Repetitive Elements Trigger RIG-I-like Receptor Signaling that Regulates the Emergence of Hematopoietic Stem and Progenitor Cells.

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.

Lineage regulators direct BMP and Wnt pathways to cell-specific programs during differentiation and regeneration.

BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, whereas expression of the erythroid regulator GATA1 directs SMAD1 loss on nonerythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity.

DDX41-associated susceptibility to myeloid neoplasms.

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.

Author Correction: Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Inflammation in Development and Aging: Insights from the Zebrafish Model.

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.

Ddx41 inhibition of DNA damage signaling permits erythroid progenitor expansion in zebrafish.

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.

Histopathologic features of breast cancer in Li-Fraumeni syndrome.

Breast cancer is the most common malignancy in female patients with Li-Fraumeni syndrome (LFS), a rare autosomal dominant hereditary syndrome characterized by germline TP53 mutations. Recent studies have shown that the majority of these tumors are estrogen receptor (ER) positive with frequent HER2 co-expression. However, the morphologic features of these tumors have not been as well studied as other germline-associated breast cancers. We evaluated the pathologic features of 27 invasive and in situ carcinomas from patients with known germline TP53 mutations collected through the Li-Fraumeni Consortium. Overall, 60% of cases were HER2 positive and 44% showed ER co-expression. Most DCIS was high nuclear grade with central necrosis and associated periductal fibrosis and lymphocytic response. Invasive carcinomas were mostly of ductal type (NOS), modified Scarff-Bloom-Richardson (mSBR) high grade, with marked nuclear atypia and high mitotic rate. Prominent tumor infiltrating lymphocytes, syncytial growth pattern, or pushing borders were not seen in these tumors. High p53 IHC expression was seen in tumors from individuals with germline TP53 missense mutations whereas little or no protein expression (<1% nuclear expression, null pattern) was seen in tumors from carriers of non-missense mutations. In this study, we report in detail the morphologic features of invasive and in situ carcinomas in LFS. We found that these tumors share features with cancers harboring somatic TP53 mutations but are distinct from BRCA-associated breast cancers.

Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment.

Haematopoietic stem and progenitor cell (HSPC) transplant is a widely used treatment for life-threatening conditions such as leukaemia; however, the molecular mechanisms regulating HSPC engraftment of the recipient niche remain incompletely understood. Here we develop a competitive HSPC transplant method in adult zebrafish, using in vivo imaging as a non-invasive readout. We use this system to conduct a chemical screen, and identify epoxyeicosatrienoic acids (EETs) as a family of lipids that enhance HSPC engraftment. The pro-haematopoietic effects of EETs were conserved in the developing zebrafish embryo, where 11,12-EET promoted HSPC specification by activating a unique activator protein 1 (AP-1) and runx1 transcription program autonomous to the haemogenic endothelium. This effect required the activation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, specifically PI(3)Kγ. In adult HSPCs, 11,12-EET induced transcriptional programs, including AP-1 activation, which modulate several cellular processes, such as migration, to promote engraftment. Furthermore, we demonstrate that the EET effects on enhancing HSPC homing and engraftment are conserved in mammals. Our study establishes a new method to explore the molecular mechanisms of HSPC engraftment, and discovers a previously unrecognized, evolutionarily conserved pathway regulating multiple haematopoietic generation and regeneration processes. EETs may have clinical application in marrow or cord blood transplantation.

Developmental HSC Microenvironments: Lessons from Zebrafish.

Hematopoietic stem cells (HSCs) posses the ability to maintain the blood system of an organism from birth to adulthood. The behavior of HSCs is modulated by its microenvironment. During development, HSCs acquire the instructions to self-renew and differentiate into all blood cell fates by passing through several developmental microenvironments. In this chapter, we discuss the signals and cell types that inform HSC decisions throughout ontogeny with a focus on HSC specification, mobilization, migration, and engraftment.

Signaling axis involving Hedgehog, Notch, and Scl promotes the embryonic endothelial-to-hematopoietic transition.

During development, the hematopoietic lineage transits through hemogenic endothelium, but the signaling pathways effecting this transition are incompletely characterized. Although the Hedgehog (Hh) pathway is hypothesized to play a role in patterning blood formation, early embryonic lethality of mice lacking Hh signaling precludes such analysis. To determine a role for Hh signaling in patterning of hemogenic endothelium, we assessed the effect of altered Hh signaling in differentiating mouse ES cells, cultured mouse embryos, and developing zebrafish embryos. In differentiating mouse ES cells and mouse yolk sac cultures, addition of Indian Hh ligand increased hematopoietic progenitors, whereas chemical inhibition of Hh signaling reduced hematopoietic progenitors without affecting primitive streak mesoderm formation. In the setting of Hh inhibition, induction of either Notch signaling or overexpression of Stem cell leukemia (Scl)/T-cell acute lymphocytic leukemia protein 1 rescued hemogenic vascular-endothelial cadherin(+) cells and hematopoietic progenitor formation. Together, our results reveal that Scl overexpression is sufficient to rescue the developmental defects caused by blocking the Hh and Notch pathways, and inform our understanding of the embryonic endothelial-to-hematopoietic transition.

BET bromodomain inhibition targets both c-Myc and IL7R in high-risk acute lymphoblastic leukemia.

We investigated the therapeutic potential of JQ1, an inhibitor of the BET class of human bromodomain proteins, in B-cell acute lymphoblastic leukemia (B-ALL). We show that JQ1 potently reduces the viability of B-ALL cell lines with high-risk cytogenetics. Among the most sensitive were lines with rearrangements of CRLF2, which is overexpressed in ~ 10% of B-ALL. CRLF2 heterodimerizes with the IL7 receptor (IL7R) and signals through JAK2, JAK1, and STAT5 to drive proliferation and suppress apoptosis. As previously observed, JQ1 induced the down-regulation of MYC transcription, the loss of BRD4 at the MYC promoter, and the reduced expression of c-Myc target genes. Strikingly, JQ1 also down-regulated IL7R transcription, depleted BRD4 from the IL7R promoter, and reduced JAK2 and STAT5 phosphorylation. Genome-wide expression profiling demonstrated a restricted effect of JQ1 on transcription, with MYC and IL7R being among the most down-regulated genes. Indeed, IL7R was the only cytokine receptor in CRLF2-rearranged B-ALL cells significantly down-regulated by JQ1 treatment. In mice xenografted with primary human CRLF2-rearranged B-ALL, JQ1 suppressed c-Myc expression and STAT5 phosphorylation and significantly prolonged survival. Thus, bromodomain inhibition is a promising therapeutic strategy for B-ALL as well as other conditions dependent on IL7R signaling.

Nucleic acid-induced inflammation on hematopoietic stem cells.

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.

Stromal cell-derived factor-1 and hematopoietic cell homing in an adult zebrafish model of hematopoietic cell transplantation.

In mammals, stromal cell-derived factor-1 (SDF-1) promotes hematopoietic cell mobilization and migration. Although the zebrafish, Danio rerio, is an emerging model for studying hematopoietic cell transplantation (HCT), the role of SDF-1 in the adult zebrafish has yet to be determined. We sought to characterize sdf-1 expression and function in the adult zebrafish in the context of HCT. In situ hybridization of adult zebrafish organs shows sdf-1 expression in kidney tubules, gills, and skin. Radiation up-regulates sdf-1 expression in kidney to nearly 4-fold after 40 Gy. Assays indicate that zebrafish hematopoietic cells migrate toward sdf-1, with a migration ratio approaching 1.5 in vitro. A sdf-1a:DsRed2 transgenic zebrafish allows in vivo detection of sdf-1a expression in the adult zebrafish. Matings with transgenic reporters localized sdf-1a expression to the putative hematopoietic cell niche in proximal and distal renal tubules and collecting ducts. Importantly, transplant of hematopoietic cells into myelosuppressed recipients indicated migration of hematopoietic cells to sdf-1a-expressing sites in the kidney and skin. We conclude that sdf-1 expression and function in the adult zebrafish have important similarities to mammals, and this sdf-1 transgenic vertebrate will be useful in characterizing the hematopoietic cell niche and its interactions with hematopoietic cells.

CD48 on hematopoietic progenitors regulates stem cells and suppresses tumor formation.

The proliferation and differentiation of adult stem cells is balanced to ensure adequate generation of differentiated cells, stem cell homeostasis, and guard against malignant transformation. CD48 is broadly expressed on hematopoietic cells but excluded from quiescent long-term murine HSCs. Through its interactions with CD244 on progenitor cells, it influences HSC function by altering the BM cytokine milieu, particularly IFNγ. In CD48-null mice, the resultant misregulation of cytokine signaling produces a more quiescent HSC, a disproportionate number of short-term progenitors, and hyperactivation of Pak1, leading to hematologic malignancies similar to those found in patients with X-linked lymphoproliferative disease. CD48 plays a vital role as an environmental sensor for regulating HSC and progenitor cell numbers and inhibiting tumor development.

Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis.

Haematopoietic stem cell (HSC) homeostasis is tightly controlled by growth factors, signalling molecules and transcription factors. Definitive HSCs derived during embryogenesis in the aorta-gonad-mesonephros region subsequently colonize fetal and adult haematopoietic organs. To identify new modulators of HSC formation and homeostasis, a panel of biologically active compounds was screened for effects on stem cell induction in the zebrafish aorta-gonad-mesonephros region. Here, we show that chemicals that enhance prostaglandin (PG) E2 synthesis increased HSC numbers, and those that block prostaglandin synthesis decreased stem cell numbers. The cyclooxygenases responsible for PGE2 synthesis were required for HSC formation. A stable derivative of PGE2 improved kidney marrow recovery following irradiation injury in the adult zebrafish. In murine embryonic stem cell differentiation assays, PGE2 caused amplification of multipotent progenitors. Furthermore, ex vivo exposure to stabilized PGE2 enhanced spleen colony forming units at day 12 post transplant and increased the frequency of long-term repopulating HSCs present in murine bone marrow after limiting dilution competitive transplantation. The conserved role for PGE2 in the regulation of vertebrate HSC homeostasis indicates that modulation of the prostaglandin pathway may facilitate expansion of HSC number for therapeutic purposes.

Hematopoietic Stem and Progenitor Cell Identification and Transplantation in Zebrafish.

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.

SF3B1 mutation and ATM deletion codrive leukemogenesis via centromeric R-loop dysregulation.

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.