HOXB4 overexpression induces unique in vivo and in vitro expansion of hemopoietic stem cells (HSCs) without causing leukemia. Very little is known about the molecular basis underlying HOXB4-induced ...HSC self-renewal. We now report the in vitro proliferation and in vivo expansion capacity of primary bone marrow (BM) cells engineered to overexpress selected HOXB4 point mutants lacking either the capacity to directly bind DNA (HOXB4(A)), or to cooperate with members of the PBX family (HOXB4(W→G)) in DNA binding. The DNA binding–incompetent HOXB4 mutant failed to enhance the proliferation activity of transduced BM populations in vitro and HSC expansion in vivo. In contrast, the HOXB4(W→G) mutant conferred a pronounced in vitro proliferation advantage to the transduced BM populations, and dramatically enhanced their in vivo regenerative potential. We also demonstrate a correlation between HOXB4 protein levels and in vitro proliferative capacity of primary BM cells. Our observations thus suggest that the capacity of HOXB4 to induce HSC expansions is DNA-binding dependent and does not require direct HOX/PBX interaction, and sets the stage for identifying HOXB4-dependent targets involved in HSC expansion.
Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely ...characterized. Using a modified Cre-loxP-based system, we now report the creation and characterization of a collection of ∼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity.
The products of PBX homeobox genes, which were initially discovered in reciprocal translocations occurring in human leukemias, have been shown to cooperate in the in vitro DNA binding with HOX ...proteins. Despite the growing body of data implicating Hox genes in the development of various cancers, little is known about the role of HOX-PBX interactions in the regulation of proliferation and induction of transformation of mammalian cells. We build on the existing model of Hox-induced transformation of Rat-1 cells to show that both cellular transformation and proliferation induced by Hoxb4 and Hoxb3 are greatly modulated by the levels of available PBX1 present in these cells. Furthermore, we show that the transforming capacity of these two HOX proteins depends on their conserved tetrapeptide and homeodomain regions which mediate binding to PBX and DNA, respectively. Taken together, results of this study demonstrate that cooperation between HOX and PBX proteins modulates cellular proliferation and strongly suggest that cooperative DNA binding by these two groups of proteins represent the basis for Hox-induced cellular transformation.
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.
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.
•GPR56 is a novel LSC marker for the majority of AML samples.•GPR56 expression levels correlate with genetic risk groups and clinical outcome in AML.
In this study, we show the high frequency of spontaneous γδ T-cell leukemia (T-ALL) occurrence in mice with biallelic deletion of enhancer of zeste homolog 2 (Ezh2). Tumor cells show little residual ...H3K27 trimethylation marks compared with controls. EZH2 is a component of the PRC2 Polycomb group protein complex, which is associated with DNA methyltransferases. Using next-generation sequencing, we identify alteration in gene expression levels of EZH2 and acquired mutations in PRC2-associated genes (DNMT3A and JARID2) in human adult T-ALL. Together, these studies document that deregulation of EZH2 and associated genes leads to the development of mouse, and likely human, T-ALL.
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.
In this session we will present results from our chemo-genomic screens designed to categorize more accurately human acute myeloid leukemia (AML) subsets based on both their genetic make-up (RNA and ...exon Next Generation Sequencing) and their response to clinically-approved chemicals. The success of this project relies on our recent development of newly defined culture conditions that support the majority of leukemia stem cells in short-term cultures (Pabst et. al., submitted) and availability of a large collection of clinically annotated specimens (J.H., BCLQ). Integration of chemical and genetic data is possible through our novel bioinformatics tools developed for this purpose (Lemieux et. al., submitted). In addition to the generation of more accurate prognostic tools, these studies set the stage for drug repositioning and personalized medicine for human AML.
No relevant conflicts of interest to declare.
Enhancer of zeste homolog 2 (EZH2) catalyzes di- and trimethylation of lysine 27 on histone H3 (H3K27me2/3) and establishes chromatin marks associated with gene silencing. We and others have recently ...shown that Ezh2 and its partners act as tumour suppressor genes in mouse and likely human lymphoblastic leukemia. Moreover some studies also suggest that Ezh2 is strongly required during B and T cell differentiation. However, the function of EZH2 during these processes remains unclear. For functional study we exploited an Ezh2 conditional knockout mouse model. The Cre-mediated deletion generates a mutated Ezh2Δ allele and abrogates production of EZH2 protein. Upon gene inactivation we monitored T-cell maturation and cancer development. We found that Ezh2 inactivation induces a block at the DN3-DN4 transition of TCRab+T-cells while TCRγδ T-cells were increased by 5 fold compared to wild type animals. Cell cycle analysis revealed increase in the proportions of TCRγδ+T-cells in the G2 phase compared to TCRβ+T-cells and wild type controls. This observation suggested a possibility of G2/M checkpoint activation resulting from either improper DNA replication, or a non-repaired DNA damage. Moreover we found that the Ezh2 deficient TCRγδ+ leukemia were prone to genomic instability. A majority of leukemias analyzed were aneuploid, and ∼50% were near-tetraploid. These observations were confirmed by Spectral Karyotyping (SKY), which also enabled detection of several chromosomal rearrangements. Consistent with these observations, analysis of global gene expression data from various RNA-Seq-derived datasets revealed that the genes having the highest correlation factor with Ezh2 are involved in cell division, DNA replication and DNA damage repair. Together, these studies show that Ezh2 is an essential regulator of the TCRγδ T-cell state, and prevents T-cell transformation, likely through regulation of DNA replication, cell division or DNA damage repair.
No relevant conflicts of interest to declare.
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