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.

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.

scholarly journals In Vivo crispr Screening Identifies GLUT1 As a Key Metabolic Regulator of MLL-AF9-Driven Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3439-3439
Author(s):  
Maria Rodriguez Zabala ◽  
Ramprasad Ramakrishnan ◽  
Katrin Reinbach ◽  
Leal Oburoglu ◽  
Somadri Ghosh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Glucose Uptake ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Acute Myeloid

Abstract Disease relapse in patients with acute myeloid leukemia (AML) is associated with a failure of current treatments to eradicate leukemia stem cells (LSCs), a self-renewing population of cells responsible for disease progression and maintenance. Thus, novel therapeutic strategies designed to specifically target LSCs while sparing normal hematopoietic stem cells are needed. To identify dependencies in LSCs that may reveal new treatment opportunities, we performed an in vivo CRISPR/Cas9 dropout screen in the widely used MLL-AF9-driven AML murine model. The pooled lentiviral CRISPR library was designed to target 960 genes encoding cell surface proteins expressed on MLL-AF9 AML cells as these are accessible for therapeutic targeting. The facilitated glucose transporter member 1(GLUT1), a major mediator of cellular glucose uptake, emerged as the highest ranked dependency in the screen, with all 6 sgRNAs depleted more than 10-fold in vivo. Consistent with the results from the screen, validation experiments confirmed that sgRNA-mediated GLUT1 disruption in c-Kit +Cas9 +dsRed +MLL-AF9 cells led to a 5-fold reduction in the establishment of leukemia in both the bone marrow and spleen of recipient mice. In line with these in vivo observations, leukemia cells expressing GLUT1 sgRNAs were rapidly depleted over time in an ex vivo competition assay (p<0.0001). GLUT1 disruption also led to a marked increase in mean survival from 28 to 73 days in mice transplanted with sorted GLUT1 sgRNA-expressing leukemia cells relative to controls. Notably, while GLUT1 loss did not affect apoptosis or cell-cycle state, it led to a more than two-fold increase in the surface expression of the myeloid differentiation marker Gr-1 (p=0.0002). Interestingly, knockdown of GLUT1 lead to reduced mRNA expression levels of key downstream genes of MLL-driven leukemia Meis1 (p<0.0001) and Hoxa9 (p=0.0013) , both of which are commonly downregulated upon differentiation. These findings suggest that GLUT1 ablation arrests AML cell growth at least in part via accelerated differentiation and attenuated cell proliferation. Given GLUT1-mediated glucose transfer constitutes the first rate-limiting step for glucose metabolism, we assessed the metabolic profile of MLL-AF9 AML cells following loss of GLUT1. Bioenergetic profiling revealed that the rate of glycolysis was significantly decreased upon GLUT1 knockdown, as measured by a decrease in extracellular acidification rate (ECAR), glucose uptake, hexokinase activity and extracellular lactate production. To further assess the feasibility of GLUT1 inhibition as a therapy for AML patients, we treated murine cKit +MLL-AF9 leukemia cells with BAY-876, a potent and highly selective GLUT1 inhibitor. BAY-876 impaired tumor growth following 24hr (IC 50 60.3 nM) and 48hr (IC 50 68.8 nM) treatment ex vivo in a dose-dependent manner. Interestingly, the inhibitory effect on the counterpart healthy bone marrow c-Kit + cells was significantly weaker (24hr IC 50 347.7 nM; 48hr IC 50 258.4nM), indicating selective targeting of LSCs. To test the efficacy of BAY-876 as an anti-leukemic agent in vivo, sublethally irradiated mice were transplanted with c-Kit +MLL-AF9 AML cells and 3 days post-injection, were randomised into two groups (Veh n=4; BAY-876 n=6) and orally treated with either vehicle or 4mg/kg of BAY-876 daily. Following 10 days of treatment, mice were sacrificed and leukemia burden was assessed. Notably, substantially lower levels of leukemia cells in the bone marrow (p=0.0095), spleen (p=0.0095), and peripheral blood (p=0.036) were observed in the BAY-876 treatment group with no significant loss of body weight. Consistent with these findings, the average spleen weight was reduced by 66% upon BAY-876 treatment (p=0.0136). Collectively, we demonstrate that MLL-AF9-driven AML cells are dependent on GLUT1 for continued growth and survival. Targeting of GLUT1 downregulates glycolysis and induces cellular differentiation. We report that genetic or pharmacological inhibition of GLUT1 is sufficient to impair leukemic growth in vitro and in vivo, highlighting a potential therapeutic opportunity for disarming intrinsic metabolic dependencies of LSCs. Ongoing studies are aimed at translating these findings to the human disease and exploring combinatorial therapies that may act synergistically to overcome mechanisms of therapy resistance and metabolic plasticity. 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 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.

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