Ezh1 Inhibits Commitment to Hemogenic Fate and HSPC Formation during Vertebrate Development

Understanding how hematopoietic stem cells (HSCs) are specified from mesodermal precursors is essential to the goal of generating patient-specific HSCs capable of multi-potent long-term function. HSCs are born from hemogenic endothelium in select arterial niches during embryonic development through a transdifferentiation process turned endothelial-to-hematopoietic transistion (EHT). Despite increasing efforts to recapitulate this process in vitro, current differentiation protocols largely fail to produce long-lived multi-lineage progenitors from human induced pluripotent stem cell (iPSC) sources. Recently, an in vitro loss-of-function screen in human hematopoietic progenitors identified the Polycomb group protein, Enhancer of Zeste Homolog 1 (EZH1), as a regulator of definitive hematopoietic commitment, as assayed by acquisition of lymphoid competence. To determine the mechanism by which Ezh1 regulates HSPC fate in vivo we employed functional knockdown and epistasis investigations using the zebrafish model. Morpholino-mediated knockdown of ezh1 promoted expression of the conserved HSC markers runx1 and c-myb in the ventral wall of the dorsal aorta (VDA) at 36 hours post fertilization (hpf), as assessed by whole mount in situ hybridization (WISH); additionally, expression of the lymphoid marker rag1 was found to be enhanced at 120 hpf, as assayed by WISH and fluorescent activated cell sorting (FACS), in line with our in vitro observations. An impact on HSPCs was confirmed and quantified by qPCR for runx1 (**p < 0.01) and FACS using the CD41:GFP reporter line (**p < 0.01), indicating significantly increased HSPC number. Importantly, this enhancement in HSPC production had no effect on gross vascular morphology of the niche as determined by confocal microscopy for flk1. Assessment of arterial versus venous fate indicated that while the latter was unchanged in morphant embryos, expression of the arterial markers, epbrinb2a, dll4, dlc and tbx20, was strongly reduced by WISH and qPCR (**p < 0.01, *p < 0.05, **p < 0.01, and **** p < 0.001, respectively). In contrast, markers of hemogenic commitment, gata2b, and scl/flk1, were significantly increased, suggesting that loss of ezh1 enhanced hematopoietic potential at the expense of maintaining arterial fate. Profiling of single-cell RNA-sequencing data obtained from sorted populations of E10.5 mouse embryos revealed EZH1 to be more highly expressed in cells undergoing the endothelial-to-hematopoietic transition, consistent with a role of EZH1 in regulating arterial verses hematopoietic fate. Gene set enrichment analysis (GSEA) from our prior in vitro studies revealed the Notch pathway to be significantly altered following EZH1 knockdown. As Notch signaling has been implicated in both arterial specification and HSC emergence, we next examined the potential role of Notch signaling in ezh1 knockdown-mediated HSPC expansion. Consistent with a hypothesized interaction, differential regulation of Notch ligands and receptors was observed in ezh1 morphants compared to wild-type siblings; specifically, expression of arterial ligands, dll4 and dlc were decreased, while hematopoietic ligands and receptors, jag1a and notch1a were enhanced. Notably, the effect on Notch signaling was specific to ezh1 knockdown, as ezh2 loss shows a distinct pattern and temporal impact, reducing HSC production rather than enhancing it, consistent with recent reports. The strong conservation of ezh1-mediated regulation of HSC number, and our identification of its mechanistic role at the level of Notch receptor/ligand interactions, position zebrafish as a platform to identify chemical mediators that can be used to regulate ezh1 function during in vitro differentiation to unlock multi-lineage HSC commitment of human iPSC for therapeutic application. Disclosures Daley: Epizyme, Inc: Other: Equity & Consulting Fees; 28/7 Therapeutics: Other: Equity & Consulting Fees.

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Notch pathway activation targets AML-initiating cell homeostasis and differentiation

Journal of Experimental Medicine ◽

10.1084/jem.20121484 ◽

2013 ◽

Vol 210 (2) ◽

pp. 301-319 ◽

Cited By ~ 94

Author(s):

Camille Lobry ◽

Panagiotis Ntziachristos ◽

Delphine Ndiaye-Lobry ◽

Philmo Oh ◽

Luisa Cimmino ◽

...

Keyword(s):

Tumor Suppressor ◽

Notch Signaling ◽

Myeloproliferative Neoplasms ◽

Notch Pathway ◽

Notch Signaling Pathway ◽

Notch Receptor ◽

Pathway Activation ◽

Function Models

Notch signaling pathway activation is known to contribute to the pathogenesis of a spectrum of human malignancies, including T cell leukemia. However, recent studies have implicated the Notch pathway as a tumor suppressor in myeloproliferative neoplasms and several solid tumors. Here we report a novel tumor suppressor role for Notch signaling in acute myeloid leukemia (AML) and demonstrate that Notch pathway activation could represent a therapeutic strategy in this disease. We show that Notch signaling is silenced in human AML samples, as well as in AML-initiating cells in an animal model of the disease. In vivo activation of Notch signaling using genetic Notch gain of function models or in vitro using synthetic Notch ligand induces rapid cell cycle arrest, differentiation, and apoptosis of AML-initiating cells. Moreover, we demonstrate that Notch inactivation cooperates in vivo with loss of the myeloid tumor suppressor Tet2 to induce AML-like disease. These data demonstrate a novel tumor suppressor role for Notch signaling in AML and elucidate the potential therapeutic use of Notch receptor agonists in the treatment of this devastating leukemia.

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Notch Signaling Is Necessary and Sufficient for Runx1-Dependent Stem Cell Induction in the Embryonic Aorta-Gonad-Mesonephros (AGM) Region.

Blood ◽

10.1182/blood.v104.11.4161.4161 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4161-4161

Author(s):

Caroline Erter Burns ◽

Leonard I. Zon

Keyword(s):

Stem Cell ◽

Notch Signaling ◽

Cell Fate ◽

Notch Pathway ◽

Notch Receptor ◽

Dorsal Aorta ◽

Loss Of Function ◽

Cardinal Vein ◽

Hematopoietic Stem

Abstract Vertebrate hematopoiesis can be divided into two embryonic phases: a short primitive wave predominantly generating erythrocytes and a definitive (fetal/adult) wave producing long-term hematopoietic stem cells (HSCs). The definitive wave occurs in the embryonic aorta-gonad-mesonephros (AGM) region through the asymmetric induction of HSCs from the ventral, but not dorsal, aortic endothelial wall. Since Notch signaling is critical for orchestrating a variety of developmental cell fate choices from invertebrates to humans and has been implicated in affecting the differentiation of some hematopoietic lineages, we analyzed whether the Notch pathway regulates definitive HSC induction in vivo. The zebrafish mutant mindbomb harbors a mutation in an essential E3 ligase that ubiquitylates Delta, which in turn allows the Notch intercellular domain to be released and activate downstream target gene transcription. Thus, in the absence of Mindbomb function Notch signaling does not occur. We found that although mindbomb mutants show normal primitive hematopoiesis, definitive c-myb and runx1 HSC expression is lacking. Since embryos injected with synthetic morpholinos designed to inhibit proper splicing of runx1 RNA ( runx morphants) show the same hematopoietic phenotype as mindbomb mutants, we next addressed the epistatic relationship between notch and runx1 using classic gain-of-function and loss-of-function analyses. In runx1 morphants expression of a notch receptor, notch3, and a delta ligand, deltaC, in the developing dorsal aorta was normal. Moreover, injection of runx1 RNA rescued HSCs in the AGM of mindbomb mutants. Together, these results suggest that Runx1 functions downstream of Notch in promoting HSC fate. We next analyzed whether a constitutively activated form of Notch (NICD) is sufficient for HSC specification in the AGM using an inducible binary transgenic system. Zebrafish carrying the heat-shock promoter driving the activator gal4 were mated to animals carrying 6 gal4 -responsive tandem upstream activating sequences (UAS) driving NICD. At the 10 somite-stage the embryos were heat-shocked at 37°C for 1 hour to activate NICD throughout the double transgenic animals. Surprisingly, expression of both HSC markers, c-myb and runx1, were expanded from their normal restricted domain in the ventral endothelium to the entire circumference of the dorsal aorta. Most interestingly, the presence of ectopic c-myb and runx1 transcripts were observed in the developing post-cardinal vein, a vessel that normally does not produce HSCs. These data imply that activation of the Notch pathway generates increased numbers of HSCs in vivo. When runx1 RNA is injected into wild-type embryos a similar expansion of c-myb transcripts is seen throughout the entire dorsal aorta and post-cardinal vein, further indicating that Runx1 functions downstream of Notch in HSC induction. In summary, discovery of the molecular programs essential and sufficient for fetal/adult hematopoietic ontogeny will lead to a further understanding of the physiologic and pathologic processes regulating stem cell homeostasis and translate into more effective therapies for blood disorders.

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TLR3 or TLR4 Activation Enhances MSC-Mediated Treg Generation Via Notch Signaling

Blood ◽

10.1182/blood.v126.23.3604.3604 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3604-3604

Author(s):

Iran Rashedi ◽

Alejandro Gomez-Aristizábal ◽

Xinghua Wang ◽

Sowmya Viswanathan ◽

Armand Keating

Keyword(s):

Notch Signaling ◽

Conflicts Of Interest ◽

Notch Pathway ◽

Poly I:C ◽

Cell Contact ◽

Human Mscs ◽

Cd4 Cells ◽

Control Mscs

Abstract Mesenchymal stromal cells (MSCs) are used as cell therapy for a variety of disorders, largely because of their immunosuppressive and regenerative functions by exerting immune effects via direct and indirect interactions with many types of immune cells. MSCs recruit and promote the generation of regulatory T cells (Tregs) both in vitro and in vivo. Toll-like receptors (TLRs), known for roles in innate and adaptive immunity, are involved in numerous pathological conditions, including graft-versus-host disease (GVHD). Several TLRs, especially TLR3 and TLR4, are highly expressed on MSCs and affect immunomodulatory functions and possibly, therapeutic potency. Indeed, two distinct anti- and pro-inflammatory MSC phenotypes have been reported after activation of TLR3 and TLR4, respectively. The role of TLRs on MSC-mediated Treg generation, however, is not known. In this study, we investigated the role of TLR3 and TLR4 in the MSC-mediated generation of Tregs in an allogeneic co-culture model. Data for each experiment were collected from 1 PBMC donor and 3 MSC donors. We found that pre-activation of TLR3 and TLR4 by their ligands (poly I:C for TLR3, LPS for TLR4) enhanced the generation of Tregs by MSCs: 1.2 ± 0.2% in CD4+ cells cultured alone, 3.9 ± 0.3% in co-culture with control MSCs, 6.04 ± 0.1% in co-culture with TLR3-activated MSCs and 6.6 ± 0.4% in co-culture with TLR4-activated MSCs. siRNA-mediated silencing of TLR3 and TLR4 reduced Tregs by 51.7% and 61.8% in co-culture with poly I:C- and LPS-primed MSCs, respectively. Treg levels for the poly I:C-activated group were 6.3 ± 0.2% for co-cultures with control MSCs, 5.2 ± 0.3% for MSCs treated with scrambled RNA and 3 ± 0.3% for MSCs treated with TLR3-siRNA. For the LPS-activated group, Treg levels were 6.7 ± 0.3% with control MSCs, 5.7 ± 0.5% with MSCs treated with scrambled RNA and 2.5 ± 0.3% for MSCs treated with TLR4-siRNA. MSC-mediated Treg induction required cell-cell contact as conditioned media (CM) from TLR-activated or control MSCs failed to induce Tregs among CD4+ enriched cells: 4.75 ± 0.1% in direct co-culture vs 2.72 ± 0.3%, P= 0.004 in CM from control MSCs, 6.35 ± 0.2% in direct co-culture vs 2.97 ± 0.2%, P=0.0008 in CM from TLR3-activated MSCs, 6.7 ± 0.3% in direct co-culture vs 3.2 ± 0.3, P=0.001 in CM from the TLR4-activated group. We showed that the notch pathway is activated in CD4+ cells co-cultured with TLR-activated, but not control MSCs, and inhibition of notch signaling reduced MSC-mediated Tregs in co-cultures with TLR3- and TLR4-activated, but not control MSCs: 4.75 ± 0.1% vs 3.76 ± 0.4%, P=0.09 in control MSCs, 6.35 ± 0.2% vs 4.43 ± 0.3%, P=0.012 in TLR3-activated MSCs, 6.7 ± 0.3% vs 3.97 ± 0.1%, P=0.001 in TLR4-activated MSCs. Our data show a new role for TLR3 and TLR4 in the immunoregulatory function of human MSCs, and indicate the involvement of notch signaling as a mechanism for the further induction of Tregs in TLR3- and TLR4-activated MSCs. These studies have implications for the use of TLR-activated MSCs in the enhanced generation of Tregs such as for the treatment of acute GVHD. Disclosures No relevant conflicts of interest to declare.

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The zebrafish reveals dependence of the mast cell lineage on Notch signaling in vivo

Blood ◽

10.1182/blood-2011-10-385989 ◽

2012 ◽

Vol 119 (15) ◽

pp. 3585-3594 ◽

Cited By ~ 21

Author(s):

Sahar I. Da'as ◽

Andrew J. Coombs ◽

Tugce B. Balci ◽

Chloe A. Grondin ◽

Adolfo A. Ferrando ◽

...

Keyword(s):

Mast Cell ◽

Notch Signaling ◽

Notch Pathway ◽

Cell Development ◽

Notch Receptor ◽

Receptor Expression ◽

Specific Marker ◽

Dependent Manner ◽

Hematopoietic Stem

We used the opportunities afforded by the zebrafish to determine upstream pathways regulating mast cell development in vivo and identify their cellular origin. Colocalization studies demonstrated zebrafish notch receptor expression in cells expressing carboxypeptidase A5 (cpa5), a zebrafish mast cell-specific marker. Inhibition of the Notch pathway resulted in decreased cpa5 expression in mindbomb mutants and wild-type embryos treated with the γ-secretase inhibitor, Compound E. A series of morpholino knockdown studies specifically identified notch1b and gata2 as the critical factors regulating mast cell fate. Moreover, hsp70::GAL4;UAS::nicd1a transgenic embryos overexpressing an activated form of notch1, nicd1a, displayed increased cpa5, gata2, and pu.1 expression. This increase in cpa5 expression could be reversed and reduced below baseline levels in a dose-dependent manner using Compound E. Finally, evidence that cpa5 expression colocalizes with lmo2 in the absence of hematopoietic stem cells revealed that definitive mast cells initially delineate from erythromyeloid progenitors. These studies identify a master role for Notch signaling in vertebrate mast cell development and establish developmental origins of this lineage. Moreover, these findings postulate targeting the Notch pathway as a therapeutic strategy in mast cell diseases.

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Expression of Hes-1 (Notch-1 Effector) during “In Vitro” Normal Erythroid Differentiation.

Blood ◽

10.1182/blood.v106.11.4287.4287 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4287-4287

Author(s):

Maria D. Cappellini ◽

Ilaria V. Libani ◽

Elisabetta Calzavara ◽

Luisa Ronzoni ◽

Raffaella Chiaramonte ◽

...

Keyword(s):

Stem Cells ◽

Notch Signaling ◽

Cell Fate ◽

Notch Pathway ◽

Erythroid Differentiation ◽

Notch Receptor ◽

Erythroid Progenitors ◽

Notch 1 ◽

Multipotent Stem Cells

Abstract Hematopoiesis involves highly regulated proliferation and differentiation during which a small number of multipotent stem cells give rise to differentiated progenies. In several developmental systems stem cells fate is influenced by soluble molecules acting via cell-cell interaction, including those mediate by the Notch receptor family. Members of Notch family transmembrane receptors are found on primitive hematopoietic precursors, suggesting a role for Notch signaling in mammalian blood cells development. Notch signaling regulates cell fate controlling asymmetric cell division during stem/progenitor cell differentiation. A previous study on K562 cell line showed that Notch signaling inhibits erythroid/megakaryocytic development by suppressing GATA-1 activity. Furthermore there are evidences that Notch is expressed in early murine erythroid precursors. Probably Notch signaling in these uncommittted precursors may lead to enhanced survival, preserving multilineage potential. The role of Notch pathway during human adult erytropiesis has not been described. The aim of this study is to investigate the modulation of Notch activity during “in vitro” human erythropiesis. Human CD34+ from perpheral blood of normal adult subjects were differentiate “in vitro” for two weeks by the addiction of IL-3, SCF and Epo. This method of colture reproduces all stages of adult erythropoiesis. We analized the modulation of the expression of Notch-1, of its effector Hes-1 and of several erythoid specific genes, at different stages of differentiation using real time PCR. Our analysis shows that Hes-1 expression, which indicates the activation of Notch-1 pathway, is very high in the early steps of differentiation (BFU-E, CFU-E) while in the late stages rapidly decreases to undetectable levels. The Notch-1 gene expression doesn’t seem to be modulated way, but we didn’t invstigate the protein levels yet. These data suggest that Notch pathway is involved in the early stages of erythroid differentiation where it may enhance erythroid progenitors survival up to CFU-E, as hypothisized in mouse model, preventing them from apoptotic stimuli and promoting their proliferation. Involvement of Notch-1 signaling in preventing erythroid progenitors from apoptosis during erythroid differentiation could be important in some erythropoietic disorders such as b-Thalassemia syndromes or diserythropoietic anemias.

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Notch Signaling By Either Notch1 or Notch2 Mediates Expansion of AGM-Derived Long-Term HSC Populations in Vitro

Blood ◽

10.1182/blood.v124.21.2897.2897 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2897-2897

Author(s):

Brandon K Hadland ◽

Barbara Varnum-Finney ◽

Randall T Moon ◽

Michael Gustave Poulos ◽

Jason M. Butler ◽

...

Keyword(s):

Early Stage ◽

Fetal Liver ◽

Notch Pathway ◽

Receptor Activation ◽

Notch Receptor ◽

Gamma Secretase ◽

Hematopoietic Stem ◽

Notch Activation

Abstract Long-term, adult-engrafting hematopoietic stem cells (HSC) first emerge from hemogenic endothelial (HE) precursors in the context of embryonic arterial vessels such as the dorsal aorta of the AGM (Aorta-Gonad-Mesonephros region), a process which requires Notch1 receptor signaling. However, a possible subsequent role for Notch receptor activation during the unique period of substantial HSC expansion in embryonic development remains less well defined. Here, we show that endothelial cells derived from the murine embryonic AGM region (AGM-EC) or fetal liver (FL-EC) provide an in vitro substrate for generation/maturation of HSC from HE/HSC-precursor populations derived from early stage murine embryos, which lack adult-engraftment capacity prior to co-culture. Notably, these EC substrates, endogenously expressing Notch ligands of the Jagged and Delta families, also support subsequent numerical expansion of AGM-derived HSC, expressing Notch1 and Notch2 receptors, as determined by limit dilution transplantation analysis. Consistent with a requirement for Notch activation in this process, phenotypic HSC expansion during EC co-culture is blocked by gamma-secretase mediated Notch pathway inhibition. Furthermore, we show that in vitro Notch activation by immobilized ligand Delta1, together with cytokines and inhibition of the TGF-beta pathway, is sufficient to increase the number of AGM-derived HSC in the absence of EC stroma. Expansion of phenotypic hematopoietic stem/progenitor cells generated by culture on Delta1 is inhibited by antibody-mediated blockade of the combination of Notch1 and Notch2, but not by either Notch1 or Notch2 alone. Consistent with this, Notch receptor-specific activation by either immobilized Notch1 or Notch2 antibody is sufficient to support AGM-derived HSC in vitro in preliminary experiments. Altogether, these studies suggest a role for Notch pathway activation by either Notch1 or Notch2 in supporting embryonic-stage HSC expansion subsequent to initial Notch1-mediated HSC specification. Disclosures Moon: Fate Therapeutics: co-founder Other. Rafii:Angiocrine Biosciences: Founder Other.

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Displaced Niche Location and Decreased Quiescence Maintenance of Hematopoietic Stem Cells Due to Dysregulation of Notch Adhesive Interaction with Stromal Environment in Mice with Notch O-Fucosylation Deficiency

Blood ◽

10.1182/blood.v120.21.29.29 ◽

2012 ◽

Vol 120 (21) ◽

pp. 29-29

Author(s):

Weihuan Wang ◽

Xiaoran Huang ◽

Jay Myers ◽

Yiwei Wang ◽

Wei Xin ◽

...

Keyword(s):

Stem Cell ◽

Notch Signaling ◽

Conflicts Of Interest ◽

Loss Of Function ◽

Ligand Interaction ◽

Hematopoietic Stem ◽

Notch Ligand ◽

Endosteal Niche

Abstract Abstract 29 O-fucose modification and Fringe mediated O-fucose extension of Notch EGF-like repeats is essential for Notch binding with Notch ligand and Notch signaling transactivation. Previously we have shown that mice with conditional deficiency of Notch O-fucose modification develop a myeloproliferative disorder (MPD) with some mice displaying features of MPD-like leukemia. We disclosed that this MPD is mainly contributed by the cell-autonomous loss of response of myeloid progenitors bearing non-fucosylated Notch to Notch ligand induced suppression of granulo-monocytic differentiation. More recently, several Notch loss-of-function mutations have been identified in human chronic myelomonocytic leukemia (CMML). To gain a better insight of the role of Notch loss-of-function in stem cell dysfunction and MPD progression, here we studied the significance of O-fucose deficiency of Notch on progenitor proliferation and survival, and on HSC quiescence maintenance and niche location. We used a mouse model of pan-Notch signaling loss-of-function by Mx-Cre1 induced deficiency of Pofut1, an enzyme that modifies EGF O-fucosylation of all 4 Notch receptors. Pofut1-null hematopoietic stem and progenitor cells (HSPCs) had enhanced myeloid specification and proliferation in vitro, and displayed an increased activation of ERK and Stat5 in response to IL3 and GM-CSF when compared to the control HSPCs. The enhanced myeloid specification of Pofut1-null HSCs could be rescued by either activated Notch1 or Notch2. In addition, the HSPCs from Pofut1-null marrow and spleen displayed a 30% reduction of apoptosis. However, the increased proliferation and survival of Pofut1-null HSPCs were only partially reversed by the blocking of G-CSF, a cytokine that was up-regulated in the serum of Pofut1-null mice, supporting a role of cell-autonomous mechanism in its contribution to the increased proliferation and survival of Pofut1-null HSPCs. In line with this notion, we found that Pofut1-null mice had ∼ 50% increase in frequencies of the multi-potential progenitors (MPP) and the short-term self-renewable HSC (ST-HSC) but a 70% reduction of the more primitive long-term self-renewable HSC (LT-HSC). This change of HSC frequency was accompanied by an increased HSC cell cycling and a loss of adhesion to Notch ligand-expressing stromal cells despite that the Pofut1-null HSCs had a normal chemotactic response to SDF-1 and normal expression of CXCR4 as well as integrin adhesion molecules. Consistent with these findings, frequencies of circulating and splenic-residing HSCs were increased in Pofut1-null mice. To explore the mechanism by which loss of O-fucose of Notch regulates the stem cell activity in the bone marrow niches, we performed two-photon intravital microscopy to visualize the niche location of transplanted HSCs. We found that the Pofut1-null HSCs were positioned further from the endosteal niche and the niche supporting osteoblasts, when compared to control HSCs. In addition, Pofut1-null HSCs were not responsive to the inhibition of HSC expansion imparted by the osteoblasts in an in vitro co-culture assay. In summary, loss of O-fucosylation of Notch not only results in skewed myeloid specification and differentiation, but also promotes HSC proliferation and suppresses HSC quiescence. We conclude that the HSC phenotypes observed in mice with Pofut1 deficiency result as a consequence of the displacement of HSCs expressing non-fucosylated Notch from the suppressive endosteal niche that is otherwise enhanced by the adhesion between HSCs with the niche supporting cells through Notch and Notch ligand interaction. Disclosures: No relevant conflicts of interest to declare.

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The role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

Nature Communications ◽

10.1038/s41467-021-24418-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Brianna J. Klein ◽

Anagha Deshpande ◽

Khan L. Cox ◽

Fan Xuan ◽

Mohamad Zandian ◽

...

Keyword(s):

Critical Role ◽

Cellular Transformation ◽

Acute Leukemias ◽

Hematopoietic Stem ◽

Nucleosome Core ◽

Stem And Progenitor Cells ◽

Transforming Activity

AbstractChromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

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Notch Signaling Regulation in HCC: From Hepatitis Virus to Non-Coding RNAs

Cells ◽

10.3390/cells10030521 ◽

2021 ◽

Vol 10 (3) ◽

pp. 521

Author(s):

Catia Giovannini ◽

Francesca Fornari ◽

Fabio Piscaglia ◽

Laura Gramantieri

Keyword(s):

Notch Signaling ◽

Cell Fate ◽

Hepatitis Virus ◽

Notch Pathway ◽

Notch Receptor ◽

Gamma Secretase ◽

Stem Cell Maintenance ◽

Pathway Activation ◽

Promising Therapeutic Approach ◽

Conserved Genes

The Notch family includes evolutionary conserved genes that encode for single-pass transmembrane receptors involved in stem cell maintenance, development and cell fate determination of many cell lineages. Upon activation by different ligands, and depending on the cell type, Notch signaling plays pleomorphic roles in hepatocellular carcinoma (HCC) affecting neoplastic growth, invasion capability and stem like properties. A specific knowledge of the deregulated expression of each Notch receptor and ligand, coupled with resultant phenotypic changes, is still lacking in HCC. Therefore, while interfering with Notch signaling might represent a promising therapeutic approach, the complexity of Notch/ligands interactions and the variable consequences of their modulations raises concerns when performed in undefined molecular background. The gamma-secretase inhibitors (GSIs), representing the most utilized approach for Notch inhibition in clinical trials, are characterized by important adverse effects due to the non-specific nature of GSIs themselves and to the lack of molecular criteria guiding patient selection. In this review, we briefly summarize the mechanisms involved in Notch pathway activation in HCC supporting the development of alternatives to the γ-secretase pan-inhibitor for HCC therapy.

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Crosstalk between NOTCH and AKT signaling during murine megakaryocyte lineage specification

Blood ◽

10.1182/blood-2011-01-328567 ◽

2011 ◽

Vol 118 (5) ◽

pp. 1264-1273 ◽

Cited By ~ 47

Author(s):

Melanie G. Cornejo ◽

Vinciane Mabialah ◽

Stephen M. Sykes ◽

Tulasi Khandan ◽

Cristina Lo Celso ◽

...

Keyword(s):

Stem Cell ◽

Notch Signaling ◽

Signaling Pathway ◽

Hematopoietic Stem Cell ◽

Stem Cell Differentiation ◽

Notch Signaling Pathway ◽

Developmental Pathways ◽

Lineage Specification ◽

Hematopoietic Stem

Abstract The NOTCH signaling pathway is implicated in a broad range of developmental processes, including cell fate decisions. However, the molecular basis for its role at the different steps of stem cell lineage commitment is unclear. We recently identified the NOTCH signaling pathway as a positive regulator of megakaryocyte lineage specification during hematopoiesis, but the developmental pathways that allow hematopoietic stem cell differentiation into the erythro-megakaryocytic lineages remain controversial. Here, we investigated the role of downstream mediators of NOTCH during megakaryopoiesis and report crosstalk between the NOTCH and PI3K/AKT pathways. We demonstrate the inhibitory role of phosphatase with tensin homolog and Forkhead Box class O factors on megakaryopoiesis in vivo. Finally, our data annotate developmental mechanisms in the hematopoietic system that enable a decision to be made either at the hematopoietic stem cell or the committed progenitor level to commit to the megakaryocyte lineage, supporting the existence of 2 distinct developmental pathways.

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