Cytosine Arabinoside Chemotherapy Does Not Enrich For Leukemic Stem Cells In Xenotransplantation Model Of Human Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1651-1651 ◽  
Author(s):  
Fabienne de Toni ◽  
Robin L. Perry ◽  
Estelle Saland ◽  
Mayumi Sugita ◽  
Marion David ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Frequent Relapses ◽  
Cell Cycle Status ◽  
Acute Myeloid

Abstract Despite a high rate of complete remission after treatment with conventional genotoxic agents, the overall survival of patients with acute myeloid leukemia (AML) is poor due to frequent relapses caused by the chemoresistance of rare leukemic stem cells (LSCs, also called Scid-Leukemia Initiating Cells). This unfavorable situation leads to a strong need to characterize those cells in order to target them with new specific therapies. Using a robust immunodeficient mouse model (NOD/LtSz-scid IL2Rγchainnull or NSG), we have previously shown that these LSCs were rare and not restricted to the CD34+CD38- immature compartment. This phenotypical heterogeneity of LSCs suggests that pharmacological targeting of LSC will not work if solely based on their cell surface markers. A better understanding of the mechanisms underlying the in vivo chemoresistance is required for the development of innovative targeted therapies. Aracytine (Ara-C, a pyrimidine analog), the most clinically used chemotherapeutic agents for AML patients, inhibits DNA synthesis and, therefore, targets and kills cycling AML cells in S phase of the cell cycle. Based on this mechanism of action, we hypothesized that Ara-C treatment will spare and enrich quiescent LSCs in vivo. We analyzed the response to Ara-C and residual disease in NSG mice engrafted with primary AML cells from 13 patients in two clinical centers (University of Pennsylvania, Philadelphia, USA and Purpan Hospital, Toulouse, France). A sub-lethal treatment of 60 mg/kg Ara-C given daily for five days induced a 5- to 50- fold reduction of peripheral blood blasts and total tumor burden in spleen and bone marrow in all patients tested. For 5 patients, we observed relapse within 4 to 6 weeks post-chemotherapy. Surprisingly, residual viable cells after Ara-C treatment showed no significant enrichment in quiescent cells and CD34+CD38- cells for the majority of primary samples tested (12 and 10 out of 13 total tested, respectively). Of note, the largest fraction (70-90%) of leukemic cells is in G0/G1 phase (including 0.5-20% in G0) in untreated engrafted mice. Moreover, we observed no significant changes in cell cycle profile of residual leukemic cells during the time course of the disease progression for 3 out of 4 patients. Finally, we assessed the frequency of LSCs in Ara-C-treated and control mice using transplantation and limiting dilution analysis in secondary recipients. Interestingly, we observed that Ara-C treatment did not increase the frequency of SL-ICs in residual cells, suggesting that blasts and LSC were equally sensitive to Ara-C in vivo. Our results show that sub-lethal regimen of Ara-C does not lead to enrichment of LSCs and induces cell death of both leukemic bulk and stem/progenitor cells independently of their cell cycle status probably through another in vivo mechanism such as apoptosis, autophagy or necroptosis. This study also suggests that further characterization of chemoresistant leukemic cells beyond phenotype and cell cycle status must rely on more functional properties in order to better elucidate the molecular basis of resistance in AML. Disclosures: Perry: MERCK: Employment. Carroll:Leukemia and Lymphoma Society: Research Funding. Sarry:AFFICHEM SA: Membership on an entity’s Board of Directors or advisory committees.

The Target Receptor Siglec-3 (CD33) Is Expressed on AML Stem Cells in a Majority of All Patients with AML.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4324-4324
Author(s):  
Alexander W. Hauswirth ◽  
Stefan FLorian ◽  
Maria-Theresa Krauth ◽  
Gerit-Holger Schernthaner ◽  
Edgar Selzer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Staining Technique ◽  
Scid Mice ◽  
Leukemic Cells ◽  
Cytostatic Drug ◽  
Leukemic Stem Cells ◽  
Targeting Therapy ◽  
Acute Myeloid

Abstract The cell surface antigen Siglec-3 = CD33 is becoming increasingly important as target of therapy in acute myeloid leukemia (AML). In particular, a conjugate consisting of the humanized CD33 antibody P67.6 (gemtuzumab) and the cytostatic drug calicheamicin has been developed for clinical use and was found to work as an effective antileukemic agent (Mylotarg®) in patients with CD33+ AML. In normal myelopoiesis, expression of CD33 is restricted to advanced stages of differentiation, whereas primitive stem cells do not express CD33 (Siglec-3). In line with this notion, CD33-targeting therapy is a non-myeloablative approach. In the present study, we asked whether leukemic stem cells in patients with AML express CD33. For this purpose, a multicolor-staining technique was applied in eleven patients with AML. Leukemic stem cells were defined as CD34+/CD38−/CD123+ cells. In all patients in whom the majority of myeloblasts expressed CD33 (=CD33+ AML, n=8), the AML progenitor cells reacted with the CD33 antibody P67.6. Repopulation experiments utilizing NOD/SCID mice confirmed that the AML stem cells in these patients reside within the CD33+ subpopulation of leukemic cells. Moreover, AML stem cells (CD34+/CD38−/CD123+ cells) highly purified (>98% purity) from patients with (CD33+) AML by cell sorting, were found to express CD33 mRNA in RT-PCR analyses. To demonstrate that AML stem cells can also reside within the CD33-negative fraction of the AML clone, we purified CD33-negative cells in a patient with AML in whom a majority of leukemic stem cells were found to lack CD33. In this particular patient, the CD33-negative cells were found to repopulate NOD/SCID mice with leukemias in the same way as the entire leukemic clone did. The CD33 antigen was neither detectable on CD34+/CD38− cells in the normal bone marrow nor on leukemic stem cells in patients with CD33-negative AML. In summary, our data show that leukemic stem cells in patients with CD33+ AML frequently express the target receptor CD33. This observation is in favor of novel treatment concepts employing CD33-targeting antibodies (Mylotarg®) in acute myeloid leukemia.

scholarly journals Novel Therapeutics That Can Target and Eradicate Leukemic Stem Cells in Acute Myeloid Leukemia: Investigating FDA-Approved Anti-Inflammatory Compounds

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5052-5052
Author(s):  
Isabella Iasenza ◽  
Meaghan Boileau ◽  
Andrea Neumann ◽  
Héloïse Frison ◽  
Mark D. Minden ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Mechanism Of Action ◽  
Myeloid Leukemia ◽  
Terminal Differentiation ◽  
Dependent Manner ◽  
Leukemic Stem Cells ◽  
Anti Inflammatory ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is an aggressive form of blood cancer defined by the uncontrolled proliferation of immature myeloblast cells in the blood and bone marrow, leading to hematopoietic failure. The 5-year survival rate is 28% in patients aged 20 years and older and 64% in patients aged 19 years and younger (SEER 2019). A large portion of these patients succumb to the disease partially due to the chemo-resistant nature of leukemic stem cells (LSCs). Hence, novel therapies targeting unique LSC biology that spare hematopoietic stem cells (HSCs) are needed to eliminate and avoid reoccurrence of this disease. We had previously identified FDA-approved anti-inflammatory glucocorticoids mometasone, halcinonide, and budesonide as compounds that induce terminal differentiation of the LSC (CD34+CD38-) and progenitor cell (CD34+CD38+) populations to leukemic blast cells (CD15+CD34-) in refractory human AML (Laverdière & Boileau et al., Blood Can. J. 2018). Following the paradigm of successful differentiation treatment in AML (acute promyelocytic leukemia with all-trans retinoic acid), the effects and mechanism of action of the glucocorticoids on LSCs need to be further investigated for other AML subtypes. Furthermore, dexamethasone, a glucocorticoid currently used to successfully treat acute lymphoblastic leukemia (ALL), is being studied in a Phase II clinical trial for induction and post-remission chemotherapy in older patients with de novo or therapy-related AML (clinicalTrials.gov, NCT03609060). To identify the subtypes of AML that are sensitive to steroid-induced LSC differentiation, we began by screening a panel of cell lines (F36P, MOLM-13, Kasumi-6, Kasumi-1 and K562) and observed that only Kasumi-1, a pediatric leukemia carrying the t(8;21) mutation leading to the fused RUNX1-RUNX1T1 gene, was responsive to glucocorticoid treatment, although without differentiation. This is consistent with the finding of Simon et al. who observed a loss of bulk AML cells in RUNX1 AML samples following dexamethasone treatment (Simon et al., Clin Cancer Res. 2017). However, we observed expansion of bulk cells following differentiation of LSCs in primary AML, indicating different mechanisms of steroid response in different samples: differentiation of LSCs or overall loss of AML cells. We will further investigate these compounds in a panel of 10 genetically defined primary AML samples to classify which oncogenetic drivers or subtypes of AML are linked to sensitivity to the three glucocorticoids, including which drive cell death vs LSC differentiation. We will perform ex vivo and in vivo studies of the glucocorticoids to assess the extent of engraftment in treated versus DMSO treated samples. This additional data will be presented at the annual meeting. In addition, to explore the mechanism of action of these steroids in AML, we investigated the roles of the cytokines interleukin-3 (IL-3), interleukin-6 (IL-6), stem cell factor (SCF), granulocyte colony stimulating factor (GCSF), thrombopoietin (TPO) and FMS-like tyrosine kinase 3 ligand (FLT3L), used to culture AML, on the differentiation effects induced by the glucocorticoids. We observed that only FLT3L was required for the complete differentiation of LSCs. In summary, we have observed that the three glucocorticoid steroids (mometasone, halcinonide, and budesonide), as well as dexamethasone to a lesser extent, can induce two different responses in a sample-dependent manner: terminal differentiation of LSCs or overall cell loss. We have also observed that the differentiation response requires FLT3L for maturation of the AML cells. Our current studies involve in vivo and genomic assays to determine the effect on functional LSCs and the genetic markers of sensitivity and we will present these results. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement.

Identification of Leukemic Stem Cells in Acute Myeloid Leukemia Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5309-5309
Author(s):  
Manal M W Elmasry ◽  
Alaa Elhaddad
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Mononuclear Phagocytes ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Two Samples ◽  
Acute Myeloid

Abstract Background: Acute myeloblastic leukemia (AML) can be viewed as newly formed, abnormal hemopoeitic tissue initiated by few leukemic stem cells (LSCs). Recognizing the LSC and identifying their behavior, plays a pivotal role in the approach of a targeted therapy.Colony-stimulating factor 1 (CSF-1), also known as M-CSF, is a protein ligand that acts on the CSF1R promotes mononuclear phagocytes survival, proliferation and differentiation. Aim of the work: Defining the self-renewing [Thy1-, CD34+, CD38-] LICs in AML cases before and after induction chemotherapy as a predictor for relapse and to determine how CSF1R (Fms) and CD34 markers affect the growth and survival of human leukemic cells in the CD38- Thy1- population. Patients and methods: This study was carried out on 30 samples from the peripheral blood of adult patients with de-novo acute myeloid leukemia. The majority of the patients were monocytic AML Samples were sorted into four populations (Fms+CD34-, Fms+CD34+, Fms-CD34+ and Fms-CD34-) according to the surface markers of the cells. Cells were cultured on mouse stromal cells transfected with a plasmid containing human CSF-1. Samples were cultured using Iscove's modified Dulbecc's medium (IMDM).The cultures were assessed for survival of leukemic cells in days. Results: The mean survival in days of the cells was 13.9 before chemotherapy and 14.1 after chemotherapy. The difference in growth was insignificant (p>0.05). The Fms-CD34+ population in all but two samples tested had the longest survival time in culture. Conclusion: Our results suggest that leukemic stem cells may survive chemotherapy mainly due to their quiescence. Human CSF-1 was shown to increase the number of leukemic cells in co-culture with mouse stroma after 5 weeks. A novel leukemic stem cell (Fms-CD34+) has been identified and is the cell responsible for the growth and maintenance of the leukemic bulk. Disclosures No relevant conflicts of interest to declare.

scholarly journals Pharmacologic Targeting of LIN28/Let-7 in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4072-4072 ◽  
Author(s):  
Martina M Roos ◽  
Michelle Li ◽  
Pang Amara ◽  
John P Chute
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Small Molecule ◽  
Myeloid Leukemia ◽  
Driver Genes ◽  
Oncogenic Driver ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy with high relapse rates and mortality due to the outgrowth of chemotherapy-resistant leukemic stem cells (LSCs). Thus, the development of novel therapeutic strategies capable of eradicating human AML represents a major area of unmet medical need. The RNA binding protein, LIN28, is a known driver of many cancer stem cells, AML included, wherein overexpression of LIN28 correlates with reduced patient survival. LIN28 blocks the function of the let-7 microRNA family, which exert tumor suppressive effects by repressing oncogenes and cell cycle regulators including MYC, RAS and CyclinD. Thus, LIN28 is an attractive mechanistic target for the purpose of inhibiting AML LSCs. Using a targeted high-throughput screen, we identified a class of small molecules which selectively block the LIN28/let-7 interaction (Roos et al., ACS Chem Biol, 2016). Preliminary studies demonstrate that a lead small molecule markedly impairs the proliferation and clonogenic capacity of human AML cell lines and primary patient AML samples. In vivo, systemic administration of a lead small molecule LIN28/let-7 inhibitor decreases leukemic tumor burden, reduces LSC numbers and significantly improves animal survival. Mechanistic studies revealed that targeted inhibition of the LIN28/let-7 axis restores let-7 microRNA levels in AML LSCs and subsequently inhibits a panoply of key oncogenic driver genes, including the NF-ĸB pathway, a hallmark for LSC proliferation. Furthermore, AML cell lines and primary patient cells treated with the LIN28/let-7 small molecule inhibitor showed a block at the G1/S phase interface and significantly decreased cell cycle progression. Consequently, LIN28/let-7 inhibition leads to LSC differentiation and ultimately leukemic cell death. In summary, we demonstrate for the first time the drugability of the LIN28/let-7 axis in vivo and reveal a novel pharmacological means to suppress a multitude of oncogenic driver genes in human AML. These results suggest that small molecule inhibition of LIN28/let-7 has high therapeutic potential as a new class of targeted therapies for AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rspo-LGR4 Cooperates with HOXA9 to Sustain Self-Renewal in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2669-2669
Author(s):  
Nunki Hassan ◽  
Basit Salik ◽  
Alastair Duly ◽  
Jenny Yingzi Wang
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Myeloid Differentiation ◽  
Beta Catenin ◽  
Leukemic Stem Cells ◽  
Self Renewal ◽  
Differentiation Block ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is associated with high relapse rates and poor survival, with limited response to conventional cancer therapy and lacking effective targeting of highly self-renewing leukemic stem cells (LSCs). The mechanism underlying the high self-renewal activity of LSCs that determines the aggressiveness of disease remains poorly understood. Although we and others have previously demonstrated the clinical significance of aberrant WNT/β-catenin signaling in AML (Science, 327:1650-1653, 2010; Cancer Cell, 18:606-618, 2010), its pharmacologically tractable components essential for the regulation of LSC self-renewal have not yet been determined. Our studies discover, for the first time, a critical link between R-spondin (RSPO)-LGR4/HOXA9 and WNT/β-catenin pathways in AML LSCs. Microarray data analysis of 183 AML patient samples showed a significant positive correlation between expression of LGR4 and HOXA9 (r=0.546, P<0.0001). LGR4 exerted a cell-of-origin-specific function in promoting aberrant self-renewal and AML progression in vivo through cooperating with HOXA9, a poor prognostic predictor. We observed that LGR4 itself was not able to fully transform normal hematopoietic stem/progenitor cells (HSPCs), but instead cooperated with HOXA9 in HSPCs to accelerate disease onset producing a highly aggressive short latency AML in vivo. LGR4 and HOXA9 were epigenetically upregulated and their coexpression was an essential determinant of RSPO-LGR4 oncogenic activity. RSPO/WNT3 ligands could serve as stem cell growth factors to sustain myeloid differentiation block and to promote proliferation of CD34+ LSC-enriched subpopulations in primary AML patient specimens co-expressing LGR4 and HOXA9. Conversely, CRISPR/Cas9-mediated knockout of LGR4 not only suppressed RSPO/WNT3 signals and markedly decreased nuclear active β-catenin, but also reduced tumor burden in a patient-derived xenograft (PDX) mouse model of relapsed AML. Importantly, this study is the first to demonstrate that pharmacological inhibition of RSPO3-LGR4 signaling by a clinical-grade anti-RSPO3 monoclonal antibody induced LSC differentiation and consequently prevented tumor growth in AML PDX mice but did not affect normal human stem cell compartment in NSG mice. Together, our findings support a critical role for RSPO-LGR4 in the Wnt/β-catenin signaling pathway to promote AML leukemogenesis. Aberrant activation of RSPO-LGR4 is crucial for enhancing the self-renewal potential and myeloid differentiation block, which contribute to an aggressive leukemia phenotype through cooperating with HOXA9. Genetic and pharmacological targeting of this pathway impairs LSC self-renewal and survival and impedes AML development in murine models and patient-derived xenografts, highlighting the therapeutic value of targeting RSPO-LGR4 signaling in AML. References: Wang Y, et al. The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML. Science. 2010;327:1650-1653. Yeung J, et al. Beta-catenin mediates the establishment and drug resistance of MLL leukemic stem cells. Cancer Cell. 2010;18:606-618. Disclosures No relevant conflicts of interest to declare.

scholarly journals Evidence for malignant transformation in acute myeloid leukemia at the level of early hematopoietic stem cells by cytogenetic analysis of CD34+ subpopulations

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 2906-2912 ◽  
Author(s):  
D Haase ◽  
M Feuring-Buske ◽  
S Konemann ◽  
C Fonatsch ◽  
C Troff ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Malignant Transformation ◽  
Cell Sorting ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is a heterogenous disease according to morphology, immunophenotype, and genetics. The retained capacity of differentiation is the basis for the phenotypic classification of the bulk population of leukemic blasts and the identification of distinct subpopulations. Within the hierarchy of hematopoietic development and differentiation it is still unknown at which stage the malignant transformation occurs. It was our aim to analyze the potential involvement of cells with the immunophenotype of pluripotent stem cells in the leukemic process by the use of cytogenetic and cell sorting techniques. Cytogenetic analyses of bone marrow aspirates were performed in 13 patients with AML (11 de novo and 2 secondary) and showed karyotype abnormalities in 10 cases [2q+, +4, 6p, t(6:9), 7, +8 in 1 patient each and inv(16) in 4 patients each]. Aliquots of the samples were fractionated by fluorescence-activated cell sorting of CD34+ cells. Two subpopulations, CD34+/CD38-(early hematopoietic stem cells) and CD34+/CD38+ (more mature progenitor cells), were screened for karyotype aberations as a marker for leukemic cells. Clonal abnormalities and evaluable metaphases were found in 8 highly purified CD34+/CD38-populations and in 9 of the CD34+/CD38-specimens, respectively. In the majority of cases (CD34+/CD38-, 6 of 8 informative samples; CD34+/CD38+, 5 of 9 informative samples), the highly purified CD34+ specimens also contained cytogenetically normal cells. Secondary, progression-associated chromosomal changes (+8, 12) were identified in the CD34+/CD38-cells of 2 patients. We conclude that clonal karyotypic abnormalities are frequently found in the stem cell-like (CD34+/CD38-) and more mature (CD34+/CD38+) populations of patients with AML, irrespective of the phenotype of the bulk population of leukemic blasts and of the primary or secondary character of the leukemia. Our data suggest that, in AML, malignant transformation as well as disease progression may occur at the level of CD34+/CD38-cells with multilineage potential.

scholarly journals Anti-MY9-blocked-ricin: an immunotoxin for selective targeting of acute myeloid leukemia cells

Blood ◽  
1991 ◽  
Vol 77 (11) ◽  
pp. 2404-2412 ◽  
Author(s):  
DC Roy ◽  
JD Griffin ◽  
M Belvin ◽  
WA Blattler ◽  
JM Lambert ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Short Exposure ◽  
Continuous Exposure ◽  
Dependent Manner ◽  
Acute Myeloid

Abstract The use of immunotoxins (IT) to selectively destroy acute myeloid leukemia (AML) cells in vivo or in vitro is complicated by both the antigenic similarity of AML cells to normal progenitor cells and the difficulty of producing a sufficiently toxic conjugate. The monoclonal antibody (MoAb) anti-MY9 is potentially ideal for selective recognition of AML cells because it reacts with an antigen (CD33) found on clonogenic AML cells from greater than 80% of cases and does not react with normal pluripotent stem cells. In this study, we describe an immunotoxin that is selectively active against CD33+ AML cells: Anti- MY9-blocked-Ricin (Anti-MY9-bR), comprised of anti-MY9 conjugated to a modified whole ricin that has its nonspecific binding eliminated by chemical blockage of the galactose binding domains of the B-chain. A limiting dilution assay was used to measure elimination of HL-60 leukemic cells from a 20-fold excess of normal bone marrow cells. Depletion of CD33+ HL-60 cells was found to be dependent on the concentration of Anti-MY9-bR and on the duration of incubation with IT at 37 degrees C. More than 4 logs of these leukemic cells were specifically depleted following short exposure to high concentrations (10(-8) mol/L) of Anti-MY9-bR. Incubation with much lower concentrations of Anti-MY9-bR (10(-10) mol/L), as compatible with in vivo administration, resulted in 2 logs of depletion of HL-60 cells, but 48 to 72 hours of continuous exposure were required. Anti-MY9-bR was also shown to be toxic to primary AML cells, with depletion of greater than 2 logs of clonogenic cells following incubation with Anti- MY9-bR 10(-8) mol/L at 37 degrees C for 5 hours. Activity of Anti-MY9- bR could be blocked by unconjugated Anti-MY9 but not by galactose. As expected, Anti-MY9-bR was toxic to normal colony-forming unit granulocyte-monocyte (CFU-GM), which expresses CD33, in a concentration- and time-dependent manner, and also to burst-forming unit-erythroid and CFU-granulocyte, erythroid, monocyte, megakaryocyte, although to a lesser extent. When compared with anti-MY9 and complement (C′), Anti- MY9-bR could be used in conditions that provided more effective depletion of AML cells with substantially less depletion of normal CFU- GM. Therefore, Anti-MY9-bR may have clinical utility for in vitro purging of AML cells from autologous marrow when used at high IT concentrations for short incubation periods. Much lower concentrations of Anti-MY9-bR that can be maintained for longer periods may be useful for elimination of AML cells in vivo.

Detection, isolation, and stimulation of quiescent primitive leukemic progenitor cells from patients with acute myeloid leukemia (AML)

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3142-3149 ◽  
Author(s):  
Yinghui Guan ◽  
Brigitte Gerhard ◽  
Donna E. Hogge
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Active Cell ◽  
Leukemic Cells ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Acute Myeloid

Abstract Although many acute myeloid leukemia (AML) colony-forming cells (CFCs) and long-term culture–initiating cells (LTC-ICs) directly isolated from patients are actively cycling, quiescent progenitors are present in most samples. In the current study,3H-thymidine (3H-Tdr) suicide assays demonstrated that most NOD/SCID mouse leukemia-initiating cells (NOD/SL-ICs) are quiescent in 6 of 7 AML samples. AML cells in G0, G1, and S/G2+M were isolated from 4 of these samples using Hoechst 33342/pyroninY staining and cell sorting. The progenitor content of each subpopulation was consistent with the 3H-Tdr suicide results, with NOD/SL-ICs found almost exclusively among G0 cells while the cycling status of AML CFCs and LTC-ICs was more heterogeneous. Interestingly, after 72 hours in serum-free culture with or without Steel factor (SF), Flt-3 ligand (FL), and interleukin-3 (IL-3), most G0 AML cells entered active cell cycle (percentage of AML cells remaining in G0 at 72 hours, 1.2% to 37%, and 0% to 7.6% in cultures without and with growth factors [GFs], respectively) while G0 cells from normal lineage-depleted bone marrow remained quiescent in the absence of GF. All 4 AML samples showed evidence of autocrine production of 2 or more of SF, FL, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, 3 of 4 samples contained an internal tandem duplication of theFLT3 gene. In summary, quiescent leukemic cells, including NOD/SL-ICs, are present in most AML patients. Their spontaneous entry into active cell cycle in short-term culture might be explained by the deregulated GF signaling present in many AMLs.

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