Acute Myeloid Leukemia Stem Cells Cells Are Rare and Heterogeneous in Human Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 390-390 ◽  
Author(s):  
Jean-Emmanuel Sarry ◽  
Kathleen Murphy ◽  
Gwenn-ael Danet-Desnoyers ◽  
Martin Carroll
Keyword(s):  
Stem Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Leukemic Stem Cells ◽  
Cell Compartment ◽  
Nsg Mice ◽  
Cd38 Expression ◽  
Cell Fractions ◽  
Limiting Dilution ◽  
Acute Myeloid

Abstract Abstract 390 Human leukemic stem cells are hypothesized to be rare, restricted to phenotypically immature hematopoietic cells and capable of incomplete differentiation. However, recent work in other tumors has challenged this hypothesis. We used a robust xenotransplantation model based on NOD-SCID-IL-2Rγcnull (NSG) mice to better characterize the frequency and heterogeneity of human SCID leukemia initiating cells (SL-IC). We performed an extensive analysis on primary specimens from 11 AML patients. First, we determined the frequency of SL-IC in un-fractionated AML specimens using transplantation (i.v.) in adult NSG mice for 12 weeks and limiting dilution analysis. Our results indicate that SL-IC are rare cells in primary AML and that the frequency of SL-IC varies greatly from patient to patient: one SL-IC per 0.14 to 4.5 × 106 mononuclear cells. Normal hematopoietic stem cells (HSC) are phenotypically characterized as lineage-, CD34+, CD38- and SL-IC were initially described as being restricted to the CD34+38- compartment. To determine in this model if SL-IC are restricted to this immature cell compartment, we sorted AML cells based on surface staining for a lineage cocktail, CD34 and CD38 expression. CD38+ cells were further sorted by expression of CD45RA and CD123. In contrast to previous results, mice injected with cells from multiple different fractions engrafted including fractions with a mature cell phenotype. Although some fractions did not engraft from individual patients, engrafting cells were found in multiple compartments from all individuals studied. For each engrafting fraction, the AML cells found in mice 12 to 16 weeks post-transplant had the same phenotypic heterogeneity (defined by expression of lineage, CD34 and CD38) as observed in primary specimens consistent with either de-differentiation or lineage infidelity for these cell surface markers. Secondary transplant experiments demonstrated that each engrafting fraction contains self-renewing leukemic stem cells. In order to compare the frequencies of SL-IC in each fraction, we sorted 4 different subsets (based on lineage and CD38 expression) from 1 AML patient and performed limiting dilution analysis (LDA) in NSG mice. SL-IC were detected in each subset, but their frequency was 10-fold higher (1 in 38,000 cells) in Lin-CD38- fractions compared to other fractions and un-fractionated samples. However, as Lin-CD38- cells represent only 3% of all leukemic cells, only 34% of SL-IC were present in this fraction. By comparison, the Lin+CD38+ cell compartment has a SL-IC frequency of 1 in 106 cells but 25% of SL-IC are found in this compartment. Overall, this data demonstrate that human AML stem cells are rare but they are not restricted to immature cell fractions. Rather, leukemic stem cells can be found at different frequencies in all cell fractions. These results suggest that efforts to therapeutically target leukemic stem cells specifically may require re-evaluation. Disclosures: Carroll: Cephalon consultancy: Consultancy; Sanofi Aventis Corporation: Research Funding; Kyowa Hakko Kirin Pharmaceutical: Research Funding.

scholarly journals Anti-Leukemic Activity of Daratumumab in Acute Myeloid Leukemia Cells and Patient-Derived Xenografts

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2312-2312 ◽  
Author(s):  
Cedric Dos Santos ◽  
Shan Xiaochuan ◽  
Zhou Chenghui ◽  
Georges Habineza Ndikuyeze ◽  
Joshua Glover ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Cell Killing ◽  
Nsg Mice ◽  
Cd38 Expression ◽  
Acute Myeloid

Abstract Daratumumab is a human antibody that binds to CD38 on the cell surface and induces cell killing by multiple mechanisms including complement mediated cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and apoptosis. In pre-clinical and clinical studies, daratumumab has been shown to effectively kill multiple myeloma (MM) cells and to enhance the potency of other treatments against MM. The purpose of the study was to investigate in vitro and in vivo efficacy of daratumumab against 9 acute myeloid leukemia (AML) cell lines and patient-derived samples. First, we evaluated the expression of CD38, complement inhibitory proteins (CIP) CD46, CD55, CD59, and FcgR1 (CD64) on AML cell lines (n=9), AML patient cells (n=10) and healthy donor bone marrow using flow cytometry. CD38 enumeration showed a substantial variation between cell lines (12,827±19, 320 molecules/cell) and between AML patients (11,560±8, 175 molecules/cell), while CD38 expression was more consistent in bone marrow (BM) from healthy donors (1,176±355 molecules/cell). The daratumumab-induced apoptosis observed in cell lines (MOLM-13, MOLM-16, MV-4-11, NB4) in vitro was not correlated with CD38 expression levels. Daratumumab induced minimal ADCC (5-20%) and low levels of (2-5%) CDC mediated cell killing in 6 AML cell lines tested. We did not observe a direct correlation between CD38 expression and ADCC, CDC, nor between CDC and CIP expression. Interestingly, treatment of two human Acute Promyelocytic Leukemia (M3) cell lines HL-60 and NB-4 with all-trans retinoic acid (ATRA) induced a 10-30-fold increase in CD38 expression, suggesting that ATRA could be used in combination with daratumumab. While we, and others, have shown that pre-incubation of primary AML cells with anti-CD38 antibodies inhibits engraftment in NSG mice, we aimed at evaluating the anti-leukemic activity of daratumumab in a therapeutic xenograft model using 3 different AML patients. NSG mice (10/group/patient) were transplanted with T cell-depleted AML cells and BM aspirates were collected 4-6 weeks later to assess leukemia burden in each mouse prior to treatment. Animals were untreated (Ctrl) or received daratumumab (10 mg/kg), or IgG1 isotype once a week for five weeks. We assessed AML burden (% huCD45+ CD33+) in BM, spleen (SPL) and peripheral blood (PB) within 5 days after the last treatment. First, we evaluated an AML (#3406, FLT3-ITD, see figure) with high expression of CD38 (13,445 molecules/cell) and low CD64 (489/cell) was evaluated. Daratumumab significantly reduced leukemia burden in SPL and PB, but had no effect in BM. The same daratumumab-induced reduction in peripheral blasts and lack of effect in BM was observed in 2 other AML patient xenografts (#7577, M1 IDH mutant/FLT3-ITD with 6,529 CD38 molecules/cell; #8096, M2 with 335 CD38 molecules/cell). Interestingly, we observed that daratumumab treatment led to a drastic reduction in CD38 surface expression in AML blasts including in BM, indicating that daratumumab efficiently targeted CD38 in bone marrow blasts. Our results suggest that the bone marrow microenvironment can impair the anti-leukemic activity of daratumumab observed in other tissues. Ongoing xenograft studies are testing whether induction with chemotherapy (Ara-C+doxorubicin), or with other agents disrupting the bone marrow microenvironment, can enhance the anti-leukemic activity of daratumumab. Figure 1: Effect of daratumumab treatment on AML 3406 leukemia burden: Figure 1:. Effect of daratumumab treatment on AML 3406 leukemia burden: Disclosures Dos Santos: Janssen R&D: Research Funding. Xiaochuan:Janssen R&D: Research Funding. Doshi:Janssen R&D: Employment. Sasser:Janssen R&D: Employment. Danet-Desnoyers:Janssen R&D: Research Funding.

scholarly journals Quantitative Detection of DNMT3A R882H Mutation in Course of Treatment of Acute Myeloid Leukemia Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4970-4970
Author(s):  
Olga Blau ◽  
Rimma Berenstein ◽  
Claudia Baldus ◽  
Nikola Suckert ◽  
Kathrin Rieger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Quantitative Detection ◽  
Leukemic Stem Cells ◽  
Donor Chimerism ◽  
Leukemic Clone ◽  
Acute Myeloid

Abstract Introduction: DNMT3A mutation is one of the most common somatic mutations in acute myeloid leukemia (AML) patients with normal karyotype. The most frequent mutation is located at R882 codon in the methyltransferase domain. Because of prognostic significance and high stability during the disease evolution, DNMT3A mutations might represent highly informative biomarkers for prognosis and outcome of disease. Methods: Using allele-specific PCR with a Blocking reagent (ASB-PCR assay) for the quantitative detection of DNMT3A R882H mutation, we analyzed 350 follow-up samples from 28 AML patients in complete remission (CR) after induction and consolidation treatment and allogeneic stem cell transplantation (alloSCT). Seventeen patients included in the follow-up analysis harbored a NPM1 mutation. Using a well-established marker for the detection of minimal residual disease (MRD) allowed to estimate the stability of DNMT3A mutation in CR and complete molecular remission (molCR). In addition, we analyzed FLT3, IDH1, and IDH2 mutations in diagnostic and follow up samples and donor chimerism after alloSCT. Results: We found the persistence of DNMT3A R882H mutations in all patients in CR after standard therapy. On the contrary, after alloSCT, DNMT3A R882H mutation was not found in patients with CR and complete donor chimerism. In relapse of leukemia, an increasing of both NPM1 and DNMT3A mutated alleles were shown all cases. Conclusion: Persistence of DNMT3A mutation after standard chemotherapy could indicate the origin of mutation in the early pre-leukemic stem cells. The loss of correlation between NPM1 and DNMT3A in CR could be associated with leukemic clone evolution. It is impotant to note, that the removal of mutated leukemic stem cells after allo SCT indicates therapeutic options allo SCT for high risk AML patients. Increased of both DNMT3A and NPM1 mutated alleles in relapse indicates the presence of both mutations, at least partly, in the same leukemic clone. We conclude that quantitative detection of DNMT3A R882H mutations at different time points of AML disease could provide additional information about the role of mutations in development and progression of AML. Disclosures Blau: BMS: Honoraria; MSD: Honoraria; Celgene: Honoraria, Research Funding; AMGEN: Honoraria; JAZZ Pharma: Honoraria; Shire: Honoraria; Janssen: Honoraria, Research Funding; Baxalta: Honoraria.

scholarly journals Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT) Activity Selectively Targets Human Acute Myeloid Leukemia Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3932-3932
Author(s):  
Amit Subedi ◽  
Qiang Liu ◽  
David Sharon ◽  
Changjiang Xu ◽  
Veronique Voisin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Cell Line ◽  
Clinical Outcomes ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Therapeutic Window ◽  
Cd38 Expression ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow associated with poor clinical outcomes. Conventional chemotherapies are effective in debulking the leukemic burden in most AML patients. However, a small population of disease-sustaining leukemic stem cells (LSCs) frequently persists and contributes to relapsed disease. Novel therapies that eradicate LSCs have the potential to improve clinical outcomes in AML. To discover novel anti-LSC agents, we performed a high-throughput flow cytometry-based drug screen of 1,220 compounds against a primary AML sample (8227). This sample harbors distinct subsets defined by CD34 and CD38 expression, and LSC activity assayed by xenotransplantation is restricted to the CD34+CD38- fraction. Through this screen, we identified compounds that selectively depleted the CD34+CD38- fraction including four structurally-unrelated NAMPT inhibitors (FK866, STF-118804, GMX1778, and KPT-9274). These inhibitors also depleted the LSC-enriched CD34+CD38- fraction in two other primary AML samples, indicating that the effect was not unique to 8227 cells. To further evaluate their impact on LSCs in 8227, we measured the expression of 104 genes that were previously found to be differentially expressed between LSC+ and LSC- cell fractions isolated from patient samples. Treatment with NAMPT inhibitors reduced the correlation between the measured LSC gene signature and the LSC+ reference profile, providing additional evidence for their anti-LSC activity. To determine whether the selective loss of CD34+CD38- cells was due to cell death or differentiation, we sorted subsets of 8227 cells based on CD34 and CD38 expression and treated each fraction with FK866. NAMPT inhibition preferentially triggered apoptosis as measured by Annexin V staining in the CD34+CD38- and CD34+CD38+ fractions over the CD34- fraction. We did not observe significant changes in the expression of CD34, CD38, or other myeloid differentiation markers (CD14 and CD15) in the remaining viable cells. Our subsequent mechanistic studies focused on KPT-9274 because it is the furthest along in clinical development. NAMPT is the rate-limiting enzyme in the NAD+ salvage pathway that converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), a direct NAD+ precursor. To confirm a decrease in intracellular NAD+ with KPT-9274 treatment, we introduced expression of genetically-encoded biosensors for measuring NAD+ levels in different cellular compartments in an AML cell line. KPT-9274 treatment for 15 hours lowered the free NAD+ pool in the cytosol and mitochondria but not in nucleus. To determine whether the drop in NAD+ was necessary for the effects of KPT-9274 on LSCs, we supplemented the primary AML samples with nicotinamide riboside (NR) which can be directly converted to NMN, thereby bypassing the requirement for NAMPT activity to generate NAD+. The addition of NR completely rescued the effects of KPT-9274 on the CD34+CD38- fraction. Niacin can also generate NAD+ through an alternative pathway that depends on nicotinic acid phosphoribosyltransferase (NAPRT). However, niacin supplementation failed to rescue the effects of NAMPT inhibition which correlated with the lack of NAPRT expression in LSC-enriched CD34+CD38- cells. Next, we studied the effects of KPT-9274 on normal CD34+ hematopoietic stem and progenitor cells (HSPCs) isolated from human cord blood. Although HSPCs were sensitive to the pro-apoptotic effects of KPT-9274, their survival was fully rescued by both NR and niacin. The rescue by niacin correlated with a higher expression of NAPRT in HSPCs. As the blood concentration of niacin is ~1,000 fold higher than that of NR, KPT-9274 is predicted to have a favorable therapeutic window in vivo. To demonstrate its in vivo activity, we treated immunodeficient NOD/SCID/IL2Rγ-null (NSG) mice engrafted with a luciferase-tagged AML cell line (OCI-AML3) with KPT-9274 at a dose of 150 or 250 mg/kg/day or vehicle control for 50 consecutive days by oral administration. KPT-9274 treatment significantly lowered leukemia burden and prolonged survival in both dosing cohorts. In summary, our results indicate that NAMPT inhibition represents an effective approach to target human LSCs through reduction in intracellular NAD+ levels and induction of apoptosis. Our data provide the preclinical rationale for investigating the use of KPT-9274 in AML clinical trials. Disclosures Chan: Genentech: Research Funding; Celgene: Research Funding; AbbVie: Research Funding.

scholarly journals 871 Construction and evaluation of interleukin 3 (IL3)-zetakine redirected cytolytic T Cells for the treatment of CD123 expressing acute myeloid leukemia

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A912-A912
Author(s):  
Rebecca Moeller ◽  
Julian Scherer ◽  
Sadik Kassim
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Jurkat Cells ◽  
Leukemic Stem Cells ◽  
Cd69 Expression ◽  
Acute Myeloid

BackgroundAcute Myeloid Leukemia (AML) is an aggressive bone marrow malignancy, characterized by the presence of leukemic blasts in the peripheral blood of patients. Poor AML prognoses1 are largely attributable to high rates of disease relapse, of which CD123+ leukemic stem cells (LSCs) are the primary cause.2 3 CD123, the alpha-chain of the IL3 cytokine receptor,6 has been identified as a favorable therapeutic AML target, overexpressed in both LSCs and blasts.4 5 We sought to direct T cells to CD123+ AML cells via cell surface tethered IL3 (termed ”IL3-zetakine”).7 The use of a zetakine instead of a chimeric antigen receptor (CAR) construct enables structure-guided site-directed mutagenesis to increase binding affinity and alter target cell signaling without detrimental T cell hyperactivation.MethodsZetakine constructs were designed using IL3 sequences bound to a transmembrane domain and intracellular costimulatory and CD3z signaling domains. The constructs were transduced into Jurkat cells with lentiviral vectors (LVV). T cell activation via CD69 expression was assessed via flow cytometry of sorted IL3 zetakine-positive Jurkat cells after co-culture with MOLM13 AML cells. Lead constructs were selected based on initial transduction percentage and activation response. In vitro functionality of each IL3 zetakine was tested with LVV transduced primary T cells by flow cytometry.ResultsZetakine constructs yielded a wide range of transduction percentages in Jurkat cells (0 – 98%) prior to sorting. In co-cultures with CD123+ MOLM13 AML cells, Jurkat cells expressing wildtype IL3 constructs lacking a costimulatory domain induced the highest level of CD69 expression (18.7% CD69+ T cells) in an antigen-specific manner (5.3-fold increase of CD69+ T cells over those cultured with MOLM13 CD123KO cells). The K110E mutant IL3 was reported to exhibit a 40-fold increased affinity over wildtype,8 but it showed no detectable zetakine function. However, additional mutant IL3 zetakines increased Jurkat cell activation up to 5.8-fold. Antigen-specific increases in CD69, as well as CD25, surface expression were also observed with zetakine-transduced primary T cells co-cultured with MOLM13 cells, in addition to target cell killing comparable to antibody-based CD123CAR T-cells.ConclusionsThis work establishes IL3 zetakines as a viable alternative to traditional CD123-targeted CAR constructs. Structure-guided IL3 zetakine mutants with altered affinity and activation profiles will further our understanding of CD123-specific cytotoxicity modulation without inducing acute T cell hyperactivation and exhaustion. These results indicate the ability of IL3 zetakine-expressing T cells to kill CD123-expressing AML cells and illustrate the potential of this novel class of therapeutics.ReferencesGanzel C, et al. Very poor long-term survival in past and more recent studies for relapsed AML patients: the ECOG-ACRIN experience. American journal of hematology 2018:10.1002/ajh.25162.Shlush LI, et al. Tracing the origins of relapse in acute myeloid leukaemia to stem cells. Nature 2017;547(7661):104–108.Hanekamp D, Cloos J, Schuurhuis GJ. Leukemic stem cells: identification and clinical application. International Journal of Hematology 2017;105(5):549–557.Bras AE, et al. CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping. Cytometry part B: Clinical Cytometry 2019;96(2):134–142.Sugita M, Guzman ML. CD123 as a therapeutic target against malignant stem cells. Hematology/Oncology clinics of North America 2020;34(3):553–564.Mingyue S, et al. CD123: a novel biomarker for diagnosis and treatment of leukemia. Cardiovascular & Hematological Disorders-Drug Targets 2019;19(3):195–204.Kahlon KS, et al. Specific recognition and killing of glioblastoma multiforme by interleukin 13-zetakine redirected cytolytic T cells. Cancer Res 2004;64(24):9160–6.Bagley CJ, et al. A discontinuous eight-amino acid epitope in human interleukin-3 binds the alpha-chain of its receptor. J Biol Chem 1996;271(50):31922–8.

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