The Notch Signaling Pathway as a Suppressor of Myeloid Transformation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-13-SCI-13
Author(s):  
Iannis Aifantis
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Notch Signaling ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Notch Pathway ◽  
Stem Cell Differentiation ◽  
Lineage Tracing ◽  
Specific Gene ◽  
Hematopoietic Stem

Abstract Abstract SCI-13 Notch signaling is a central regulator of differentiation in a variety of organisms and tissue types. Its activity is controlled by the multi-subunit γ-secretase complex (γSE) complex. Although Notch signaling can play both oncogenic and tumor suppressor roles in solid tumors, in the hematopoietic system, it is exclusively oncogenic, notably in T cell acute lymphoblastic leukemia (T-ALL), a disease characterized by Notch1 activating mutations. We identified somatic inactivating Notch pathway mutations in a fraction of chronic myelomonocytic leukemia (CMML) patients. Inactivation of Notch signaling in mouse hematopoietic stem cells (HSC) results in an aberrant accumulation of granulocyte/monocyte progenitors (GMP), extramedullary hematopoieisis and the induction of CMML-like disease. Transcriptome analysis reveals that Notch signaling regulates an extensive myelomonocytic-specific gene signature, through the direct suppression of gene transcription by the Notch target Hes1. These studies identify a novel role for Notch signaling during early hematopoietic stem cell differentiation and suggest that the Notch pathway can play both tumor-promoting and suppressive roles within the same tissue. These observations also suggest that Notch activity is not simply a promoter of the T cell lineage in the thymus but that Notch signaling thresholds could regulate commitment and/or survival of distinct hematopoietic lineages in the bone marrow. To address these issues in vivo, we have generated Notch receptor lineage tracing and activity reporter genetic tools. Analysis of these animal models identified unique novel functions for the Notch pathway during early bone marrow hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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.

scholarly journals Notch Signaling in the Bone Marrow Lymphopoietic Niche

2021 ◽  
Vol 12 ◽  
Author(s):  
Kilian Sottoriva ◽  
Kostandin V. Pajcini
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Notch Signaling ◽  
Signaling Pathway ◽  
Lymphoblastic Leukemia ◽  
Notch Signaling Pathway ◽  
Model Systems ◽  
Activation Mechanism ◽  
Cell Contact ◽  
Hematopoietic Stem

Lifelong mammalian hematopoiesis requires continuous generation of mature blood cells that originate from Hematopoietic Stem and Progenitor Cells (HSPCs) situated in the post-natal Bone Marrow (BM). The BM microenvironment is inherently complex and extensive studies have been devoted to identifying the niche that maintains HSPC homeostasis and supports hematopoietic potential. The Notch signaling pathway is required for the emergence of the definitive Hematopoietic Stem Cell (HSC) during embryonic development, but its role in BM HSC homeostasis is convoluted. Recent work has begun to explore novel roles for the Notch signaling pathway in downstream progenitor populations. In this review, we will focus an important role for Notch signaling in the establishment of a T cell primed sub-population of Common Lymphoid Progenitors (CLPs). Given that its activation mechanism relies primarily on cell-to-cell contact, Notch signaling is an ideal means to investigate and define a novel BM lymphopoietic niche. We will discuss how new genetic model systems indicate a pre-thymic, BM-specific role for Notch activation in early T cell development and what this means to the paradigm of lymphoid lineage commitment. Lastly, we will examine how leukemic T-cell acute lymphoblastic leukemia (T-ALL) blasts take advantage of Notch and downstream lymphoid signals in the pathological BM niche.

Direct Inhibition of the Notch Transactivation Complex with Synthetic Constrained Peptides in T-Cell Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2819-2819 ◽  
Author(s):  
Raymond Moellering ◽  
Melanie Cornejo ◽  
Jennifer Rocknik ◽  
Michael Hancock ◽  
Christina DelBianco ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Notch Signaling ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Notch Pathway ◽  
Stem Cell Differentiation ◽  
Gamma Secretase ◽  
Clinical Resistance ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Notch signaling represents a central pathway regulating hematopoiesis, stem cell differentiation, and malignant transformation in human cancer. Activation of highly conserved Notch1 receptors results in cleavage and release of an intracellular domain (ICN1). Following translocation to the nucleus, ICN1 forms a ternary complex with the transcriptional repressor CSL (CBF-1, Suppressor of Hairless and Lag-1) bound to cognate DNA. This event triggers a repressor-to-activator switch, as an interfacial groove is formed which recruits the Mastermind-Like (MAML1) co-activator protein. Activating mutations in NOTCH1 are found in more than 50% of patients with T-Cell Acute Lymphoblastic Leukemia (T-ALL), promoting protein stability and establishing a direct link to disease pathogenesis. Pharmacologic efforts to target the Notch pathway in T-ALL have been directed at gamma secretase, a regulatory enzyme in Notch activation. Recently, the observed clinical resistance to gamma secretase inhibitors has been explained, in part, by additional mutations in the Notch-targeting ubiquitin ligase, Fbxw7, which further increases oncoprotein stability. Therefore, direct inhibitors of ICN1 function are highly desirable. Drawing upon insights afforded by the resolved crystal structure of the DNA-bound ICN1:MAML1:CSL complex, we synthesized a series of hydrocarbon stapled alpha-helical peptides targeting Notch (SAHNs) based on minimal motifs of the MAML protein predicted to engage the composite ICN1:CSL interface. Direct, high-affinity binding to purified components of the Notch complex was confirmed using surface plasmon resonance (SPR). Nuclear access of SAHN1 was confirmed using quantitative epifluorescent and confocal microscopy. Intracellular association with ICN1 and CSL was established using bidirectional affinity chromatography. Using a novel CSL-responsive reporter construct, we observed inhibition of endogenous Notch transactivation by SAHN1 in T-ALL cell lines. Furthermore, SAHN1 induces a dose-dependent knockdown of endogenous Notch1 target genes including HES1, HEY1 and cMYC in T-ALL cell lines. Remarkably, inhibition of Notch signaling by SAHN1 confers selective cytotoxicity at 48 hours in a panel of T-ALL cell lines with known mutations in NOTCH, including those resistant to gamma secretase inhibitors. Supporting an on-target mechanism of action, we have prepared a damaged analogue of SAHN1 containing a two-residue rearrangement (SAHN1D). SAHN1D possesses reduced binding affinity for the Notch complex and despite comparable intracellular access, SAHN1D lacks both transcriptional and cytotoxic effects on cultured T-ALL cell lines in vitro. Efficacy studies have also been performed in vivo using a novel murine model of T-ALL. In summary, we report here the design, biochemical characterization and translational rationale supporting the first direct inhibitor of the Notch transactivation complex in T-ALL.

Fetal Vs. Adult Hematopoietic Progenitors Respond Differently to NOTCH1: Implications for Pediatric Vs. Adult T-ALL.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2381-2381
Author(s):  
Vincenzo Giambra ◽  
Sonya H Lam ◽  
Amy Ng ◽  
Claudia Benz ◽  
Olena O Shevchuk ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Lymphoblastic Leukemia ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Old Adult ◽  
Adult Bone

Abstract Abstract 2381 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of T-cell progenitors which affects both children and adults. Whereas pediatric T-ALL is curable in 80–90% of cases, only 40% of adults with T-ALL survive beyond 5 years. Fetal liver and adult bone marrow-derived hematopoietic stem/progenitor cells (HSPCs) are known to differ in terms of their gene expression programs and functional properties. Despite this work, the extent to which differential programming of fetal and adult HSPCs may impact the biology of their respective leukemias in children and adults remains unexplored. NOTCH1 is a prominent oncogene in T-ALL and activated by mutation in over 50% of cases. The retroviral NOTCH1 mouse bone marrow transplant model of T-ALL is well established; however, most investigators use 8–12 week-old adult mice as bone marrow donors for these experiments and thus these studies could be interpreted as having modeled adult disease. In order to explore the possibility that fetal program HSPCs would more accurately model pediatric disease, we transduced lineage-negative fetal liver HSPCs with activated NOTCH1 (ΔE) retrovirus, transplanted them into syngeneic (C57BL/6) recipients, and compared the behavior of the resulting leukemias to those generated from lineage-negative 8-week-old adult bone marrow HSPCs. Primary transplant recipient mice developed nearly identical T-ALL disease in terms of penetrance, latency, disease distribution/burden, and immunophenotype. Serial transplantation of these leukemias into secondary recipients, however, revealed stark differences in that whereas “adult” leukemias were readily transplantable, “fetal” leukemias were largely non-transplantable. In order to quantitate leukemia-initiating cell (LIC) frequencies in these two situations, we performed secondary transplants into highly permissive, immunodeficient (NOD/Scid/Il2rg−/−) recipients at limiting dilution and observed fetal leukemias to exhibit 500-fold lower LIC activity than adult leukemias (1 in ∼4500 cells vs. 1 in ∼9 cells, respectively). To identify potential mechanisms that might underlie this difference in LIC activity, we compared the behaviors of fetal liver vs. adult bone marrow-derived HSPCs shortly after transduction with NOTCH1(ΔE) virus. Interestingly, NOTCH1 induced fetal HSPCs to cycle rapidly whereas adult HSPCs were largely quiescent. We also noted that non-transduced cells in fetal HSPC cultures were also cycling rapidly, and through a series of fetal/adult mixing and conditioned media experiments, we determined that NOTCH1 induces an autocrine IGF1 signaling circuit in fetal, but not adult HSPCs. This observation was also confirmed to hold true for CD34+ human cord blood vs. adult bone marrow HSPCs. Expression profiling/qRT-PCR and chromatin immunoprecipitation (ChIP) studies further revealed NOTCH1 to induce IGF1 transcription and altered chromatin structure (increased H3K4me3 and decreased H3K27me3 marks) specifically in fetal, but not adult HSPCs. These findings suggest that developmental stage-specific programming in fetal vs. adult progenitors underlies their differential responses to oncogenic NOTCH1 signaling, and also the biological aggressiveness of resulting leukemias. Therapeutic targeting of adult-specific pathways may thus achieve improved clinical responses in adults with T-ALL and perhaps also the minority of pediatric patients with more aggressive, possibly “adult-like” disease. Disclosures: No relevant conflicts of interest to declare.

IMMU-50. GLIOBLASTOMA MANIPULATES HEMATOPOIETIC STEM CELL DIFFERENTIATION OUTCOMES AND DRIVES ALTERED IMMUNE RECONSTITUTION

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi104-vi104
Author(s):  
Bayli DiVita Dean ◽  
Tyler Wildes ◽  
Joseph Dean ◽  
David Shin ◽  
Connor Francis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Progenitor Cells ◽  
Rna Sequencing ◽  
Cell Fate ◽  
Immune Reconstitution ◽  
Stem Cell Differentiation ◽  
Cell Fate Determination ◽  
Hematopoietic Stem ◽  
Fate Determination

Abstract INTRODUCTION Bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) give rise to the cellular components of the immune system. Unfortunately, immune reconstitution from HSPCs are negatively impacted by solid cancers, including high-grade gliomas. For example, an expansion of myeloid progenitor cells has been previously described across several cancers that originate outside the CNS. A similar expansion of MDSCs coupled with diminished T cell function has also been described in the peripheral blood of patients with newly-diagnosed GBM. Alterations in both lymphoid and myeloid compartments due to CNS malignancy led us to determine how intracranial gliomas impact HSPCs in both their capacity to reconstitute the immune compartment and in their cell fate determination. This is important to better understand the impact of gliomas on immunity and how we can leverage these findings to better develop cellular immunotherapeutics. METHODS HSPCs were isolated from bone marrow of C57BL/6 mice with orthotopic KR158B glioma, or age-matched naïve mice. Experiments were conducted to compare relative changes in: gene expression (RNA-sequencing), precursor frequencies, cell fate determination, and cellular function of cells derived from HSPCs of glioma-bearing mice. RESULTS RNA-sequencing revealed 700+ genes whose expression was significantly up- or downregulated in HSPCs from glioma-bearing mice, particularly those involved with stemness and metabolic activity. Importantly, HSPCs from glioma-bearing mice expressed upregulation of genes involved in myelopoiesis relative to naïve mice. This was coupled with an expansion of granulocyte macrophage precursors (GMPs), the progenitors to gMDSCs. Next, differentiation assays revealed that HSPCs from glioma-bearing mice had higher propensity of differentiating into MDSC under homeostatic conditions relative to controls both in vitro and in vivo. Furthermore, mice bearing intracranial gliomas possess an expansion of MDSCs which are more suppressive on T cell proliferation and hinders T cell-mediated tumor cell killing relative to MDSCs derived from naïve control mice.

scholarly journals CongenitalSTAT3mutation Mediates Primary Persistent Polymorphic Inflammatory Activation and T Cell Leukemogenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1054-1054 ◽  
Author(s):  
Hongxing Liu
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
Lymph Nodes ◽  
Conflicts Of Interest ◽  
Sh2 Domain ◽  
Janus Kinase ◽  
Gingival Hyperplasia ◽  
Hematopoietic Stem ◽  
T Cell Leukemogenesis

Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways play a pivotal role in inflammation and immunity, among which, JAK/STAT3 pathway is the most potent and leads the crosstalk of immunity and oncogenesis. Somatic STAT3 activatingmutations have been found in about 40% of T cell large granular lymphocytic leukemia (T-LGLL) patients, most of which are located in exon 21 which encodes Src homology 2 (SH2) domain leading to the increased activity of aberrant STAT3 protein and the upregulation of its transcriptional targets. While germline STAT3activatingmutations represent a newly defined entity of immune dysregulations named infantile-onset multisystem autoimmune disease-1 (ADMIO1, #MIM 615952). Both the two diseases are rare and poorly understood. Here, we report a pedigree including a proband, a six-year-old girl, primarily manifesting as thrombocytopenia and lymphadenopathy and her father diagnosed as T-LGLL with pure red cell aplastic anemia without autoimmune disorders preceding or during his disease course. Morphology of the bone marrow smears of the proband indicated normal hyperplasia without evident dyspepsia or increased blast cells. However, the vacuoles in monocytes and the density and size of granules in neutrophils increased, and megaloblast transformation was observed in some neutrophils. (Fig. 1A, 1B) Biopsy of an enlarged lymph node showed the reactive follicular hyperplasia. (Fig. 1C) Whole exon sequencing and pedigree analysis of the family revealed the germline STAT3 c.833G>A/p.R278Hmutation harbored by the proband which originated de novo from her father who additionally carried a germline TAL1G62Rmutation and somatically accumulated an FLT3-ITD mutation. (Fig. 2) Through single-cell RNA sequencing, we also found the increase of circulating CD8+ T cells and the decrease of NK cells of the proband. (Fig. 3) The STAT3 target genes were generally overactivated, and the expression of cytokines decreased in transcription level. In the genes participating in JAK/STATs pathways, the expression of JAK3, STAT1, and STAT3was up-regulated significantly. (data not shown) Immunophenotype of the proband by flow cytometry confirmed change in immunocyte compartments, (Fig. 4) but the serum cytokine concentrations measured by flow cytometry yielded controversial results, that most of cytokines were moderately elevated, and IL-1β, IL-5, TNF-α, and IFN-γ were of the most evident. (data not shown) During the treatment and follow-up, Cyclosporin A (CsA) was efficient in maintaining her circulating platelets in the range of 166×109/L to 302×109/L, but the enlarged lymph nodes and hepatosplenomegaly had no response. Eleven months later, CsA was replaced by tacrolimusfor the severe gingival hyperplasia, which has efficiently stabilized her platelets count and normalized the enlarged lymph nodes, liver, and spleen. On the contrary, in the three and a half years' span of illness, the father was refractory to CsA and methotrexate (MTX), moreover, lethal bone marrow suppression was induced by one course of fludarabine. For the high level of HLA-I and HLA-II antibodies in the circulation, plantlets transfusions were only efficient after plasmapheresis. The father eventually died from pulmonary and gastrointestinal infection due to the failure of maternal HLA-haploidentical hematopoietic stem cell transplantation (HSCT). We comprehensively elaborated the immunophenotype of the proband and thoroughly elucidated the genetic alternations of the father which led to the T cell leukemogenesis, which brought new insight on these two rare diseases and highlighted a more scrupulous therapeutic strategy in T-LGLL with congenital mutations. Figure 1 Disclosures No relevant conflicts of interest to declare.

RhoA Is An Essential Regulator of Mitosis and Survival During Hematopoietic Stem Cell Differentiation to Multipotent Progenitors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1273-1273
Author(s):  
Xuan Zhou ◽  
Jaime Meléndez ◽  
Yuxin Feng ◽  
Richard Lang ◽  
Yi Zheng
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Intracellular Signaling ◽  
Conflicts Of Interest ◽  
Stem Cell Differentiation ◽  
Conditional Knockout ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Differentiated Cells ◽  
Multipotent Progenitors

Abstract Abstract 1273 The maintenance and differentiation of hematopoietic stem cells (HSC) are critical for blood cell homeostasis, which is tightly regulated by a variety of factors. In spite of extensive investigation of HSC biology, however, the mechanism of regulation of HSC and progenitor cell division, particularly the unique molecular events controlling the mitosis process during HSC differentiation, remains unclear. RhoA GTPase is a critical intracellular signaling nodal that has been implicated in signal transduction from cytokines, chemokines, wnt/notch/shh, and adhesion molecules to impact on cell adhesion, migration, cell cycle progression, survival and gene expression. Recent mouse genetic studies in keratinocytes and embryonic fibroblast cells showed that RhoA is a key regulator of mitosis. By using an interferon-inducible RhoA conditional knockout mouse model (Mx-cre;RhoAlox/lox), we have made the discovery that RhoA plays an indispensible role in primitive hematopoietic progenitor differentiation through the regulation of mitosis and survival. RhoA deficient mice die at ∼10 days because of hematopoietic failure, as evidenced by a loss of bone marrow, splenocyte and PB blood cells. Syngenic as well as reverse transplant experiments demonstrate that these effects are intrinsic to the hematopoietic compartment. RhoA loss results in pancytopenia associated with a rapid exhaustion of the lin−c-kit+ (LK) phenotypic progenitor population (within 4 days after two polyI:C injections). Meanwhile, the lin−c-kit+sca1+ (LSK) primitive cell compartment is transiently increased in BM after RhoA deletion due to a compensatory loss of quiescence and increased cell cycle. Interestingly, we find that within the LSK population, there is a significant accumulation of LSKCD34+Flt2− short-term HSCs (ST-HSC) and a corresponding decrease in frequency of LSKCD34+Flt2+ multipotent progenitors (MPPs). Consistent with these phenotypes, the LK and more differentiated hematopoietic cell populations of RhoA knockout mice show an increased apoptosis while the survival activities of LSK and more primitive compartments of WT and RhoA KO mice remain comparable. These data suggest that RhoA plays an indispensible role in the step of ST-HSCs differentiation to MPP cells, possibly through the regulation of MPP cell survival. This hypothesis is further supported by a competitive transplantation experiment. Deletion of RhoA in a competitive transplantation model causes an extinction of donor derived (CD45.2+) differentiated cells (myeloid, erythroid, T and B cells) in the peripheral blood. Interestingly, bone marrow CD45.2+ LSK cells are only marginally affected by deletion of RhoA and RhoA−/− LSK cells are able to engraft into 2nd recipient, whereas CD45.2+ LK and more differentiated cells are mostly eliminated after RhoA deletion. This effect is associated with a decrease in the survival of CD45.2+ RhoA−/− LK, but not LSK cells. Further in vitro culture of isolated lin− progenitors demonstrates that RhoA deficiency results in a failure of cytokinesis, causing an accumulation of multinucleated cells, further suggesting that RhoA is essential for the cytokinesis of hematopoietic progenitors. Surprisingly, the well-defined Rho downstream target, actomyosin machinery, does not appear to be affected by RhoA knockout. We are further exploring the mechanism of RhoA contribution to the differentiation of HSCs by dissecting the signaling and functional relationship of RhoA regulated survival activity and cell cycle mitosis in early hematopoietic progenitors. Disclosures: No relevant conflicts of interest to declare.

Comparative Analysis on Cellular Components of Bone Marrow Microenvironment in Normal and Aberrant Hematopoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4808-4808
Author(s):  
Young-Ho Lee ◽  
Young-hee Kwon ◽  
Kyoujung Hwang ◽  
Hyunju Jun ◽  
Byungbae Park ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Conflicts Of Interest ◽  
T Cell Subsets ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Stromal Cell Derived Factor ◽  
And Control ◽  
Cellular Components

Abstract Abstract 4808 Background: It is now evident that hematopoietic stem cells (HSCs) reside preferentially at the endosteal region within the bone marrow (BM) where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fate. We investigated the microenvironmental differences including osteoblastic activities and HSC components in myeloproliferative (chronic myeloid leukemia, CML) and hypogenerative disease (aplastic anemia, AA) as well as normal control (NC). Methods: The immunohistochemistry for osteonectin, osteocalcin, stromal cell derived factor (SDF, CXCL12), T cell, T helper/inducer cell, T suppressor/cytotoxic cell, hematopoietic stem/progenitor (CD34, CD117) and megakaryocytes was performed on BM biopsy specimens from 10 AA patients, 10 CML patients and 10 NC (lymphoma without BM involvement). The positive cells for immunohistochemical stainings except osteocalcin on each slide were calculated on 10 high power fields (HPF, ×400), and then corrected by the cellularity. The positive cells for osteocalcin were counted on the peritrabecular line on each slide, and then corrected by the mean length measured. Results: The CD34+ cells (p=0.012) and megakaryocytes (p<0.0001) were significantly lower in AA than in NC, but CD117+ cells was comparable in AA, CML, and control samples. The osteonectin+ cells (p=0.0003) were lower in CML than in AA and NC, however the osteocalcin+ cells showed wide variation (0-903/2035um) and no significant difference. The SDF+ cells (p<0.0001) was significantly higher in AA and very lower in CML, compared with NC. The counts for T cell and T cell subsets were significantly lower in CML than in NC, and higher in AA than in NC (p<0.0001). Conclusions: Cellular components of BM microenvironment in 2 hematologic diseases representative of myeloproliferation (CML) and hyporegeneration (AA) respectively are quite different. Further studies would be required to explore the role of these components for hematopoiesis and the rationale for therapeutic application. Disclosures: No relevant conflicts of interest to declare.

Effects of G-CSF On Acute Lymphoblastic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2564-2564
Author(s):  
Jordan Basnett ◽  
Adam Cisterne ◽  
Kenneth F Bradstock ◽  
Linda J Bendall
Keyword(s):  
Bone Marrow ◽  
Disease Progression ◽  
Microarray Analysis ◽  
Environmental Changes ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Stem ◽  
Childhood All ◽  
Extracellular Matrix Components ◽  
The Impact

Abstract Abstract 2564 G-CSF is commonly used to treat chemotherapy-induced neutropenia and for the mobilization of hematopoietic stem cells for transplantation in patients with leukemia. Administration of G-CSF has profound effects on the bone marrow microenvironment including the cleavage of molecules required for the maintenance of lymphopoiesis, including CXCL12 and VLA-4. We have recently reported that G-CSF results in the dramatic suppression of B-lymphopoiesis. This, together with previous reports by ourselves, and others, showing that disruption of CXCL12 or VLA-4 slow the progression of B-lineage ALL lead us to consider that G-CSF may similarly antagonize the progression of ALL. To explore this possibility, we examined the impact of G-CSF administration on six human ALL xenografts using a NOD/SCID mouse model. Mice were engrafted without radiation and G-CSF commenced when 1% of the bone marrow consisted of ALL cells. G-CSF was administered twice daily for 10 days, at which time all animals were culled and leukemia assessed in the blood, bone marrow and spleens. Surprisingly G-CSF was found to increase disease progression in two of xenografts investigated (1345 and 0398, referred to as G-CSF responsive xenografts hereafter), while the remainder demonstrated a small reduction in leukemia, with one showing a statistical significant decrease. No evidence for a direct mitogenic effect of G-CSF could be demonstrated in any of the xenografts using exogenous G-CSF in vitro cultures in the presence or absence of human or murine stromal support. Consistent with these findings, and previous reports, little to no G-CSF receptor was detected by flow cytometry or microarray analysis of xenografts. Microarray analysis of the xenografts revealed significant differences in gene expression between the G-CSF responsive xenografts and the remainder of the samples. A total of 83 genes were expressed at a higher level and 127 genes at a lower level in the G-CSF responsive xenografts. The more highly expressed genes included cell cycle regulators (eg cyclin A1), adhesion molecules (eg ALCAM), extracellular matrix components and surface receptors. Perhaps the most interesting was the exclusive expression of the acetylcholine receptor (cholinergic receptor, nicotinic, beta 4, nAChRb4) in the G-CSF responsive cases. Analysis of a large public dataset of childhood ALL samples revealed significantly higher expression of this gene in ALL samples with rearranged MLL (p<0.03). However, small numbers of cases in all ALL subgroups had greater than an 2 fold higher expression compared to normal B cell progenitors. The role of nAChR in the response of ALL cells to micro-environmental changes induced by G-CSF remains to be determined, however, nAChR has known roles in cell proliferation and inhibition of apoptosis. Furthermore G-CSF is known to induce acetylcholine production in other tissues. In summary, G-CSF inhibited leukemia progression in the majority of patient xenografts, however, in a subset of samples G-CSF accelerated disease progression. Clinically, G-CSF administration to ALL patients has not been associated with any major adverse outcomes. However our data suggest that a small subset of patients may experience accelerated disease. Identification of features associated with adverse responses to G-CSF will permit the identification of patients for whom G-CSF may present a risk for increased disease progression. Disclosures: No relevant conflicts of interest to declare.

Dnmt3a Deletion and FLT3-ITD Cooperate in a Mouse Model of T-Lymphoblastic Leukemia (T-ALL).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2428-2428
Author(s):  
Liubin Yang ◽  
Min Luo ◽  
Mira Jeong ◽  
Choladda V. Curry ◽  
Grant Anthony Challen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Notch Pathway ◽  
Leukemic Transformation ◽  
Aberrant Dna Methylation

Abstract Abstract 2428 Aberrant DNA methylation repeatedly has been implicated in cancer development. DNA methyltransferase (DNMT) 3A, which mediates de novo DNA methylation, was found to be mutated in 20% of patients with acute myeloid leukemia and 10% of patients with myelodysplastic syndrome. Recently, mutations associated with myeloid malignancies such as DNMT3A and FLT3 have also been uncovered in patients with early T-cell precursor lymphoblastic leukemia (ETP-ALL) (Neumann et al., 2012; Van Vlierberghe et al., 2011; Zaremba et al., 2012). ETP-ALL is a type of very high-risk ALL associated with myeloid/stem cell gene expression signature and myeloid markers. We have demonstrated that Dnmt3a deletion in mouse causes increased self-renewal of hematopoietic stem cells and an impairment of differentiation (Challen et al., 2011). Dnmt3a loss also produces aberrant methylation associated with oncogenes and tumor suppressor genes. Yet, whether aberrant DNA methylation can drive leukemia remains unknown. As Dnmt3a deletion alone was insufficient for malignancy, secondary mutations are likely necessary for leukemic transformation. Because FLT3 internal tandem duplication (ITD) frequently co-exist with DNMT3A mutations in acute leukemias, we hypothesized that Dnmt3a-loss may cooperate with FLT3-ITD to promote leukemic transformation; and we established a mouse model to test this. Deletion of conditional Dnmt3a with Mx1-cre was induced by injections of pIpC. Subsequently, bone marrow from Dnmt3a-deleted (Dnmt3aKO) donor mice was transduced with MSCV-FLT3-ITD-GFP retrovirus or MSCV-GFP control and transplanted into lethally irradiated recipients. The mice were monitored monthly for development of malignancies by complete blood count and peripheral blood analysis by flow cytometry and followed for disease latency. Moribund mice were sacrificed and analyzed with peripheral blood smears, histology, and immunophenotyping. Dnmt3a deletion with overexpression of FLT3-ITD caused rapid onset T-ALL in 6/8 mice (n=6) with a median latency of 78 days compared to 121 days in WT mice (n=4) overexpressing FLT3-ITD (p&lt;0.0001 Log-rank Mantel-Cox Test) (See figure). Mice from both groups exhibited leukocytosis, splenomegaly, and thymomegaly with high GFP expression detected by FACS. Even after we transduced bone marrow cells enriched for myeloid progenitor and stem cells, Dnmt3a deletion again accelerated T-ALL with median survival of 89 days (n=9) versus 110 days in WT-FLT3-ITD (n=10) mice. T-ALL was observed in 2/4 WT-FLT3-ITD mice and 5/6 Dnmt3aKO-FLT3-ITD mice analyzed (p&lt;0.0001 Log-rank Mantel-Cox Test). By flow cytometry, two distinct types of T-ALL were observed in the bone marrow of Dnmt3a deleted leukemic mice: one was characterized by a double positive population (DP) of CD4+CD8+ lympoblasts (1/6) and another early immature T-cell-like type of CD4-CD8-CD44+CD25-CD11bloCD117+ lymphoblasts (4/6). Gene expression analysis by RT-PCR in the early immature T-ALL showed downregulation of Notch-pathway genes (such as Notch1, Notch 3, Deltex, Hes1) and upregulation of stem cell-associated genes Lyl1 and Scl1, suggesting an ETP-like T-ALL. The ETP-like ALL phenotype has not been seen in WT mice overexpressing FLT3-ITD. The opposite gene expression pattern was seen in the DP population with upregulation of Notch-pathway genes. Furthermore, the DP leukemia was transplantable to secondary recipients within 2 weeks. Whether ETP-like ALL can be transplanted is still under investigation. We are also currently studying the changes in global CpG methylation among the leukemias that have Dnmt3a loss, FLT3-ITD overexpression, and control and also anticipate data from transcriptome analysis by RNA-Seq. These data suggest that stem or progenitor bone marrow cells primed by early loss of Dnmt3a are transformed into DP T-ALL and ETP-like ALL fueled by the overexpression of the oncogene FLT3-ITD. The ETP-like ALL phenotype has not been seen previously in WT mice overexpressing FLT3-ITD, suggesting that Dnmt3a ablation is required. The Dnmt3a-deleted-FLT3-ITD mice with T-ALL is, to our knowledge, the first animal model of human immature T-cell leukemia. This model can enhance our understanding of the pathogenesis of ETP-like ALL with respect to aberrant DNA methylation and will serve as a powerful tool to test novel therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.

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