An RNAi Screen Reveals a Pattern of Addiction to Epigenetic Pathways That Distinguishes MLL-AF9 Leukemia From Normal Hematopoietic Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 65-65
Author(s):  
Christopher Vakoc ◽  
Johannes Zuber ◽  
Scott Lowe ◽  
Eric Wang ◽  
Amy Rappaport ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Model ◽  
Chromatin Structure ◽  
Therapeutic Benefit ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Hematopoietic Stem ◽  
H3k27 Methylation ◽  
Polycomb Complex

Abstract Abstract 65 Epigenetic pathways act to control gene expression in a heritable fashion without altering DNA sequence, typically involving the control of chromatin structure. Several lines of evidence implicate the involvement of epigenetics in the pathogenesis of human cancer, however it remains uncertain to what extent manipulating epigenetic pathways can fully rectify malignant cellular states for therapeutic benefit. To systematically explore this issue, we have developed a novel RNAi screening pipeline that can distinguish the epigenetic requirements for normal and malignant hematopoiesis. This was achieved by first constructing a custom shRNA library targeting all known enzymatic complexes that regulate chromatin structure (1100 shRNAs in total). Next, each shRNA was delivered systematically to cells derived from a mouse model of chemotherapy-resistant acute myeloid leukemia driven by the oncogenes MLL-AF9 and NRAS, or to several non-transformed hematopoietic cell lines of different lineages. shRNAs were scored for their capacity to differentially inhibit growth of leukemic cells without influencing growth of non-leukemic cells. Each of the identified genes was then evaluated in vivo for its influence on normal reconstitution of the hematopoietic system following transplantation of shRNA-infected hematopoietic stem and progenitor cells into lethally irradiated recipient mice. Each gene was likewise suppressed in leukemia cells in vivo using both constitutive and conditional RNAi vectors. The net result of the in vivo testing was identification of 6 genes encoding different regulators of chromatin structure whose suppression provides therapeutic benefit in a mouse model of therapy-resistant AML, without significantly influencing the production of normal blood lineages. In support of the accuracy of our screening protocol, one of the identified genes from the screen encodes the protein Menin, a known MLL-AF9 cofactor essential for disease initiation and shown to be dispensable for steady-state hematopoiesis in knockout mice. Two of the identified hits in our screen are the genes Eed and Suz12, which encode two subunits of the PRC2 Polycomb complex (Eed and Suz12), which catalyzes histone H3K27 methylation to suppress gene expression. Inhibiting PRC2 function in MLL-AF9 leukemia cells leads to monocytic differentiation, as revealed by FACS, RT-qPCR, and cell morphology analysis. Microarray experiments coupled with chromatin immunoprecipitation identified a core program of myeloid fate determinants that are suppressed by PRC2 via H3K27 methylation. In addition, we observe that MLL-AF9 directly occupies several Polycomb gene promoters to upregulate their expression in leukemic cells. Our findings highlight an unexpected alliance between the MLL-AF9 oncogene (a Trithorax protein) and PRC2 (a Polycomb complex), which act together to block myeloid differentiation in AML. Our findings also highlight the utility of employing RNAi in vivo to identify novel therapeutic targets in otherwise chemotherapy-resistant disease models. Disclosures: No relevant conflicts of interest to declare.

Cellular and Molecular Targets of MLL-AF9 in a Novel Conditional Mouse Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1280-1280
Author(s):  
Vaia Stavropoulou ◽  
Susanne Kaspar ◽  
Laurent Brault ◽  
Sabine Juge ◽  
Stefano Morettini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Mouse Model ◽  
Prognostic Markers ◽  
Bone Marrow Cells ◽  
Median Latency ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Abstract 1280 Previous studies have shown that the expression of several leukemia-associated mixed lineage leukemia (MLL) fusion genes transformed human and mouse bone marrow cells in vitro and in vivo. In order to dissect the molecular and cellular targets of the MLL-AF9 fusion, we generated a novel inducible doxycycline (DOX)-regulated transgenic mouse model. Conditional ex vivo activation of MLL-AF9 induced aberrant self-renewal and impaired differentiation of long-term or short-term hematopoietic stem (LT-HSC and ST-HSC), common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) cells in a fully reversible manner. Direct activation of the fusion in vivo or after transplantation of transgenic bone marrow cells into irradiated hosts induced an aggressive and transplantable disease after a median latency of 80days characterized as acute myelo-monocytic leukemia closely mimicking the human disease. Fusion gene expression and leukemia induction was DOX dosage dependent and reversible upon DOX removal. Activation of MLL-AF9 in isolated LT-HSC or GMP cells in vitro or in vivo resulted in the accumulation of immature blast-like cells with similar immunophenotypes. However, MLL-AF9-expressing stem and progenitor cells displayed distinct properties such as colony formation, differentiation and resistance to chemotherapeutic drugs. Turning-off the fusion resulted in multi-lineage differentiation of LT-HSC-derived cells, whereas GMP-derived cells were limited to mature macrophages and granulocytes suggesting partial maintenance of their original identity. In line with these in vitro observations, lower cell numbers of transplanted LT-HSCs induced a more aggressive leukemia with a significantly shorter latency as compared to ST-HSC, CMP or GMPs. Immunophenotypically 15% of the LT-HSC derived leukemias displayed a CMP–like phenotype and had a median latency of 37d (“early”) whereas the rest of the cases displayed a GMP-like phenotype with a median latency of 73d (“late”). In contrast, only GMP-like phenotypes and longer latencies were observed upon transplanting ST-HSCs (75d), CMPs (72d) or GMPs (100d). Transplantation of blasts from “early” LT-HSC- and GMP-derived leukemias into secondary recipients induced the disease after similar latency, however, cytarabine (Ara-C) treatment significantly delayed only the disease induced by GMP- but not by LT-HSC-derived blasts. Gene expression profiling in immortalized pre-leukemic cells revealed down-regulation of over 300 genes, including several well-known MLL targets such as Meis1, HoxA5, HoxA9 and HoxA10 upon reducing the levels of MLL-AF9 expression. Likewise, we observed a global decrease in histone H3 lysine 79 dimethylation consistent with a Dot1l function in MLL-AF9 driven leukemia. LT-HSC-derived (“early”) blasts displayed distinct genetic signatures with > 400 genes highly and > 1300 genes lowly expressed (p001 fc1.5), clearly separating them from the GMP-derived blasts. Evi-1 and Erg, two prognostic markers in patient-derived gene signatures, stood out among these genes. The aggressive “early” LT-derived murine leukemias showed high Evi-1 and Erg expression levels (Evi-1 high, Erg high) as compared to the “late” LT-derived (Evi-1 low, Erg high) or the GMP-derived leukemias (Evi-1 low, Erg low). These observations suggest that the previously reported poor prognosis associated with elevated EVI-1 and/or ERG expression might directly reflect the cell of origin of the disease. We are currently exploiting our highly informative MLL-AF9 disease model to evaluate the functional relevance of novel origin-dependent MLL-AF9 target genes and to identify novel prognostic markers and therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inhibition of Kirsten-Ras reduces fibrosis and protects against renal dysfunction in a mouse model of chronic folic acid nephropathy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Lucy J. Newbury ◽  
Jui-Hui Wang ◽  
Gene Hung ◽  
Bruce M. Hendry ◽  
Claire C. Sharpe
Keyword(s):  
Folic Acid ◽  
Kidney Disease ◽  
Mouse Model ◽  
Renal Dysfunction ◽  
Antisense Oligonucleotides ◽  
Underlying Mechanism ◽  
Therapeutic Benefit ◽  
End Stage

Abstract Chronic Kidney Disease is a growing problem across the world and can lead to end-stage kidney disease and cardiovascular disease. Fibrosis is the underlying mechanism that leads to organ dysfunction, but as yet we have no therapeutics that can influence this process. Ras monomeric GTPases are master regulators that direct many of the cytokines known to drive fibrosis to downstream effector cascades. We have previously shown that K-Ras is a key isoform that drives fibrosis in the kidney. Here we demonstrate that K-Ras expression and activation are increased in rodent models of CKD. By knocking down expression of K-Ras using antisense oligonucleotides in a mouse model of chronic folic acid nephropathy we can reduce fibrosis by 50% and prevent the loss of renal function over 3 months. In addition, we have demonstrated in vitro and in vivo that reduction of K-Ras expression is associated with a reduction in Jag1 expression; we hypothesise this is the mechanism by which targeting K-Ras has therapeutic benefit. In conclusion, targeting K-Ras expression with antisense oligonucleotides in a mouse model of CKD prevents fibrosis and protects against renal dysfunction.

scholarly journals Hyperactive Nras and Dose Reduction of Tet2 Collaborate to Dys-Regulate Hematopoietic Stem Cells and Promote Leukemogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1204-1204
Author(s):  
Xi Jin ◽  
Tingting Qin ◽  
Nathanael G Bailey ◽  
Meiling Zhao ◽  
Kevin B Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Double Mutant ◽  
Mutant Mice ◽  
Cytokine Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Single Mutant ◽  
Tet2 Mutation

Abstract Activating mutations in RAS and somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) are frequently detected in hematologic malignancies. Global genomic sequencing revealed the co-occurrence of RAS and TET2 mutations in chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias (AMLs), suggesting that the two mutations collaborate to induce malignant transformation. However, how the two mutations interact with each other, and the effects of co-existing RAS and TET2 mutations on hematopoietic stem cell (HSC) function and leukemogenesis, remains unknown. In this study, we generated conditional Mx1-Cre+;NrasLSL-G12D/+;Tet2fl/+mice (double mutant) and activated the expression of mutant Nras and Tet2 in hematopoietic tissues with poly(I:C) injections. Double mutant mice had significantly reduced survival compared to mice expressing only NrasG12D/+ or Tet2+/-(single mutants). Hematopathology and flow-cytometry analyses showed that these mice developed accelerated CMML-like phenotypes with higher myeloid cell infiltrations in the bone marrow and spleen as compared to single mutants. However, no cases of AML occurred. Given that CMML is driven by dys-regulated HSC function, we examined stem cell competitiveness, self-renewal and proliferation in double mutant mice at the pre-leukemic stage. The absolute numbers of HSCs in 10-week old double mutant mice were comparable to that observed in wild type (WT) and single mutant mice. However, double mutant HSCsdisplayed significantly enhanced self-renewal potential in colony forming (CFU) replating assays. In vivo competitive serial transplantation assays using either whole bone marrow cells or 15 purified SLAM (CD150+CD48-Lin-Sca1+cKit+) HSCs showed that while single mutant HSCs have increased competitiveness and self-renewal compared to WT HSCs, double mutants have further enhanced HSC competitiveness and self-renewal in primary and secondary transplant recipients. Furthermore, in vivo BrdU incorporation demonstrated that while Nras mutant HSCs had increased proliferation rate, Tet2 mutation significantly reduced the level of HSC proliferation in double mutants. Consistent with this, in vivo H2B-GFP label-retention assays (Liet. al. Nature 2013) in the Col1A1-H2B-GFP;Rosa26-M2-rtTA transgenic mice revealed significantly higher levels of H2B-GFP in Tet2 mutant HSCs, suggesting that Tet2 haploinsufficiency reduced overall HSC cycling. Overall, these findings suggest that hyperactive Nras signaling and Tet2 haploinsufficiency collaborate to enhance HSC competitiveness through distinct functions: N-RasG12D increases HSC self-renewal, proliferation and differentiation, while Tet2 haploinsufficiency reduces HSC proliferation to maintain HSCs in a more quiescent state. Consistent with this, gene expression profiling with RNA sequencing on purified SLAM HSCs indicated thatN-RasG12D and Tet2haploinsufficiencyinduce different yet complementary cellular programs to collaborate in HSC dys-regulation. To fully understand how N-RasG12D and Tet2dose reduction synergistically modulate HSC properties, we examined HSC response to cytokines important for HSC functions. We found that when HSCs were cultured in the presence of low dose stem cell factor (SCF) and thrombopoietin (TPO), only Nras single mutant and Nras/Tet2 double mutant HSCs expanded, but not WT or Tet2 single mutant HSCs. In the presence of TPO and absence of SCF, HSC expansion was only detected in the double mutants. These results suggest that HSCs harboring single mutation of Nras are hypersensitive to cytokine signaling, yet the addition of Tet2 mutation allows for further cytokine independency. Thus, N-RasG12D and Tet2 dose reduction collaborate to promote cytokine signaling. Together, our data demonstrate that hyperactive Nras and Tet2 haploinsufficiency collaborate to alter global HSC gene expression and sensitivity to stem cell cytokines. These events lead to enhanced HSC competitiveness and self-renewal, thus promoting transition toward advanced myeloid malignancy. This model provides a novel platform to delineate how mutations of signaling molecules and epigenetic modifiers collaborate in leukemogenesis, and may identify opportunities for new therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

scholarly journals Regulation of lamin properties and functions: does phosphorylation do it all?

Open Biology ◽  
2015 ◽  
Vol 5 (11) ◽  
pp. 150094 ◽  
Author(s):  
Magdalena Machowska ◽  
Katarzyna Piekarowicz ◽  
Ryszard Rzepecki
Keyword(s):  
Gene Expression ◽  
Experimental Data ◽  
Chromatin Structure ◽  
In Silico ◽  
Building Blocks ◽  
Model Organisms ◽  
Intracellular Signalling Pathway ◽  
Evolutionarily Conserved ◽  
Structure Regulation

The main functions of lamins are their mechanical and structural roles as major building blocks of the karyoskeleton. They are also involved in chromatin structure regulation, gene expression, intracellular signalling pathway modulation and development. All essential lamin functions seem to depend on their capacity for assembly or disassembly after the receipt of specific signals, and after specific, selective and precisely regulated interactions through their various domains. Reversible phosphorylation of lamins is crucial for their functions, so it is important to understand how lamin polymerization and interactions are modulated, and which sequences may undergo such modifications. This review combines experimental data with results of our in silico analyses focused on lamin phosphorylation in model organisms to show the presence of evolutionarily conserved sequences and to indicate specific in vivo phosphorylations that affect particular functions.

scholarly journals Gene-edited stem cells enable CD33-directed immune therapy for myeloid malignancies

2019 ◽  
pp. 201819992 ◽  
Author(s):  
Florence Borot ◽  
Hui Wang ◽  
Yan Ma ◽  
Toghrul Jafarov ◽  
Azra Raza ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Residual Disease ◽  
Immune Therapy ◽  
Leukemic Cells ◽  
Sequencing Analysis ◽  
Hematopoietic Stem ◽  
Genetic Ablation ◽  
Targeted Immunotherapy

Antigen-directed immunotherapies for acute myeloid leukemia (AML), such as chimeric antigen receptor T cells (CAR-Ts) or antibody-drug conjugates (ADCs), are associated with severe toxicities due to the lack of unique targetable antigens that can distinguish leukemic cells from normal myeloid cells or myeloid progenitors. Here, we present an approach to treat AML by targeting the lineage-specific myeloid antigen CD33. Our approach combines CD33-targeted CAR-T cells, or the ADC Gemtuzumab Ozogamicin with the transplantation of hematopoietic stem cells that have been engineered to ablate CD33 expression using genomic engineering methods. We show highly efficient genetic ablation of CD33 antigen using CRISPR/Cas9 technology in human stem/progenitor cells (HSPC) and provide evidence that the deletion of CD33 in HSPC doesn’t impair their ability to engraft and to repopulate a functional multilineage hematopoietic system in vivo. Whole-genome sequencing and RNA sequencing analysis revealed no detectable off-target mutagenesis and no loss of functional p53 pathways. Using a human AML cell line (HL-60), we modeled a postremission marrow with minimal residual disease and showed that the transplantation of CD33-ablated HSPCs with CD33-targeted immunotherapy leads to leukemia clearance, without myelosuppression, as demonstrated by the engraftment and recovery of multilineage descendants of CD33-ablated HSPCs. Our study thus contributes to the advancement of targeted immunotherapy and could be replicated in other malignancies.

scholarly journals EXTH-16. HUMAN PLACENTAL HEMATOPOIETIC STEM CELL DERIVED NATURAL KILLER CELLS FOR GLIOBLASTOMA IMMUNOTHERAPY

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi85-vi85
Author(s):  
Shuyang He ◽  
Joseph Gleason ◽  
Nassir Habboubi ◽  
Robert Hariri ◽  
Xiaokui Zhang
Keyword(s):  
Mouse Model ◽  
Clinical Symptoms ◽  
Quantitative Polymerase Chain Reaction ◽  
Phase 1 ◽  
Human Telomerase Reverse Transcriptase ◽  
Hematopoietic Stem ◽  
Safety And Efficacy ◽  
Nsg Mice ◽  
Nsg Mouse

Abstract Taniraleucel (CYNK-001) is an allogeneic, off-the-shelf cell therapy enriched for CD56+/CD3- NK cells expanded from placental CD34+ cells. CYNK-001 exhibits in vitro cytotoxicity against various cancer cell types, including glioblastoma multiforme (GBM), and secretes cytokines during co-culture with cancer cells. To evaluate in vivo anti-GBM activity, safety and persistence of CYNK-001, we conducted two studies in the non-obese diabetic (NOD)-scid IL2Rgammanull (NSG) immune deficient mouse models. First, CYNK-001 in vivo anti-GBM activity was assessed in a U-87MG orthotopic NSG mouse model. Luciferase-expressing U-87MG cells were stereotactically injected into the cranium of NSG mice at Day 0. Repeated dosing of 0.5x106 CYNK-001 cells at Day 14 and Day 25 by intracranial (IC) injection showed a statistically significant reduction of Bioluminescence Imaging (BLI) compared to the PBS control. Furthermore, intravenous (IV) and intracerebroventricular (ICV) routes of administration were evaluated compared to IC. CYNK-001 administered with IC resulted in a greater reduction of BLI than IV and ICV. Second, a single-dose toxicity study was conducted in naïve NSG mice to assess the safety and persistence of CYNK-001 following an IC injection. IC administration of 1×106 CYNK-001 was well tolerated, and no adverse clinical symptoms were observed. Quantitative polymerase chain reaction (qPCR) analysis for the human telomerase reverse transcriptase (hTERT) gene revealed that CYNK-001 persisted in the brain up to seven days. Our studies demonstrated that CYNK-001with IC administration appears safe and well tolerated in naïve as well as U-87MG tumor bearing NSG mice. Furthermore, CYNK-001 anti-tumor activity was exhibited in a GBM orthotopic NSG mouse model. Taken together, our data support a safety and efficacy evaluation of CYNK-001 in patients with GBM. A Phase 1 study in adult patients with recurrent/refractory GBM is planned to start this year evaluating the safety and efficacy of CYNK-001 with both IV and IC administrations.

Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia

Blood ◽  
2011 ◽  
Vol 117 (14) ◽  
pp. 3737-3747 ◽  
Author(s):  
Dirk Heckl ◽  
Daniel C. Wicke ◽  
Martijn H. Brugman ◽  
Johann Meyer ◽  
Axel Schambach ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Aplastic Anemia ◽  
Bone Marrow Cells ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Egfp Reporter

AbstractThpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl−/−) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl−/− bone marrow cells into Mpl−/− mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl−/− cells had increased long-term repopulating potential, with a marked increase in lineage−Sca1+cKit+ cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage−Sca1+cKit+ cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

scholarly journals Curcumin and Its Analogue Induce Apoptosis in Leukemia Cells and Have Additive Effects with Bortezomib in Cellular and Xenograft Models

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
L. I. Nagy ◽  
L. Z. Fehér ◽  
G. J. Szebeni ◽  
M. Gyuris ◽  
P. Sipos ◽  
...  
Keyword(s):  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Great Promise ◽  
Human Leukemia ◽  
Human Leukemia Cell Line ◽  
Apoptotic Effects

Combination therapy of bortezomib with other chemotherapeutics is an emerging treatment strategy. Since both curcumin and bortezomib inhibit NF-κB, we tested the effects of their combination on leukemia cells. To improve potency, a novel Mannich-type curcumin derivative, C-150, was synthesized. Curcumin and its analogue showed potent antiproliferative and apoptotic effects on the human leukemia cell line, HL60, with different potency but similar additive properties with bortezomib. Additive antiproliferative effects were correlated well with LPS-induced NF-κB inhibition results. Gene expression data on cell cycle and apoptosis related genes, obtained by high-throughput QPCR, showed that curcumin and its analogue act through similar signaling pathways. In correlation with in vitro results similar additive effect could be obsereved in SCID mice inoculated systemically with HL60 cells. C-150 in a liposomal formulation given intravenously in combination with bortezomib was more efficient than either of the drugs alone. As our novel curcumin analogue exerted anticancer effects in leukemic cells at submicromolar concentration in vitro and at 3 mg/kg dose in vivo, which was potentiated by bortezomib, it holds a great promise as a future therapeutic agent in the treatment of leukemia alone or in combination.

Flt3 Ligand Negatively Regulates In Vitro Migration, Intracellular Signaling Activated by SDF-1α/CXCR4 and In Vivo Homing of Hematopoietic Progenitor Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2674-2674
Author(s):  
Seiji Fukuda ◽  
Hal E. Broxmeyer ◽  
Louis M. Pelus
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Leukemia Cells ◽  
Flt3 Ligand ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

Abstract The Flt3 receptor tyrosine kinase (Flt3) is expressed on primitive normal and transformed hematopoietic cells and Flt3 ligand (FL) facilitates hematopoietic stem cell mobilization in vivo. The CXC chemokine SDF-1α(CXCL12) attracts primitive hematopoietic cells to the bone marrow microenvironment while disruption of interaction between SDF-1α and its receptor CXCR4 within bone marrow may facilitate their mobilization to the peripheral circulation. We have previously shown that Flt3 ligand has chemokinetic activity and synergistically increases migration of CD34+ cells and Ba/F3-Flt3 cells to SDF-1α in short-term migration assays; this was associated with synergistic phosphorylation of MAPKp42/p44, CREB and Akt. Consistent with these findings, over-expression of constitutively active ITD (internal tandem duplication) Flt3 found in patients with AML dramatically increased migration to SDF-1α in Ba/F3 cells. Since FL can induce mobilization of hematopoietic stem cells, we examined if FL could antagonize SDF-1α/CXCR4 function and evaluated the effect of FL on in vivo homing of normal hematopoietic progenitor cells. FL synergistically increased migration of human RS4;11 acute leukemia cells, which co-express wild-type Flt3 and CXCR4, to SDF-1α in short term migration assay. Exogenous FL had no effect on SDF-1α induced migration of MV4-11 cells that express ITD-Flt3 and CXCR4 however migration to SDF-1α was partially blocked by treatment with the tyrosine kinase inhibitor AG1296, which inhibits Flt3 kinase activity. These results suggest that FL/Flt3 signaling positively regulates SDF-1α mediated chemotaxis of human acute leukemia cells in short-term assays in vitro, similar to that seen with normal CD34+ cells. In contrast to the enhancing effect of FL on SDF-1α, prolonged incubation of RS4;11 and THP-1 acute myeloid leukemia cells, which also express Flt3 and CXCR4, with FL for 48hr, significantly inhibited migration to SDF-1α, coincident with reduction of cell surface CXCR4. Similarly, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulates CXCR4 expression, inhibits SDF-1α-mediated phosphorylation of MAPKp42/p44, CREB and Akt and impairs migration to SDF-1α. Despite reduction of surface CXCR4, CXCR4 mRNA and intracellular CXCR4 in Ba/F3-Flt3 cells were equivalent in cells incubated with or without FL, determined by RT-PCR and flow cytometry after cell permeabilization, suggesting that the reduction of cell surface CXCR4 expression is due to accelerated internalization of CXCR4. Furthermore, incubation of Ba/F3-Flt3 cells with FL for 48hr or over-expression of ITD-Flt3 in Ba/F3 cells significantly reduced adhesion to VCAM1. Consistent with the negative effect of FL on in vitro migration and adhesion to VCAM1, pretreatment of mouse bone marrow cells with 100ng/ml of FL decreased in vivo homing of CFU-GM to recipient marrow by 36±7% (P<0.01), indicating that FL can negatively regulate in vivo homing of hematopoietic progenitor cells. These findings indicate that short term effect of FL can provide stimulatory signals whereas prolonged exposure has negative effects on SDF-1α/CXCR4-mediated signaling and migration and suggest that the FL/Flt3 axis regulates hematopoietic cell trafficking in vivo. Manipulation of SDF-1α/CXCR4 and FL/Flt3 interaction could be clinically useful for hematopoietic cell transplantation and for treatment of hematopoietic malignancies in which both Flt3 and CXCR4 are expressed.

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