Eric Deneault

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.

GPR56 identifies primary human acute myeloid leukemia cells with high repopulating potential in vivo

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.

RNA-Seq reveals spliceosome and proteasome genes as most consistent transcripts in human cancer cells.

Accurate quantification of gene expression by qRT-PCR relies on normalization against a consistently expressed control gene. However, control genes in common use often vary greatly between samples, especially in cancer. The advent of Next Generation Sequencing technology offers the possibility to better select control genes with the least cell to cell variability in steady state transcript levels. Here we analyze the transcriptomes of 55 leukemia samples to identify the most consistent genes. This list is enriched for components of the proteasome (ex. PSMA1) and spliceosome (ex. SF3B2), and also includes the translation initiation factor EIF4H, and many heterogeneous nuclear ribonucleoprotein genes (ex. HNRNPL). We have validated the consistency of our new control genes in 1933 cancer and normal tissues using publically available RNA-seq data, and their usefulness in qRT-PCR analysis is clearly demonstrated.

Identification of non–cell-autonomous networks from engineered feeder cells that enhance murine hematopoietic stem cell activity

In a previous gain-of-function screen, we identified 18 nuclear factors that enhance mouse hematopoietic stem cell (HSC) activity in vitro. Of these factors, the majority was believed to augment HSC function intrinsically. In the current study, we investigated the mechanisms of action of the previously identified agonists of HSC activity and tested whether human HSCs are also responsive to these factors. Our results unexpectedly revealed that the majority of the identified factors confer a competitive advantage to mouse HSCs in a non-cell-autonomous manner. Five of these factors, namely FOS, SPI1, KLF10, TFEC, and PRDM16, show robust transcriptional cross-regulation and are often associated with osteoclastogenesis. These findings define at least one novel non-cell-autonomous network in engineered niches. Surprisingly, and in contrast to their important effect on mouse HSCs, all engineered niches failed to significantly enhance the activity of human HSCs. This last finding further supports a lack of conservation in determinants that control HSC self-renewal in mouse versus human cells.