Search

Abstract: The leukemic stem cell (LSC) score LSC-17 based on a stemness-related gene expression signature is an indicator of poor disease outcome in acute myeloid leukemia (AML). However, it is not known whether "niche anchoring" of LSC affects disease evolution. To address this issue, we conditionally inactivated the adhesion molecule JAM-C (Junctional Adhesion Molecule-C) expressed by hematopoietic stem cells (HSCs) and LSCs in an inducible mixed-lineage leukemia (iMLL)-AF9-driven AML mouse model. Deletion of Jam3 (encoding JAM-C) before induction of the leukemia-initiating iMLL-AF9 fusion resulted in a shift from long-term to short-term HSC expansion, without affecting disease initiation and progression. In vitro experiments showed that JAM-C controlled leukemic cell nesting irrespective of the bone marrow stromal cells used. RNA sequencing performed on leukemic HSCs isolated from diseased mice revealed that genes upregulated in Jam3-deficient animals belonged to activation protein-1 (AP-1) and tumor necrosis factor α (TNF-α)/NF-κB pathways. Human orthologs of dysregulated genes allowed to identify a score that was distinct from, and complementary to, the LSC-17 score. Substratification of patients with AML using LSC-17 and AP-1/TNF-α genes signature defined 4 groups with median survival ranging from <1 year to a median of "not reached" after 8 years. Finally, coculture experiments showed that AP-1 activation in leukemic cells was dependent on the nature of stromal cells. Altogether, our results identify the AP-1/TNF-α gene signature as a proxy of LSC anchoring in bone marrow niches, which improves the prognostic value of the LSC-17 score. This trial was registered at www.ClinicalTrials.gov as #NCT02320656., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

FLT3, DNMT3A , and NPM1 are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon cooccurrence. Here we show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia-specific GPR56 high CD34 low immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor ( HLF ). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from DNMT3A/NPM1 double-mutated subclones. Moreover, in DNMT3A R882-mutated patients, CpG hypomethylation at the HLF transcription start site correlated with high HLF mRNA expression, which was itself associated with poor survival. Loss of HLF via CRISPR/Cas9 significantly reduced the CD34 + GPR56 + LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. HLF knockout cells were more actively cycling when freshly harvested from mice, but rapidly exhausted when reintroduced in culture. RNA sequencing of primary human triple-mutated AML cells after shRNA-mediated HLF knockdown revealed the NOTCH target Hairy and Enhancer of Split 1 ( HES1 ) and the cyclin-dependent kinase inhibitor CDKN1C/p57 as novel targets of HLF, potentially mediating these effects. Overall, our data establish HLF as a novel LSC regulator in this genetically defined high-risk AML subgroup., (© 2019 by The American Society of Hematology.)

Prognostic markers that capture leukemia stem cell (LSC) activity can be useful for the risk stratification of acute myeloid leukemia (AML) patients. In a recent issue of Nature, Ng et al. (2016) develop a prognostic score based on a 17-gene expression signature of LSCs to predict outcome in AML patients., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non-cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy., Competing Interests: The authors have declared that no conflict of interest exists.

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy, which is initiated and driven by a rare fraction of leukemia stem cells (LSCs). Despite the difficulties of identifying a common LSC phenotype, there is increasing evidence that high expression of stem cell gene signatures is associated with poor clinical outcome. Identification of functionally distinct subpopulations in this disease is therefore crucial to dissecting the molecular machinery underlying LSC self-renewal. Here, we combined next-generation sequencing technology with in vivo assessment of LSC frequencies and identified the adhesion G protein-coupled receptor 56 (GPR56) as a novel and stable marker for human LSCs for the majority of AML samples. High GPR56 expression was significantly associated with high-risk genetic subgroups and poor outcome. Analysis of GPR56 in combination with CD34 expression revealed engraftment potential of GPR56(+)cells in both the CD34(-)and CD34(+)fractions, thus defining a novel LSC compartment independent of the CD34(+)CD38(-)LSC phenotype., (© 2016 by The American Society of Hematology.)

Purpose of Review: Hematopoietic stem and progenitor cell (HSPC) transplantation is frequently used in the treatment of hematological diseases. The outcome of the procedure is strongly influenced by the quantity of injected cells, especially if low cell numbers are infused as frequently encountered with cord blood transplants. Ex-vivo expansion of cord blood HSPCs would increase cell numbers, thus accelerating engraftment and reducing infectious complications and transplant-related mortality. In addition, expansion would maximize accessibility to better HLA-matched units, further improving patients' outcome. Similarly, in-vitro maintenance or expansion of leukemic stem cells (LSCs) would enable research into the much awaited targeted therapies that spare normal hematopoietic stem cells (HSCs). Here, we review recent findings on small molecules (excluding biologicals) regulating the activity of normal and leukemic stem cells and provide insights into basic science and clinical implications., Recent Findings: High-throughput screening of small molecules active on primary hematopoietic cells has led to the identification of two potent series of chemical compounds, best exemplified by StemRegenin1 and UM171, that both expand HSPCs. Current data suggest that the aryl hydrocarbon receptor antagonist StemRegenin1 is most active on primitive normal hematopoietic progenitors and LSCs and that UM171 expands long-term normal HSCs., Summary: Small molecules are clinically useful and powerful tools for expanding HSPCs. They are also of potential value for dissecting the still elusive regulatory networks that govern self-renewal of human HSCs.

Leukemic stem cells (LSCs) are considered a major cause of relapse in acute myeloid leukemia (AML). Defining pathways that control LSC self-renewal is crucial for a better understanding of underlying mechanisms and for the development of targeted therapies. However, currently available culture conditions do not prevent spontaneous differentiation of LSCs, which greatly limits the feasibility of cell-based assays. To overcome these constraints we conducted a high-throughput chemical screen and identified small molecules that inhibit differentiation and support LSC activity in vitro. Similar to reports with cord blood stem cells, several of these compounds suppressed the aryl-hydrocarbon receptor (AhR) pathway, which we show to be inactive in vivo and rapidly activated ex vivo in AML cells. We also identified a compound, UM729, that collaborates with AhR suppressors in preventing AML cell differentiation. Together, these findings provide newly defined culture conditions for improved ex vivo culture of primary human AML cells.

We recently generated 2 phenotypically similar Hoxa9+Meis1 overexpressing acute myeloid leukemias that differ by their in vivo biologic behavior. The first leukemia, named FLA2, shows a high frequency of leukemia stem cells (LSCs; 1 in 1.4 cells), whereas the second, FLB1, is more typical with a frequency of LSCs in the range of 1 per several hundred cells. To gain insights into possible mechanisms that determine LSC self-renewal, we profiled and compared the abundance of nuclear and cytoplasmic proteins and phosphoproteins from these leukemias using quantitative proteomics. These analyses revealed differences in proteins associated with stem cell fate, including a hyperactive p38 MAP kinase in FLB1 and a differentially localized Polycomb group protein Ezh2, which is mostly nuclear in FLA2 and predominantly cytoplasmic in FLB1. Together, these newly documented proteomes and phosphoproteomes represent a unique resource with more than 440 differentially expressed proteins and 11 543 unique phosphopeptides, of which 80% are novel and 7% preferentially phosphorylated in the stem cell-enriched leukemia., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.

The determinants of normal and leukemic stem cell self-renewal remain poorly characterized. We report that expression of the reactive oxygen species (ROS) scavenger glutathione peroxidase 3 (GPx3) positively correlates with the frequency of leukemia stem cells (LSCs) in Hoxa9+Meis1-induced leukemias. Compared with a leukemia with a low frequency of LSCs, a leukemia with a high frequency of LSCs showed hypomethylation of the Gpx3 promoter region, and expressed high levels of Gpx3 and low levels of ROS. LSCs and normal hematopoietic stem cells (HSCs) engineered to express Gpx3 short hairpin RNA (shRNA) were much less competitive in vivo than control cells. However, progenitor cell proliferation and differentiation was not affected by Gpx3 shRNA. Consistent with this, HSCs overexpressing Gpx3 were significantly more competitive than control cells in long-term repopulation experiments, and overexpression of the self-renewal genes Prdm16 or Hoxb4 boosted Gpx3 expression. In human primary acute myeloid leukemia samples, GPX3 expression level directly correlated with adverse prognostic outcome, revealing a potential novel target for the eradication of LSCs.

The stem cell-intrinsic model of self-renewal via asymmetric cell division (ACD) posits that fate determinants be partitioned unequally between daughter cells to either activate or suppress the stemness state. ACD is a purported mechanism by which hematopoietic stem cells (HSCs) self-renew, but definitive evidence for this cellular process remains open to conjecture. To address this issue, we chose 73 candidate genes that function within the cell polarity network to identify potential determinants that may concomitantly alter HSC fate while also exhibiting asymmetric segregation at cell division. Initial gene-expression profiles of polarity candidates showed high and differential expression in both HSCs and leukemia stem cells. Altered HSC fate was assessed by our established in vitro to in vivo screen on a subcohort of candidate polarity genes, which revealed 6 novel positive regulators of HSC function: Ap2a2, Gpsm2, Tmod1, Kif3a, Racgap1, and Ccnb1. Interestingly, live-cell videomicroscopy of the endocytic protein AP2A2 shows instances of asymmetric segregation during HSC/progenitor cell cytokinesis. These results contribute further evidence that ACD is functional in HSC self-renewal, suggest a role for Ap2a2 in HSC activity, and provide a unique opportunity to prospectively analyze progeny from HSC asymmetric divisions.

The molecular mechanisms regulating self-renewal of leukemia stem cells remain poorly understood. Here we report the generation of 2 closely related leukemias created through the retroviral overexpression of Meis1 and Hoxa9. Despite their apparent common origin, these clonal leukemias exhibit enormous differences in stem cell frequency (from 1 in 1.4, FLA2; to 1 in 347, FLB1), suggesting that one of these leukemias undergoes nearly unlimited self-renewal divisions. Using next-generation RNA-sequencing, we characterized the transcriptomes of these phenotypically similar, but biologically distinct, leukemias, identifying hundreds of differentially expressed genes and a large number of structural differences (eg, alternative splicing and promoter usage). Focusing on ligand-receptor pairs, we observed high expression levels of Sdf1-Cxcr4; Jagged2-Notch2/1; Osm-Gp130; Scf-cKit; and Bmp15-Tgfb1/2. Interestingly, the integrin beta 2-like gene (Itgb2l) is both highly expressed and differentially expressed between our 2 leukemias (∼ 14-fold higher in FLA2 than FLB1). In addition, gene ontology analysis indicated G-protein-coupled receptor had a much higher proportion of differential expression (22%) compared with other classes (∼ 5%), suggesting a potential role regulating subtle changes in cellular behavior. These results provide the first comprehensive transcriptome analysis of a leukemia stem cell and document an unexpected level of transcriptome variation between phenotypically similar leukemic cells.

Polycomb Group (PcG) proteins are transcriptional repressors that epigenetically modify chromatin and participate in the establishment and maintenance of cell fates. These proteins play important roles in both stem cell self-renewal and in cancer development. Our understanding of their mechanism of action has greatly advanced over the past 10 years, but many unanswered questions remain. In this review, we present the currently available experimental data that connect PcG protein function with some of the key processes which govern somatic stem cell activity. We also highlight recent studies suggesting that a delicate balance in PcG gene dosage is crucial for proper stem cell homeostasis and prevention of cancer stem cell development., (Copyright 2010 Elsevier Inc. All rights reserved.)

An emerging concept in the field of cancer biology is that a rare population of 'tumour stem cells' exists among the heterogeneous group of cells that constitute a tumour. This concept, best described with human leukaemia, indicates that stem cell function (whether normal or neoplastic) might be defined by a common set of critical genes. Here we show that the Polycomb group gene Bmi-1 has a key role in regulating the proliferative activity of normal stem and progenitor cells. Most importantly, we provide evidence that the proliferative potential of leukaemic stem and progenitor cells lacking Bmi-1 is compromised because they eventually undergo proliferation arrest and show signs of differentiation and apoptosis, leading to transplant failure of the leukaemia. Complementation studies showed that Bmi-1 completely rescues these proliferative defects. These studies therefore indicate that Bmi-1 has an essential role in regulating the proliferative activity of both normal and leukaemic stem cells.