Nadine Mayotte

NUP98–HOXA9 expression in hemopoietic stem cells induces chronic and acute myeloid leukemias in mice

Here we describe hemopoietic chimeras serving as a mouse model for NUP98–HOXA9‐induced leukemia, which reproduced several of the phenotypes observed in human disease. Mice transplanted with bone marrow cells expressing NUP98–HOXA9 through retroviral transduction acquire a myeloproliferative disease (MPD) and eventually succumb to acute myeloid leukemia (AML). The NUP98 portion of the fusion protein was shown to be responsible for transforming a clinically silent pre‐leukemic phase observed for Hoxa9 into a chronic, stem cell‐derived MPD. The co‐expression of NUP98–HOXA9 and Meis1 accelerated the transformation of MPD to AML, identifying a genetic interaction previously observed for Hoxa9 and Meis1. Our findings demonstrate the presence of overlapping yet distinct molecular mechanisms for MPD versus AML, illustrating the complexity of leukemic transformation.

Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal

Human adult stem cell expansion Transfused blood saves lives. Despite the widespread use of this critical resource, it is difficult to increase blood cell numbers outside of the body. By screening thousands of small compounds, Fares et al. identify a molecule that expands human stem cell numbers in cord blood. The researchers generate many variations of that molecule and show that one such compound provides even greater human blood cell expansion. If researchers can provide increased numbers of stem cells and progenitor cells, cord blood should find even greater use in the clinic. Science, this issue p. 1509 The self-renewal of human hematopoietic stem cells in vitrois enhanced by the pyrimidoindole derivative UM171. The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds, UM171 being the prototype, is independent of suppression of the aryl hydrocarbon receptor, which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy.

An RNAi Screen Identifies Msi2 and Prox1 as Having Opposite Roles in the Regulation of Hematopoietic Stem Cell Activity

In this study, we describe an in vivo RNA interference functional genetics approach to evaluate the role of 20 different conserved polarity factors and fate determinants in mouse hematopoietic stem cell (HSC) activity. In total, this screen revealed three enhancers and one suppressor of HSC-derived reconstitution. Pard6a, Prkcz, and Msi2 shRNA-mediated depletion significantly impaired HSC repopulation. An in vitro promotion of differentiation was observed after the silencing of these genes, consistent with their function in regulating HSC self-renewal. Conversely, Prox1 knockdown led to in vivo accumulation of primitive and differentiated cells. HSC activity was also enhanced in vitro when Prox1 levels were experimentally reduced, identifying it as a potential antagonist of self-renewal. HSC engineered to overexpress Msi2 or Prox1 showed the reverse phenotype to those transduced with corresponding shRNA vectors. Gene expression profiling studies identified a number of known HSC and cell cycle regulators as potential downstream targets to Msi2 and Prox1.

The Competitive Nature of HOXB4-Transduced HSC Is Limited by PBX1: The Generation of Ultra-Competitive Stem Cells Retaining Full Differentiation Potential

We previously showed that HOXB4 is a potent stimulator of hematopoietic stem cell (HSC) proliferation in vivo and ex vivo. As a result, HOXB4 overexpressing HSCs are 20- to 50-times more competitive than untransduced cells when transplanted into mice. By knocking down the expression of PBX1 (PBX1(K.D.)) in HOXB4 overexpressing cells, we now present the possibility of generating HSCs that are >20-times more competitive than those that overexpress HOXB4. The differentiation activity of these cells appears intact, since they competitively contributed to the reconstitution of normal myeloid and lymphoid compartments in vivo. We also show that the in vivo expansion of HOXB4-PBX1(K.D.)-expressing HSCs regenerated normal stem cell pools and did not lead to HSC levels above those detected in unmanipulated mice. The vigorous competitive nature of these cells in vivo compared to HOXB4-transduced HSCs suggests the existence of a distinct, non-cell autonomous mechanism that limits the expansion of HOXB4-transduced hemopoietic stem cells in mice.

RNA-seq analysis of 2 closely related leukemia clones that differ in their self-renewal capacity

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.

Asymmetric segregation and self-renewal of hematopoietic stem and progenitor cells with endocytic Ap2a2

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.

RNAi screen identifies Jarid1b as a major regulator of mouse HSC activity

Histone methylation is a dynamic and reversible process proposed to directly impact on stem cell fate. The Jumonji (JmjC) domain-containing family of demethylases comprises 27 members that target mono-, di-, and trimethylated lysine residues of histone (or nonhistone) proteins. To evaluate their role in regulation of hematopoietic stem cell (HSC) behavior, we performed an in vivo RNAi-based functional screen and demonstrated that Jarid1b and Jhdm1f play opposing roles in regulation of HSC activity. Decrease in Jarid1b levels correlated with an in vitro expansion of HSCs with preserved long-term in vivo lymphomyeloid differentiation potential. Through RNA sequencing analysis, Jarid1b knockdown was associated with increased expression levels of several HSC regulators (Hoxa7, Hoxa9, Hoxa10, Hes1, Gata2) and reduced levels of differentiation-associated genes. shRNA against Jhdmlf, in contrast, impaired hematopoietic reconstitution of bone marrow cells. Together, our studies identified Jarid1b as a negative regulator of HSC activity and Jhdmlf as a positive regulator of HSC activity.

A role for GPx3 in activity of normal and leukemia stem cells

High levels of glutathione peroxidase 3 (GPx3) expression correlate with adverse prognosis in acute myeloid leukemia, and enhance activity of long-term repopulating hematopoietic stem cells in mice.

Entinostat Prevents Leukemia Maintenance in a Collaborating Oncogene‐Dependent Model of Cytogenetically Normal Acute Myeloid Leukemia

The incidence of refractory acute myeloid leukemia (AML) is on the increase due in part to an aging population that fails to respond to traditional therapies. High throughput genomic analysis promises better diagnosis, prognosis, and therapeutic intervention based on improved patient stratification. Relevant preclinical models are urgently required to advance drug development in this area. The collaborating oncogenes, HOXA9 and MEIS1, are frequently co‐overexpressed in cytogenetically normal AML (CN‐AML), and a conditional transplantation mouse model was developed that demonstrated oncogene dependency and expression levels comparable to CN‐AML patients. Integration of gene signatures obtained from the mouse model and a cohort of CN‐AML patients using statistically significant connectivity map analysis identified Entinostat as a drug with the potential to alter the leukemic condition toward the normal state. Ex vivo treatment of leukemic cells, but not age‐matched normal bone marrow controls, with Entinostat validated the gene signature and resulted in reduced viability in liquid culture, impaired colony formation, and loss of the leukemia initiating cell. Furthermore, in vivo treatment with Entinostat resulted in prolonged survival of leukemic mice. This study demonstrates that the HDAC inhibitor Entinostat inhibits disease maintenance and prolongs survival in a clinically relevant murine model of cytogenetically normal AML. STEM Cells2013;31:1434–1445

EPCR expression marks UM171-expanded CD34+ cord blood stem cells

A small subset of human cord blood CD34+ cells express endothelial protein C receptor (EPCR/CD201/PROCR) when exposed to the hematopoietic stem cell (HSC) self-renewal agonist UM171. In this article, we show that EPCR-positive UM171-treated cells, as opposed to EPCR-negative cells, exhibit robust multilineage repopulation and serial reconstitution ability in immunocompromised mice. In contrast to other stem cell markers, such as CD38, EPCR expression is maintained when cells are introduced in culture, irrespective of UM171 treatment. Although engineered overexpression of EPCR fails to reproduce the effects of UM171 on HSC activity, its expression is required for the repopulating activity of human HSCs. Altogether, our results indicate that EPCR is a reliable and cell culture-compatible marker of UM171-expanded human cord blood HSCs.