensus was not achieved on glucagon, fentanyl or midazolam.There was at least 75% agreement for having biopsy forceps (standard and jumbo), polypectomy snares, sclerotherapy needles and agent for a variceal bleed, band ligation kit, multipolar electrocautery probes, heater probe catheter, endoscopic clips, hemostatic powder and retrieval devices. Consensus was not achieved on having a monopolar probe, sclerotherapy agent for a nonvariceal bleed and balloon tamponade devices.Additional details on the results of the survey are provided in the Supplementary Appendix.Through a structured consensus process, this study looked at what should be included on an after-hours endoscopy cart. The expert panel was comprised of experienced gastroenterologists who perform therapeutic endoscopy after-hours. Thus, this position paper provides some clarity on an important clinical topic based on expert opinion. To our knowledge, no prior studies have assessed what is needed on an after-hours endoscopy cart.The first question addressed standard equipment. Not unexpectedly, consensus was achieved for the basic supplies needed to perform endoscopy including sterile saline, sterile water, endoscope lubricant various syringes, bite blocks, a water pump with foot peddle, formalin jars for biopsy specimens, digital photo and printing capability and an overtube. Overtubes are essential as they may be needed to protect the airway during removal of foreign objects. Furthermore, they allow easy passage of the endoscope for removing multiple objects while protecting the esophageal mucosa from sharp objects.Consensus was not achieved on having simethicone, a suction or vacuum pump and a large bore nasogastric tube. The utility of a nasogastric tube for suspected gastrointestinal bleeding is debatable and has fallen out of clinical practice in many centres. Its use has not been shown to reduce mortality, length of hospital stay, surgery or transfusion requirement . If needThe second question evaluated medications. Consensus was achieved for hyoscine butylbromide and epinephrine on an overnight cart. Hyoscine works by causing smooth muscle relaxation to reduce spasms in the gastrointestinal tract. Cardiac monitoring is essential when administering this drug as it can be associated with tachycardia and hypotension. Epinephrine injection therapy favours hemostasis by creating local tamponade and possibly by causing local vasospam. Consensus was not achieved on having erythromycin and metoclopramide on the travel cart. Prokinetics are used to improve mucosal visualization at the time of gastroscopy. A meta-analysis of erythromycin versus placebo in patients with upper gastrointestinal bleeding demonstrated the incidence of an empty stomach at the time of endoscopy was significantly increased in those who received erythromycin . In addiConsensus was not achieved as to the need for storing glucagon, fentanyl or midazolam on the endoscopy cart. Sedation medications are usually kept in a secure area as per hospital policy and thus having them on an endoscopy travel cart is ill-advised and may be against institutional policies. As most overnight endoscopy is performed in the emergency department, operating room or intensive care unit, these medications are readily available. Glucagon can be used to attempt to relax the esophagus in the setting of a food bolus. Studies have shown mixed results and if anything glucagon would have been attempted prior to requesting an after-hours procedure ,12.In the third question, consensus was achieved for having biopsy forceps (standard and jumbo), polypectomy snares, sclerotherapy needle, band ligation kit, multipolar electrocautery probes, heater probe catheter, endoscopic clips, a hemostatic powder kit and retrieval devices. Tc-325 is the only approved hemostatic powder in Canada and is an inorganic biologically inert compound that has granules which, when in contact with moisture in the gastrointestinal tract, become coherent and adhesive to an actively bleeding surface, serving as a mechanical barrier for hemostasis . Tc-325 Consensus was not achieved on having a monopolar probe and balloon tamponade devices. As per the Baveno VI Consensus, balloon tamponade should only be used in refractory esophageal variceal bleeding as a temporary bridge until definitive treatment can be instituted . EndoscoConsensus was achieved for having a sclerotherapy agent for a variceal bleed (for example tetradecyl sulfate or cyanoacrylate). Consensus was not achieved for having a sclerotherapy agent for a nonvariceal bleed . Data exist to support the use of sclerosant injection in both variceal and nonvariceal bleeds . HoweverWe acknowledge the limitations of this position paper. The recommendations were developed through a consensus method and thus represents the opinion of the participants. Generalizability may be restricted as there were a limited number of participants and a limited number of centres from across Canada. Despite these issues, our paper serves to increase awareness and promote conversations among stakeholders to ensure appropriate resources are available. Not only should the endoscopy cart be stocked appropriately, all health care personnel involved in performing the procedure should know how to use and trouble shoot problems with the equipment.In summary, after-hours endoscopy is commonly performed. Through a structured consensus process, we provide guidance for what should be on an after-hours endoscopy cart. Standardization of equipment may help improve safety and quality of after-hours endoscopic procedures.


This paper aims at reviewing the works published in the last five years (2016–2020) on polymer nanocomposites based on epoxy resins. The different nanofillers successfully added to epoxies to enhance some of their characteristics, in relation to the nature and the feature of each nanofiller, are illustrated. The organic–inorganic hybrid nanostructured epoxies are also introduced and their strong potential in many applications has been highlighted. The different methods and routes employed for the production of nanofilled/nanostructured epoxies are described. A discussion of the main properties and final performance, which comprise durability, of epoxy nanocomposites, depending on chemical nature, shape, and size of nanoparticles and on their distribution, is presented. It is also shown why an efficient uniform dispersion of the nanofillers in the epoxy matrix, along with strong interfacial interactions with the polymeric network, will guarantee the success of the application for which the nanocomposite is proposed. The mechanisms yielding to the improved properties in comparison to the neat polymer are illustrated. The most important applications in which these new materials can better exploit their uniqueness are finally presented, also evidencing the aspects that limit a wider diffusion. The epoxy resin family comprises thermosetting polymers characterized by many outstanding characteristics, such as excellent mechanical strength and stiffness, chemical and corrosion resistance, durability, and stability if exposed to different environmental agents, low shrinkage upon curing process, excellent adhesion to different materials . They arThe already good properties of epoxy resins can be further enhanced with the addition of one or more nanosized phases, obtaining the so-called nanocomposite, or with the realization of a system composed by an organic  phase strictly interconnected at the nano-scale level to an inorganic one, the so-called organic–inorganic (O–I) hybrid systems. O–I hybrid materials are, in fact, often referred to as structures characterized by nano-scale repeating distances between the organic phase and the inorganic one .Along with many withstanding properties, epoxy resins also display some weaknesses, such as very low values of toughness , low thermal/electrical conductivity, high coefficients of thermal expansion, and deficiencies that somehow restrict their use in some more demanding applications . These properties can be on-demand modified, improved, or adjusted to address specific requirements again with the addition of preformed nano-sized constituents, i.e., the nanofillers.Nanofillers can be defined as materials of different chemical nature that have at least one dimension between 1 and 100 nm, produced as 0D, 1D, 2D, and 3D nanomaterials .The addition of nanoparticles, possessing different chemical structures, dimensions, and shape, can improve or properly modify specific characteristics of the hosting epoxy matrix; these remarkable improvements arise from the very high surface/volume ratio of the nanomaterials, leading to multipurpose “smart” functional nanocomposite. It must also be bared in mind that the chemical composition of the particle surface, which can be different from the internal one, has a strong effect on the interaction between the nanofiller and the epoxy matrix.In addition to their chemical composition, the size and the aspect ratio of the nanofillers as well as their loading content in the epoxy matrix are all key parameters that severely affect the final performance of the nanocomposite . The nanThe shape and the features of the nanofiller will also define the process employed for the production of the nanocomposite. The nanophases are typically dispersed in the (un-cured liquid) epoxy matrix in very low percentages (most likely never above 10 wt.%), taking into account that the best performance is always obtained at low loads of the nanofiller. This solution also allows for reduction of both the aggregation effect of the nanoparticles and the final cost of the nanocomposite. International research is particularly active in this field, as witnessed by a huge number of papers that appeared in the last five years on this challenging topic. We found, in fact, 2027 papers on the Scopus database looking for the keyword “epoxy nanocomposite”. Over time, the number of published papers per year shows an increasing trend a. From tA comprehensive review of all the papers published in the last five years on the polymer nanocomposites based on epoxy resins is outside of our purposes. Rather, this manuscript aims at giving an overview on the different trendy nanofillers that have been successfully added to epoxies to enhance some of their characteristics, in relation to the nature and the feature of each nanofiller. Another class of nanostructured epoxies, i.e., the organic–inorganic hybrid one, is introduced, highlighting its strong potential in many applications. Differently from other recent reviews focused on specific properties of the nanocomposites ,8,9,10 oDifferent nanoparticles have been recently experimented as fillers able to enhance/modify properties and characteristics of epoxy resins. They belong to different chemical families, i.e., carbon-based fillers, metal oxides, and clay nanoparticles, just to mention a few of them.−10 to 10−14 S/cm [This family of nanofillers includes several compounds already widely used at very low contents in epoxy-based nanocomposites intended for many different applications, due to a mix of desirable properties, such as excellent physical and mechanical characteristics, hardness, high thermal and electrical conductivity  and outstanding electrical (with a conductivity of 6 × 105 S/m), mechanical , thermal (conductivity around 5000 W/m·K), and barrier properties [2/g), may represent a feasible alternative to functionalized/unmodified carbon nanotubes also when enhancements in mechanical, electrical, and thermal properties are mostly required for a conventional epoxy [3N4), an advanced derivative of graphene, is able to provide even greater anticorrosion performance to waterborne epoxy due to the strong shielding effect of the surface modified g-C3N4 nanosheets [Graphene , with itoperties . Its addoperties ,21,22,23al epoxy . Graphitnosheets . In the nosheets . A substCarbon nanotubes (CNTs), characterized by low density and high aspect ratio (diameter even less than 1 nm and length of dozens of nanometers), are among the most promising nanoreinforcements for many polymers, including the epoxy resins, due to their capacity to positively modify the mechanical, electrical, rheological (key parameter for the processability), and thermal properties of the hosting polymeric matrix, even at very low percentages (0.1–0.2 wt.%); the possibility to use very low amounts of nanofiller is desirable in order to limit self-aggregation of the nanoparticles ,29. On t2O4 nanoparticles (average diameter around 30 nm) are very appealing for electrical/electromagnetic applications, also due to the high percolation threshold that can be achieved in MnFe2O4 [A variant of CNTs is represented by multi-walled carbon nanotubes (MWCNTs), possessing an average outer diameter from 5 to 50 nanometers and a length of 0.5–200 µm, that demonstrated to give the epoxy resin, and the relative FRPs, excellent mechanical, thermal, and durability characteristics ,35,36,37 MnFe2O4 . Finally MnFe2O4 .Nanodiamond is another nanomaterial incorporated into epoxy resins to enhance the mechanical and tribological properties of the composite . FunctioMXenes is a family of 2D transition metal carbides, carbonitrides, and nitrides. Due to their outstanding mechanical, thermal, and electrical properties, these multilayered compounds have been employed to realize multifunctional epoxy-based nanocomposites characterized by enhanced electrical and thermal conductivity, self-healing characteristics, excellent toughness, and mechanical properties, even at low filler percentages (1 wt.%) ,48,49,502O3 is certainly one of the most widely used to manufacture polymer nanocomposites with enhanced thermal and mechanical performance. A further favorable advantage resides in the lower production costs of alumina nanoparticles if compared to carbon-based nanofillers and nanotitania. The average size (diameter) of this nanofiller, which can be found as nanospheres or nanorods , can benterface ,54,55. Tnterface ,56,57. ADue to their strong adhesion exerted to a wide range of surfaces and their good chemical resistance, epoxy resins are frequently used as anticorrosive and protective coatings for metals. The addition of a nanofiller in an epoxy-based coating can be a good choice to enhance the scratch resistance of the coating, thus improving the durability and the performance of the coating. On the other hand, the special shape/size of the nanofiller can be used to carry corrosion inhibitors, released on the basis of the surrounding pH value, the direct addition of which in the epoxy resin can lead to a decrease in their efficiency . This is2), a nanofiller for coatings that is especially popular for its high corrosion resistance, chemical stability, and non-toxicity coupled with affordable costs. The addition to epoxy of titanium dioxide nanotubes, i.e., nanocarriers encapsulating an organic inhibitor for corrosion, is able to impart improved resistance to corrosion to the final coatings [2 in improving the durability of an epoxy matrix is also confirmed in other studies concerning composites based on glass fibers (GFRP); the addition of 0.1 wt.% of this nanofiller is able to reduce the coefficient of water diffusion by 9%, with additional improvements in flexural and interlaminar strengths [Titania is the common name of titanium dioxide  able to improve the most important functional properties of the coatings for metals, such as abrasion and scratch resistance, especially when their surfaces are properly modified [SiOmodified . The incmodified . The appmodified . Referrimodified . Furthermodified .Nanostructured organic–inorganic (O–I) “hybrid” systems containing nanosilica can be formed directly in the epoxy network during its development, with a morphology consisting of co-continuous organic domains chemically linked to the inorganic phase. These systems gained a large success due to their outstanding synergistic properties derived from the beneficial combination of nanosized phases into a bulk epoxy, differing in chemical composition, physical properties, and morphology of the final phases. They can find an advantageous application, for instance, in the field of “cold-cured” adhesives, i.e., those able to set and harden at ambient temperature, due to a marked improvement in properties, in particular the glass transition temperature and durability ,68.ZnO-based nanofillers are used for reinforcement of epoxy matrices; increased surface hardness  and enha3O4 nanoparticles (diameter ranging from 10 to 50 nm) have been successfully experimented with to produce magnetic epoxy-based nanocomposites for a wide range of applications, such as microwave absorbing elements, microelectromechanical devices, micro-sensors, and systems to store/harvest energy [3O4 content has a positive effect on the capacity to store energy, on magnetic properties, and on microwave absorption performance [Finally, Fet energy ,72. A diformance .Originally employed mainly in thermoplastic nanocomposites, nanoclays are now also widely employed in epoxy systems with different functions, i.e., as a thermal stabilizer, to supply electrical and optical properties, flame retardancy, biocompatibility, and as scavengers for pollutants. Nanoclays, in addition, are able to confer enhanced mechanical properties, corrosion resistance , and fir2Si2O5(OH)4) nanotubes (HNTs), display inner/outer diameters around 12–15/50–60 nm and a length in the range 0.5–10 μm. HNTs are characterized by outstanding biocompatibility, thermal stability, and mechanical strength, and constitute promising nanofillers for the development of epoxy-based nanocomposite for many different applications [2) and outer (mainly Al2O3) surfaces of the nanotubes. Low contents of halloysite nanotubes (up to 1 wt.%) are preferred, leading to increased cross-linking density in comparison to the pristine epoxy, while at higher loads , the advancement of curing reactions is somehow hindered.Halloysite  layer during a fire, limiting further advancement of flame, with low emissions of smoke . Aluminum hypophosphite nanoparticles (possessing a diameter lower than 60 nm) are phosphorus-based compounds that can be used as a flame retardant additive for epoxy resins, even at very low percentages (up to 0.5 wt.% of epoxy) and without any superficial modification . PhosphoSimilarly, the addition of nanoaluminum diethylphosphinate to low amounts of functionalized MWCNTs is able to enhance the fire resistance of epoxy resin nanocomposites .Tg [Several recent studies in the literature describe the addition of boehmite, i.e., colloidal plate-like crystalline nanoparticles (with average thickness around 15 nm), in epoxy resins. Boehmite (γ−AlO(OH) is a mineral aluminum hydroxide in which the Al cations are placed between octahedral oxygen layers. It has been demonstrated that this stiff nanofiller supplies a noticeable reinforcing effect to the host epoxy matrix, with enhancements in elastic modulus and strength as well as in fracture toughness and hardness ,83,84. I2O3 nanoparticles can be incorporated in the epoxy matrix along with BN platelets; the formation of a sort of bridge between the different nanofillers is able to enhance the thermal conductivity of the nanocomposite, creating a conductive pathway [Boron nitride (BN) can be added in forms of nanosheets (65–75 nm thick) in epoxy matrices to impart high thermal conductivity to these polymers, due to the high thermal conductivity (around 300 W/m·K) of BN in isolation [ pathway . With a  pathway . Finally pathway .Aluminum nitride (AlN) spherical nanoparticles (average diameter of 50 nm) were added to epoxy resin in the view to enhance its electrical properties; these nanocomposites display, additionally, a greater thermal resistance . Among nanofillers experimented for the fabrication of piezoelectric nanosensors and energy harvesting devices, produced in the form of epoxy-based FRP elements, antimony sulfoiodide (SbSI) nanowires are worthy to be mentioned due to their high value of electromechanical and piezoelectric coefficients .Polymer-based nanocomposites differ from conventional (macro/micro) composite macromolecular materials, especially for the substantial larger interfacial area between the matrix and the reinforcing nanophase(s)  and for the outstanding aspect ratio of the nanofiller. A high outer surface of the nanoreinforcement implies that the addition of a relatively small amount of this filler in the polymeric (epoxy) matrix enables appreciable modifications in the macroscopic properties of the nanostructured material.The success of the nanocomposite, in terms of achievement of optimized properties, on the other hand, relies on an effective dispersion of the nanophase(s) in the epoxy matrix . To thisThe enhancement in the final properties of epoxy nanocomposites is, then, basically governed by two factors, namely the dispersion of the nanofiller and its interaction with the matrix . Therefo2 nanoparticles [Often, the functionalization of the surface of a nanofiller is achieved by treatment with silane solutions, such as in the case of nanoclays , graphenarticles . The silarticles ,94.In the case of CNT, MWCNT, graphene oxide nanoparticles, and surface functionalization can also be obtained through a covalent attachment of amine functional groups on the nanofiller surfaces ,28,95. TPlasma treatment has been successfully employed to functionalize the surface of carbon-based nanofillers, such as CNTs, GNP (graphene nanoplatelets) ,106, andAmong the others, surface functionalization treatment employing ozone can be performed under an air atmosphere to provide oxygen radicals on the surface of nanofillers; this can be achieved at reasonable costs and by employing simple equipment . As an eIn some cases, the chemical compounds employed to functionalize the surface of the nanofiller can additionally act as curing and/or toughening agents for the epoxy matrix, thus modifying the kinetics as well as further improving some characteristics of the nanocomposite, in particular the energy absorption, which is a highly desirable feature in some applications, for instance, in the field of adhesives .The organic–inorganic nanostructured epoxy-silica hybrids are produced employing the hydrolysis–condensation technology typical of ceramic materials. In these nanosystems, the inorganic phase is composed of siloxane nanodomains, i.e., nanometric silica with hydroxyl groups produced by the sol-gel method concurrently to the cross-linking reactions of the organic (epoxy) phase . It is tTg, or even enhance both of them. According to their physical and chemical properties, in fact, the nanoparticles can influence the chemistry and the rate of the curing reactions and, in turn, the resulting cross-linking density and glass transition range. It is fundamental, therefore, to monitor the advancement of cure in relation to the selected process parameters able to guarantee the formation of an optimum nanostructure within the epoxy-based nanocomposite.The design stage of epoxy-based nanocomposites able to meet specific requirements includes the selection of a proper nanofiller or an appropriate organic–inorganic hybrid nanostructure and the identification of suitable curing conditions, in order to achieve a microstructure of the nanocomposite able to supply the desired characteristics/properties. On one hand, the key parameter governing an effective adaptation of the nanofilled epoxy microstructure to the intended application is its cross-linking density developed during curing according to appropriate reaction kinetics. Depending on the nanofiller chemical nature and physical characteristics, its addition in the epoxy can sterically hinder curing reactions, thus leading to a decrease in cross-linking density and in Tg (about 20 °C) [Tg greater than 8%) [Tg have also been reported for epoxy-based nanocomposites containing epoxy-grafted silica (up to 5–6% at a filler content of 2.5 wt.%) [Tg have been found upon the incorporation of nano-Al2O3 particles (greater than 10 °C) at a 0.1 wt.% loading [Tg (up to 3 °C) have been measured upon addition of boehmite nanoparticles at low concentration levels  [Tg of the epoxy matrix are the result of different events, namely the restrictions in epoxy chain movements, due to the presence of nanoparticles, and the change in cross-linking density of the matrix resin. As already underlined, the functional groups employed to modify the surface of the nanofiller, with the aim of avoiding the agglomeration of the particles and improving their dispersion in the epoxy matrix, may also affect  the cross-linking process of the resin and, in turn, the glass transition temperature of the nanocomposite [The addition to a conventional (cured at a moderate temperature) DGEBA (diglycidyl ether of bisphenol A) resin of boehmite nanoparticles (3 wt.%) with organically modified surfaces has brought increased kinetics of cross-linking reactions with appreciable growths in t 20 °C) . An incrthan 8%) . Increas.5 wt.%)  and nano.5 wt.%) . On the  loading ; more li 1 wt.%) . The difomposite . As an eomposite .The incorporation of nanoparticles in epoxy resins allows modification of many properties. In comparison to the neat polymer, enhanced mechanical properties ,118,119,2) appreciably improve the barrier properties [Shape, size, and dispersion of the nanoparticles, more than the type of nanofiller, influence the final properties of the composite. In fact, nanoparticle aggregation is able to create defects into the matrix which may lead to micro-cracks ,128,131.operties ,124,125,operties ,132. Synoperties ,106,117,The flexural and tensile strengths of the epoxy nanocomposites are higher (even above 20%) than those measured in the pure resin ,129. In Epoxy resins may potentially contain micro-pores generated during the manufacturing process. A certain reduction of voids and pores can be obtained by nanofiller loading. The added nanoparticles can also restrict the movement of polymer molecules under mechanical loads. Both these phenomena result in increases in mechanical properties . Where aA large amount of energy, therefore, is required to allow the crack to pass the uniformly dispersed nanofillers and to propagate until the fracture failure ,101,113.In addition, when nanofiller agglomeration occurs , the interchain interactions during the cure of the polymer may be hindered, yielding reductions in the tensile and flexural strengths ,129. It is worth mentioning that the mechanical properties of nanofilled epoxy resins significantly decline as a consequence of water absorption, even at low amounts of absorbed water. This behavior has been ascribed to the weakening of the interface between the epoxy matrix and the nanofillers which hinders the transfer of the stresses under mechanical loads ,110.0.01 Hz) is significantly higher in comparison to the neat epoxy resin [Neat epoxy resins usually exhibit low corrosion protection because of their proneness to defect formation and shrinkage, as well as because of their poor flexibility. Well-dispersed nanofillers are able to promote barrier and anti-corrosion performance in epoxy coatings ,121. Corxy resin ,125,135.xy resin ,122,125.xy resin .− and OH−), can further improve the barrier properties against corrosive agents [The highest adhesion strength exhibited by the nanofilled epoxy coatings allows achieving excellent anti-corrosion performance ,134,137.e agents ,137.The addition of functionalized nanoparticles has been found effective in reducing the tendency to aggregate, thus enhancing the properties of the nanocomposite, especially the corrosion resistance of epoxy coatings ,124,137.The reduced barrier performance against corrosion can also shorten the nanocomposite’s service life . In factContact between the particles of nanofiller is necessary to form thermal conduction pathways; in this condition, the heat flow is promoted and the thermal conductivity increases ,16,86,87Additionally, the high electrical conductivity in these nanocomposites is promoted by an effective direct electrical contact between the nanoparticles ,110 Fig. Such a The addition of nanofillers to epoxy resins enhance the thermal stability of the material. In most cases, higher final decomposition temperatures  and imprThe extraordinary chemical and physical properties of a wide variety of experimental nano-epoxies enable a wide range of applications, varying from coatings to structural adhesives, from FRP matrix to electrical/electronic components. Epoxy nanocomposites have found advantageous application in many industrial fields ,120 suchThe nanofilled epoxy coatings display exceptional corrosion resistance performance and superior barrier properties due to the presence of the nanofiller that can limit the ingress of water and/or potentially harmful chemicals. The light weight and the high corrosion resistance of epoxy nanocomposites make these materials particularly advantageous in the aeronautical and automotive fields. The reinforcing effect of the nanofiller can also improve the superficial characteristics of the coating  with a substantial improvement in dirt resistance. When optical clarity and transparency are important features for the superficial treatment, a wide selection of nanoparticles, with a size smaller than the wavelength of visible light, is available in order to ensure these characteristics.In the field of adhesives, the presence of well-dispersed nanoparticles in epoxy enhances the adhesion to different inorganic substrates, even when the cross-linking reactions of the resin are carried out in an uncontrolled environment . This represents a distinctive advantage in the field of adhesive resins employed in building industry where, for economical and practical reasons, the cure at high temperatures is not possible. The addition of carefully selected nanophases in an epoxy matrix for FRP is also capable to increase the bond at fiber/matrix interface, limiting debonding under applied stress, and the fracture toughness. Furthermore, the nanofiller can also supply different functional properties to fiber-reinforced composite for specific applications, along with greater resistance to environmental agents and fire. Once again, all these aspects are particularly desirable in the field of  construction.Although the epoxy nanocomposites are more frequently employed because of their good electrical properties, they are also suggested as insulating materials ,149. SomNanostructured hybrid epoxies containing interpenetrating silica nanodomains represent a very promising solution to overcome limits and deficiencies displayed by typical epoxy-based adhesives and coatings, especially in terms of greater glass transition temperatures, superior resistance to weathering, and to harsh external conditions. Generally speaking, all nanofilled epoxy materials display improved durability than the pristine epoxy against water/moisture, temperatures, harsh environments, and fire (the addition of nanofillers can prevent flashover and the spreading of flame), allowing an extension of the lifetime of the applications in which the materials are employed.As illustrated in the previous discussion, the effectiveness of an epoxy-based nanocomposite, realized with a functional nanomaterial, mainly depends on the chemical nature and the composition of the nanofiller and on the production/application methods. The latter, in particular, are established once adequate procedures and technologies have been identified to obtain optimal performance and characteristics of the nanocomposite. Consequently, greater production temperatures, longer processing times, use of toxic chemicals, and expensive techniques may be necessary, with processes having stricter purity requirements and lower yields: all these choices are feasible only for the production of high values components . Furthermore, some methods/procedures are quite difficult to transfer from laboratory to industrial scale.Different technological limits can be experienced in the production of polymer-based nanocomposites. The importance of the achievement of a fine and uniform dispersion of the nanofiller in the resin matrix has been widely discussed. The most widely used and economic method for the preparation of a nanocomposite is to simply disperse the nanofiller in the liquid resin, but this procedure is not always effective. In addition, when very low amounts of nanoparticles are added to the epoxy, it is still possible to employ the traditional methods for the production/application of the nanocomposite. On the other hand, since the viscosity of the nanofiller/resin suspension appreciably grows at high loadings of the nanoparticles, more sophisticated and expensive techniques must be used in the latter cases for the production of nanocomposites . Once agAnalogously, the experimental routes proposed for the functionalization of the nanofiller surface frequently request specially customized or finely synthesized chemicals or solvents while the adoption of cheap and commercially available chemical products would be more favored by industry, thus accelerating the scale-up manufacture.Last, but not least, several concerns are associated with health issues and environmental impacts of such nanomaterials during their whole “life”, according to life cycle analysis (LCA) concepts. In the production of nanofillers, there is wide use of toxic compounds, acid/basic chemicals, and organic solvents. Furthermore, most of the formulations are proprietary of the suppliers, being not completely revealed their composition and potential hazards on humans, animals, and the environment . The cheTg, and degree of cross-linking of the epoxy nanocomposite are also influenced by the kind of nanofiller, its content, and dimensional features. The huge potential of epoxy-based nanocomposites in many different applications has been described, providing some examples of the possible industrial uses of these nanomaterials. The main technological limits in the production of epoxy-based nanocomposites and their impact on human health and environment have been finally illustrated.In this paper, an overview of the very recent papers (from 2016) that have appeared in the literature dealing with the main current developments and progress in the field of epoxy-based nanocomposites has been presented. The different nanofillers able to provide  improved properties to epoxies or to mitigate some of their well-known deficiencies have been introduced, highlighting the role played by the chemical nature of the nanoparticles, their size and aspect ratio, and their content in the epoxy. A different class of nanocomposite, i.e., the organic–inorganic hybrid systems, with the organic epoxy phase interconnected at the nano-scale level to the inorganic one, has been also illustrated. The reviewed papers have demonstrated that the development of an excellently performing nanocomposite based on epoxy resin, as well as on any other polymeric matrix, relies on the uniform dispersion of nanoparticles and on their interactions with the epoxy matrix; chemical modifications of the surface of the nanofillers allow achievement of these goals, but they are also able to alter the curing kinetics of the epoxy resin. The rate of advancement of cross-linking reactions, the final Smart epoxy nanocomposites have, then, proven to be invaluable materials for advanced applications in many fields; the exploitation of these nanomaterials in further new and demanding applications has probably not yet been revealed. However, research and development efforts should be still spent to produce safer materials, with new properties and excellent functional abilities. Thus, the production and application processes of such polymer-based nanomaterials must be carefully analyzed, identifying any route to optimize operational and safety segments.


Patients suffering from chronic lymphocytic leukemia (CLL) display highly diverse clinical courses ranging from indolent cases to aggressive disease, with genetic and epigenetic features resembling this diversity. Here, we developed a comprehensive approach combining a variety of molecular and clinical data to pinpoint translocation events disrupting long-range chromatin interactions and causing cancer-relevant transcriptional deregulation. Thereby, we discovered a B cell specific cis-regulatory element restricting the expression of genes in the associated locus, including PRMT5 and DAD1, two factors with oncogenic potential. Experimental PRMT5 inhibition identified transcriptional programs similar to those in patients with differences in PRMT5 abundance, especially MYC-driven and stress response pathways. In turn, such inhibition impairs factors involved in DNA repair, sensitizing cells for apoptosis. Moreover, we show that artificial deletion of the regulatory element from its endogenous context resulted in upregulation of corresponding genes, including PRMT5. Furthermore, such disruption renders PRMT5 transcription vulnerable to additional stimuli and subsequently alters the expression of downstream PRMT5 targets. These studies provide a mechanism of PRMT5 deregulation in CLL and the molecular dependencies identified might have therapeutic implementations. Stratification of patients into clinical risk groups is mainly based on the mutational status of the immunoglobulin variable regions, with IGHV-unmutated CLLs displaying a worse prognosis than IGHV-mutated cases1, but also changes in transcriptional networks have successfully been used to assign patients to such groups2. These different approaches together with recent therapeutic improvements like the application of monoclonal antibodies and kinase inhibitors improved prognosis, yet the overall outcome remains diverse and CLL mostly an incurable disease5.Among hematopoietic malignancies chronic lymphocytic leukemia (CLL), a disorder of mature B cells, represents one of the most common forms of neoplasms with an incidence of 4–5 cases in 100,000 per year according to the National Cancer Institute of USA 18. This allowed us to distinguish genes in close proximity to breakpoints that might be affected, from those with unaffected regulatory interactions, on which aberrations therefore most likely do not have an effect. This identified ~4,600 disrupted interactions affecting ~1,700 unique genes, 318 of which exhibited alterations in their expression larger than two times the interquartile range (IQR) for that gene across all patients. Out of these 318 genes, we found that 47 genes were recurrently deregulated by at least one IQR in two or more patients.To evaluate effects of structural variations on gene expression and tumor progression, we applied an integrative approach to find factors with deregulated expression in cancer patients which might contribute to the disease Fig. . Specifi2 with publicly available clinical and expression data and divided patients into high or low expressing groups separated by the median expression value for each of the 47 candidate factors. Survival analysis (Log-rank test) indicated 6 factors for which statistical differences in overall survival (OS) could be observed  were associated with the induction of several genes, like DAD1 and PRMT5 above our IQR threshold Fig. . These gme3 Fig. . While mels Fig. . In addiels Fig. . We founels Fig. . With th−12)  in the combined dataset Fig. . Approxi12) Fig. , indicat22  with the same expression data, we found significant transcriptional reduction of the HALLMARK gene sets for MYC- and E2F-target genes as well as those responsible for the G2/M checkpoint, similarly in our inhibitor data and the CLLE cohort Fig. . Despiteity Fig. . We nextity Fig. , supportTo further categorize regulated genes within the combined dataset of HG-3 and PGA-1 cells, we employed Reactome Pathway analysis and found expression changing genes mainly related to processes of genomic replication as well as DNA-repair/-maintenance and checkpoint control Fig. . To furtAs checkpoint and DNA repair pathways were predicted to have reduced activity, we focussed on BRCA1, as a central regulator of repair processes. Inhibition of PRMT5 caused both, reduced levels of BRCA1 mRNA as well as protein Figs.  and 3c. Finding cases of aberrant PRMT5 expression linked to the disruption of a corresponding regulatory unit in CLL, we aimed to recapitulate these genomic events to unravel a potential effect of this region on the expression of the corresponding genes. As we found the regulatory landscape in this locus to be highly similar in normal B cells, CLL donors and cell lines   Fig. . Among t-6) Fig. .In summary, the downstream molecular effects in cells that have genomically activated levels of PRMT5 resembled at least partly the expression pattern in CLL donors with high PRMT5 expression and inversely reflected what we observed upon PRMT5 inhibition.27 we successfully pinpointed cancer-relevant regulons disrupted uniquely in CLL and identified corresponding candidate genes overexpressed in the respective donors that contributed to overall prognosis. By including clinical as well as cell type specific regulatory data, we extended previous approaches, characterizing aberrant expression patterns related to non-coding genomic alterations and involved in tumor progression31. Among the identified candidates, we focused on the PRMT5 containing locus and a possible mechanism of its deregulation.Through integrative analysis of multiple kinds of large scale and patient derived data13, it displays oncogenic functions in various cancers33. PRMT5 regulating mechanisms involve CDK4-activity34; more recently BCR-signaling in germinal center B cells and in cases of diffuse large B cell lymphoma (DLBCL) has been described to trigger increased PRMT5 expression35. As the molecular events at the PRMT5 locus and how they deregulate associated expression during cellular transformation remain still elusive, our findings might represent a more common mechanism of PRMT5 deregulation in B cell lymphomas. This might have remained unnoticed so far, due to the lack of whole genome studies in patient cohorts large enough to detect this disruption of the upstream regulatory site. Due to its prominent role in hematopoiesis, PRMT5 might contribute to lineage definition, and as such a gene it might show high regulatory complexity via multiple enhancer interactions37.While a crucial function of PRMT5 has been described in hematopoietic progenitors and during the germinal center reaction39. Additionally, the loss of this upstream regulatory region apparently increased the sensitivity of the locus to transcriptional perturbations caused by particular external stimuli, adding another potential layer of transcriptional control. Downstream transcriptional regulation mediated by PRMT5 showed diverging effects with processes of stress response and DNA-replication maintained by PRMT5 and lymphocyte related pathways suppressed. Among the genes activated upon PRMT5 inhibition were PIK3IP1, FCRL5 and KLF2, which became correspondingly repressed with higher PRMT5 levels. In B cells these genes have mainly been associated with differentiation processes, therefore this could indicate a restriction of lineage commitment mediated by PRMT541.In line with our observations of concordant PRMT5 and DAD1 deregulation, regulatory complexity within a locus can increase further by simultaneous interactions of different genes with the same regulatory site43. Blocking PRMT5 activity in our case, however, resulted in a reduction of BRCA1 levels as well as of additional components of different repair pathways. Thereby, we could prime cells to undergo apoptosis upon additional treatment with the PARP-inhibitor Veliaparib. Inhibitors of Poly(ADP-ribose) polymerases have been used to exploit DNA-repair defects and as therapeutic strategy in BRCA-deficient cancers45. Sensitizing cells for such inhibitors through the inhibition of PRMT5 might provide additional possibilities of treatment. Also, PRMT5 maintained the expression of the AP1 protein BATF3, which has been described as an upstream MYC-activator in Hodgkin lymphomas and T cell leukemia27 and is known to cause lymphomas of mature B cells in mice46. Our thorough transcriptional analysis identified further molecular interactions with the MYC transcriptional network, supported by additional analysis of clinical data, showing CLL patients with high PRMT5 expression to display enhanced MYC activity. In turn high MYC levels seemed to protect from the effects of PRMT5 inhibition in our system. MYC has long been known as a crucial oncogene in multiple tumor types, including several B-NHLs, like DLBCL or Burkitt lymphoma49. In CLL few cases of inactivated MYC-antagonists, like MGA, PTPN11 or FUBP1 have been described50 postulating an important function of the MYC network in this disease. We identified MXD4, an additional antagonist of MYC being repressed by PRMT5, thereby providing a possible explanation for the observed requirement of MYC driven tumors for PRMT5 expression10, beyond its role in maintaining MYC and CCND1 expression via activating WNT-signaling51. In turn, signaling via AKT/MYC forms a feedforward-loop enhancing PRMT5 function35, fitting our observation of high MYC levels apparently having a protective effect on the physiological consequences of PRMT5 inhibition in CLL and MCL cell lines.Besides the expression of growth enhancing pathways, we found PRMT5 to maintain DNA repair pathways, which is in line with reports of PRMT5 affecting DNA repair by influencing the splicing of repair factorsPRMT5 gained increasing attention due to its oncogenic functions, while little is known about its initial deregulation during cellular transformation. By reconstructing an event of genomic deregulation for PRMT5 in CLL, we validated our strategy to identify deregulated cancer driving factors in a highly cell type specific manner and contributed to further understanding of PRMT5 function in chronic lymphocytic leukemia.2 unless stated otherwise and authenticated by fingerprinting at the DSMZ , cultivated as follows and split every 2–3 days: DAUDI (ACC-78), HG-3 (ACC-765), JEKO-1 (ACC-553), JVM-2 (ACC-12), JVM-13 (ACC-13), MINO (ACC-687), PGA-1 (ACC-766), RAMOS (ACC-603), REC-1 (ACC-584), VAL (ACC-586), WA-C3CD5+ (ACC-769) cells were grown in RPMI 1640 medium with 10% fetal calf serum (FCS) and MEC-1 (ACC-497) in Iscove’s MDM with 10% FCS. 293 T (ACC-635) cells were grown in Dulbecco’s MEM with 10% FCS and at 10% CO2. Inhibition of PRMT5 enzymatic activity was achieved with the indicated doses of EPZ015666  for 96 h. Additional treatments were performed with the following chemicals: Veliparib , SAHA , JQ1 , Chaetocin .All used cell lines were held at 37 °C and 5% CO4 cells per well and cell line were seeded in triplicates into 96 well plates and confluence of the whole well was monitored over a total of 96 h every 6 h with the IncuCyte live cell imager (Essen BioScience). Metabolic analyses were performed with a standardized MTT-assay -2, 5-diphenyltetrazolium bromide; Sigma). Cells were grown in the presence of MTT for 4 h and measured in a Spark microplate-reader (Tecan). Induction of apoptosis was monitored with the IncuCyte Caspase-3/7 Red Reagent  for the indicated time and according to manufacturer’s instructions.For the analysis of growth rates 1052 . Lentiviral particles were generated by simultaneous transfection of the gRNA construct with pCMV-VSV-G53  and psPAX2, a kind gift of D. Trono’s lab  into 293 T cells with SuperFect (Qiagen). Target PGA-1 cells were spin-infected on two consecutive days at 800 g for 30 min at 30 °C in the presence of 3 μg/mL polybrene. Selection of transduction positive clones was done by the addition of 0.2 µg/mL puromycin (Sigma). Serial dilutions were performed to isolate individual clones, which were screened for the correct rearrangement with direct PCR (TaKaRa)  and phosphatase inhibitors (Roche). Protein concentrations were measured with Coomassie Blue Bradford Reagent  and equal amounts of protein were separated on 6–15% polyacrylamide gels and transferred onto PVDF membranes using a mini-Protean system (Biorad). Membranes were blocked in 5% BSA or 3% dry milk powder in TBS + 0.5% Tween-20 and incubated with the following primary antibodies overnight: rabbit polyclonal PRMT5 ; rabbit polyclonal DAD1 ; rabbit polyclonal OXA1L ; rabbit polyclonal MXD4 ; mouse monoclonal c-Myc ; rabbit polyclonal BRCA1 ; mouse monoclonal GAPDH , mouse monoclonal alpha-tubulin . Washing and staining with either anti-rabbit or anti-mouse secondary antibodies linked to HRP  followed. Detection was done with Clarity Max Western ECL reagent (Biorad) and the digital ChemoStar Imager (INTAS).Western blot quantifications were performed using the ImageJ software by measuring the mean intensity of each band, performing background subtraction and normalization to the indicated control protein band. Where indicated, the derived values were further divided by the respective control condition. Each experiment was done in independent replicates as indicated and median values are shown together with corresponding SD.For the analysis of transcript abundance, total RNA was isolated with RNAeasy Kit (Qiagen) and 1 µg of total RNA was subjected to reverse transcription using the Superscript II First strand synthesis kit with random primers (Invitrogen). Semi-quantitative PCR was performed for the indicated genes in a 7500 Fast Real-time PCR System (Applied Biosystems) with the SsoFast EvaGreen Supermix (Biorad). Primers are listed in Supplemental Table 19: around 200.000 cells per reaction were incubated overnight at 4 °C in antibody-buffer containing 0.05% digitonin (Millipore) and one of the following antibodies: rabbit polyclonal CTCF , rabbit polyclonal H3K4me3 , rabbit polyclonal H3K27ac , rabbit polyclonal H3K27me3 , after three washsteps, samples were incubated with proteinA-NMase fusion protein (a kind gift of Epicypher) for 1 h at 4 °C. Cleavage around bound target proteins was done at 0 °C for 5 min by addition of calciumchlorid to a final concentration of 10 mM. After removal of the buffer, reaction was stopped by adding 20 mM EGTA in STOP buffer and subsequent release of cut DNA-fragments at 37 °C, followed by phenol-chloroform extraction. For global analysis of binding-sites, 5 ng precipitated DNA bound to each factor of interest was subjected to library preparation with the KAPA Hyperprep Kit . Indexed adapters were used to pool samples for 75 bp single-end runs on an Illumina NextSeq500. Following sequencing, fastq generation and demultiplexing was done via bcl2fastq .Cleavage Under Target and Release Using Nuclease (CUT&RUN) was done according to the protocol published by the Henikoff lab with slight modifications54 to the Gencode Homo sapiens genome (v26) and converted/sorted via samtools (0.1.19)55. For expression analysis the reads were counted to each gene via HTSeq-count python script (0.8.0)56. Data was processed and analyzed in the R/Bioconductor environment (3.4.4/3.6). Normalization, estimation of dispersions and testing for differentially expressed genes based on a test assuming negative binomial data distribution was computed via DESeq. 2 (1.18.1)57 and enriched by ensembl v89 annotations. Batch effects were considered in the gene expression model and p-values were FDR-corrected. GO annotation and Reactome Pathway analysis were done with the R/bioconductor packages clusterProfiler (3.12.0)58 and ReactomePA (1.28.0)59, respectively. Additional pathway analysis was done using Ingenuity Pathway Analysis (Qiagen) and GSEA60. Prediction of MYC activity using the DoRhotEA algorithm was described previously23. Graphical representation of genomic loci and data from NGS was generated with ggplot2 (3.2.0) and GVIZ (1.28.0)61.Following sequencing and fastq generation, demultiplexing was done via bcl2fastq . Reads were trimmed via fastq-mcf (ea-utils 1.04.807) and quality controlled via FastQC (v0.11.5). For gene expression and CUT&RUN-seq analysis reads were aligned by STAR (2.5.3a)https://dcc.icgc.org/api/v1/download?fn=/release_22/Projects/CLLE-ES/). Regions of genomic aberrations were extracted by extending each chromosomal breakpoint by +/− 50 bp, maintaining respective donor IDs. Genomic coordinates were transferred to hg38 with the UCSC-liftover tool. Similarly, promoter-interactions from PrHi-C were taken from supplementary data from Javierre et al.18 and genomic coordinates of individual regions were transferred to the same genomic build. Candidate genes were called, if genomic breakpoints located within promoter-interactions of individual genes. For each of such genes, we calculated the DESeq2-normalized median expression and its interquartile range (IQR) across all donors for which data on structural variations was available. Subsequently, we filtered for donors with expression changes in such candidate genes > 2x IQR and with a normalized expression> 10. Additional filtering was done by checking the occurrence of similar disrupted promoter-interactions in at least one additional donor with the corresponding expressional change > 1x IQR. Finally, the derived list of candidates was checked for prognostic effects. Therefore, all donors of the ICGC-CLLE cohort (n = 291), together with the donors described in Herold et al.2 (n = 107) were grouped according to their expression level of each of the candidate genes and overall survival in the groups of high versus low expressing donors was estimated by Log-rank test, setting a Benjamini-Hochberg corrected padj <5 * 10−2 as threshold and visualized as Kaplan-Meier plots .Data on structural variations from the ICGC CLLE project (v22) was downloaded from the ICGC-web portal (Sequencing data generated during this study has been deposited at the European Nucleotide Archive and entries can be found via ArrayExpress under E-MTAB-8219 for RNA-seq data and E-MTAB-8203 for CUT&RUN-seq.Supplementary information.Supplementary information 2.