Notch Signaling Is Required for Mast Cell Development In the Zebrafish and May Represent a Novel Therapeutic Strategy In Systemic Mastocytosis

Abstract Abstract 930 We have been exploiting the advantages provided by the zebrafish system to elucidate the molecular pathways regulating mast cell (MC) development in vertebrates and to model human MC diseases, such as systemic mastocytosis (SM). SM is a pre-leukemic myeloproliferative disease that results from perturbed MC development and proliferation. We have previously described zebrafish MC equivalents; demonstrated the significance of carboxypeptidase A5 (cpa5) as a zebrafish MC-specific marker; and established pu.1 and gata2 as essential transcription factors for early MC development (Dobson et al., Blood 2008). More recent co-localization studies suggest that definitive MCs originate from erythroid myeloid progenitors (EMPs), but unlike other myeloid lineages are uniquely dependent on Notch pathway signaling. Notch receptors and their ligands are expressed on a number of hematopoietic cells, including MCs. In zebrafish embryos, cpa5 co-localizes with the EMP marker, lmo2 at 28 hours post-fertilization (hpf) and with notch1a, notch1b and notch3 at both 28 hpf and 7 days post-fertilization (dpf). Morpholino knockdown studies specifically implicate notch1b as the key notch gene involved in MC fate determination and gata2 as an intermediary. Notch1b “morphants” exclusively display absent cpa5 expression with a concomitant decrease in gata2 expression by whole mount in situ hybridization (WISH). Furthermore, the zebrafish Notch signaling mutant, mindbomb, displays profound delay in the onset of cpa5 expression anteriorly and absent MCs in the posterior blood island at 48 hpf. Wild type embryos treated with Compound E, (CpdE), a γ-secretase inhibitor that inhibits Notch signaling, show a similar phenotype at 50 μM and complete absence of cpa5 expression at 75 μM. Embryos treated with 50 μM CpdE show decreased gata2 expression, but wild type pu.1 and gata1 expression, suggesting a particular sensitivity of the MC lineage to Notch pathway inhibition potentially mediated through gata2. By extension, these findings suggest the Notch pathway may serve as a prospective therapeutic target in MC diseases like SM. We have established a transgenic zebrafish model of SM that ubiquitously expresses the human c-KIT D816V mutation under the zebrafish β-actin promoter. Beginning at 9 months of age, adult fish develop a number of skin and visceral lesions, some of which have been found to contain an abundance of MCs. Transgenic embryos lack a developmental phenotype but demonstrate evidence of increased caspase-3 mediated apoptosis as well as increased cell proliferation by 5-bromo-2-deoxyuridine (BrdU) assay, suggesting additional mutations are required to progress to SM. These studies have provided new insights into the role of Notch signaling in MC development and the opportunity to use the zebrafish as an in vivo model to identify and evaluate novel therapeutic strategies in MC diseases. Disclosures: No relevant conflicts of interest to declare.

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Notch Signaling Is Required for Mast Cell Development in the Zebrafish.

Blood ◽

10.1182/blood.v114.22.3588.3588 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3588-3588

Author(s):

Sahar Da'as ◽

Lauren C. Klein ◽

Adolfo A. Ferrando ◽

Jason N. Berman

Keyword(s):

Mast Cell ◽

Transcription Factors ◽

Notch Signaling ◽

Notch Pathway ◽

Human Condition ◽

Specific Marker ◽

Wild Type ◽

Hematopoietic Stem ◽

Cell Fate Decisions

Abstract Abstract 3588 Poster Board III-525 The molecular pathways regulating mast cell (MC) development in vertebrates remain to be elucidated. The Notch signaling pathway is highly conserved in all metazoans and has been implicated in regulating hematopoietic stem cell induction and lineage cell fate decisions. Notch receptors and their ligands are expressed in a number of hematopoietic cells, including MCs. We were the first to identify zebrafish MC equivalents (Dobson et al., Blood 2008) and examine vertebrate MC transcriptional regulation in vivo. These studies demonstrated the significance of carboxypeptidase A 5 (cpa5) as a zebrafish MC-specific marker. Co-localization studies reveal zebrafish notch3 (a homologue of human NOTCH3) is expressed in a proportion of cpa5 positive cells in 7 day old embryos. Moreover, the zebrafish Notch signaling mutant, mind bomb, displays profound loss of cpa-5 expression, as do wild type zebrafish embryos treated with Compound E (Cpd E), a gamma-secretase inhibitor that blocks Notch signaling. We previously identified pu.1 and gata2 as essential transcription factors for early MC development. Interestingly, we observed a dose-dependent response, with reduced cpa5 and gata2 but preserved pu.1 expression at 50 μM Cpd E, compared with profound decreased expression of all these factors, as well as gata1 and mpo at 75 μM Cpd E. These data suggest a particular role for Notch signaling in regulating MC development, as well as a potentially broader role in regulating the myeloid and erythroid lineages. These studies are currently being validated through reciprocal experiments overexpressing notch mRNA in wild type embryos and rescue experiments overexpressing the notch intracellular domain and the above-mentioned transcription factors in Notch deficient embryos (mind bomb and Cpd E treated). We have also developed a transgenic zebrafish line expressing the human c-KIT D816V mutation found in systemic mastocytosis, which exhibits increased mast cells at the expense of erythroid cells, features in keeping with the human condition. These transgenic fish provide an opportunity to examine if Notch pathway inhibition alone, or in combination with other therapies, such as those targeting the c-KIT kinase, have a therapeutic impact in this condition. Parallel approaches in a human mastocytosis cell line are also being undertaken. These studies promise key insight into the role of Notch signaling in MC development and the opportunity to use the zebrafish as an in vivo model for identifying novel therapeutic strategies in MC diseases. Disclosures: Ferrando: Merck, Pfizer: Research Funding.

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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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Using the Zebrafish As a Tool for Modeling Systemic Mastocytosis,

Blood ◽

10.1182/blood.v118.21.3208.3208 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3208-3208

Author(s):

Tugce B Balci ◽

Andrew J Coombs ◽

Chloe Grondin ◽

Sahar I Da'as ◽

Ian Chute ◽

...

Keyword(s):

Mast Cells ◽

Notch Signaling ◽

Systemic Mastocytosis ◽

Notch Pathway ◽

Notch Signalling ◽

Blue Staining ◽

Transgenic Zebrafish ◽

Dependent Manner ◽

Transgenic Embryos

Abstract Abstract 3208 The zebrafish system provides many advantages in investigating intricate molecular pathways regulating vertebrate blood cell development and disease in vivo. We have been exploiting these assets to elucidate normal mast cell (MC) function and previously described the structural and functional characteristics of zebrafish MC equivalents (Dobson et al, Blood 2008, Da'as et al, Dev Comp Imm 2011). We have used this knowledge to develop transgenic zebrafish models of systemic mastocytosis (SM). SM is a pre-leukemic myeloproliferative disease that results from perturbed MC development and proliferation. Our recent studies have suggested that zebrafish MCs are uniquely dependent on Notch pathway signaling in contrast to that observed for other myeloid cell populations. Whole mount in situ hybridization (WISH) studies on the zebrafish Notch signaling mutant, mindbomb and notch1b “morphant” embryos both displayed decreased to absent carboxypeptidase A5 (cpa5) positive mast cells by WISH. Furthermore, wild type embryos treated with Compound E (CpdE), a γ-secretase inhibitor that inhibits Notch signaling, showed a similar phenotype. Given the role for Notch signalling in normal MC development, we wanted to see if driving the Notch pathway would result in a phenotype reminiscent of SM. Through a heat-shock inducible Gal4-UAS based system; we now demonstrate that over-expression of Notch signalling results in increased cpa5 positive mast cells in embryos as observed by WISH at 30, 36 and 48 hours post fertilization (hpf). Importantly, we were able to inhibit this increase and even reduce MC numbers below baseline levels in a dose-dependent manner using CpdE. Concurrently, we have established a transgenic zebrafish model of SM that ubiquitously expresses the human c-KIT D816V mutation under the zebrafish β-actin promoter. Beginning at 9 months of age, adult fish develop a number of skin and visceral lesions, many of which have been found to contain an abundance of MCs as identified by toluidine blue staining and tryptase immunohistochemistry. Transgenic embryos lack a developmental phenotype but demonstrate evidence of decreased phospho-histone H3 (pH3) signaling, suggesting additional mutations are required to progress to SM. In support of this G2/M arrest phenotype, microarray studies conducted on transgenic embryos revealed upregulation of p53 and cyclin G1. These studies have provided new insights into the role of Notch signaling in MC development and the opportunity to use the zebrafish as an in vivo model to identify and evaluate novel therapeutic strategies in MC diseases. Disclosures: No relevant conflicts of interest to declare.

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Notch Expression Expands the Pre-Malignant Pool of T-Cell Acute Lymphoblastic Leukemia Clones without Affecting Self-Renewal

Blood ◽

10.1182/blood.v116.21.3161.3161 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3161-3161

Author(s):

Jessica S Blackburn ◽

Sali Liu ◽

David M. Langenau

Keyword(s):

T Cell ◽

Notch Signaling ◽

Primary Tumor ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Notch Pathway ◽

Leukemic Cells ◽

Distinct Advantage ◽

Rodent Models ◽

Self Renewal

Abstract Abstract 3161 60% of human T-cell acute lymphoblastic leukemias (T-ALL) harbor NOTCH1 activating mutations, making it the most commonly mutated oncogene in T-ALL. Notch signaling is critical for T cell development, and activating Notch mutations are found in all subtypes of T-ALL, suggesting that Notch deregulation may be a dominant initiating event in human disease. In human and rodent models of T-ALL, Notch directly induces cMyc expression. However, cMyc over expression cannot completely rescue Notch inhibition, suggesting that Notch may have other important roles in T-ALL progression. Classic viral insertion screens in mice have indentified that insertional activation of Notch1 is common in Myc induced T-cell malignancies, suggesting that Notch imparts a distinct advantage to leukemic clones independent of cMyc. Notch-induced transgenic zebrafish models of T-ALLs are unique in that Notch signaling does not induce cMyc expression, allowing new opportunities to determine the function of Notch which are independent of cMyc. As with rodent models, the co-expression of Notch and cMyc in zebrafish T cells significantly enhanced T-ALL progression compared to cMyc or Notch alone (p<0.001). However, Notch co-expression with Myc did not enhance proliferation, alter cell cycle kinetics, or modify apoptosis in leukemic cells when compared to Myc alone expressing T-ALLs. Moreover, clonality assays using RT-PCR analyses for T-cell receptor-beta rearrangements indicate that Notch collaborates with Myc to increase the number of T-ALL clones contained within the primary tumor by 2–4 fold when compared to single transgenic animals that express only cMyc. Following serial transplantation, a large portion of T-ALL clones present in primary Notch/Myc leukemias are not found in transplanted animals. This starkly contrasts to results seen in Myc-alone expressing T-ALLs where all primary clones are capable of engraftment and reinitiation of leukemia. Paradoxically, transplant animals developing T-ALL from leukemias that coexpress Notch and Myc have similar numbers of clones as found in primary Myc-induced leukemias. Primary Myc-induced T-ALLs express high levels of endogenous scl and lmo2, recapitulating the most common and treatment resistant subtype in human T-ALL. By contrast, T-ALLs that co-express Notch and Myc fail to upregulate any of the T-ALL oncogenes; however, following transplantation into recipient animals, double transgenic Notch/Myc leukemias now express high levels of scl and lmo2. Finally, large scale limiting dilution cell transplantation analyses using syngeneic zebrafish demonstrated that Notch does not collaborate with Myc to increase self-renewal of leukemia initiating cells (LICs). Primary T-ALLs expressing both Notch and Myc have 10-fold less leukemia-initiating frequency when compared to T-ALLs that express only cMyc; however, following serial passaging, these Notch/Myc leukemias exhibit similar leukemia-initiating frequency as Myc-induced T-ALLs. Taken together, our data supports a model where Notch expands a pool of pre-malignant T-ALL clones within the primary tumor, a subset of which acquire additional mutations to confer a fully transformed phenotype. By contrast, Myc alone is insufficient to increase the overall pool of pre-malignant clones but confers a fully-transformed phenotype to leukemic cells accounting for the longer latency likely reflecting acquisition of additional genetic changes in clones. Our data may explain why a subset of relapse human T-ALLs develop disease from an underrepresented clone found in the primary leukemia. Primary human T-ALLs likely have a large pool of premalignant clones resulting from NOTCH-pathway activation that are unable to self-renew and thus, cannot give rise to relapsed T-ALL. Disclosures: No relevant conflicts of interest to declare.

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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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Histone Deacetylase Inhibitor SAHA Mediates Epigenetic Silencing of KIT D816V Mutated Systemic Mastocytosis Primary Mast Cells and Selective Apoptosis of Mutated Mast Cells

Blood ◽

10.1182/blood.v126.23.2834.2834 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2834-2834 ◽

Cited By ~ 2

Author(s):

Hani Abdulkadir ◽

Jennine Grootens ◽

Matilda Kjellander ◽

Eva Hellstrom Lindberg ◽

Gunnar Nilsson ◽

...

Keyword(s):

Cell Death ◽

Mast Cell ◽

Mast Cells ◽

Histone Deacetylase ◽

Histone Deacetylase Inhibitors ◽

Conflicts Of Interest ◽

Systemic Mastocytosis ◽

Specific Treatment ◽

Epigenetic Silencing ◽

Kit Receptor

Abstract Systemic mastocytosis (SM) is a myeloproliferative disease for which there is currently no specific therapy. Over 90% of the patients carry the D816V point mutation that renders the KIT receptor constitutively active. In the current study, we assessed the sensitivity of mast cell line HMC1.2 and primary SM patient mast cells to histone deacetylase inhibitors, and found that SAHA is most efficient. SAHA induced a rapid downregulation of KIT mRNA, with a subsequent reduction in total KIT protein as well as cell surface KIT. This was followed by major mast cell apoptosis. Primary SM patient mast cells cultured ex vivo were even more sensitive to SAHA than HMC1.2 cells, whereas healthy subject mast cells were unaffected. There was a correlation between cell death and SM disease severity, where cell death was more pronounced in the case of aggressive disease, with almost 100% cell death among mast cells from the mast cell leukemia patient. Using ChIP qPCR, we found that the level of active chromatin mark H3K18ac/totalH3 decreased significantly in the KIT region, due to an increase in H3 density. This epigenetic silencing was specific to the KIT region and not seen in control genes upstream and downstream of KIT. Primary analysis of ChIP-seq data for histone marks H3K4me3 and H3K27me3, demonstrates a downregulation of transcription factors involved in activation of KIT receptor, such as MAPK, for the SAHA treated samples. This indicates an indirect epigenetic silencing of KIT. Our results therefore demonstrate that SAHA epigenetically silences KIT, and work is ongoing to elucidate the exact mechanisms of KIT regulation. Altogether, SAHA maybe a specific treatment for SM. Disclosures No relevant conflicts of interest to declare.

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Delta/Notch signaling controls neuroepithelial morphogenesis in the zebrafish spinal cord

10.1101/517714 ◽

2019 ◽

Cited By ~ 1

Author(s):

Priyanka Sharma ◽

Vishnu Muraleedharan Saraswathy ◽

Li Xiang ◽

Maximilian Fürthauer

Keyword(s):

Spinal Cord ◽

Nervous System ◽

Notch Signaling ◽

Neural Development ◽

Notch Pathway ◽

Neural Precursor Cells ◽

Wild Type ◽

Tissue Architecture ◽

Embryonic Neurogenesis ◽

In The Wild

ABSTRACTThe morphogenesis of the nervous system requires coordinating the specification and differentiation of neural precursor cells, the establishment of neuroepithelial tissue architecture and the execution of specific cellular movements. How these aspects of neural development are linked is incompletely understood. Here we inactivate a major regulator of embryonic neurogenesis - the Delta/Notch pathway - and analyze the effect on zebrafish central nervous system morphogenesis. While some parts of the nervous system can establish neuroepithelial tissue architecture independently of Notch, Notch signaling is essential for spinal cord morphogenesis. In this tissue, Notch signaling is required to repress neuronal differentiation and promote neuroepithelial apico-basal polarity. Concomitant with a loss of their neuroepithelial properties, Notch signaling deficient cells also alter their morphogenetic behavior. In the wild-type zebrafish neural tube, cells divide at the organ midline to contribute one daughter cell to each organ half. Notch deficient animals fail to display this behavior and therefore form a misproportioned spinal cord. Taken together, our findings show that Notch signaling governs not only the cellular composition but also the morphogenetic shaping of the zebrafish spinal cord.

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Mutational Complexity Is Related To Disease Severity In Systemic Mastocytosis

Blood ◽

10.1182/blood.v122.21.4094.4094 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4094-4094

Author(s):

Juliana Schwaab ◽

Susanne Schnittger ◽

Karl Sotlar ◽

Christoph Walz ◽

Alice Fabarius ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Conflicts Of Interest ◽

Systemic Mastocytosis ◽

Single Gene ◽

Missense Mutations ◽

Molecular Feature ◽

Additional Mutation ◽

Molecular Aberrations ◽

Kit D816v

Abstract Systemic mastocytosis (SM) is a rare hematologic neoplasm characterized by abnormal accumulation of mast cells in various tissues, predominantly skin, bone marrow and visceral organs. The morphologic phenotype and extent of organ infiltration/dysfunction are basis for the subclassification of SM into indolent SM (ISM), smoldering SM (SSM), SM with associated hematologic non-mast cell disease (SM-AHNMD), aggressive SM (ASM) and mast cell leukemia (MCL). A somatic point mutation in the kinase domain of the receptor tyrosine kinase (TK) KIT at position 816 (KIT D816V) is present in >95% of patients and plays a central role in the pathogenesis and diagnosis of SM. To further explore mechanisms contributing to the clinical diversity of SM, we analyzed 39 KIT D816V mutated patients with different SM subtypes [ISM, n=10; SSM, n=2; ISM-AHNMD, n=5 (CMML, n=2; MDS/MPNu, n=3); ASM, n=1; ASM-AHNMD, n=14 (CMML, n= 5, MDS/MPNu, n=4, HES/CEL, n=2; AML, n=3); MCL, n=3; MCL-AHNMD n=4 (MDS/MPNu, n=2; HES/CEL, n=1; MDS, n=1)] for the presence of additional mutations. We applied next-generation sequencing to investigate ASXL1, CBL, IDH1/2, JAK2, KRAS, MLL-PTD, NPM1, NRAS, TP53, SRSF2, SF3B1, SETBP1, U2AF1 at mutational hotspot regions, and analyzed the complete coding regions of EZH2, ETV6, RUNX1, and TET2. Additional molecular aberrations were identified in 24/27 (89%) patients with advanced SM (SM-AHNMD, 5/5; ASM/MCL, 19/23) while only 3/12 (25%) ISM/SSM patients carried one additional mutation each (U2AF1, SETBP1, CBL) (p<0.001). In TET2, 9 missense, 4 nonsense, 13 frameshift mutations and one in-frame deletion as well as one splice site mutation were found in 15/39 (39%) patients. Ten of 15 (67%) patients carried more than one TET2 mutation. In SRSF2, missense mutations were identified in 14/39 (36%) patients which were clustered at codon 95 (P95H, n=9; P95L, n=2; P95R, n=2), with one additional mutation identified at codon 18 (V18L, n=1). In RUNX1, 10 mutations (9 missense mutations, 1 frameshift) were identified in 9/39 (23%) patients. Ten CBL mutations were identified in 8 patients (8 missense mutations, 1 duplication, 1 splice mutation) and 8 ASXL1 mutations were identified in 8 patients (7 frameshift and 1 nonsense mutation). In 13 patients, two (TET2, n=8; KRAS, n=1), three (CBL, n=2) or four mutations (TET2, n=2) were found in a single gene. Less frequently affected genes were KRAS (n=4), NRAS, JAK2, U2AF1 (n=2, each), EZH2, SETBP1, and ETV6 (n=1, each). No mutations were identified in MLL, IDH1, IDH2, NPM1, SF3B1 and TP53. In advanced SM, 21/27 patients (78%) carried ³3 mutations and 11/27 patients (41%) exhibited ³5 mutations. The median number of mutations was 4. The concurrent presence of KIT-TET2-SRSF2 (10/39, 26%), KIT-SRSF2-RUNX1 (7/39, 18%), KIT-TET2-CBL (5/39, 13%), KIT-SRSF2-ASXL1 (4/39, 10%) and KIT-TET2-ASXL1 (4/39, 10%) was strongly associated with (A)SM/MCL-AHNMD in 16/16 (100%) of patients (CMML, 7/16, 44%; MDS/MPNu, 6/16, 38%; HES/CEL, 3/16, 19%). Clinical follow-up was available for 38 patients. Six of 38 (16%) patients died, all of whom had at least one additional mutation with 5 patients positive for ³3 and 2 patients for ³5 mutations. The median survival of all 26 patients with at least one additional mutation was 12 months. In contrast, none of the 12 cases with KIT D816V mutation only died. This translated into a significant inferior survival (p=0.019) for patients with additional mutations (figure 1). We therefore conclude that, in addition to the multilineage involvement by KIT D816V, the presence of additional molecular aberrations is a new molecular feature that may contribute essentially to the abnormal phenotype and behaviour of neoplastic mast cells in advanced SM and thus to the clinical diversity and prognosis of advanced mast cell disorders. Disclosures: No relevant conflicts of interest to declare.

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Ectopic Expression of Lin28 Biases Myeloid Differentiation to Promote an Immature Mast Cell State and Is Implicated in Aggressive Mastocytosis

Blood ◽

10.1182/blood.v124.21.5571.5571 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5571-5571

Author(s):

Leo D. Wang ◽

Phi Nguyen ◽

Robert G Rowe ◽

Tata Nageswara Rao ◽

George Q. Daley ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Conflicts Of Interest ◽

Systemic Mastocytosis ◽

Expression Patterns ◽

Ectopic Expression ◽

In Vitro Assays ◽

Human Mast Cell ◽

Adult Mice

Abstract Dysregulated mast cell development leads to systemic mastocytosis, a clinically variable but often devastating family of hematologic disorders. Lin28 is a heterochronic gene and pluripotency factor implicated in many types of malignancy, and prior studies suggest that Lin28 expression can restore a fetal hematopoietic program in adult mice. However, the role of Lin28 in hematologic malignancy remains controversial. In our study, we induced expression of Lin28 in adult mice using a doxycycline-responsive transgenic system. Lin28 induction caused marked mast cell accumulation in target organs such as the skin and peritoneal cavity. In vitro assays revealed a skewing of myeloid commitment in Lin28-expressing hematopoietic progenitors, with increased levels of Lin28 in common myeloid progenitors and basophil-mast cell progenitors altering gene expression patterns to favor cell fate choices that enhance mast cell specification. In addition, Lin28-induced mast cells appeared phenotypically and functionally immature, and in vitro assays suggested a slowing of mast cell terminal differentiation in the context of Lin28 upregulation. Finally, interrogation of human mast cell leukemia samples revealed upregulation of LIN28 in abnormal mast cells from patients with aggressive systemic mastocytosis (ASM). This work identifies Lin28 as a novel regulator of innate immune function and a new protein of interest in mast cell disease. Disclosures No relevant conflicts of interest to declare.

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A Novel DLL1-Anti-CD19 Chimeric Protein Effectively Targets Notch Activation to B-Cell Acute Lymphoblastic Leukemia and Induces Cell Death

Blood ◽

10.1182/blood.v126.23.4833.4833 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4833-4833

Author(s):

Rebecca E Wiersma ◽

Sankaranarayanan Kannan ◽

Srinivas S. Somanchi ◽

Lizhi Zeng ◽

Patrick A. Zweidler-McKay

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

Notch Signaling ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Notch Pathway ◽

Chimeric Protein ◽

Notch Ligand ◽

Cell Counts ◽

Notch Receptors

Abstract Background: Acute lymphoblastic leukemia (ALL) is the most prevalent form of cancer in children, and those who relapse continue to have poor survival. Therefore, the development of improved and specifically targeted treatment options is vital. The Notch pathway has been shown to act as a tumor suppressor in B-ALL via cell type specific induction of growth-inhibiting and pro-apoptotic pathways. In this study, we aim to therapeutically activate Notch signaling in B-ALL via targeting the Notch ligand DLL1 to B cells using an anti-CD19 scFv chimeric protein. Methods: A soluble chimeric protein composed of the extracellular domain of the Notch ligand DLL1 linked to a validated anti-CD19 scFv was isolated from HEK-293 producer cells. Human B-ALL (SB, Nalm6) and T-ALL (Jurkat) lines were treated with DLL1-anti-CD19scFv chimeric protein, and expression of the Notch target gene HES1 and effects on cell growth and survival were measured. Results: Both B- and T-ALL lines express Notch1 and Notch2 receptors on the cell surface. Exposure of B-ALL cells to DLL1-anti-CD19scFv chimeric protein led to an increase of Notch signaling, measured via 3-14 fold increase in HES1 mRNA expression. As expected, surface expression of the Notch receptors decreased upon chimera exposure, as Notch receptors are cleaved and destroyed upon activation. Importantly, exposure of the B-ALL lines to the chimeric protein led to a maximum 60% decrease in cell counts over 3-4 days, in contrast to T-ALL, where exposure did not effect growth significantly. Conclusions: This study demonstrates that Notch signaling can be feasibly activated in human B-ALL cells through a soluble DLL1-anti-CD19 scFv chimeric protein. Activation of Notch signaling via this method leads to growth inhibition and cell death in B-ALL, but not T-ALL cells. Based on our findings, we suggest that this soluble DLL1-anti-CD19 chimera may be a potential therapeutic approach for B-ALL, and further in vivo testing is warranted. Disclosures No relevant conflicts of interest to declare.

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