ABSTRACT
Plants are sessile and have to cope with environmentally induced damage through modification of growth and defense pathways. How tissue regeneration is triggered in such responses and whether this involves stem cell activation is an open question. The stress hormone jasmonate (JA) plays well-established roles in wounding and defense responses. JA also affects growth, which is hitherto interpreted as a trade-off between growth and defense. Here, we describe a molecular network triggered by wound-induced JA that promotes stem cell activation and regeneration. JA regulates organizer cell activity in the root stem cell niche through the RBR-SCR network and stress response protein ERF115. Moreover, JA-induced ERF109 transcription stimulates CYCD6;1 expression, functions upstream of ERF115, and promotes regeneration. Soil penetration and response to nematode herbivory induce and require this JA-mediated regeneration response. Therefore, the JA tissue damage response pathway induces stem cell activation and regeneration and activates growth after environmental stress.
Subject(s)
Cyclopentanes/metabolism , Oxylipins/metabolism , Plant Roots/metabolism , Stem Cells/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Cyclins/metabolism , Gene Expression Regulation, Plant/genetics , Herbivory , Indoleacetic Acids/metabolism , Regeneration/physiology , Signal Transduction/physiology , Stress, Physiological , Transcription Factors/metabolismABSTRACT
The interaction of RB with chromatin is key to understanding its molecular functions. Here, for first time, we identify the full spectrum of chromatin-bound RB. Rather than exclusively binding promoters, as is often described, RB targets three fundamentally different types of loci (promoters, enhancers, and insulators), which are largely distinguishable by the mutually exclusive presence of E2F1, c-Jun, and CTCF. While E2F/DP facilitates RB association with promoters, AP-1 recruits RB to enhancers. Although phosphorylation in CDK sites is often portrayed as releasing RB from chromatin, we show that the cell cycle redistributes RB so that it enriches at promoters in G1 and at non-promoter sites in cycling cells. RB-bound promoters include the classic E2F-targets and are similar between lineages, but RB-bound enhancers associate with different categories of genes and vary between cell types. Thus, RB has a well-preserved role controlling E2F in G1, and it targets cell-type-specific enhancers and CTCF sites when cells enter S-phase.
Subject(s)
Chromatin , Retinoblastoma Protein , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromatin/genetics , E2F Transcription Factors/genetics , E2F Transcription Factors/metabolism , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , Promoter Regions, Genetic , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , Transcription Factor AP-1/geneticsABSTRACT
Cells escape the need for mitogens at a restriction point several hours before entering S phase. The restriction point has been proposed to result from CDK4/6 initiating partial Rb phosphorylation to trigger a bistable switch whereby cyclin E-CDK2 and Rb mutually reinforce each other to induce Rb hyperphosphorylation. Here, using single-cell analysis, we unexpectedly found that cyclin E/A-CDK activity can only maintain Rb hyperphosphorylation starting at the onset of S phase and that CDK4/6 activity, but not cyclin E/A-CDK activity, is required to hyperphosphorylate Rb throughout G1 phase. Mitogen removal in G1 results in a gradual loss of CDK4/6 activity with a high likelihood of cells sustaining Rb hyperphosphorylation until S phase, at which point cyclin E/A-CDK activity takes over. Thus, it is short-term memory, or transient hysteresis, in CDK4/6 activity following mitogen removal that sustains Rb hyperphosphorylation, demonstrating a probabilistic rather than an irreversible molecular mechanism underlying the restriction point.
Subject(s)
Cell Proliferation , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/metabolism , Epithelial Cells/drug effects , G1 Phase Cell Cycle Checkpoints , Mitogens/pharmacology , Animals , Cell Line , Dose-Response Relationship, Drug , Epithelial Cells/enzymology , Fibroblasts/drug effects , Fibroblasts/enzymology , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/enzymology , Humans , Mice , Models, Biological , Phosphorylation , Retinoblastoma Binding Proteins/metabolism , Signal Transduction , Time Factors , Ubiquitin-Protein Ligases/metabolismABSTRACT
E2F1, E2F2, and E2F3A, the three activators of the E2F family of transcription factors, are key regulators of the G1/S transition, promoting transcription of hundreds of genes critical for cell-cycle progression. We found that during late S and in G2, the degradation of all three activator E2Fs is controlled by cyclin F, the substrate receptor of 1 of 69 human SCF ubiquitin ligase complexes. E2F1, E2F2, and E2F3A interact with the cyclin box of cyclin F via their conserved N-terminal cyclin binding motifs. In the short term, E2F mutants unable to bind cyclin F remain stable throughout the cell cycle, induce unscheduled transcription in G2 and mitosis, and promote faster entry into the next S phase. However, in the long term, they impair cell fitness. We propose that by restricting E2F activity to the S phase, cyclin F controls one of the main and most critical transcriptional engines of the cell cycle.
Subject(s)
Cell Cycle/genetics , Cyclins/genetics , E2F1 Transcription Factor/genetics , E2F2 Transcription Factor/genetics , E2F3 Transcription Factor/genetics , SKP Cullin F-Box Protein Ligases/genetics , Transcription, Genetic , Cell Line, Tumor , Cyclins/metabolism , E2F1 Transcription Factor/metabolism , E2F2 Transcription Factor/metabolism , E2F3 Transcription Factor/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Gene Expression Regulation , Genetic Fitness , HEK293 Cells , HeLa Cells , Humans , Mutation , Osteoblasts/cytology , Osteoblasts/metabolism , Proteolysis , SKP Cullin F-Box Protein Ligases/metabolism , Signal Transduction , UbiquitinationABSTRACT
Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb∆K4 and Rb∆K7 knock-in mice, in which either four or all seven phosphorylation sites in the C-terminal region of pRb, respectively, have been abolished by Ser/Thr-to-Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb∆K4 mice exhibit telomere attrition but no other abnormalities, Rb∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb∆K7 mice is insulin-sensitive and associated with failure of quiescent pancreatic ß-cells to re-enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence-associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre-treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re-entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK-inhibitor therapeutics, diabetes, and longevity.
Subject(s)
Aging/physiology , Ascorbic Acid/pharmacology , Diabetes Mellitus, Experimental/prevention & control , Retinoblastoma Protein/metabolism , Animals , Cellular Senescence/drug effects , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/pathology , E2F1 Transcription Factor/metabolism , Embryonic Development/genetics , Female , Fibroblasts/drug effects , Gene Knock-In Techniques , Insulin-Secreting Cells/pathology , Mice , Phosphorylation , Pregnancy , Retinoblastoma Protein/genetics , Telomere/geneticsABSTRACT
The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling the expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila melanogaster. We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase (Pgk), being mostly affected. The loss of E2F regulation on the Pgk gene led to a decrease in glycolytic flux, tricarboxylic acid cycle intermediates levels, adenosine triphosphate (ATP) content, and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in PgkΔE2F mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in PgkΔE2F mutants. Moreover, PgkΔE2F animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression, and development in the PgkΔE2F animals underscore the importance of E2F regulation on a single E2F target, Pgk.
Subject(s)
Drosophila Proteins , Drosophila , E2F Transcription Factors , Phosphoglycerate Kinase , Animals , Chromatin , Drosophila/genetics , E2F Transcription Factors/genetics , Phosphoglycerate Kinase/genetics , Phosphoglycerate Kinase/metabolism , Promoter Regions, Genetic , Drosophila Proteins/genetics , Drosophila Proteins/metabolismABSTRACT
Living organisms possess mechanisms to safeguard genome integrity. To avoid spreading mutations, DNA lesions are detected and cell division is temporarily arrested to allow repair mechanisms. Afterward, cells either resume division or respond to unsuccessful repair by undergoing programmed cell death (PCD). How the success rate of DNA repair connects to later cell fate decisions remains incompletely known, particularly in plants. The Arabidopsis thaliana RETINOBLASTOMA-RELATED1 (RBR) protein and its partner E2FA, play both structural and transcriptional functions in the DNA damage response (DDR). Here we provide evidence that distinct RBR protein interactions with LXCXE motif-containing proteins guide these processes. Using the N849F substitution in the RBR B-pocket domain, which specifically disrupts binding to the LXCXE motif, we show that these interactions are dispensable in unchallenging conditions. However, N849F substitution abolishes RBR nuclear foci and promotes PCD and growth arrest upon genotoxic stress. NAC044, which promotes growth arrest and PCD, accumulates after the initial recruitment of RBR to foci and can bind non-focalized RBR through the LXCXE motif in a phosphorylation-independent manner, allowing interaction at different cell cycle phases. Disrupting NAC044-RBR interaction impairs PCD, but their genetic interaction points to opposite independent roles in the regulation of PCD. The LXCXE-binding dependency of the roles of RBR in the DDR suggests a coordinating mechanism to translate DNA repair success to cell survival. We propose that RBR and NAC044 act in two distinct DDR pathways, but interact to integrate input from both DDR pathways to decide upon an irreversible cell fate decision.
Subject(s)
Arabidopsis Proteins , Arabidopsis , DNA Damage , DNA Repair , Amino Acid Motifs , Apoptosis , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/geneticsABSTRACT
Although lengthening of the cell cycle and G1 phase is a generic feature of tissue maturation during development, the underlying mechanism remains poorly understood. Here, we develop a time-lapse imaging strategy to measure the four cell cycle phases in single chick neural progenitor cells in their endogenous environment. We show that neural progenitors are widely heterogeneous with respect to cell cycle length. This variability in duration is distributed over all phases of the cell cycle, with the G1 phase contributing the most. Within one cell cycle, each phase duration appears stochastic and independent except for a correlation between S and M phase duration. Lineage analysis indicates that the majority of daughter cells may have a longer G1 phase than mother cells, suggesting that, at each cell cycle, a mechanism lengthens the G1 phase. We identify that the CDC25B phosphatase known to regulate the G2/M transition indirectly increases the duration of the G1 phase, partly through delaying passage through the restriction point. We propose that CDC25B increases the heterogeneity of G1 phase length, revealing a previously undescribed mechanism of G1 lengthening that is associated with tissue development.
Subject(s)
Neural Stem Cells , Cell Cycle/physiology , Cell Division , G1 Phase/physiology , cdc25 Phosphatases/genetics , cdc25 Phosphatases/metabolismABSTRACT
Most retinoblastomas develop from maturing cone precursors in response to biallelic RB1 loss and are dependent on cone maturation-related signaling. Additionally, â¼2% lack RB1 mutations but have MYCN amplification (MYCNA), N-Myc protein overexpression, and more rapid and invasive growth, yet the MYCNA retinoblastoma cell of origin and basis for its responses to deregulated N-Myc are unknown. Here, using explanted cultured retinae, we show that ectopic N-Myc induces cell cycle entry in cells expressing markers of several retinal types yet induces continuous proliferation and tumorigenesis only in cone precursors. Unlike the response to RB1 loss, both immature cone arrestin-negative (ARR3-) and maturing ARR3+ cone precursors proliferate, and maturing cone precursors rapidly dedifferentiate, losing ARR3 as well as L/M-opsin expression. N-Myc-overexpressing retinal cells also lose cell lineage constraints, occasionally coexpressing the cone-specific RXRγ with the rod-specific NRL or amacrine-specific AP2α and widely coexpressing RXRγ with the progenitor and Müller cell-specific SOX9 and retinal ganglion cell-specific BRN3 and GAP43. Mechanistically, N-Myc induced Cyclin D2 and CDK4 overexpression, pRB phosphorylation, and SOX9-dependent proliferation without a retinoma-like stage that characterizes pRB-deficient retinoblastoma, despite continuous p16INK4A expression. Orthotopic xenografts of N-Myc-overexpressing retinal cells formed tumors with retinal cell marker expression similar to those in MYCN-transduced retinae and MYCNA retinoblastomas in patients. These findings demonstrate the MYCNA retinoblastoma origin from immature and lineage-deconstrained cone precursors, reveal their opportunistic use of an undifferentiated retinal progenitor cell feature, and illustrate that different cancer-initiating mutations cooperate with distinct developmental stage-specific cell signaling circuitries to drive retinoblastoma tumorigenesis.
Subject(s)
Carcinogenesis , N-Myc Proto-Oncogene Protein , Retinal Cone Photoreceptor Cells , Retinal Neoplasms , Retinoblastoma , Carcinogenesis/genetics , Cell Cycle , Humans , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Retinal Cone Photoreceptor Cells/metabolism , Retinal Cone Photoreceptor Cells/pathology , Retinal Neoplasms/metabolism , Retinal Neoplasms/pathology , Retinoblastoma/metabolism , Retinoblastoma/pathologyABSTRACT
The RB1 gene is frequently mutated in human cancers but its role in tumorigenesis remains incompletely defined. Using an induced pluripotent stem cell (iPSC) model of hereditary retinoblastoma (RB), we report that the spliceosome is an up-regulated target responding to oncogenic stress in RB1-mutant cells. By investigating transcriptomes and genome occupancies in RB iPSCderived osteoblasts (OBs), we discover that both E2F3a, which mediates spliceosomal gene expression, and pRB, which antagonizes E2F3a, coregulate more than one-third of spliceosomal genes by cobinding to their promoters or enhancers. Pharmacological inhibition of the spliceosome in RB1-mutant cells leads to global intron retention, decreased cell proliferation, and impaired tumorigenesis. Tumor specimen studies and genome-wide TCGA (The Cancer Genome Atlas) expression profile analyses support the clinical relevance of pRB and E2F3a in modulating spliceosomal gene expression in multiple cancer types including osteosarcoma (OS). High levels of pRB/E2F3aregulated spliceosomal genes are associated with poor OS patient survival. Collectively, these findings reveal an undiscovered connection between pRB, E2F3a, the spliceosome, and tumorigenesis, pointing to the spliceosomal machinery as a potentially widespread therapeutic vulnerability of pRB-deficient cancers.
Subject(s)
Bone Neoplasms , Carcinogenesis , E2F3 Transcription Factor , Gene Expression Regulation, Neoplastic , Induced Pluripotent Stem Cells , Osteosarcoma , Retinoblastoma Binding Proteins , Spliceosomes , Ubiquitin-Protein Ligases , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Carcinogenesis/genetics , E2F3 Transcription Factor/genetics , E2F3 Transcription Factor/metabolism , Genes, Retinoblastoma , Humans , Induced Pluripotent Stem Cells/metabolism , Mutation , Osteosarcoma/genetics , Osteosarcoma/pathology , Retinal Neoplasms/genetics , Retinoblastoma/genetics , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Spliceosomes/genetics , Spliceosomes/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Retinoblastoma protein is central in signaling networks of fundamental cell decisions such as proliferation and differentiation in all metazoans and cancer development. Immunostaining and biochemical evidence demonstrated that during interphase retinoblastoma protein is in the nucleus and is hypophosphorylated, and during mitosis is in the cytoplasm and is hyperphosphorylated. The purpose of this study was to visualize in vivo in a non-diseased tissue, the dynamic spatial and temporal nuclear exit toward the cytoplasm of this protein during mitosis and its return to the nucleus to obtain insights into its potential cytosolic functions. Using high-resolution time-lapse images from confocal microscopy, we tracked in vivo the ortholog in plants the RETINOBLASTOMA RELATED (RBR) protein tagged with Green Fluorescent Protein (GFP) in Arabidopsis thaliana's root. RBR protein exits from dense aggregates in the nucleus before chromosomes are in prophase in less than 2 min, spreading outwards as smaller particles projected throughout the cytosol during mitosis like a diffusive yet controlled event until telophase, when the daughter's nuclei form; RBR returns to the nuclei in coordination with decondensing chromosomal DNA forming new aggregates again in punctuated larger structures in each corresponding nuclei. We propose RBR diffused particles in the cytoplasm may function as a cytosolic sensor of incoming signals, thus coordinating re-aggregation with DNA is a mechanism by which any new incoming signals encountered by RBR may lead to a reconfiguration of the nuclear transcriptomic context. The small RBR diffused particles in the cytoplasm may preserve topologic-like properties allowing them to aggregate and restore their nuclear location, they may also be part of transient cytoplasmic storage of the cellular pre-mitotic transcriptional context, that once inside the nuclei may execute both the pre mitosis transcriptional context as well as new transcriptional instructions.
ABSTRACT
RB1 deficiency leads to retinoblastoma (Rb), the most prevalent intraocular malignancy. Tumor-associated macrophages (TAMs) are related to local inflammation disorder, particularly by increasing cytokines and immune escape. Microglia, the unique resident macrophages for retinal homeostasis, are the most important immune cells of Rb. However, whether RB1 deficiency affects microglial function remain unknown. In this study, microglia were successfully differentiated from Rb patient- derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), and then we investigated the function of RB1 in microglia by live imaging phagocytosis assay, immunofluorescence, RNA-seq, qRT-PCR, ELISA and retina organoids/microglia co-culturing. RB1 was abundantly expressed in microglia and predominantly located in the nucleus. We then examined the phagocytosis ability and secretion function of iMGs in vitro. We found that RB1 deficiency did not affect the expression of microglia-specific markers or the phagocytic abilities of these cells by live-imaging. Upon LPS stimulation, RB1-deficient microglia displayed enhanced innate immune responses, as evidenced by activated MAPK signaling pathway and elevated expression of IL-6 and TNF-α at both mRNA and protein levels, compared to wildtype microglia. Furthermore, retinal structure disruption was observed when retinal organoids were co-cultured with RB1-deficient microglia, highlighting the potential contribution of microglia to Rb development and potential therapeutic strategies for retinoblastoma.
Subject(s)
Induced Pluripotent Stem Cells , Retinal Neoplasms , Retinoblastoma , Humans , Retinoblastoma/genetics , Retinoblastoma/metabolism , Retinoblastoma/pathology , Microglia/metabolism , Induced Pluripotent Stem Cells/metabolism , Retina , Retinal Neoplasms/genetics , Retinal Neoplasms/metabolism , Retinal Neoplasms/pathologyABSTRACT
Studies are lacking on long-term effects among retinoblastoma patients in low- and middle-income countries. Therefore, we examined cause-specific mortality in a retrospective cohort of retinoblastoma patients treated at Antonio Candido de Camargo Cancer Center (ACCCC), São Paulo, Brazil from 1986 to 2003 and followed up through December 31, 2018. Vital status and cause of death were ascertained from medical records and multiple national databases. We estimated overall and cause-specific survival using the Kaplan-Meier survival method, and estimated standardized mortality ratios (SMRs) and absolute excess risk (AER) of death. This cohort study included 465 retinoblastoma patients (42% hereditary, 58% nonhereditary), with most (77%) patients diagnosed at advanced stages (IV or V). Over an 11-year average follow-up, 80 deaths occurred: 70% due to retinoblastoma, 22% due to subsequent malignant neoplasms (SMNs) and 5% to non-cancer causes. The overall 5-year survival rate was 88% consistent across hereditary and nonhereditary patients (p = .67). Hereditary retinoblastoma patients faced an 86-fold higher risk of SMN-related death compared to the general population (N = 16, SMR = 86.1, 95% CI 52.7-140.5), corresponding to 42.4 excess deaths per 10,000 person-years. This risk remained consistent for those treated with radiotherapy and chemotherapy (N = 10, SMR = 90.3, 95% CI 48.6-167.8) and chemotherapy alone (N = 6, SMR = 80.0, 95% CI 35.9-177.9). Nonhereditary patients had only two SMN-related deaths (SMR = 7.2, 95% CI 1.8-28.7). There was no excess risk of non-cancer-related deaths in either retinoblastoma form. Findings from this cohort with a high proportion of advanced-stage patients and extensive chemotherapy use may help guide policy and healthcare planning, emphasizing the need to enhance early diagnosis and treatment access in less developed countries.
ABSTRACT
Retinoblastoma (RB) is the most common intraocular malignant tumor in children, primarily attributed to the bi-allelic loss of the RB1 gene in the developing retina. Despite significant progress in understanding the basic pathogenesis of RB, comprehensively unravelling the intricate network of genetics and epigenetics underlying RB tumorigenesis remains a major challenge. Conventional clinical treatment options are limited, and despite the continuous identification of genetic loci associated with cancer pathogenesis, the development of targeted therapies lags behind. This review focuses on the reported genomic and epigenomic alterations in retinoblastoma, summarizing potential therapeutic targets for RB and providing insights for research into targeted therapies.
ABSTRACT
BACKGROUND: Retinoblastoma is the most common intraocular malignant tumor occurring among children, with an incidence rate of 1/15â 000. This study built a joinpoint regression model to assess the incidence trend of retinoblastoma from 2004 to 2015 and constructed a nomogram to predict the overall survival (OS) in children. MATERIALS AND METHODS: Patients less than 19 years diagnosed with retinoblastoma from 2004 to 2015 were selected from the SEER database. Joinpoint regression analysis (version 4.9.0.0) was performed to evaluate the trends in retinoblastoma incidence rates from 2004 to 2015. Cox Regression Analysis was applied to investigate prognostic risk factors that influence OS. RESULTS: Joinpoint regression revealed that retinoblastoma incidence exhibited no significant increase or decrease from 2004 to 2015. As per the multiple Cox regression, tumor size, laterality, and residence (rural-urban continuum code) were correlated with OS and were used to construct a nomogram. The nomogram exhibited a good C-index of 0.71 (95% CI, 0.63 to 0.79), and the calibration curve for survival probability demonstrated that the predictions corresponded well with actual observations. CONCLUSIONS AND RELEVANCE: A prognostic nomogram integrating the risk factors for retinoblastoma was constructed to provide comparatively accurate individual survival predictions. If validated, this type of assessment could be used to guide therapy in patients with retinoblastoma.
Subject(s)
Retinal Neoplasms , Retinoblastoma , Child , Humans , Prognosis , Nomograms , Incidence , Retinoblastoma/epidemiology , Retinal Neoplasms/epidemiology , SEER ProgramABSTRACT
BACKGROUND: GD2-directed immunotherapy is highly effective in the treatment of high-risk neuroblastoma (NB), and might be an interesting target also in other high-risk tumors. METHODS: The German-Austrian Retinoblastoma Registry, Essen, was searched for patients, who were treated with anti-GD2 monoclonal antibody (mAb) dinutuximab beta (Db) in order to evaluate toxicity, response and outcome in these patients. Additionally, we evaluated anti-GD2 antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in retinoblastoma cell lines in vitro. Furthermore, in vitro cytotoxicity assays directed against B7-H3 (CD276), a new identified potential target in RB, were performed. RESULTS: We identified four patients with relapsed stage IV retinoblastoma, who were treated with Db following autologous stem cell transplantation (ASCT). Two out of two evaluable patients with detectable tumors responded to immunotherapy. One of these and another patient who received immunotherapy without residual disease relapsed 10 and 12 months after start of Db. The other patients remained in remission until last follow-up 26 and 45 months, respectively. In vitro, significant lysis of RB cell lines by ADCC and CDC with samples from patients and healthy donors and anti-GD2 and anti-CD276-mAbs were demonstrated. CONCLUSION: Anti-GD2-directed immunotherapy represents an additional therapeutic option in high-risk metastasized RB. Moreover, CD276 is another target of interest.
Subject(s)
Hematopoietic Stem Cell Transplantation , Retinal Neoplasms , Retinoblastoma , Humans , Retinoblastoma/therapy , Transplantation, Autologous , Neoplasm Recurrence, Local , Immunotherapy , Gangliosides , B7 AntigensABSTRACT
Early treatment of retinoblastoma (RB) has significantly improved clinical outcomes. N6-methyladenosine (m6A) methylation is crucial for cancer progression. Thus, we investigated the role of FTO-dependent demethylation in RB and its underlying mechanisms. The biological behavior of RB cells was analyzed using cell counting kit-8, colony formation analysis, transwell assay, flow cytometry, and western blot analysis. m6A modification was evaluated using methylated RNA immunoprecipitation and dual-luciferase reporter assays, and E2F3 stability was assessed using Actinomycin D. The roles of FTO and E2F3 were also elucidated in vivo. These results indicated that FTO was highly expressed in RB cells with low m6A levels. FTO knockdown inhibited RB cell growth, migration, invasion, and epithelial-mesenchymal transition and arrested the cell cycle at the G0/G1 phase. Mechanistically, FTO interference promoted m6A methylation of E2F3, which was recognized by YTHDF2, thereby reducing mRNA stability. E2F3 overexpression partially rescued the effects of FTO knockdown on biological behavior. Moreover, FTO knockdown reduced tumor weight, tumor volume, ki67 expression, and tumor cell infiltration by mediating E2F3. Taken together, FTO silencing inhibited the malignant processes of RB by suppressing E2F3 in an m6A-YTHD2-dependent manner. These findings suggest that FTO is a novel therapeutic target for RB.
Subject(s)
Alpha-Ketoglutarate-Dependent Dioxygenase FTO , E2F3 Transcription Factor , Retinal Neoplasms , Retinoblastoma , Humans , Adenosine , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism , Cell Cycle , E2F3 Transcription Factor/genetics , E2F3 Transcription Factor/metabolism , Retinoblastoma/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
PURPOSE: To describe the clinical presentation and treatment outcomes of children who received a diagnosis of retinoblastoma in 2017 throughout Asia. DESIGN: Multinational, prospective study including treatment-naïve patients in Asia who received a diagnosis of retinoblastoma in 2017 and were followed up thereafter. PARTICIPANTS: A total of 2112 patients (2797 eyes) from 96 retinoblastoma treatment centers in 33 Asian countries. INTERVENTIONS: Chemotherapy, radiotherapy, enucleation, and orbital exenteration. MAIN OUTCOME MEASURES: Enucleation and death. RESULTS: Within the cohort, 1021 patients (48%) were from South Asia (SA), 503 patients (24%) were from East Asia (EA), 310 patients (15%) were from Southeast Asia (SEA), 218 patients (10%) were from West Asia (WA), and 60 patients (3%) were from Central Asia (CA). Mean age at presentation was 27 months (median, 23 months; range, < 1-261 months). The cohort included 1195 male patients (57%) and 917 female patients (43%). The most common presenting symptoms were leukocoria (72%) and strabismus (13%). Using the American Joint Committee on Cancer Staging Manual, Eighth Edition, classification, tumors were staged as cT1 (n = 441 [16%]), cT2 (n = 951 [34%]), cT3 (n = 1136 [41%]), cT4 (n = 267 [10%]), N1 (n = 48 [2%]), and M1 (n = 129 [6%]) at presentation. Retinoblastoma was treated with intravenous chemotherapy in 1450 eyes (52%) and 857 eyes (31%) underwent primary enucleation. Three-year Kaplan-Meier estimates for enucleation and death were 33% and 13% for CA, 18% and 4% for EA, 27% and 15% for SA, 32% and 22% for SEA, and 20% and 11% for WA (P < 0.0001 and P < 0.0001), respectively. CONCLUSIONS: At the conclusion of this study, significant heterogeneity was found in treatment outcomes of retinoblastoma among the regions of Asia. East Asia displayed better outcomes with higher rates of globe and life salvage, whereas Southeast Asia showed poorer outcomes compared with the rest of Asia. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.
Subject(s)
Retinal Neoplasms , Retinoblastoma , Child , Humans , Male , Female , Infant , Child, Preschool , Retinoblastoma/diagnosis , Retinoblastoma/epidemiology , Retinoblastoma/therapy , Retinal Neoplasms/diagnosis , Retinal Neoplasms/epidemiology , Retinal Neoplasms/therapy , Prospective Studies , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Treatment Outcome , Asia/epidemiology , Retrospective Studies , Eye EnucleationABSTRACT
PURPOSE: To study the long-term efficacy of intravitreal topotecan (IVT) for vitreous seeds in eyes with retinoblastoma and risk factors for their recurrence. DESIGN: Retrospective, non-comparative, interventional study. PARTICIPANTS: Ninety-one eyes of 90 patients with retinoblastoma treated between January 2013 and April 2019. METHODS: Patients with recurrent or refractory vitreous seeds after completion of intravenous or intra-arterial chemotherapy were treated with IVT (30 µg/0.15 ml) by the safety-enhanced technique. The injection was repeated every 4 weeks until the regression of seeds. Patients with a minimum follow-up of 12 months were included in the analysis. MAIN OUTCOME MEASURES: Primary outcome measures were vitreous seed regression and eye salvage. Secondary outcomes were risk factors for vitreous seed recurrence after treatment with IVT, vision salvage, and complications of IVT. RESULTS: The median age of the patients was 18 months, with most having group D (n = 58 [64%]) and group E (n = 26 [29%]) retinoblastoma. Vitreous seeds were refractory in 46 eyes (51%) and recurrent in 45 eyes (49%). A total of 317 IVT injections were administered, with the median being 3 injections. The median number of IVT injections required was 2.5 injections for dust, 3 injections for sphere, and 5 injections for cloud morphologic features. Recurrence of vitreous seeds after IVT was seen in 17 eyes (19%) at a mean follow-up of 7.9 months. At a mean follow-up 34 months, vitreous seed regression was achieved in 88 eyes (97%) and eye salvage was achieved in 77 eyes (85%). Older age (P = 0.018) and recurrence of retinal tumor (15/17 eyes; P < 0.01) significantly increased the risk of vitreous seed recurrence. Cataract was the most common complication seen in 17 eyes (9%). CONCLUSIONS: Intravitreal topotecan at an every 3- to 4-week regimen is effective against both refractory and recurrent vitreous seeds. The vitreous seed morphologic features correspond to the number of injections required for regression. Increasing age and recurrence of retinal tumor increase the risk of vitreous seed recurrence after treatment with IVT. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.
ABSTRACT
BACKGROUND: Management guidelines and corresponding survival data for patients with recurrent retinoblastoma (RB) are lacking. This study aimed to summarize the clinical characteristics of patients with recurrent RB and analyze their survival outcomes. METHODS: We retrospectively analyzed 68 patients with recurrent RB who underwent treatment in our institution from January 2016 to December 2020. Patients were grouped according to location of recurrence: intraocular, orbital, and distant metastasis. RESULTS: The male:female ratio was 1.3:1 and the median age at recurrence was 37.5 months (range, 30.3-62.8). The number of patients in the intraocular recurrence, orbital recurrence, and metastasis groups was 13 (19.1%), 23 (33.8%), and 32 (47.1%), respectively. Thirty patients died, 36 were alive at last follow-up, and two were lost to follow-up. Eye enucleation was performed in 94.1% of patients. Five-year overall survival in patients with intraocular recurrence, orbital recurrence, and metastasis was 84.6%, 69.6%, and 31.3%, respectively (P = 0.001). Most deaths occurred within 2 years of recurrence. Presence of high-risk pathological factors, central nervous system invasion, and absence of combination therapy were independent predictors of worse 5-year overall survival. CONCLUSION: The rate of eye preservation in survivors of recurrent RB was very low. Although 5-year overall survival in patients who underwent treatment for intraocular and orbital recurrence was high, it was low in those with metastasis. RB patients may need lifelong follow-up for recurrence and secondary malignancy.