Actin Polymerization Controls the Organization of WASH Domains at the Surface of Endosomes

Sorting of cargoes in endosomes occurs through their selective enrichment into sorting platforms, where transport intermediates are generated. The WASH complex, which directly binds to lipids, activates the Arp2/3 complex and hence actin polymerization onto such sorting platforms. Here, we analyzed the role of actin polymerization in the physiology of endosomal domains containing WASH using quantitative image analysis. Actin depolymerization is known to enlarge endosomes. Using a novel colocalization method that is insensitive to the heterogeneity of size and shape of endosomes, we further show that preventing the generation of branched actin networks induces endosomal accumulation of the WASH complex. Moreover, we found that actin depolymerization induces a dramatic decrease in the recovery of endosomal WASH after photobleaching. This result suggests a built-in turnover, where the actin network, i.e. the product of the WASH complex, contributes to the dynamic exchange of the WASH complex by promoting its detachment from endosomes. Our experiments also provide evidence for a role of actin polymerization in the lateral compartmentalization of endosomes: several WASH domains exist at the surface of enlarged endosomes, however, the WASH domains coalesce upon actin depolymerization or Arp2/3 depletion. Branched actin networks are thus involved in the regulation of the size of WASH domains. The potential role of this regulation in membrane scission are discussed

Tension and Robustness in Multitasking Cellular Networks

Cellular networks multitask by exhibiting distinct, context-dependent dynamics. However, network states (parameters) that generate a particular dynamic are often sub-optimal for others, defining a source of “tension” between them. Though multitasking is pervasive, it is not clear where tension arises, what consequences it has, and how it is resolved. We developed a generic computational framework to examine the source and consequences of tension between pairs of dynamics exhibited by the well-studied RB-E2F switch regulating cell cycle entry. We found that tension arose from task-dependent shifts in parameters associated with network modules. Although parameter sets common to distinct dynamics did exist, tension reduced both their accessibility and resilience to perturbation, indicating a trade-off between “one-size-fits-all” solutions and robustness. With high tension, robustness can be preserved by dynamic shifting of modules, enabling the network to toggle between tasks, and by increasing network complexity, in this case by gene duplication. We propose that tension is a general constraint on the architecture and operation of multitasking biological networks. To this end, our work provides a framework to quantify the extent of tension between any network dynamics and how it affects network robustness. Such analysis would suggest new ways to interfere with network elements to elucidate the design principles of cellular networks

VPS29 Is Not an Active Metallo-Phosphatase but Is a Rigid Scaffold Required for Retromer Interaction with Accessory Proteins

VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn2+ and Zn2+ in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies

Defects in ER–endosome contacts impact lysosome function in hereditary spastic paraplegia

Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER–endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell–derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER–endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration.This work was supported by grants to E. Reid: UK Medical Research Council Project Grant (MR/M00046X/1), Wellcome Trust Senior Research Fellowship in Clinical Science (082381), Project Grant from United States Spastic Paraplegia Foundation, Project Grant from Tom Wahlig Stiftung, and Project Grant form UK HSP Family Group. J.R. Edgar is supported by the Wellcome Trust (grant 086598). T. Newton and G. Pearson are supported by the Medical Research Council PhD studentships (G0800117 and MR/K50127X/1). F. Berner was supported by the National Institute for Health Research Biomedical Research Centre at Addenbrooke's Hospital. B. Winner is supported by the Tom Wahlig Advanced Fellowship, the German Federal Ministry of Education and Research (01GQ113), the Bavarian Ministry of Education and Culture, Sciences and Arts in the framework of the Bavarian Molecular Biosystems Research Network and ForIPS, and the Interdisciplinary Centre for Clinical Research (University Hospital of Erlangen, N3 and F3). Cambridge Institute for Medical Research is supported by a Wellcome Trust Strategic Award (100140) and Equipment Grant (093026)

Global Retinoblastoma Presentation and Analysis by National Income Level.

Importance: Early diagnosis of retinoblastoma, the most common intraocular cancer, can save both a child's life and vision. However, anecdotal evidence suggests that many children across the world are diagnosed late. To our knowledge, the clinical presentation of retinoblastoma has never been assessed on a global scale. Objectives: To report the retinoblastoma stage at diagnosis in patients across the world during a single year, to investigate associations between clinical variables and national income level, and to investigate risk factors for advanced disease at diagnosis. Design, Setting, and Participants: A total of 278 retinoblastoma treatment centers were recruited from June 2017 through December 2018 to participate in a cross-sectional analysis of treatment-naive patients with retinoblastoma who were diagnosed in 2017. Main Outcomes and Measures: Age at presentation, proportion of familial history of retinoblastoma, and tumor stage and metastasis. Results: The cohort included 4351 new patients from 153 countries; the median age at diagnosis was 30.5 (interquartile range, 18.3-45.9) months, and 1976 patients (45.4%) were female. Most patients (n = 3685 [84.7%]) were from low- and middle-income countries (LMICs). Globally, the most common indication for referral was leukocoria (n = 2638 [62.8%]), followed by strabismus (n = 429 [10.2%]) and proptosis (n = 309 [7.4%]). Patients from high-income countries (HICs) were diagnosed at a median age of 14.1 months, with 656 of 666 (98.5%) patients having intraocular retinoblastoma and 2 (0.3%) having metastasis. Patients from low-income countries were diagnosed at a median age of 30.5 months, with 256 of 521 (49.1%) having extraocular retinoblastoma and 94 of 498 (18.9%) having metastasis. Lower national income level was associated with older presentation age, higher proportion of locally advanced disease and distant metastasis, and smaller proportion of familial history of retinoblastoma. Advanced disease at diagnosis was more common in LMICs even after adjusting for age (odds ratio for low-income countries vs upper-middle-income countries and HICs, 17.92 [95% CI, 12.94-24.80], and for lower-middle-income countries vs upper-middle-income countries and HICs, 5.74 [95% CI, 4.30-7.68]). Conclusions and Relevance: This study is estimated to have included more than half of all new retinoblastoma cases worldwide in 2017. Children from LMICs, where the main global retinoblastoma burden lies, presented at an older age with more advanced disease and demonstrated a smaller proportion of familial history of retinoblastoma, likely because many do not reach a childbearing age. Given that retinoblastoma is curable, these data are concerning and mandate intervention at national and international levels. Further studies are needed to investigate factors, other than age at presentation, that may be associated with advanced disease in LMICs

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

The global retinoblastoma outcome study : a prospective, cluster-based analysis of 4064 patients from 149 countries

DATA SHARING : The study data will become available online once all analyses are complete.BACKGROUND : Retinoblastoma is the most common intraocular cancer worldwide. There is some evidence to suggest that major differences exist in treatment outcomes for children with retinoblastoma from different regions, but these differences have not been assessed on a global scale. We aimed to report 3-year outcomes for children with retinoblastoma globally and to investigate factors associated with survival. METHODS : We did a prospective cluster-based analysis of treatment-naive patients with retinoblastoma who were diagnosed between Jan 1, 2017, and Dec 31, 2017, then treated and followed up for 3 years. Patients were recruited from 260 specialised treatment centres worldwide. Data were obtained from participating centres on primary and additional treatments, duration of follow-up, metastasis, eye globe salvage, and survival outcome. We analysed time to death and time to enucleation with Cox regression models. FINDINGS : The cohort included 4064 children from 149 countries. The median age at diagnosis was 23·2 months (IQR 11·0–36·5). Extraocular tumour spread (cT4 of the cTNMH classification) at diagnosis was reported in five (0·8%) of 636 children from high-income countries, 55 (5·4%) of 1027 children from upper-middle-income countries, 342 (19·7%) of 1738 children from lower-middle-income countries, and 196 (42·9%) of 457 children from low-income countries. Enucleation surgery was available for all children and intravenous chemotherapy was available for 4014 (98·8%) of 4064 children. The 3-year survival rate was 99·5% (95% CI 98·8–100·0) for children from high-income countries, 91·2% (89·5–93·0) for children from upper-middle-income countries, 80·3% (78·3–82·3) for children from lower-middle-income countries, and 57·3% (52·1-63·0) for children from low-income countries. On analysis, independent factors for worse survival were residence in low-income countries compared to high-income countries (hazard ratio 16·67; 95% CI 4·76–50·00), cT4 advanced tumour compared to cT1 (8·98; 4·44–18·18), and older age at diagnosis in children up to 3 years (1·38 per year; 1·23–1·56). For children aged 3–7 years, the mortality risk decreased slightly (p=0·0104 for the change in slope). INTERPRETATION : This study, estimated to include approximately half of all new retinoblastoma cases worldwide in 2017, shows profound inequity in survival of children depending on the national income level of their country of residence. In high-income countries, death from retinoblastoma is rare, whereas in low-income countries estimated 3-year survival is just over 50%. Although essential treatments are available in nearly all countries, early diagnosis and treatment in low-income countries are key to improving survival outcomes.The Queen Elizabeth Diamond Jubilee Trust and the Wellcome Trust.https://www.thelancet.com/journals/langlo/homeam2023Paediatrics and Child Healt

Abstract 3027: Role of BCOR in retinoblastoma

Abstract Retinoblastoma, which is usually initiated by the biallelic mutational inactivation of RB1, is the most common primary eye cancer in children and is fatal if left untreated. While retinoblastoma is initially sensitive to chemotherapy, it demonstrates a strong propensity for therapeutic escape, which is a leading cause of ocular morbidity, eye loss, and reduced survival. This problem is exacerbated by a poor understanding of how retinoblastoma progresses after the initial loss of RB1. Genomic analysis of primary retinoblastoma tumors has revealed the corepressor BCOR as the second most common target of mutations in retinoblastoma. Here, we explored the role of BCOR in normal retinal development to provide insights into its role in retinoblastoma. Mass spectrometry following immunoprecipitation of wildtype BCOR in primary retinoblastoma cells revealed novel BCOR interacting proteins in the Polycomb and MiDAC epigenetic complexes. Subsequent studies revealed potential functional consequences of BCOR mutation that may promote retinoblastoma progression. This work reveals new insights the role of epigenetic deregulation in retinoblastoma progression, and they nominate new candidates for therapeutic intervention. Citation Format: Michelle Garying Zhang, Dawn Owens, Stefan Kurtenbach, Jeffim Kuznetsov, Matthew Fields, Daniel Pelaez, James W. Harbour. Role of BCOR in retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3027

Abstract 4861: The role of PRAME in promoting uveal melanoma metastasis

Abstract Uveal melanoma (UM) is the most common primary eye cancer and has a predisposition for fatal metastasis. Our group has shown that UMs can be categorized by gene expression profiling (GEP) into two molecular classes associated with metastatic risk: Class 1 (low metastatic risk) and Class 2 (high metastatic risk). While the majority of Class 2 UMs (71%) metastasize, only 38% of Class 1 UMs metastasize. Our group was the first to identify the cancer-testis antigen PRAME as a biomarker for increased metastatic risk in Class 1 UMs, and sub-categorized these tumors into Class1 PRAME+ (intermediate metastatic risk) and Class1 PRAME- (low metastatic risk) subgroups. Further we identified PRAME+ was associated with shorter time to metastasis and worse prognosis in class 2 patients. We have now shown that this PRAME expression is correlated with hypomethylation in the promoter region of the gene, suggesting a mechanism for its expression. Since we have previously shown that PRAME is associated with early metastasis in Class 1 and Class 2 UMs, we hypothesized that PRAME plays a direct role in UM metastasis. To better understand the role of PRAME in UM, we used Class1PRAME+ (F41) and Class1PRAME– (Mel290) UM cell lines to develop in vitro models to study the role of PRAME. Since F41 cells are PRAME+ and highly metastatic, we constitutively silenced PRAME with a V5 tag (F41 shPRAME-V5). For Mel290 cells, we overexpressed PRAME to see whether this cell line, which does not induce metastatic death, became more malignant. We confirmed the silencing and overexpression of PRAME in our transformed cells with western blot. For cell proliferation, we found a slight increase in the Mel290 PRAME+ cells compared to non-transformed Mel290. Strikingly, in F41 cells, knockdown of PRAME led to complete cell death. In a xenograft animal model, NSG male mice were injected with either Mel290 PRAME-V5, Mel290, F41 shPRAME-V5 or F41 TET-empty vector cells. We found that PRAME overexpressing cell lines (Mel290 PRAME-V5 and F41 TET-empty vector) induced rapid, multiple and aggressive liver metastases that led to death, whereas the non-expressing PRAME cells (Mel290 and F41 shPRAME-V5) did not present macro or micrometastasis in any cases. This data highly suggests that PRAME overexpression is involved in UM metastasis. Thus, PRAME mRNA expression is not only a prognostic biomarker but also could be an important target for treatment. Citation Format: Margaret I. Sanchez, Matthew G. Field, Jeffim N. Kuznetsov, Stefan Kurtenbach, Dien Pham, James W. Harbour. The role of PRAME in promoting uveal melanoma metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4861. doi:10.1158/1538-7445.AM2017-4861</jats:p