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 Discovery of a Novel Dihydroorotate Dehydrogenase Inhibitor That Induces Differentiation and Overcomes Ara-C Resistance of Acute Myeloid Leukemia Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2663-2663
Author(s):  
Satoshi Kitazawa ◽  
Yukiko Ishii ◽  
Keiko Makita-Suzuki ◽  
Koichi Saito ◽  
Kensuke Takayanagi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Oral Administration ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Dihydroorotate Dehydrogenase ◽  
Once Daily ◽  
Hl60 Cells ◽  
Pyrimidine Nucleotide ◽  
Acute Myeloid

Cancer initiating cells (CIC) are suggested to be responsible for drug resistance and cancer relapse that are associated with poor prognosis. Therefore, drugs effective for CIC could fulfill an unmet clinical need. We performed a drug screen with chemical libraries to find out new compounds which specifically eradicated CIC established in the previous report (Yamashita et al., Cancer Research, 2015). We obtained compounds with a carboxylic acid skeleton as hit compounds. Interestingly, FF1215T, one of the hit compounds, was shown to inhibit growths of CIC by decreasing intracellular pyrimidine nucleotide levels. Finally, we identified dihydroorotate dehydrogenase (DHODH), which was essential for de novo pyrimidine synthesis as the target of the hit compounds in a ligand fishing assay. FF1215T inhibited DHODH enzymatic activity with the 50% inhibitory concentration value of 9 nM, which showed greater potency than well-known DHODH inhibitors brequinar (12 nM), teriflunomide (262 nM), and vidofludimus (141 nM). Growing evidence suggests that DHODH is considered to be a promising target to overcome a differentiation blockade of acute myeloid leukemia (AML) cells (Sykes et al., Cell, 2016).Therefore, we explored the effect of FF1215T on AML growth and differentiation. FF1215T demonstrated growth inhibitory effect in multiple human AML cell lines such as U937, MOLM13, HL60, and MV4-11 with the 50% growth inhibition values of 90-170 nM. FF1215T decreased intracellular pyrimidine nucleotide levels, induced DNA damage marker γ-H2AX possibly due to the replication stress, and finally led to apoptosis in HL60 cells. Cell cycle analysis revealed that FF1215T treatment arrested HL60 and THP1 cells at S phase and increased sub-G1 population in these cells. In addition, our DHODH inhibitors induced upregulation of cell-surface CD11b and CD86, which are monocyte and macrophage differentiation markers, morphological changes, and phagocytic activities in several AML cells, indicating differentiation of AML cells toward monocyte and macrophage by DHODH inhibition. FF1215T also depleted UDP-GlcNAc, a substrate for Protein O-GlcNAcylation, and diminished global O-GlcNAcylation and O-GlcNAcylated protein expressions such as c-Myc, SOX2, and OCT4, which play important roles in maintenance and self renewal of stem cells. We also found that our DHODH inhibitors induced CD11b and CD86, and increased the ratio of macrophage-like cells in primary patient-derived AML cells and these effects were rescued by uridine supplementation (Fig). Inhibitions of colony formations of primary AML cells were also shown after 14 days of FF1215T treatment. In exploring the value of DHODH inhibitors in the clinic, we identified that our DHODH inhibitors worked to overcome the resistance of standard therapy Ara-C. Our DHODH inhibitors were effective against Ara-C-resistant models of HL60 cells as well as HL60 parental cells. Notably, our DHODH inhibitors synergistically inhibited growths of Ara-C-resistant THP1 cells and enhanced CD11b upregulation of THP1 cells when combined with Ara-C by activating conversion of Ara-C to its active form Ara-CTP. Next, we optimized the hit compounds and identified an orally available DHODH inhibitor FF14984T that achieved high and prolonged plasma concentrations in vivo. Oral administration of 10 and 30 mg/kg FF14984T once daily for 10 days exhibited significant anti-tumor effects in mice xenografted with HL60 cells. These treatments showed strong reduction of CTP in tumor and induction of DHO in tumor and plasma. When 30 mg/kg FF14984T was orally administrated to orthotropic MOLM13-xenografted mice once daily for 12 days, hCD45+ cells proportions in bone marrow were decreased whereas hCD11bhigh/hCD45+ ratio increased, indicating that FF14984T induced AML differentiation in vivo. Finally, oral administration of 30 mg/kg FF14984T once daily significantly prolonged survival of mice in U937 orthotropic models. Taken together, we developed a novel potent DHODH inhibitor FF14984T that induced cellular differentiation and anti-leukemic effects on cell lines and primary AML cells. FF14984T is possibly a promising therapeutic option for Ara-C-resistant AML patients that can also benefit from the combination therapy of FF14984T and Ara-C. Identifying the precise mechanism of AML differentiation by DHODH inhibitor and its effects on CIC are currently ongoing. Disclosures Kitazawa: FUJIFILM Corporation: Employment. Ishii:FUJIFILM Corporation: Employment. Makita-Suzuki:FUJIFILM Corporation: Employment. Saito:FUJIFILM Corporation: Employment. Takayanagi:FUJIFILM Corporation: Employment. Sugihara:FUJIFILM Corporation: Employment. Matsuda:FUJIFILM Corporation: Employment. Yamakawa:FUJIFILM Corporation: Employment. Tsutsui:FUJIFILM Corporation: Employment. Tanaka:FUJIFILM Corporation: Employment. Hatta:FUJIFILM Corporation: Research Funding. Natsume:FUJIFILM Corporation: Research Funding. Kondo:FUJIFILM Corporation: Research Funding. Hagiwara:FUJIFILM Coporation: Employment. Kiyoi:FUJIFILM Corporation: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; Perseus Proteomics Inc.: Research Funding.

scholarly journals Combination of BCL-2 Inhibition and Triptolide Causes Synthetic Lethality in Acute Myeloid Leukemia through Preventing Reciprocal Resistance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2083-2083
Author(s):  
Bing Xu ◽  
Yuanfei Shi ◽  
Long Liu ◽  
Bing Z Carter
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Synthetic Lethality ◽  
Leukemic Cells ◽  
Apoptotic Pathway ◽  
P53 Status ◽  
Acute Myeloid

BCL-2 inhibition exerts effective pro-apoptotic activities in acute myeloid leukemia (AML) but clinical efficacy as a monotherapy was limited in part due to the treatment-induced MCL-1 increase. Triptolide (TPL) exhibits anti-tumor activities in part by upregulating pro-apoptotic BCL-2 proteins and decreasing MCL-1 expression in various malignant cells. We hypothesized that combined BCL-2 inhibition and TPL exert synergistic anti-leukemia activities and prevent the resistance to BCL-2 inhibition in AML. We here report that TPL combined with BCL-2 inhibitor ABT-199 synergistically induced apoptosis in leukemic cells regardless of p53 status through activating the intrinsic mitochondrial apoptotic pathway in vitro. Although ABT-199 or TPL alone inhibited AML growth in vivo, the combination therapy demonstrated a significantly stronger anti-leukemic effect. Mechanistically, TPL significantly upregulated BH3 only proteins including PUMA, NOXA, BID and BIM and decreased MCL-1 but upregulated BCL-2 expression in both p53 wild type and p53 mutant AML cell lines, while the combination decreased both BCL-2 and MCL-1 and further increased BH3 only BCL-2 proteins. MCL-1 and BCL-2 increases associated with respective ABT-199 and TPL treatment and resistance were also observed in vivo. Significantly downregulating MCL-1 and elevating BH3 only proteins by TPL could not only potentially block MCL-1-mediated resistance but also enhance anti-leukemic efficacy of ABT-199. Conversely, BCL-2 inhibition counteracted the potential resistance of TPL mediated by upregulation of BCL-2. The combination further amplified the effect, which likely contributed to the synthetic lethality. This mutual blockade of potential resistance provides a rational basis for the promising clinical application of TPL and BCL-2 inhibition in AML independent of p53 status. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding.

scholarly journals Fc Receptor-Dependent Trogocytosis of CD39 Impacts Engraftment and Invasiveness of Acute Myeloid Leukemia Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3298-3298
Author(s):  
Lili Feng ◽  
Haohai Zhang ◽  
Paola de Andrade Mello ◽  
Dina Stroopinsky ◽  
Wenda Gao ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Bioluminescence Imaging ◽  
Lentiviral Vector ◽  
Advisory Committees ◽  
Acute Myeloid

Abstract Corresponding author: Dr. Simon. C. Robson ([email protected]). Introduction: CD39/ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1) is the prototypic member of the GDA1-CD39 superfamily of ectonucleotidases and modulates purinergic signaling pathways. CD39 expression has been noted in human acute myeloid leukemia (AML) and likely contributes to chemoresistance [1]. Our study reported here elucidates the impact of Cd39 on engraftment and invasiveness of AML TIB-49 cells using an immunocompetent murine experimental model. Methods: Wild-type (WT) mice and Cd39 -/- mice on C57BL/6 background were bred at Beth Israel Deaconess Medical Center. The syngeneic murine AML cell line TIB-49 (Cd39 negative in vitro) was purchased from American Type Culture Collection. For bioluminescence imaging experiments, TIB-49 cells were transduced with luciferase/mCherry using a lentiviral vector. For AML model, mice were administered with 1×10 6 TIB-49-luciferase cells intravenously via tail vein injection. For chloroma model, mice were subcutaneously inoculated with 1×10 6 TIB-49 cells in the right flank. Bioluminescence imaging of TIB-49-luciferase bearing mice was conducted with the IVIS TM 50 Imaging System. Blood, spleen and bone marrow (BM) were also collected from TIB-49 bearing AML mice for FACS (fluorescence activated cell sorting) analysis. To explore Cd39 in TIB engraftment and invasiveness, TIB-49 cells were further transduced with a lentiviral vector overexpressing mCd39 with TdTomato. WT mice were intravenously inoculated with 1×10 6 of either TIB-49-TdTomato cells or TIB-49-mCd39-TdTomato cells, and the above read-outs were determined. To investigate the potential of CD39 as a therapeutic target, we engineered anti-mouse Cd39 antibodies (αCd39 mAb) with isotype selection and removal of fucose to further promote Fc receptor (FcR) interactions. Results: Bioluminescence imaging results indicated that TIB-49 engraftment was decreased in global Cd39 -/- mice with decreased disease burdens noted relative to WT (Figure 1A). FACS analysis of blood, spleen and BM-derived cells from TIB-49 bearing AML-model mice (day 31) confirmed higher engraftment of TIB-49 cells (TdTomato+) at all sites in WT compared to Cd39 -/- mice (Figure 1B). TIB-49 cells did not express Cd39 in vitro, but TIB-49 cells harvested from spleen and BM of WT but not Cd39 -/- mice displayed high levels of Cd39. This indicated TIB-49 cells acquired Cd39 from host cells, in a process of antibody-independent trogocytosis (Figure 1C), as RT-PCR did not detect Cd39 mRNA expression in TIB-49 cells in vivo. Additionally, circulating TIB-49 cells from the blood of WT mice were Cd39 negative (Figure 1C), suggesting a role for the tumor microenvironment in mediating trogocytosis. TIB-49 cells expressing host Cd39 in WT mice spleen and BM lost Cd39 after being exposed to αCd39 mAb treatment. Cd39 translocated from TIB-49 cells to effector cells, at least in part, dependent on FcR mediated trogocytosis (Figure 1D). When Cd39 was overexpressed on TIB-49 cells (TIB-49-mCd39-TdTomato), the engraftment was boosted in WT mice in vivo when compared to TIB-49-TdTomato cells (day 19, Figure 1E) with higher levels of Cd39 expression than that observed on TIB-49-TdTomato cells in spleen and BM (day 26) (Figure 1F). Moreover, TIB-49-mCd39-TdTomato bearing mice displayed shorter survival times, when compared with TIB-49-TdTomato bearing AML mice (Figure 1G). The αCd39 mAb monotherapy had no effect on TIB-49 chloroma model growth. However, pretreatment with αCd39 mAb effectively boosted daunorubicin chemotherapeutic effects in vivo (Figure 1H and 1I). Conclusions: Our study suggests bidirectional trogocytosis between TIB-49 AML and host immune cells, which is further modulated by FcR interaction. Re-distribution of Cd39 from host to TIB-49 cells or induced high level expression contributes to engraftment and invasiveness, resulting in decreased survival. Targeting CD39 is a potential therapeutic approach, operational not only by boosting chemosensitivity but furthering anti-leukemic effects in experimental models. Disclosures: No relevant conflicts of interest to declare. References: [1] Nesrine Aroua, Emeline Boet, Margherita Ghisi, et al. Extracellular ATP and CD39 Activate cAMP-Mediated Mitochondrial Stress Response to Promote Cytarabine Resistance in Acute Myeloid Leukemia. Cancer Discov. 2020. Figure 1 Figure 1. Disclosures Stroopinsky: The Blackstone Group: Consultancy. Avigan: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Aviv MedTech Ltd: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexcel: Consultancy; Takeda: Consultancy; Sanofi: Consultancy.

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 and Purification of Leukemic Stem-Like Cells in Acute Myeloid Leukemia Cell Line

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4734-4734
Author(s):  
Miaorong She ◽  
Xingqing Niu ◽  
Xilin Chen ◽  
Guo Kunyuan ◽  
Maohua Zhou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Critical Role ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Leukemic Stem Cells ◽  
Self Renewal ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is initiated and maintained by a rare population of (leukemic stem cells) LSCs. LSCs play the central role in the relapse and refractory of AML and highlight the critical need for the new therapeutic strategies to directly target the LSC population for ultimately curing leukemia which is it is important to identify and study LSCs. However, relatively little is known about the unique molecular mechanisms of survival and self-renewal of LSCs because of very small number of LSCs in bone marrow. In this study, we investigated whether established leukemia cell lines contain LSCs. We showed that leukemia cell line contain leukemic stem-like cells which have been phenotypically restricted within the CD34+CD38− fraction. We demonstrated that CD34+CD38− cells could generate CD34+CD38+ cells in culture medium and had proliferation function. Moreover, CD34+CD38− cells had self-renewal potential both in vitro soft agar colonies formation assay and in vivo NOD/SCID mouse xenotransplant model serial transplantation. Furthermore, CD34+CD38− cells isolated from leukemia cell line were found resistant to conventional chemotherapy and NK cells-mediated cytotoxicity and these were related to up-regulation of ABCG2 and MRP-1 and antiapoptotic proteins of Bcl2. Down-regulation of NKG2D ligand also played a critical role in NK cytotoxicity resistance. Taken together, our studies provide a novel cell model for leukemic stem cells research. Our data also shed light on mechanism of double resistant to resistant to chemotherapy and NK cell immunotherapy, which was helpful for developing novel effective strategies for LSCs.

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.

The Salicylamide Derivative, Niclosamide, Inhibits CREB Function in Acute Myeloid Leukemia Cells In Vitro and In Vivo

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1647-1647
Author(s):  
Hee-Don Chae ◽  
Nick Cox ◽  
Xiaohua Zhang ◽  
Jae Wook Lee ◽  
David Morgens ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Binding Protein ◽  
Luciferase Reporter ◽  
Therapeutic Window ◽  
Hl60 Cells ◽  
Acute Myeloid

Abstract CREB (cAMP Response Element Binding protein) is a transcription factor that is overexpressed in primary Acute Myeloid Leukemia (AML) cells and is associated with a decreased event-free survival and increased risk of relapse. We previously demonstrated that CREB overexpression increases leukemia cell growth and survival. Transgenic mice overexpressing CREB in myeloid cells develop a myeloproliferative neoplasm and myelodysplasia. CREB knockdown inhibits AML cell proliferation but not normal hematopoietic stem cell activity in vivo. To demonstrate the feasibility of targeting CREB for treatment for AML, we recently described a small molecule inhibitor of CREB, N-(4-cyanophenyl)-3-hydroxy-2-naphthamide (XX-650-23), which is a compound originally based on naphthol AS-E phosphate first identified as an inhibitor of CREB interaction with its coactivator, CBP (CREB Binding Protein). To identify a lead candidate with improved potency and physicochemical properties, we performed structure-activity relationships (SAR) studies for a series of salicylamides derived from naphthol AS-E phosphate. Development of this series led to the identification of the anthelmintic niclosamide as a potent agent that suppresses cell viability of five AML cell lines (IC50= 280 nM (HL60), 340 nM (KG1), 420 nM (MOLM13), 560 nM (MV411), 360 nM (U937), without a significant decrease in colony forming activity of normal bone marrow cells up to 10 μM (18- to 36-fold therapeutic window). Niclosamide binds CBP with a KD of 22.3 nM by Surface Plasmon Resonance (Biacore) analysis. To determine whether niclosamide specifically inhibits CREB-mediated gene expression in cells, luciferase reporter gene activity under the control of a promoter containing two CRE elements was measured after treatment of niclosamide for 6 hours. Niclosamide inhibited CREB-driven luciferase activity in HL60 cells with an IC50 of 1.09 μM. We also examined the efficacy of niclosamide in an AML patient-derived xenograft (PDX) mouse model. Niclosamide significantly inhibited the progression of AML in mice injected with primary AML cells. The percentage of circulating AML cells in the peripheral blood (%), vehicle vs. niclosamide treatment 5 weeks after engraftment were 28.75 ± 3.507 vs. 0.5363 ± 0.2744 (n=8, p< 0.001, mean ± SEM). In Kaplan Meier analysis, the median survival of PDX mice was 41 days vs. 51.5 days (p = 0.0015, log-rank test). To characterize the cellular effects of niclosamide, we analyzed the DNA profile, apoptosis, DNA-damage, cell cycle regulators, and other signaling molecules using flow cytometry. Niclosamide treatment increased DNA-damaged and apoptosis populations during the G1/S cell cycle phase, which also showed reduced phosphorylated CREB levels. To examine the functional requirement of CREB, we determined the effects of CREB knockdown in HL60 cells treated with niclosamide. CREB knockdown protected HL60 cells from niclosamide treatment-mediated cytotoxic effects (IC50=670 nM for CREB knockdown vs. 200 nM for vector control cells). Furthermore, combination treatment of niclosamide with XX-650-23 in HL60 cells showed an additive antiproliferative effect, suggesting that niclosamide and XX-650-23 regulate the same targets or pathways to inhibit viability of AML cells. To further identify genes that confer resistance or sensitivity to niclosamide, we performed a functional shRNA screen using subsets of whole genomic shRNA libraries (apoptosis, motility, other cancer; 35154 elements). We identified 53 genes, including tumor necrosis factor receptor superfamily members, which when knocked downed conferred resistance to niclosamide at a 10% false discovery rate. Taken together, our results demonstrate that niclosamide is a potential drug to treat AML by inducing DNA-damage, apoptosis and cell cycle arrest through the inhibition of CREB-dependent pathways in AML cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals P09.12 Bifunctional SIRPα-CD123 fusion antibody for the elimination of acute myeloid leukemia stem cells

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A58.1-A58
Author(s):  
S Tahk ◽  
SM Schmitt ◽  
B Vick ◽  
C Augsberger ◽  
L Pascual Ponce ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Immune Escape ◽  
Nk Cell ◽  
Regulatory Protein ◽  
Target Antigen ◽  
Hematopoietic Stem ◽  
Acute Myeloid

BackgroundDespite advances in the development of novel strategies against acute myeloid leukemia (AML), treatment options are limited and most patients relapse. Relapse occurs due to the persistence of chemotherapy-resistant leukemic stem cells (LSCs), which re-initiate the outgrowth of the disease, highlighting the need of targeting LSCs to improve patient survival. LSCs are characterized by the expression of the interleukin-3 receptor α, also known as CD123. CD123 is expressed on AML blasts and LSCs, and shows a moderate expression on normal hematopoietic stem cells, claiming CD123 as a suitable target antigen. CD47 is a ubiquitously expressed immune checkpoint upregulated on LSCs where it acts as an immune escape mechanism. CD47 transmits a ‘don’t eat me’ signal upon its interaction with the signal regulatory protein alpha (SIRPα) receptor on macrophages, thus inhibiting phagocytosis. In order to efficiently eliminate LSCs, we have designed a bifunctional antibody that specifically targets CD123 and simultaneously blocks CD47. Importantly, our strategy restricts the benefits of the CD47 blockade to CD123+ AML cells. Thus, we hypothesize a lower risk for on-target off-leukemia toxicity.Materials and MethodsThe bifunctional SIRPα-CD123 antibody was generated by fusing an extracellular domain of the SIRPα receptor, which functions as the CD47 blocking domain, to the CD123 antibody. The biological activity of the SIRPα-CD123 antibody was examined using AML-derived MOLM-13 cells, primary AML patient material and patient-derived xenografted (PDX) AML cells with NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice.ResultsThe SIRPα fusion improved the binding of the bifunctional SIRPα-CD123 antibody to AML cells compared to a conventional CD123 antibody. The SIRPα-CD123 antibody enhanced the elimination of the AML-derived MOLM-13 cells by antibody-dependent cellular cytotoxicity via NK cells. Additionally, the cytotoxicity was confirmed using primary patient-derived AML cells. Furthermore, an improved cytotoxicity towards leukemia initiating AML PDX cells was observed with the SIRPα-CD123 antibody using luciferase bioluminescence in vivo imaging. With regards to the inhibition of CD47 signaling, we were able to show a blockade of CD47 on CD123+CD47+ cells by the SIRPα-CD123 antibody. Correspondingly, a significant increase in phagocytosis of primary patient-derived AML cells mediated by monocyte-derived macrophages was observed in both allogenic and autologous setting. We were also able to show a preferential binding to MOLM-13 in the presence of a 20-fold excess of red blood cells indicating a potential low on-target off-leukemia toxicity.ConclusionsThe bifunctional SIRPα-CD123 fusion antibodies target the CD123+CD47+ cells and stimulate their phagocytosis by blocking the inhibitory CD47 signal. The dual mode of action of the SIRPα-CD123 has the potential to deplete the AML LSCs through NK cell cytotoxicity and macrophage-mediated phagocytosis while restricting the CD47 related on-target off-leukemia toxicity.SupportH2020-EU grant agreement no 641549Disclosure InformationS. Tahk: None. S.M. Schmitt: None. B. Vick: None. C. Augsberger: None. L. Pascual Ponce: None. I. Jeremias: None. G. Wittmann: None. M. Subklewe: None. N.C. Fenn: None. K. Hopfner: None.

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.

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