Reciprocal regulation of Daxx and PIK3CA promotes colorectal cancer cell growth.

Upregulation of death-domain-associated protein (Daxx) is strongly associated with diverse cancer types. Among these, the clinicopathological significance and molecular mechanisms of Daxx overexpression in colorectal cancer (CRC) remain unknown. Here, we showed that Daxx expression was increased in both clinical CRC samples and CRC cell lines. Daxx knockdown significantly reduced proliferation activity in CRC cells and tumor growth in a xenograft model. Further studies revealed that Daxx expression could be attenuated by either treatment with the PIK3CA inhibitor PIK-75 or PIK3CA depletion in CRC cells. Conversely, expression of PIK3CA constitutively active mutants could increase Daxx expression. These data suggest that PIK3CA positively regulates Daxx expression. Consistently, the expression levels of PIK3CA and Daxx were positively correlated in sporadic CRC samples. Interestingly, Daxx knockdown or overexpression yielded decreased or increased levels of PIK3CA, respectively, in CRC cells. We further demonstrated that Daxx activates the promoter activity and expression of PIK3CA. Altogether, our results identify a mechanistic pathway of Daxx overexpression in CRC and suggest a reciprocal regulation between Daxx and PIK3CA for CRC cell growth.

A PIAS1 Protective Variant S510G Delays polyQ Disease Onset by Modifying Protein Homeostasis.

BACKGROUND: Polyglutamine (polyQ) diseases are dominant neurodegenerative diseases caused by an expansion of the polyQ-encoding CAG repeats in the disease-causing gene. The length of the CAG repeats is the major determiner of the age at onset (AO) of polyQ diseases, including Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3). OBJECTIVE: We set out to identify common genetic variant(s) that may affect the AO of polyQ diseases. METHODS: Three hundred thirty-seven patients with HD or SCA3 were enrolled for targeted sequencing of 583 genes implicated in proteinopathies. In total, 16 genes were identified as containing variants that are associated with late AO of polyQ diseases. For validation, we further investigate the variants of PIAS1 because PIAS1 is an E3 SUMO (small ubiquitin-like modifier) ligase for huntingtin (HTT), the protein linked to HD. RESULTS: Biochemical analyses revealed that the ability of PIAS1S510G to interact with mutant huntingtin (mHTT) was less than that of PIAS1WT , resulting in lower SUMOylation of mHTT and lower accumulation of insoluble mHTT. Genetic knock-in of PIAS1S510G in a HD mouse model (R6/2) ameliorated several HD-like deficits (including shortened life spans, poor grip strength and motor coordination) and reduced neuronal accumulation of mHTT. CONCLUSIONS: Our findings suggest that PIAS1 is a genetic modifier of polyQ diseases. The naturally occurring variant, PIAS1S510G , is associated with late AO in polyQ disease patients and milder disease severity in HD mice. Our study highlights the possibility of targeting PIAS1 or pathways governing protein homeostasis as a disease-modifying approach for treating patients with HD. © 2021 International Parkinson and Movement Disorder Society.

Expression of the AHPND Toxins PirAvp and PirBvp Is Regulated by Components of the Vibrio parahaemolyticus Quorum Sensing (QS) System.

Acute hepatopancreatic necrosis disease (AHPND) in shrimp is caused by Vibrio strains that harbor a pVA1-like plasmid containing the pirA and pirB genes. It is also known that the production of the PirA and PirB proteins, which are the key factors that drive the observed symptoms of AHPND, can be influenced by environmental conditions and that this leads to changes in the virulence of the bacteria. However, to our knowledge, the mechanisms involved in regulating the expression of the pirA/pirB genes have not previously been investigated. In this study, we show that in the AHPND-causing Vibrio parahaemolyticus 3HP strain, the pirAvp and pirBvp genes are highly expressed in the early log phase of the growth curve. Subsequently, the expression of the PirAvp and PirBvp proteins continues throughout the log phase. When we compared mutant strains with a deletion or substitution in two of the quorum sensing (QS) master regulators, luxO and/or opaR (luxOD47E, ΔopaR, ΔluxO, and ΔopaRΔluxO), our results suggested that expression of the pirAvp and pirBvp genes was related to the QS system, with luxO acting as a negative regulator of pirAvp and pirBvp without any mediation by opaRvp. In the promoter region of the pirAvp/pirBvp operon, we also identified a putative consensus binding site for the QS transcriptional regulator AphB. Real-time PCR further showed that aphBvp was negatively controlled by LuxOvp, and that its expression paralleled the expression patterns of pirAvp and pirBvp. An electrophoretic mobility shift assay (EMSA) showed that AphBvp could bind to this predicted region, even though another QS transcriptional regulator, AphAvp, could not. Taken together, these findings suggest that the QS system may regulate pirAvp/pirBvp expression through AphBvp.

Isotypes of autoantibodies against novel differential 4-hydroxy-2-nonenal-modified peptide adducts in serum is associated with rheumatoid arthritis in Taiwanese women.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disorder with systemic inflammation and may be induced by oxidative stress that affects an inflamed joint. Our objectives were to examine isotypes of autoantibodies against 4-hydroxy-2-nonenal (HNE) modifications in RA and associate them with increased levels of autoantibodies in RA patients. METHODS: Serum samples from 155 female patients [60 with RA, 35 with osteoarthritis (OA), and 60 healthy controls (HCs)] were obtained. Four novel differential HNE-modified peptide adducts, complement factor H (CFAH)1211-1230, haptoglobin (HPT)78-108, immunoglobulin (Ig) kappa chain C region (IGKC)2-19, and prothrombin (THRB)328-345, were re-analyzed using tandem mass spectrometric (MS/MS) spectra (ProteomeXchange: PXD004546) from RA patients vs. HCs. Further, we determined serum protein levels of CFAH, HPT, IGKC and THRB, HNE-protein adducts, and autoantibodies against unmodified and HNE-modified peptides. Significant correlations and odds ratios (ORs) were calculated. RESULTS: Levels of HPT in RA patients were greatly higher than the levels in HCs. Levels of HNE-protein adducts and autoantibodies in RA patients were significantly greater than those of HCs. IgM anti-HPT78-108 HNE, IgM anti-IGKC2-19, and IgM anti-IGKC2-19 HNE may be considered as diagnostic biomarkers for RA. Importantly, elevated levels of IgM anti-HPT78-108 HNE, IgM anti-IGKC2-19, and IgG anti-THRB328-345 were positively correlated with the disease activity score in 28 joints for C-reactive protein (DAS28-CRP). Further, the ORs of RA development through IgM anti-HPT78-108 HNE (OR 5.235, p < 0.001), IgM anti-IGKC2-19 (OR 12.655, p < 0.001), and IgG anti-THRB328-345 (OR 5.761, p < 0.001) showed an increased risk. Lastly, we incorporated three machine learning models to differentiate RA from HC and OA, and performed feature selection to determine discriminative features. Experimental results showed that our proposed method achieved an area under the receiver operating characteristic curve of 0.92, which demonstrated that our selected autoantibodies combined with machine learning can efficiently detect RA. CONCLUSIONS: This study discovered that some IgG- and IgM-NAAs and anti-HNE M-NAAs may be correlated with inflammation and disease activity in RA. Moreover, our findings suggested that IgM anti-HPT78-108 HNE, IgM anti-IGKC2-19, and IgG anti-THRB328-345 may play heavy roles in RA development.

SUMOylation of XRCC1 activated by poly (ADP-ribosyl)ation regulates DNA repair.

XRCC1 is an essential scaffold protein for base excision repair (BER) and helps to maintain genomic stability. XRCC1 has been indicated as a substrate for small ubiquitin-like modifier modification (SUMOylation); however, how XRCC1 SUMOylation is regulated in cells and how SUMOylated XRCC1 regulates BER activity are not well understood. Here, we show that SUMOylation of XRCC1 is regulated in cells under methyl-methanesulfonate (MMS) treatment and facilitates BER. Poly(ADP-ribose) polymerase 1 (PARP1) is activated by MMS immediately and synthesizes poly(ADP-ribose) (PAR), which in turn promotes recruitment of SUMO E3 TOPORS to XRCC1 and facilitates XRCC1 SUMOylation. A SUMOylation-defective mutant of XRCC1 had lower binding activity for DNA polymerase beta (POLB) and was linked to a lower capacity for repair of MMS-induced DNA damages. Our study therefore identified a pathway in which DNA damage-induced poly(ADP-ribosyl)ation (PARylation) promotes SUMOylation of XRCC1, which leads to more efficient recruitment of POLB to complete BER.

Tandem Mach Zehnder Directional Coupler Design and Simulation on Silicon Platform for Optical Coherence Tomography Applications.

We design and compare the splitting ratio wavelength flatness of directional coupler (DC), Mach-Zehnder directional coupler (MZDC), and tandem MZDC. All coupler responses are analyzed, and tandem MZDC performance is the best in the wavelength insensitivity compared with the other two. An MZDC with any coupling ratio could be utilized to match the maximum flatness in a 40-nm wavelength range. To extend a broad flatness range, the tandem MZDC is proposed and still follows the Mach Zehnder structure taking two MZDCs as couplers connected through a decoupled region. Unlike DC, MZDC with the flat wavelength response has a non-linear output phase. Hence, using two wavelength-insensitive MZDCs as the coupling function in a tandem MZDC could demonstrate a more extensive decoupled phase term to maximize the flat wavelength response. The tandem MZDC theoretically demonstrates the splitting ratio with 100-nm flatness in the wavelength range from 1250 nm to 1350 nm. Finally, a point spread function through the tandem MZDC shows a 24-dB signal-to-noise ratio improvement in optical coherence tomography applications.

Structural and functional roles of Daxx SIM phosphorylation in SUMO paralog-selective binding and apoptosis modulation.

Small ubiquitin-like modifier (SUMO) conjugation and interaction are increasingly associated with various cellular processes. However, little is known about the cellular signaling mechanisms that regulate proteins for distinct SUMO paralog conjugation and interactions. Using the transcriptional coregulator Daxx as a model, we show that SUMO paralog-selective binding and conjugation are regulated by phosphorylation of the Daxx SUMO-interacting motif (SIM). NMR structural studies show that Daxx (732)E-I-I-V-L-S-D-S-D(740) is a bona fide SIM that binds to SUMO-1 in a parallel orientation. Daxx-SIM is phosphorylated by CK2 kinase at residues S737 and S739. Phosphorylation promotes Daxx-SIM binding affinity toward SUMO-1 over SUMO-2/3, causing Daxx preference for SUMO-1 conjugation and interaction with SUMO-1-modified factors. Furthermore, Daxx-SIM phosphorylation enhances Daxx to sensitize stress-induced cell apoptosis via antiapoptotic gene repression. Our findings provide structural insights into the Daxx-SIM:SUMO-1 complex, a model of SIM phosphorylation-enhanced SUMO paralog-selective modification and interaction, and phosphorylation-regulated Daxx function in apoptosis.

Quantitative Analysis of Glucose Metabolic Cleavage in Glucose Transporters Overexpressed Cancer Cells by Target-Specific Fluorescent Gold Nanoclusters.

The glucose metabolism rate in cancer cells is a crucial piece of information for the cancer aggressiveness. A feasible method to monitor processes of oncogenic mutations has been demonstrated in this work. The fluorescent gold nanoclusters conjugated with glucose (glucose-AuNCs) were successfully synthesized as a cancer-targeting probe for glucose transporters (Gluts) overexpressed by U-87 MG cancer cells, which can be observed under confocal microscopy. The structural and optical characterizations of fluorescent glucose-AuNCs were confirmed by transmission electron microscope (TEM) and Fourier transform infrared spectroscopy (FTIR). The MTT assay exhibited the high biocompatibility of water-soluble glucose-AuNCs for further biomedical applications. The glucose metabolic cleavage of glucose-AuNCs by glycolytic enzymes from U-87 MG cancer cell was measured by fluorescence change of glucose-AuNCs. The fluorescence change based on the integrated area under fluorescence spectra ( A t) of glucose-AuNCs was plotted as a function of different reaction time ( t) with glycolytic enzymes. The fitted curve of A t versus t showed the first-order kinetics to explain the mechanism of glucose metabolic cleavage rate of glucose-AuNCs by glycolytic enzymes. The rate constant k could be utilized to determine the glucose metabolism rate of glucose-AuNCs for the quantitative analysis of cancer aggressiveness. Our work provides a practical application of target-specific glucose-AuNCs as a fluorescence probe to analyze the glucose metabolism in Gluts overexpressed cancer cells.

Serum ApoA4 levels predicted the progression of renal impairment in T2DM.

BACKGROUND: Among multiple causes, diabetic nephropathy (DN) is the major underlying renal disease that leads to end-stage renal disease (ESRD), and early diagnosis can effectively prevent or delay the progression to ESRD. Therefore, the current study aimed to develop noninvasive, accurate detection markers. MATERIALS & METHODS: For this study, 62 diabetes mellitus (DM) patients, 59 DN patients and 21 healthy controls (HCs) were recruited. All participants' serum samples were subjected to concavanalin (Con) A affinity chromatography, which utilizes glycoproteins to discover potential markers. RESULTS: From nano LC-MS and Western blot analysis, apolipoprotein A-IV (ApoA4) was selected which featured a gradual, almost twofold increase in the order of HC, DM and DN. In the Con A-based ELISA, the DM group was 1.91-fold higher than the HC group, while the DN group was 2.56-fold higher than the HCs and 1.33-fold higher than the DM group. In addition, significant positive correlations were observed between ApoA4 and blood urea nitrogen levels and between ApoA4 and creatine levels, while significant negative correlations were seen between serum protein levels and between serum albumin levels in comparisons of DM and DN samples. CONCLUSIONS: Serum Con A-bound ApoA4 levels were higher in the DM group than in HCs, and further increased in the DN group. Levels of ApoA4 were positively correlated with blood urea nitrogen and creatine, but negatively correlated with serum protein and albumin. This evidence supports serum Con A-bound ApoA4 as a circulating marker for predicting the progression of renal impairment in DM patients.

Ubc9 acetylation modulates distinct SUMO target modification and hypoxia response.

While numerous small ubiquitin-like modifier (SUMO) conjugated substrates have been identified, very little is known about the cellular signalling mechanisms that differentially regulate substrate sumoylation. Here, we show that acetylation of SUMO E2 conjugase Ubc9 selectively downregulates the sumoylation of substrates with negatively charged amino acid-dependent sumoylation motif (NDSM) consisting of clustered acidic residues located downstream from the core ψ-K-X-E/D consensus motif, such as CBP and Elk-1, but not substrates with core ψ-K-X-E/D motif alone or SUMO-interacting motif. Ubc9 is acetylated at residue K65 and K65 acetylation attenuates Ubc9 binding to NDSM substrates, causing a reduction in NDSM substrate sumoylation. Furthermore, Ubc9 K65 acetylation can be downregulated by hypoxia via SIRT1, and is correlated with hypoxia-elicited modulation of sumoylation and target gene expression of CBP and Elk-1 and cell survival. Our data suggest that Ubc9 acetylation/deacetylation serves as a dynamic switch for NDSM substrate sumoylation and we report a previously undescribed SIRT1/Ubc9 regulatory axis in the modulation of protein sumoylation and the hypoxia response.

Two Interrogated FBG Spectral Linewidth for Strain Sensing through Correlation.

The spectral linewidth from two cross-correlated fiber Bragg gratings (FBGs) are interrogated and characterized using a delayed self-homodyne method for fiber strain sensing. This approach employs a common higher frequency resolution instead of wavelength. A sensitivity and resolution of 166 MHz/µÎµ and 50 nε were demonstrated from 4 GHz spectral linewidth characterization on the electric spectrum analyzer. A 10 nε higher resolution can be expected through random noise analyses when the spectral linewidth from two FBG correlations is reduced to 1 GHz. Moreover, the FBG spectrum is broadened during strain and experimentally shows a 0.44 pm/µÎµ sensitivity, which is mainly caused by the photo elastic effect from the fiber grating period stretch.

Low Levels of IgG Recognizing the α-1-Antitrypsin Peptide and Its Association with Taiwanese Women with Primary Sjögren's Syndrome.

The aim of this study was to examine oxidative stress and low level of α-1-antitrypsin (A1AT) in primary Sjögren's syndrome (pSS), and evaluate the associated autoreactivity against unmodified and their 4-hydroxy-2-nonenal (HNE)-modified peptides with pSS. Two differentially expressed proteins, α-1-acid glycoprotein 1 (A1AG1) and A1AT, exhibited 2-fold differences, and their HNE modifications were identified by depleted-albumin and immunoglobulin G (IgG) serum protein, in-solution digestion, in-gel digestion, and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) from pSS patients and age-matched healthy controls (HCs). Furthermore, levels of proteins, confirmation of HNE modifications, HNE-protein adducts and autoreactivity against unmodified and their HNE-modified peptides were further validated. Levels of the HNE-protein adduct and A1AG1 were significantly higher in pSS patients than HCs, but levels of A1AT were significantly lower in pSS patients compared to HCs. Only the HNE modification of A1AT was confirmed. Our study suggests that elevated HNE-protein adduct, oxidative stress, level (odds ratio (OR) 4.877, p = 0.003), lowered A1AT level (OR 3.910, p = 0.010) and a decreased level of anti-A1AT50-63 IgG (OR 3.360, p = 0.010) showed an increased risk in pSS patients compared to HCs, respectively.

An arginine-rich motif of ring finger protein 4 (RNF4) oversees the recruitment and degradation of the phosphorylated and SUMOylated Krüppel-associated box domain-associated protein 1 (KAP1)/TRIM28 protein during genotoxic stress.

Krüppel-associated box domain-associated protein 1 (KAP1) is a universal transcriptional corepressor that undergoes multiple posttranslational modifications (PTMs), including SUMOylation and Ser-824 phosphorylation. However, the functional interplay of KAP1 PTMs in regulating KAP1 turnover during DNA damage response remains unclear. To decipher the role and cross-talk of multiple KAP1 PTMs, we show here that DNA double strand break-induced KAP1 Ser-824 phosphorylation promoted the recruitment of small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, ring finger protein 4 (RNF4), and subsequent RNF4-mediated, SUMO-dependent degradation. Besides the SUMO interacting motif (SIM), a previously unrecognized, but evolutionarily conserved, arginine-rich motif (ARM) in RNF4 acts as a novel recognition motif for selective target recruitment. Results from combined mutagenesis and computational modeling studies suggest that RNF4 utilizes concerted bimodular recognition, namely SIM for Lys-676 SUMOylation and ARM for Ser(P)-824 of simultaneously phosphorylated and SUMOylated KAP1 (Ser(P)-824-SUMO-KAP1). Furthermore, we proved that arginines 73 and 74 within the ARM of RNF4 are required for efficient recruitment to KAP1 or accelerated degradation of promyelocytic leukemia protein (PML) under stress. In parallel, results of bimolecular fluorescence complementation assays validated the role of the ARM in recognizing Ser(P)-824 in living cells. Taken together, we establish that the ARM is required for RNF4 to efficiently target Ser(P)-824-SUMO-KAP1, conferring ubiquitin Lys-48-mediated proteasomal degradation in the context of double strand breaks. The conservation of such a motif may possibly explain the requirement for timely substrate selectivity determination among a myriad of SUMOylated proteins under stress conditions. Thus, the ARM dynamically regulates the SIM-dependent recruitment of targets to RNF4, which could be critical to dynamically fine-tune the abundance of Ser(P)-824-SUMO-KAP1 and, potentially, other SUMOylated proteins during DNA damage response.

Structural analysis of poly-SUMO chain recognition by the RNF4-SIMs domain.

The E3 ubiquitin ligase RNF4 (RING finger protein 4) contains four tandem SIM [SUMO (small ubiquitin-like modifier)-interaction motif] repeats for selective interaction with poly-SUMO-modified proteins, which it targets for degradation. We employed a multi-faceted approach to characterize the structure of the RNF4-SIMs domain and the tetra-SUMO2 chain to elucidate the interaction between them. In solution, the SIM domain was intrinsically disordered and the linkers of the tetra-SUMO2 were highly flexible. Individual SIMs of the RNF4-SIMs domains bind to SUMO2 in the groove between the ß2-strand and the α1-helix parallel to the ß2-strand. SIM2 and SIM3 bound to SUMO with a high affinity and together constituted the recognition module necessary for SUMO binding. SIM4 alone bound to SUMO with low affinity; however, its contribution to tetra-SUMO2 binding avidity is comparable with that of SIM3 when in the RNF4-SIMs domain. The SAXS data of the tetra-SUMO2-RNF4-SIMs domain complex indicate that it exists as an ordered structure. The HADDOCK model showed that the tandem RNF4-SIMs domain bound antiparallel to the tetra-SUMO2 chain orientation and wrapped around the SUMO protamers in a superhelical turn without imposing steric hindrance on either molecule.

SUMOylation of Blimp-1 is critical for plasma cell differentiation.

Transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) is a master regulator of plasma cell differentiation. Here we show that Blimp-1 is covalently modified by SUMO1 at lysine 816, a modification mediated by SUMO E3 ligase PIAS1. Mutation of Blimp-1 lysine 816 reduces transcriptional repression--correlating with a reduced interaction with a histone deacetylase, HDAC2--and impairs differentiation of antibody-secreting cells. Thus, the SUMO pathway critically regulates Blimp-1 function during plasma cell differentiation.

LOXL1-AS1 contributes to metastasis in sonic-hedgehog medulloblastoma by promoting cancer stem-like phenotypes.

BACKGROUND: Medulloblastomas (MBs) are one of the most common malignant brain tumor types in children. MB prognosis, despite improvement in recent years, still depends on clinical and biological risk factors. Metastasis is the leading cause of MB-related deaths, which highlights an unmet need for risk stratification and targeted therapy to improve clinical outcomes. Among the four molecular subgroups, sonic-hedgehog (SHH)-MB harbors clinical and genetic heterogeneity with a subset of high-risk cases. Recently, long non-coding (lnc)RNAs were implied to contribute to cancer malignant progression, but their role in MB remains unclear. This study aimed to identify pro-malignant lncRNAs that have prognostic and therapeutic significance in SHH-MB. METHODS: The Daoy SHH-MB cell line was engineered for ectopic expression of MYCN, a genetic signature of SHH-MB. MYCN-associated lncRNA genes were identified using RNA-sequencing data and were validated in SHH-MB cell lines, MB tissue samples, and patient cohort datasets. SHH-MB cells with genetic manipulation of the candidate lncRNA were evaluated for metastatic phenotypes in vitro, including cell migration, invasion, sphere formation, and expressions of stemness markers. An orthotopic xenograft mouse model was used to evaluate metastasis occurrence and survival. Finally, bioinformatic screening and in vitro assays were performed to explore downstream mechanisms. RESULTS: Elevated lncRNA LOXL1-AS1 expression was identified in MYCN-expressing Daoy cells and MYCN-amplified SHH-MB tumors, and was significantly associated with lower survival in SHH-MB patients. Functionally, LOXL1-AS1 promoted SHH-MB cell migration and cancer stemness in vitro. In mice, MYCN-expressing Daoy cells exhibited a high metastatic rate and adverse effects on survival, both of which were suppressed under LOLX1-AS1 perturbation. Integrative bioinformatic analyses revealed associations of LOXL1-AS1 with processes of cancer stemness, cell differentiation, and the epithelial-mesenchymal transition. LOXL1-AS1 positively regulated the expression of transforming growth factor (TGF)-ß2. Knockdown of TGF-ß2 in SHH-MB cells significantly abrogated their LOXL1-AS1-mediated prometastatic functions. CONCLUSIONS: This study proved the functional significance of LOXL1-AS1 in SHH-MB metastasis by its promotion of TGF-ß2-mediated cancer stem-like phenotypes, providing both prognostic and therapeutic potentials for targeting SHH-MB metastasis.

RRM2 inhibition alters cell cycle through ATM/Rb/E2F1 pathway in atypical teratoid rhabdoid tumor.

BACKGROUND: Atypical teratoid rhabdoid tumor (ATRT) is an aggressive brain tumor that mainly affects young children. Our recent study reported a promising therapeutic strategy to trigger DNA damage, impede homologous recombination repair, and induce apoptosis in ATRT cells by targeting ribonucleotide reductase regulatory subunit M2 (RRM2). COH29, an inhibitor of RRM2, effectively reduced tumor growth and prolonged survival in vivo. Herein, we explored the underlying mechanisms controlling these functions to improve the clinical applicability of COH29 in ATRT. METHODS: Molecular profiling of ATRT patients and COH29-treated cells was analyzed to identify the specific signaling pathways, followed by validation using a knockdown system, flow cytometry, q-PCR, and western blot. RESULTS: Elevated E2F1 and its signaling pathway were correlated with poor prognosis. RRM2 inhibition induced DNA damage and activated ATM, which reduced Rb phosphorylation to promote Rb-E2F1 interaction and hindered E2F1 functions. E2F1 activity suppression led to decreased E2F1-dependent target expressions, causing cell cycle arrest in the G1 phase, decreased S phase cells, and blocked DNA damage repair. CONCLUSION: Our study highlights the role of ATM/Rb/E2F1 pathway in controlling cell cycle arrest and apoptosis in response to RRM2 inhibition-induced DNA damage. This provides insight into the therapeutic benefits of COH29 and suggests targeting this pathway as a potential treatment for ATRT.

SENP2 restrains the generation of pathogenic Th17 cells in mouse models of colitis.

The molecular mechanisms contributing to the regulation of Th17-mediated inflammation remain underexplored. We here report a SUMO-specific protease (SENP)2-mediated pathway induced in pathogenic Th17 cells that restricts the pathogenesis of inflammatory colitis. SENP2 regulates the maturation of small ubiquitin-like modifiers (SUMO) and recycles SUMO from the substrate proteins. We find higher levels of SENP2 in pathogenic Th17 cells. By deleting Senp2 in T-cell lineages in mice, we demonstrate that the lack of Senp2 exacerbates the severity of experimental colitis, which is linked to elevated levels of GM-CSF+IL-17A+ pathogenic Th17 cells and more severe dysbiosis of the intestinal microbiome. Adoptive transfer experiments demonstrate the cell-autonomous effect of Senp2 in restraining Th17 differentiation and colitis. The enzymatic activity of SENP2 is important for deSUMOylation of Smad4, which reduces Smad4 nuclear entry and Rorc expression. Our findings reveal a SENP2-mediated regulatory axis in the pathogenicity of Th17 cells.

Targeting of RRM2 suppresses DNA damage response and activates apoptosis in atypical teratoid rhabdoid tumor.

BACKGROUND: Atypical teratoid rhabdoid tumors (ATRT) is a rare but aggressive malignancy in the central nervous system, predominantly occurring in early childhood. Despite aggressive treatment, the prognosis of ATRT patients remains poor. RRM2, a subunit of ribonucleotide reductase, has been reported as a biomarker for aggressiveness and poor prognostic conditions in several cancers. However, little is known about the role of RRM2 in ATRT. Uncovering the role of RRM2 in ATRT will further promote the development of feasible strategies and effective drugs to treat ATRT. METHODS: Expression of RRM2 was evaluated by molecular profiling analysis and was confirmed by IHC in both ATRT patients and PDX tissues. Follow-up in vitro studies used shRNA knockdown RRM2 in three different ATRT cells to elucidate the oncogenic role of RRM2. The efficacy of COH29, an RRM2 inhibitor, was assessed in vitro and in vivo. Western blot and RNA-sequencing were used to determine the mechanisms of RRM2 transcriptional activation in ATRT. RESULTS: RRM2 was found to be significantly overexpressed in multiple independent ATRT clinical cohorts through comprehensive bioinformatics and clinical data analysis in this study. The expression level of RRM2 was strongly correlated with poor survival rates in patients. In addition, we employed shRNAs to silence RRM2, which led to significantly decrease in ATRT colony formation, cell proliferation, and migration. In vitro experiments showed that treatment with COH29 resulted in similar but more pronounced inhibitory effect. Therefore, ATRT orthotopic mouse model was utilized to validate this finding, and COH29 treatment showed significant tumor growth suppression and prolong overall survival. Moreover, we provide evidence that COH29 treatment led to genomic instability, suppressed homologous recombinant DNA damage repair, and subsequently induced ATRT cell death through apoptosis in ATRT cells. CONCLUSIONS: Collectively, our study uncovers the oncogenic functions of RRM2 in ATRT cell lines, and highlights the therapeutic potential of targeting RRM2 in ATRT. The promising effect of COH29 on ATRT suggests its potential suitability for clinical trials as a novel therapeutic approach for ATRT.

Clinical and Molecular Features in Medulloblastomas Subtypes in Children in a Cohort in Taiwan.

Medulloblastoma (MB) was classified into four molecular subgroups: WNT, SHH, group 3, and group 4. In 2017, 12 subtypes within 4 subgroups and 8 subtypes within non-WNT/non-SHH subgroups according to the differences of clinical features and biology were announced. In this study, we aimed to identify the heterogeneity of molecular features for discovering subtype specific factors linked to diagnosis and prognosis. We retrieved 70 MBs in children to perform RNA sequencing and a DNA methylation array in Taiwan. Integrated with clinical annotations, we achieved classification of 12 subtypes of pediatric MBs in our cohort series with reference to the other reported series. We analyzed the correlation of cell type enrichment in SHH MBs and found that M2 macrophages were enriched in SHH ß, which related to good outcomes of SHH MBs. The high infiltration of M2 macrophages may be an indicator of a favorable prognosis and therapeutic target for SHH MBs. Furthermore, C11orf95-RELA fusion was observed to be associated with recurrence and a poor prognosis. These results will contribute to the establishment of a molecular diagnosis linked to prognostic indicators of relevance and help to promote molecular-based risk stratified treatment for MBs in children.