BACKGROUND. FXLEARN, the first-ever large multi-site trial of effects of disease-targeted pharmacotherapy on learning, was designed to explore a new paradigm for measuring effects of mechanism-targeted treatment in fragile X syndrome (FXS). In FXLEARN, the effects of mGluR5 negative allosteric modulator (NAM) AFQ056 on language learning were evaluated in 3-6 year-old children with FXS, expected to have more learning plasticity than adults, where prior trials of mGluR5 NAMs have failed. METHODS. After a 4-month single-blind placebo lead-in, participants were randomized 1:1 to AFQ056 or placebo, with 2 months of dose optimization to the maximum tolerated dose, then 6 months of treatment during which a language learning intervention was implemented for both groups. The primary outcome was a centrally scored videotaped communication measure, the Weighted Communication Scale (WCS). Secondary outcomes were objective performance-based and parent-report cognitive and language measures. RESULTS. FXLEARN enrolled 110 participants, randomized 99, and 91 completed the placebo-controlled period. Although both groups made language progress and there were no safety issues, the change in WCS score during the placebo-controlled period was not significantly different between the AFQ056 and placebo-treated groups, nor were there any significant between-group differences in change in any secondary measures. CONCLUSION. Despite the large body of evidence supporting use of mGluR5 NAMs in animal models of FXS, this study suggests that this mechanism of action does not translate into benefit for the human FXS population and that better strategies are needed to determine which mechanisms will translate from pre-clinical models to humans in genetic neurodevelopmental disorders. TRIAL REGISTRATION. ClincalTrials.gov NCT02920892 FUNDING. This study was supported by NeuroNEXT network NIH grants U01NS096767, U24NS107200, U24NS107209, U01NS077323, U24NS107183, U24NS107168, U24NS107128, U24NS107199, U24NS107198, U24NS107166, U10NS077368, U01NS077366, U24NS107205, U01NS077179, and U01NS077352, NIH grant P50HD103526 and Novartis IIT grant AFQ056X2201T for provision of AFQ056.
Elizabeth Berry-Kravis, Leonard Abbeduto, Randi Hagerman, Christopher S. Coffey, Merit Cudkowicz, Craig A. Erickson, Andrea McDuffie, David Hessl, Lauren E. Ethridge, Flora Tassone, Walter E. Kaufmann, Katherine Friedmann, Lauren Bullard, Anne Hoffmann, Jeremy Veenstra-VanderWeele, Kevin Staley, David Klements, Michael Moshinsky, Brittney Harkey, Jeffrey D. Long, Janel Fedler, Elizabeth Klingner, Dixie J. Ecklund, Michele Costigan, Trevis Huff, Brenda Pearson
The NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome is a crucial component of the innate immune system that initiates inflammatory responses. Post-translational modifications (PTMs) of NLRP3, including ubiquitination and phosphorylation, control inflammasome activation and determine the intensity of inflammation. However, the role of other PTMs in controlling NLRP3 inflammasome activation remains unclear. This study founded that toll-like receptor (TLR) priming induced NLRP3 ISGylation (a type of PTM in which ISG15 covalently binds to the target protein) to stabilise the NLRP3 protein. Viral infection, represented by SARS-COV-2 infection, and type I IFNs induced the expression of ISG15 and the predominant E3 ISGylation ligases HECT domain- and RCC1-like domain-containing proteins (HERCs; HERC5 in humans and HERC6 in mice). HERCs promoted NLRP3 ISGylation and inhibited K48-linked ubiquitination and proteasomal degradation, resulting in the enhancement of NLRP3 inflammasome activation. Concordantly, Herc6 deficiency ameliorated NLRP3-dependent inflammation, and hyperinflammation caused by viral infection. These results illustrate the mechanism by which type I IFNs responses control inflammasome activation and viral infection-induced aberrant NLRP3 activation. This work identifies ISGylation as a PTM of NLRP3 and provides a priming target for modulating NLRP3-dependent immunopathology.
Ying Qin, Xintong Meng, Mengge Wang, Wenbo Liang, Rong Xu, Jingchunyu Chen, Hui Song, Yue Fu, Jingxin Li, Chengjiang Gao, Mutian Jia, Chunyuan Zhao, Wei Zhao
Microvillus Inclusion Disease (MVID), caused by loss-of-function mutations in the motor protein Myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid-base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex Immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking anti-diarrheal drug, Crofelemer, dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. Gamma-secretase inhibition with DAPT recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum- and glucocorticoid-induced protein kinase 2 (SGK2), and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID.
Meri Kalashyan, Krishnan Raghunathan, Haley Oller, Marie-Theres Bayer, Lissette Jimenez, Joseph T. Roland, Elena Kolobova, Susan J. Hagen, Jeffrey D. Goldsmith, Mitchell D. Shub, James R. Goldenring, Izumi Kaji, Jay R. Thiagarajah
PCIF1 can mediate the methylation of N6,2′-O-dimethyladenosine (m6Am) in mRNA. Yet, the detailed interplay between PCIF1 and the potential cofactors and its pathological significance remains elusive. Here, we demonstrated that PCIF1-mediated cap mRNA m6Am modification promoted head and neck squamous cell carcinoma (HNSCC) progression both in vitro and in vivo. CTBP2 was identified as a cofactor of PCIF1 to catalyze m6Am deposition on mRNA. CLIP-seq data demonstrated CTBP2 bound to similar mRNAs as PCIF1. We then utilized m6Am-seq method to profile mRNA m6Am site at single-base resolution and found mRNA of TET2, a well-known tumor suppressor, was a major target substrate of PCIF1-CTBP2 complex. Mechanistically, knockout of CTBP2 reduced PCIF1 occupancy on TET2 mRNA and PCIF1-CTBP2 complex negatively regulated the translation of TET2 mRNA. Collectively, our study demonstrated the oncogenic function of the epitranscriptome regulator PCIF1-CTBP2 complex, highlighting the importance of the m6Am modification in tumor progression.
Kang Li, Jie Chen, Caihua Zhang, Maosheng Cheng, Shuang Chen, Wei Song, Chunlong Yang, Rongsong Ling, Zhi Chen, Xiaocheng Wang, Gan Xiong, Jieyi Ma, Yan Zhu, Quan Yuan, Qi Liu, Liang Peng, Qianming Chen, Demeng Chen
The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. Although the molecular and cellular mechanisms that regulate mucin production and movement are beginning to be understood, the upstream epithelial signals that contribute to mucin regulation remain unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity. We used genetic mouse models and non-inflamed samples from Inflammatory Bowel Disease (IBD) patients undergoing anti-TNF therapy to assess the effect of in vivo perturbation of TNF. We found that inhibition of epithelial TNF promotes the differentiation of secretory progenitor cells into mucus-producing goblet cells. Furthermore, TNF treatment and CFTR inhibition in intestinal organoids demonstrated that TNF promotes ion transport and luminal flow via CFTR. The absence of TNF led to slower gut transit times, which we propose results from increased mucus accumulation coupled with decreased luminal fluid pumping. These findings point to a TNF-CFTR signaling axis in the adult intestine and identify epithelial-derived TNF as an upstream regulator of mucin homeostasis.
Efren A. Reyes, David Castillo-Azofeifa, Jérémie Rispal, Tomas Wald, Rachel K. Zwick, Brisa Palikuqi, Angela Mujukian, Shervin Rabizadeh, Alexander R. Gupta, James M. Gardner, Dario Boffelli, Zev J. Gartner, Ophir D. Klein
Diabetic kidney disease (DKD) can lead to end-stage kidney disease (ESKD) and mortality, however, few mechanistic biomarkers are available for high risk patients, especially those without macroalbuminuria. Urine from participants with diabetes from Chronic Renal Insufficiency Cohort (CRIC), Singapore Study of Macro-Angiopathy and Reactivity in Type 2 Diabetes (SMART2D), and the Pima Indian Study determined if urine adenine/creatinine ratio (UAdCR) could be a mechanistic biomarker for ESKD. ESKD and mortality were associated with the highest UAdCR tertile in CRIC (HR 1.57, 1.18, 2.10) and SMART2D (HR 1.77, 1.00, 3.12). ESKD was associated with the highest UAdCR tertile in patients without macroalbuminuria in CRIC (HR 2.36, 1.26, 4.39), SMART2D (HR 2.39, 1.08, 5.29), and Pima Indian study (HR 4.57, CI 1.37-13.34). Empagliflozin lowered UAdCR in non-macroalbuminuric participants. Spatial metabolomics localized adenine to kidney pathology and transcriptomics identified ribonucleoprotein biogenesis as a top pathway in proximal tubules of patients without macroalbuminuria, implicating mammalian target of rapamycin (mTOR). Adenine stimulated matrix in tubular cells via mTOR and stimulated mTOR in mouse kidneys. A specific inhibitor of adenine production was found to reduce kidney hypertrophy and kidney injury in diabetic mice. We propose that endogenous adenine may be a causative factor in DKD.
Kumar Sharma, Guanshi Zhang, Jens Hansen, Petter Bjornstad, Hak Joo Lee, Rajasree Menon, Leila Hejazi, Jian-Jun Liu, Anthony Franzone, Helen C. Looker, Byeong Yeob Choi, Roman Fernandez, Manjeri A. Venkatachalam, Luxcia Kugathasan, Vikas S. Sridhar, Loki Natarajan, Jing Zhang, Varun S. Sharma, Brian Kwan, Sushrut S. Waikar, Jonathan Himmelfarb, Katherine R. Tuttle, Bryan Kestenbaum, Tobias Fuhrer, Harold Feldman, Ian H. de Boer, Fabio C. Tucci, John Sedor, Hiddo Lambers Heerspink, Jennifer Schaub, Edgar A. Otto, Jeffrey B. Hodgin, Matthias Kretzler, Christopher R. Anderton, Theodore Alexandrov, David Cherney, Su Chi Lim, Robert G. Nelson, Jonathan Gelfond, Ravi Iyengar
Melanomas reprogram their metabolism to rapidly adapt to therapy-induced stress conditions, allowing them to persist and ultimately develop resistance. We report that a subpopulation of melanoma cells tolerate MAPK-pathway inhibitors (MAPKi) through a concerted metabolic reprogramming mediated by peroxisomes and UDP-glucose ceramide glycosyltransferase (UGCG). Compromising peroxisome biogenesis, by repressing PEX3 expression, potentiated the pro-apoptotic effects of MAPKi via an induction of ceramides, an effect limited by UGCG-mediated ceramide metabolism. Co-targeting PEX3 and UGCG selectively eliminated a subset of metabolically active, drug-tolerant CD36+ melanoma persister cells, thereby sensitizing melanoma to MAPKi and delaying resistance. Increased levels of peroxisomal genes and UGCG were found in patient-derived MAPKi-relapsed melanomas, and simultaneously inhibiting PEX3 and UGCG restored MAPKi sensitivity in multiple models of therapy resistance. Finally, combination therapy comprised of a newly identified inhibitor of the PEX3-PEX19 interaction, a UGCG inhibitor and MAPKi demonstrated potent anti-tumor activity in pre-clinical melanoma models, thus representing a promising approach for melanoma treatment.
Fan Huang, Feiyang Cai, Michael S. Dahabieh, Kshemaka Gunawardena, Ali Talebi, Jonas Dehairs, Farah El-Turk, Jae Yeon Park, Mengqi Li, Christophe Goncalves, Natascha Gagnon, Jie Su, Judith H. LaPierre, Perrine Gaub, Jean-Sébastien Joyal, John J. Mitchell, Johannes V. Swinnen, Wilson H. Miller Jr., Sonia V. del Rincón
BACKGROUND. The biology of Plasmodium vivax is markedly different to that of P. falciparum; how this shapes the immune response to infection remains unclear. To address this shortfall, we inoculated human volunteers with a clonal field isolate of P. vivax and tracked their response through infection and convalescence. METHODS. Participants were injected intravenously with blood-stage parasites and infection dynamics were tracked in real-time by quantitative PCR. Whole blood samples were used for high dimensional protein analysis, RNA-sequencing and Cytometry by Time Of Flight (CyTOF), and temporal changes in the host response to P. vivax were quantified by linear regression. Comparative analyses with P. falciparum were then undertaken using analogous datasets derived from prior controlled human malaria infection studies. RESULTS.P. vivax rapidly induced a type I inflammatory response that coincided with hallmark features of clinical malaria. This acute phase response shared remarkable overlap with that induced by P. falciparum but was significantly elevated (at RNA and protein level) leading to an increased incidence of pyrexia. In contrast, T cell activation and terminal differentiation was significantly increased in volunteers infected with P. falciparum. Heterogeneous CD4+ T cells were found to dominate this adaptive response and phenotypic analysis revealed unexpected features normally associated with cytotoxicity and autoinflammatory disease. CONCLUSION.P. vivax triggers increased systemic interferon signaling (cf P. falciparum), which likely explains its reduced pyrogenic threshold. In contrast, P. falciparum drives T cell activation far in excess of P. vivax, which may partially explain why falciparum malaria more frequently causes severe disease. TRIAL REGISTRATION. ClinicalTrials.gov NCT03797989 FUNDING. Supported by the European Union's Horizon 2020 Research and Innovation programme, the Wellcome Trust and the Royal Society.
Florian A. Bach, Diana Muñoz Sandoval, Michalina Mazurczyk, Yrene Themistocleous, Thomas A. Rawlinson, Adam C. Harding, Alison Kemp, Sarah E. Silk, Jordan R. Barrett, Nick J. Edwards, Alasdair C. Ivens, Julian C. Rayner, Angela M. Minassian, Giorgio Napolitani, Simon J. Draper, Philip J. Spence
Stimulation of adipocyte beta-adrenergic receptors (beta-ARs) induces expression of uncoupling protein 1 (UCP1), promoting non-shivering thermogenesis. Association of beta-ARs with a lysine myristoylated form of A-kinase anchoring protein 12 (AKAP12)/gravin-alpha is required for downstream signaling that culminates in UCP1 induction. Conversely, demyristoylation of gravin-alpha by histone deacetylase 11 (HDAC11) suppresses this pathway. Whether inhibition of HDAC11 in adipocytes is sufficient to drive UCP1 expression independently of beta-ARs is not known. Here, we demonstrate that adipocyte-specific deletion of HDAC11 in mice leads to robust induction of UCP1 in adipose tissue (AT), resulting in increased body temperature. These effects are mimicked by treating mice in vivo or human AT ex vivo with an HDAC11-selective inhibitor, FT895. FT895 triggers biphasic, gravin-alpha myristoylation-dependent induction of UCP1 protein expression, with a non-canonical acute response that is post-transcriptional and independent of protein kinase A (PKA), and a delayed response requiring PKA activity and new Ucp1 mRNA synthesis. Remarkably, HDAC11 inhibition promotes UCP1 expression even in models of adipocyte catecholamine resistance where beta-AR signaling is blocked. These findings define cell autonomous, multi-modal roles for HDAC11 as a suppressor of thermogenesis, and highlight the potential of inhibiting HDAC11 to therapeutically alter AT phenotype independently of beta-AR stimulation.
Emma L. Robinson, Rushita Bagchi, Jennifer L. Major, Bryan C. Bergman, Jennifer L. Matsuda, Timothy A. McKinsey
Human cancers induce a chaotic, dysfunctional vasculature that promotes tumor growth and dampens most current therapies, but the underlying mechanism has been unclear. Here we show that SPEN (split end), a transcription repressor, coordinates ribosome RNA (rRNA) synthesis in endothelial cells (ECs) and is required for physiological and tumor angiogenesis. SPEN deficiency attenuated EC proliferation and blunted retinal angiogenesis, which was attributed to p53 activation. Furthermore, SPEN knockdown activated p53 by upregulating the noncoding promoter RNA (pRNA), which represses rRNA transcription and triggers p53-mediated nucleolar stress. In human cancer biopsies, low endothelial SPEN level correlated with extended overall survival. Consistently in mice, endothelial SPEN deficiency compromised rRNA expression and repressed tumor growth and metastasis by normalizing tumor vessels, which was abrogated by p53 haploinsufficiency. rRNA gene transcription is driven by RNA polymerase I (RNPI). We found that CX-5461, an RNPI inhibitor, recapitulated the effect of Spen ablation on tumor vessel normalization, and combining CX-5461 with cisplatin substantially improved the efficacy on treating tumors in mice. Together, these results demonstrate that SPEN is required for angiogenesis by repressing pRNA to enable rRNA gene transcription and ribosomal biogenesis, and that RNPI represents a target for tumor vessel normalization therapy of cancer.
Zi-Yan Yang, Xian-Chun Yan, Jia-Yu-Lin Zhang, Liang Liang, Chun-Chen Gao, Pei-Ran Zhang, Yuan Liu, Jia-Xing Sun, Bai Ruan, Juan-Li Duan, Ruo-Nan Wang, Xing-Xing Feng, Bo Che, Tian Xiao, Hua Han
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