RESUMO
Zebrafish are an emergent animal model to study human diseases due to their significant genetic similarity to humans, swift development, and genetic manipulability. Their utility extends to the exploration of the involvement of inflammation in host defense, immune responses, and tissue regeneration. Additionally, the zebrafish model system facilitates prompt screening of chemical compounds that affect inflammation. This study explored the diverse roles of inflammatory pathways in zebrafish development and aging. Serving as a crucial model, zebrafish provides insights into the intricate interplay of inflammation in both developmental and aging contexts. The evidence presented suggests that the same inflammatory signaling pathways often play instructive or beneficial roles during embryogenesis and are associated with malignancies in adults.
Assuntos
Inflamação , Peixe-Zebra , Animais , Humanos , Peixe-Zebra/genética , Modelos Animais , Envelhecimento/genética , Transdução de SinaisRESUMO
Hematopoiesis, the process of generating blood cells, starts during development with the primitive, pro-definitive, and definitive hematopoietic waves. The first two waves will generate erythrocytes and myeloid cells, although the definitive wave will give rise to hematopoietic stem cells (HSCs) that are multipotent and can produce most of the blood cells in an adult. Although HSCs are highly proliferative during development, during adulthood they remain quiescent in the bone marrow. Inflammatory signaling in the form of interferons, interleukins, tumor necrosis factors, and others is well-established to influence both developmental and adult hematopoiesis. Here we discuss the role of specific inflammatory pathways that are induced by sensing nucleic acids. We discuss the role of RNA-sensing members of the Toll-like, Rig-I-like, nucleotide-binding oligomerization domain (NOD)-like, and AIM2-like protein kinase receptors and the DNA-sensing receptors, DEAD-Box helicase 41 (DDX41) and cGAS. The main downstream pathways of these receptors are discussed, as well as their influence on developmental and adult hematopoiesis, including hematopoietic pathologies.
Assuntos
Ácidos Nucleicos , Humanos , Adulto , Ácidos Nucleicos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Hematopoese/fisiologia , Medula Óssea , Inflamação/metabolismoRESUMO
RNA splicing factor SF3B1 is recurrently mutated in various cancers, particularly in hematologic malignancies. We previously reported that coexpression of Sf3b1 mutation and Atm deletion in B cells, but not either lesion alone, leads to the onset of chronic lymphocytic leukemia (CLL) with CLL cells harboring chromosome amplification. However, the exact role of Sf3b1 mutation and Atm deletion in chromosomal instability (CIN) remains unclear. Here, we demonstrated that SF3B1 mutation promotes centromeric R-loop (cen-R-loop) accumulation, leading to increased chromosome oscillation, impaired chromosome segregation, altered spindle architecture, and aneuploidy, which could be alleviated by removal of cen-R-loop and exaggerated by deletion of ATM. Aberrant splicing of key genes involved in R-loop processing underlay augmentation of cen-R-loop, as overexpression of the normal isoform, but not the altered form, mitigated mitotic stress in SF3B1-mutant cells. Our study identifies a critical role of splice variants in linking RNA splicing dysregulation and CIN and highlights cen-R-loop augmentation as a key mechanism for leukemogenesis.
Assuntos
Leucemia Linfocítica Crônica de Células B , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Estruturas R-Loop , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mutação , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
Deleterious germ line DDX41 variants confer risk for myeloid neoplasms (MNs) and less frequently for lymphoid malignancies, with autosomal dominant inheritance and an estimated prevalence of 3% among MNs. Germ line DDX41 variants include truncating alleles that comprise about two-thirds of all alleles, missense variants located preferentially within the DEAD-box domain, and deletion variants. The identification of a truncating allele on tumor-based molecular profiling should prompt germ line genetic testing because >95% of such alleles are germ line. Somatic mutation of the wild-type DDX41 allele occurs in about half of MNs with germ line DDX41 alleles, typically in exons encoding the helicase domain and most frequently as R525H. Several aspects of deleterious germ line DDX41 alleles are noteworthy: (1) certain variants are common in particular populations, (2) MNs develop at older ages typical of de novo disease, challenging the paradigm that inherited cancer risk always causes disease in young people, (3) despite equal frequencies of these variants in men and women, men progress to MNs more frequently, suggesting a gender-specific effect on myeloid leukemogenesis, and (4) individuals with deleterious germ line DDX41 variants develop acute severe graft-versus-host disease after allogeneic hematopoietic cell transplantation with wild-type donors more than others unless they receive posttransplant cyclophosphamide, suggesting a proinflammatory milieu that stimulates donor-derived T cells. Biochemical studies and animal models have identified DDX41's ability to interact with double-stranded DNA and RNA:DNA hybrids with roles in messenger RNA splicing, ribosomal RNAs or small nucleolar RNAs processing, and modulation of innate immunity, disruption of which could promote inflammation and drive tumorigenesis.
Assuntos
RNA Helicases DEAD-box , Leucemia Mieloide , Transtornos Mieloproliferativos , Animais , Feminino , RNA Helicases DEAD-box/genética , Mutação em Linhagem Germinativa , Transtornos Mieloproliferativos/genética , RNA Mensageiro , Humanos , MasculinoRESUMO
The zebrafish as a model organism is well known for its versatile genetics, rapid development, and straightforward live imaging. It is an excellent model to study hematopoiesis because of its highly conserved ontogeny and gene regulatory networks. Recently developed highly specific transgenic reporter lines have allowed direct imaging and tracking of hematopoietic stem and progenitor cells (HSPCs) in live zebrafish. These reporter lines can also be used for fluorescence-activated cell sorting (FACS) of HSPCs. Similar to mammalian models, HSPCs can be transplanted to reconstitute the entire hematopoietic system of zebrafish recipients. However, the zebrafish provides unique advantages to study HSPC biology, such as transplants into embryos and high-throughput chemical screening. This chapter will outline the methods needed to identify, isolate, and transplant HSPCs in zebrafish.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais Geneticamente Modificados , Mamíferos/metabolismoRESUMO
Hematopoietic stem and progenitor cells (HSPCs) sustain lifelong hematopoiesis. Mutations of pre-mRNA splicing machinery, especially splicing factor 3b, subunit 1 (SF3B1), are early lesions found in malignancies arising from HSPC dysfunction. However, why splicing factor deficits contribute to HSPC defects remains incompletely understood. Using zebrafish, we show that HSPC formation in sf3b1 homozygous mutants is dependent on STAT3 activation. Clinically, mutations in SF3B1 are heterozygous; thus, we explored if targeting STAT3 could be a vulnerability in these cells. We show that SF3B1 heterozygosity confers heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Cells carrying mutations in other splicing factors or treated with splicing modulators are also more sensitive to STAT3 inhibition. Mechanistically, we illustrate that STAT3 inhibition exacerbates aberrant splicing in SF3B1 mutant cells. Our findings reveal a conserved vulnerability of splicing factor mutant HSPCs that could allow for their selective targeting in hematologic malignancies.
Assuntos
Hematopoese , Peixe-Zebra , Camundongos , Humanos , Animais , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Peixe-Zebra/metabolismo , Hematopoese/genética , Splicing de RNA/genética , Células-Tronco Hematopoéticas/metabolismo , Mutação/genética , Fosfoproteínas/metabolismo , Fator de Transcrição STAT3/genéticaRESUMO
DEAD-box Helicase 41 (DDX41) is a member of the DExD/H-box helicase family that has a variety of cellular functions. Of note, germline and somatic mutations in the DDX41 gene are prevalently found in myeloid malignancies. Here, we present a comprehensive and analytic review covering relevant clinical, translational and basic science findings on DDX41. We first describe the initial characterisation of DDX41 mutations in patients affected by myelodysplastic syndromes, their associated clinical characteristics, and current treatment modalities. We then cover the known cellular functions of DDX41, spanning from its discovery in Drosophila as a neuroregulator through its more recently described roles in inflammatory signalling, R-loop metabolism and snoRNA processing. We end with a summary of the identified basic functions of DDX41 that when perturbed may contribute to the underlying pathology of haematologic neoplasms.
Assuntos
RNA Helicases DEAD-box , Neoplasias Hematológicas , Síndromes Mielodisplásicas , Humanos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismoRESUMO
Background: Mutations in the SF3B1 splicing factor are commonly seen in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), yet the specific oncogenic pathways activated by mis-splicing have not been fully elucidated. Inflammatory immune pathways have been shown to play roles in the pathogenesis of MDS, though the exact mechanisms of their activation in splicing mutant cases are not well understood. Methods: RNA-seq data from SF3B1 mutant samples was analyzed and functional roles of interleukin-1 receptor-associated kinase 4 (IRAK4) isoforms were determined. Efficacy of IRAK4 inhibition was evaluated in preclinical models of MDS/AML. Results: RNA-seq splicing analysis of SF3B1 mutant MDS samples revealed retention of full-length exon 6 of IRAK4, a critical downstream mediator that links the Myddosome to inflammatory NF-kB activation. Exon 6 retention leads to a longer isoform, encoding a protein (IRAK4-long) that contains the entire death domain and kinase domain, leading to maximal activation of NF-kB. Cells with wild-type SF3B1 contain smaller IRAK4 isoforms that are targeted for proteasomal degradation. Expression of IRAK4-long in SF3B1 mutant cells induces TRAF6 activation leading to K63-linked ubiquitination of CDK2, associated with a block in hematopoietic differentiation. Inhibition of IRAK4 with CA-4948, leads to reduction in NF-kB activation, inflammatory cytokine production, enhanced myeloid differentiation in vitro and reduced leukemic growth in xenograft models. Conclusions: SF3B1 mutation leads to expression of a therapeutically targetable, longer, oncogenic IRAK4 isoform in AML/MDS models. Funding: This work was supported by Cincinnati Children's Hospital Research Foundation, Leukemia Lymphoma Society, and National Institute of Health (R35HL135787, RO1HL111103, RO1DK102759, RO1HL114582), Gabrielle's Angel Foundation for Cancer Research, and Edward P. Evans Foundation grants to DTS. AV is supported by Edward P. Evans Foundation, National Institute of Health (R01HL150832, R01HL139487, R01CA275007), Leukemia and Lymphoma Society, Curis and a gift from the Jane and Myles P. Dempsey family. AP and JB are supported by Blood Cancer UK (grants 13042 and 19004). GC is supported by a training grant from NYSTEM. We acknowledge support of this research from The Einstein Training Program in Stem Cell Research from the Empire State Stem Cell Fund through New York State Department of Health Contract C34874GG. MS is supported by a National Institute of Health Research Training and Career Development Grant (F31HL132420).
Genes contain blocks of code that tell cells how to make each part of a protein. Between these blocks are sections of linking DNA, which cells remove when they are preparing to use their genes. Scientists call this process 'splicing'. Cells can splice some genes in more than one way, allowing them to make different proteins from the same genetic code. Mutations that affect the splicing process can change the way cells make their proteins, leading to disease. For example, the myelodysplastic syndromes are a group of blood cancers often caused by mutations in splicing proteins, such as SF3B1. The disorder stops blood cells from maturing and causes abnormal inflammation. So far, the link between splicing, blood cell immaturity, inflammation and cancer is not clear. To find out more, Choudhary, Pellagatti et al. looked at the spliced genetic code from people with myelodysplastic syndromes. Mutations in the splicing protein SF3B1 changed the way cells spliced an important signalling molecule known as IRAK4. Affected cells cut out less genetic code and made a longer version of this signalling protein, named IRAK4-Long. This altered protein activated inflammation and stopped blood cells from maturing. Blocking IRAK4-Long reversed the effects. It also reduced tumour formation in mice carrying affected human cells. The molecule used to block IRAK4, CA-4948 also known as Emavusertib is currently being evaluated in clinical trials for myelodysplastic syndromes and other types of blood cancer. The work of Choudhary, Pellagatti et al. could help scientists to design genetic tests to predict which patients might benefit from this treatment.
Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Criança , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação , Síndromes Mielodisplásicas/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Isoformas de Proteínas/metabolismo , Splicing de RNARESUMO
DEAD-box Helicase 41 (DDX41) is a recently identified factor mutated in hematologic malignancies whose function in hematopoiesis is unknown. Using an in vivo model of Ddx41 deficiency, we unveiled a critical role for this helicase in regulating erythropoiesis. We demonstrated that loss of ddx41 leads to anemia caused by diminished proliferation and defective differentiation of erythroid progenitors. Mis-expression and alternative splicing of cell cycle genes is rampant in ddx41 mutant erythroid progenitors. We delineated that the DNA damage response is activated in mutant cells resulting in an Ataxiatelangiectasia mutated (ATM) and Ataxia-telangiectasia and Rad3-related (ATR)-triggered cell cycle arrest. Inhibition of these kinases partially suppressed ddx41 mutant anemia. These findings establish a critical function for Ddx41 in promoting healthy erythropoiesis via protection from genomic stress and delineate a mechanistic framework to explore a role for ATM and ATR signaling in DDX41-mutant hematopoietic pathologies.
Assuntos
Dano ao DNA , Peixe-Zebra , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , RNA Helicases DEAD-box/genética , Eritropoese/genética , Transdução de Sinais , Peixe-Zebra/metabolismoRESUMO
PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) are formed embryonically during a dynamic developmental process and later reside in adult hematopoietic organs in a quiescent state. In response to their changing environment, HSCs have evolved diverse mechanisms to cope with intrinsic and extrinsic challenges. This review intends to discuss how HSCs and other stem cells co-opted DNA and RNA innate immune pathways to fine-tune developmental processes. RECENT FINDINGS: Innate immune receptors for nucleic acids like the RIG-I-like family receptors and members of DNA sensing pathways are expressed in HSCs and other stem cells. Even though the "classic" role of these receptors is recognition of foreign DNA or RNA from pathogens, it was recently shown that cellular transposable element (TE) RNA or R-loops activate such receptors, serving as endogenous triggers of inflammatory signaling that can shape HSC formation during development and regeneration. SUMMARY: Endogenous TEs and R-loops activate RNA and DNA sensors, which trigger distinct inflammatory signals to fine-tune stem cell decisions. This phenomenon could have broad implications for diverse somatic stem cells, for a variety of diseases and during aging.
RESUMO
Hematopoietic stem cells (HSCs) are rare cells that arise in the embryo and sustain adult hematopoiesis. Although the functional potential of nascent HSCs is detectable by transplantation, their native contribution during development is unknown, in part due to the overlapping genesis and marker gene expression with other embryonic blood progenitors. Using single-cell transcriptomics, we define gene signatures that distinguish nascent HSCs from embryonic blood progenitors. Applying a lineage-tracing approach to selectively track HSC output in situ, we find significantly delayed lymphomyeloid contribution. An inducible HSC injury model demonstrates a negligible impact on larval lymphomyelopoiesis following HSC depletion. HSCs are not merely dormant at this developmental stage, as they showed robust regeneration after injury. Combined, our findings illuminate that nascent HSCs self-renew but display differentiation latency, while HSC-independent embryonic progenitors sustain developmental hematopoiesis. Understanding these differences could improve de novo generation and expansion of functional HSCs.
Assuntos
Células-Tronco Embrionárias/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Linhagem da Célula , Autorrenovação Celular , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/citologia , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Camundongos , Análise de Célula Única , Transcriptoma , Peixe-ZebraRESUMO
Hematopoietic stem and progenitor cells (HSPCs) arise during embryonic development and are essential for sustaining the blood and immune systems throughout life. Tight regulation of HSPC numbers is critical for hematopoietic homeostasis. Here, we identified DEAD-box helicase 41 (Ddx41) as a gatekeeper of HSPC production. Using zebrafish ddx41 mutants, we unveiled a critical role for this helicase in regulating HSPC production at the endothelial-to-hematopoietic transition. We determined that Ddx41 suppresses the accumulation of R-loops, nucleic acid structures consisting of RNA:DNA hybrids and ssDNAs whose equilibrium is essential for cellular fitness. Excess R-loop levels in ddx41 mutants triggered the cGAS-STING inflammatory pathway leading to increased numbers of hemogenic endothelium and HSPCs. Elevated R-loop accumulation and inflammatory signaling were observed in human cells with decreased DDX41, suggesting possible conservation of mechanism. These findings delineate that precise regulation of R-loop levels during development is critical for limiting cGAS-STING activity and HSPC numbers.
Assuntos
Embrião não Mamífero/citologia , Células-Tronco Hematopoéticas/citologia , Estruturas R-Loop , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Diferenciação Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Embrião não Mamífero/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Hematopoietic stem cell transplantation (HSCT) is a treatment for many malignant, congenital, and acquired hematologic diseases. Some outstanding challenges in the HSCT field include the paucity of immunologically-matched donors, our inability to effectively expand hematopoeitic stem cells (HSCs) ex vivo, and the high infection risk during engraftment. Scientists are striving to develop protocols to generate, expand, and maintain HSCs ex vivo, however these are not yet ready for clinical application. Given these problems, advancing our understanding of HSC specification, regulation, and differentiation in preclinical models is essential to improve the therapeutic utility of HSCT. In this review, we link biomedical researchers and transplantation clinicians by discussing the potential therapeutic implications of recent fundamental HSC research in model organisms. We consider deficiencies in current HSCT practice, such as problems achieving adequate cell dose for successful and rapid engraftment, immense inflammatory cascade activation after myeloablation, and graft-vs-host disease. Furthermore, we discuss recent advances in the field of HSC biology and transplantation made in preclinical models of zebrafish, mouse, and nonhuman primates that could inform emerging practice for clinical application.
Assuntos
Modelos Animais de Doenças , Transplante de Células-Tronco Hematopoéticas , Animais , Doença Enxerto-Hospedeiro , Células-Tronco Hematopoéticas , Camundongos , Primatas , Condicionamento Pré-Transplante , Peixe-ZebraRESUMO
Transplantation is the most common assay for measuring the in vivo functionality of hematopoietic stem cells (HSCs). Although various HSC transplantation strategies have been developed in zebrafish, they are underutilized because of challenges related to immune matching and preconditioning toxicity. To circumvent these limitations, we developed a simple and robust transplantation model using HSC-deficient hosts. Homozygous runx1W84X mutants are devoid of definitive hematopoietic cells, including HSCs and adaptive immune cells; thus, they require no preconditioning regimen for transplantation. Marrow cell transplantation into runx1-mutant zebrafish 2 days after fertilization significantly improved their survival to adulthood and resulted in robust, multilineage, long-lasting, serially repopulating engraftment. Furthermore, we demonstrated that engraftment into runx1 homozygous mutants was significantly higher than into runx1 heterozygotes, demonstrating that the improved transplantation success is attributable to the empty HSC niche in mutants and not just the embryonic environment. Competitive transplantation of marrow cells into runx1 mutants revealed a stem cell frequency similar to that of murine marrow cells, which demonstrates the utility of this model for quantifying HSC function. The streamlined approach and robustness of this assay will help broaden its feasibility for future high-throughput transplantation experiments in zebrafish and will enable further novel discoveries in the biology of HSCs.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Peixe-Zebra , Animais , Medula Óssea , Transplante de Medula Óssea , Células-Tronco Hematopoéticas , CamundongosRESUMO
Inflammatory signaling is required for hematopoietic stem and progenitor cell (HSPC) development. Here, we studied the involvement of RIG-I-like receptors (RLRs) in HSPC formation. Rig-I or Mda5 deficiency impaired, while Lgp2 deficiency enhanced, HSPC emergence in zebrafish embryos. Rig-I or Mda5 deficiency reduced HSPC numbers by inhibiting inflammatory signals that were in turn enhanced in Lgp2 deficient embryos. Simultaneous reduction of Lgp2 and either Rig-I or Mda5 rescued inflammatory signals and HSPC numbers. Modulating the expression of the signaling mediator Traf6 in RLR deficient embryos restored HSPC numbers. Repetitive element transcripts could be detected in hemogenic endothelial cells and HSPCs, suggesting a role as RLR ligands. Indeed, ectopic expression of repetitive elements enhanced HSPC formation in wild-type, but not in Rig-I or Mda5 deficient embryos. Manipulation of RLR expression in mouse fetal liver HSPCs indicated functional conservation among species. Thus, repetitive elements transcribed during development drive RLR-mediated inflammatory signals that regulate HSPC formation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/metabolismo , Sequências Repetitivas de Ácido Nucleico , Transdução de Sinais , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Animais , Biomarcadores , Montagem e Desmontagem da Cromatina , Elementos de DNA Transponíveis , Suscetibilidade a Doenças , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Imunidade Inata , Imuno-Histoquímica , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , RNA Helicases/deficiência , RNA Helicases/genética , Proteínas de Ligação a RNA/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Ácido Valproico/farmacologia , Peixe-ZebraRESUMO
Genome-wide association studies identify genomic variants associated with human traits and diseases. Most trait-associated variants are located within cell-type-specific enhancers, but the molecular mechanisms governing phenotypic variation are less well understood. Here, we show that many enhancer variants associated with red blood cell (RBC) traits map to enhancers that are co-bound by lineage-specific master transcription factors (MTFs) and signaling transcription factors (STFs) responsive to extracellular signals. The majority of enhancer variants reside on STF and not MTF motifs, perturbing DNA binding by various STFs (BMP/TGF-ß-directed SMADs or WNT-induced TCFs) and affecting target gene expression. Analyses of engineered human blood cells and expression quantitative trait loci verify that disrupted STF binding leads to altered gene expression. Our results propose that the majority of the RBC-trait-associated variants that reside on transcription-factor-binding sequences fall in STF target sequences, suggesting that the phenotypic variation of RBC traits could stem from altered responsiveness to extracellular stimuli.