Vitamin C-induced epigenomic remodelling in IDH1 mutant acute myeloid leukaemia.

The genomes of myeloid malignancies are characterized by epigenomic abnormalities. Heterozygous, inactivating ten-eleven translocation 2 (TET2) mutations and neomorphic isocitrate dehydrogenase (IDH) mutations are recurrent and mutually exclusive in acute myeloid leukaemia genomes. Ascorbic acid (vitamin C) has been shown to stimulate the catalytic activity of TET2 in vitro and thus we sought to explore its effect in a leukaemic model expressing IDH1R132H. Vitamin C treatment induced an IDH1R132H-dependent reduction in cell proliferation and an increase in expression of genes involved in leukocyte differentiation. Vitamin C induced differentially methylated regions that displayed a significant overlap with enhancers implicated in myeloid differentiation and were enriched in sequence elements for the haematopoietic transcription factors CEBPß, HIF1α, RUNX1 and PU.1. Chromatin immunoprecipitation sequencing of PU.1 and RUNX1 revealed a significant loss of PU.1 and increase of RUNX1-bound DNA elements accompanied by their demethylation following vitamin C treatment. In addition, vitamin C induced an increase in H3K27ac flanking sites bound by RUNX1. On the basis of these data we propose a model of vitamin C-induced epigenetic remodelling of transcription factor-binding sites driving differentiation in a leukaemic model.

Meis2 as a critical player in MN1-induced leukemia.

Meningioma 1 (MN1) is an independent prognostic marker for normal karyotype acute myeloid leukemia (AML), with high expression linked to all-trans retinoic acid resistance and poor survival. MN1 is also a potent and sufficient oncogene in murine leukemia models, strongly dependent on the MEIS1/AbdB-like HOX protein complex to transform common myeloid progenitors, block myeloid differentiation, and promote leukemic stem cell self-renewal. To identify key genes and pathways underlying leukemic activity, we functionally assessed MN1 cell phenotypic heterogeneity, revealing leukemic and non-leukemic subsets. Using gene expression profiling of these subsets combined with previously published comparisons of full-length MN1 and mutants with varying leukemogenic activity, we identified candidate genes critical to leukemia. Functional analysis identified Hlf and Hoxa9 as critical to MN1 in vitro proliferation, self-renewal and impaired myeloid differentiation. Although critical to transformation, Meis1 knockdown had little impact on these properties in vitro. However, we identified Meis2 as critical to MN1-induced leukemia, with essential roles in proliferation, self-renewal, impairment of differentiation and disease progression in vitro and in vivo. Here, we provide evidence of phenotypic and functional hierarchy in MN1-induced leukemic cells, characterise contributions of Hlf, Hoxa9 and Meis1 to in vitro leukemic properties, and reveal Meis2 as a novel player in MN1-induced leukemogenesis.

A novel AHI-1-BCR-ABL-DNM2 complex regulates leukemic properties of primitive CML cells through enhanced cellular endocytosis and ROS-mediated autophagy.

Tyrosine kinase inhibitor (TKI) therapies induce clinical remission with remarkable effects on chronic myeloid leukemia (CML). However, very few TKIs completely eradicate the leukemic clone and persistence of leukemic stem cells (LSCs) remains challenging, warranting new, distinct targets for improved treatments. We demonstrated that the scaffold protein AHI-1 is highly deregulated in LSCs and interacts with multiple proteins, including Dynamin-2 (DNM2), to mediate TKI-resistance of LSCs. We have now demonstrated that the SH3 domain of AHI-1 and the proline rich domain of DNM2 are mainly responsible for this interaction. DNM2 expression was significantly increased in CML stem/progenitor cells; knockdown of DNM2 greatly impaired their survival and sensitized them to TKI treatments. Importantly, a new AHI-1-BCR-ABL-DNM2 protein complex was uncovered, which regulates leukemic properties of these cells through a unique mechanism of cellular endocytosis and ROS-mediated autophagy. Thus, targeting this complex may facilitate eradication of LSCs for curative therapies.

Constitutive IRF8 expression inhibits AML by activation of repressed immune response signaling.

Myeloid differentiation is blocked in acute myeloid leukemia (AML), but the molecular mechanisms are not well characterized. Meningioma 1 (MN1) is overexpressed in AML patients and confers resistance to all-trans retinoic acid-induced differentiation. To understand the role of MN1 as a transcriptional regulator in myeloid differentiation, we fused transcriptional activation (VP16) or repression (M33) domains with MN1 and characterized these cells in vivo. Transcriptional activation of MN1 target genes induced myeloproliferative disease with long latency and differentiation potential to mature neutrophils. A large proportion of differentially expressed genes between leukemic MN1 and differentiation-permissive MN1VP16 cells belonged to the immune response pathway like interferon-response factor (Irf) 8 and Ccl9. As MN1 is a cofactor of MEIS1 and retinoic acid receptor alpha (RARA), we compared chromatin occupancy between these genes. Immune response genes that were upregulated in MN1VP16 cells were co-targeted by MN1 and MEIS1, but not RARA, suggesting that myeloid differentiation is blocked through transcriptional repression of shared target genes of MN1 and MEIS1. Constitutive expression of Irf8 or its target gene Ccl9 identified these genes as potent inhibitors of murine and human leukemias in vivo. Our data show that MN1 prevents activation of the immune response pathway, and suggest restoration of IRF8 signaling as therapeutic target in AML.

Ex vivo expansion of normal and chronic myeloid leukemic stem cells without functional alteration using a NUP98HOXA10homeodomain fusion gene.

HOX genes have been implicated as regulators of normal and leukemic stem cell functionality, but the extent to which these activities are linked is poorly understood. Previous studies revealed that transduction of primitive mouse hematopoietic cells with a NUP98HOXA10homeodomain (NA10HD) fusion gene enables a subsequent rapid and marked expansion in vitro of hematopoietic stem cell numbers without causing their transformation or deregulated expansion in vivo. To determine whether forced expression of NA10HD in primitive human cells would have a similar effect, we compared the number of long-term culture-initiating cells (LTC-ICs) present in cultures of lenti-NA10HD versus control virus-transduced CD34(+) cells originally isolated from human cord blood and chronic phase (CP) chronic myeloid leukemia (CML) patients. We found that NA10HD greatly increases outputs of both normal and Ph(+)/BCR-ABL(+) LTC-ICs, and this effect is particularly pronounced in cultures containing growth factor-producing feeders. Interestingly, NA10HD did not affect the initial cell cycle kinetics of the transduced cells nor their subsequent differentiation. Moreover, immunodeficient mice repopulated with NA10HD-transduced CP-CML cells for more than 8 months showed no evidence of altered behavior. Thus, NA10HD provides a novel tool to enhance both normal and CP-CML stem cell expansion in vitro, without apparently altering other properties.

NUP98-HOXA10hd-expanded hematopoietic stem cells efficiently reconstitute bone marrow of mismatched recipients and induce tolerance.

Gene therapy as well as methods capable of returning cells to a pluripotent state (iPS) have enabled the correction of genetic deficiencies in syngenic adult progenitors, reducing the need for immunosuppression in cell therapy approaches. However, in diseases involving mutations that lead to the complete lack of a protein, such as Duchenne muscular dystrophy, the main immunogens leading to rejection of transplanted cells are the therapeutic proteins themselves. In these cases even iPS cells would not circumvent the need for immunosuppression, and alternative strategies must be developed. One such potential strategy seeks to induce immune tolerance using hematopoietic stem cells originated from the same donor or iPS line from which the therapeutic progenitors are derived. However, donor hematopoietic stem cells (HSCs) are available in limiting numbers and embryonic stem (ES) cell-derived HSCs engraft poorly in adults. While these limitations have been circumvented by ectopic expression of HOXB4, overexpression of this protein is associated with inefficient lymphoid reconstitution. Here we show that adult HSCs expanded with a NUP98- HOXA10hd fusion protein sustain long-term engraftment in immunologically mismatched recipients and generate normal numbers of lymphoid cells. In addition, NUP98-HOXA10hd-expanded cells induce functional immune tolerance to a subsequent transplant of myogenic progenitors immunologically matched with the transplanted HSCs.

miRNA*: a passenger stranded in RNA-induced silencing complex?

Processing of the pre-microRNA (pre-miRNA) through Dicer1 generates a miRNA duplex, consisting of a miRNA and miRNA* strand (also termed guide strand and passenger strand, respectively). Despite the general consensus that miRNA*s have no regulatory activity, recent publications have provided evidence that the abundance, possible function, and physiological relevance of miRNA*s have been underestimated. This review provides an account of our current understanding of miRNA* origination and activity, mounting evidence for their unique functions and regulatory mechanisms, and examples of specific miRNA*s from the literature.

Comprehensive microRNA expression profiling of the hematopoietic hierarchy.

The hematopoietic system produces a large number of highly specialized cell types that are derived through a hierarchical differentiation process from a common stem cell population. miRNAs are critical players in orchestrating this differentiation. Here, we report the development and application of a high-throughput microfluidic real-time quantitative PCR (RT-qPCR) approach for generating global miRNA profiles for 27 phenotypically distinct cell populations isolated from normal adult mouse hematopoietic tissues. A total of 80,000 RT-qPCR assays were used to map the landscape of miRNA expression across the hematopoietic hierarchy, including rare progenitor and stem cell populations. We show that miRNA profiles allow for the direct inference of cell lineage relations and functional similarity. Our analysis reveals a close relatedness of the miRNA expression patterns in multipotent progenitors and stem cells, followed by a major reprogramming upon restriction of differentiation potential to a single lineage. The analysis of miRNA expression in single hematopoietic cells further demonstrates that miRNA expression is very tightly regulated within highly purified populations, underscoring the potential of single-cell miRNA profiling for assessing compartment heterogeneity.

Additional sex combs-like 1 belongs to the enhancer of trithorax and polycomb group and genetically interacts with Cbx2 in mice.

The Additional sex combs (Asx) gene of Drosophila behaves genetically as an enhancer of trithorax and polycomb (ETP) in displaying bidirectional homeotic phenotypes, suggesting that is required for maintenance of both activation and silencing of Hox genes. There are three murine homologs of Asx called Additional sex combs-like1, 2, and 3. Asxl1 is required for normal adult hematopoiesis; however, its embryonic function is unknown. We used a targeted mouse mutant line Asxl1(tm1Bc) to determine if Asxl1 is required to silence and activate Hox genes in mice during axial patterning. The mutant embryos exhibit simultaneous anterior and posterior transformations of the axial skeleton, consistent with a role for Asxl1 in activation and silencing of Hox genes. Transformations of the axial skeleton are enhanced in compound mutant embryos for the polycomb group gene M33/Cbx2. Hoxa4, Hoxa7, and Hoxc8 are derepressed in Asxl1(tm1Bc) mutants in the antero-posterior axis, but Hoxc8 expression is reduced in the brain of mutants, consistent with Asxl1 being required both for activation and repression of Hox genes. We discuss the genetic and molecular definition of ETPs, and suggest that the function of Asxl1 depends on its cellular context.

Complementary and independent function for Hoxb4 and Bmi1 in HSC activity.

Determinants regulating short- and long-term repopulating hematopoietic stem cell (STR-HSC and LTR-HSC) self-renewal remain largely uncharacterized. To gain further insights into HSC self-renewal, we investigated possible genetic interactions between two well-recognized regulators of this process: Bmi1 and Hoxb4. Using complementation and overexpression strategies in mouse HSCs, we document that Bmi1 is not required for the in vivo expansion of fetal HSCs but is essential for the long-term maintenance of adult HSCs. Importantly, we show that Hoxb4 overexpression induces an expansion of Bmi1(-/-)STR-HSCs leading to a rescue of their repopulation defect. In contrast to Hoxb4, we also show that Bmi1 fails to induce HSC expansion ex vivo. Consistent with these results, we report high levels of Angptl3 and Cbx7 in Hoxb4- and Bmi1-transduced cells, respectively. Together, these results support the emerging concept that fate and sustainability of this fate are two critical components of self-renewal in adult stem cells such as HSCs.

Leukemogenic properties of NUP98-PMX1 are linked to NUP98 and homeodomain sequence functions but not to binding properties of PMX1 to serum response factor.

PMX1 is a member of a non-clustered homeobox gene family, not normally expressed in hematopoietic cells, and first identified for its role in enhancing the binding of the serum response factor (SRF) to the serum responsive element (SRE). PMX1 has never been linked to leukemia on its own, raising the possibility of unique mechanisms underlying the oncogenicity of NUP98-PMX1. To elucidate the leukemogenic potential of NUP98-PMX1, we compared the effects of PMX1 and NUP98-PMX1 and, through strategic mutations, the involvement of the SRE in NUP98-PMX1-mediated leukemia. NUP98-PMX1, but not PMX1, had potent ability to impair differentiation, promote proliferation of myeloid progenitors, induce lethal myeloproliferative disease and to activate a number of genes previously linked to leukemic stem cells. Similar to NUP98-HOX fusions, the transforming potential of NUP98-PMX1 required the NUP98 portion and DNA-binding capability of the PMX1 homeodomain and collaborated with Meis1 to induce more rapid onset myeloproliferative-like myeloid leukemia. The transforming activity of NUP98-PMX1 was independent of its ability to interact with SRF. These findings provide novel evidence of the contributory role of the NUP98 sequence in conferring leukemogenic properties on a partner gene and point to common leukemogenic pathways for NUP98-PMX1 and NUP98-clustered HOX fusions.

Hox genes in hematopoiesis and leukemogenesis.

Gene expression analyses, gene targeting experiments and retroviral overexpression studies in the murine bone marrow transplantation model have provided strong correlative evidence for the involvement of clustered Hox genes in normal hematopoiesis. The data strongly support the hypothesis that the role of Hox genes in normal hematopoiesis is primarily at the level of hematopoietic stem cell function. A large body of evidence now links Hox genes to leukemic transformation including dysregulated HOX expression in leukemic patient samples, their involvement as oncogenic fusion proteins with NUP98 and their requirement for the oncogenicity of Mll fusions. In recent years, much attention has been devoted to the identification and characterization of leukemic stem cells. Given the documented role of Hox genes in hematopoiesis and leukemogenesis, we propose that Hox-dependent pathways are closely linked to the self-renewal program crucial to the origin and function of leukemic stem cells.

Transplantable cell lines generated with NUP98-Hox fusion genes undergo leukemic progression by Meis1 independent of its binding to DNA.

Hox genes have been identified in chromosomal translocations involving the nucleoporin gene NUP98. Though the resulting chimeric proteins directly participate in the development of leukemia, the long latency and monoclonal nature of the disease support the requirement for secondary mutation(s), such as those leading to overexpression of Meis1. Models to identify such events and to study leukemic progression are rare and labor intensive. Herein, we took advantage of the strong transforming potential of NUP98-HOXD13 or NUP98-HOXA10 to establish preleukemic myeloid lines from bone marrow cells that faithfully replicate the first step of Hox-induced leukemogenesis. These lines contain early granulomonocytic progenitors with extensive in vitro self-renewal capacity, short-term myeloid repopulating activity and low propensity for spontaneous leukemic conversion. We exploit such lines to show that Meis1 efficiently induces their leukemic progression and demonstrate a high frequency of preleukemic cells in the cultures. Furthermore, we document that the leukemogenic potential of Meis1 is independent of its direct binding to DNA and likely reflects its ability to increase the repopulating capacity of the preleukemic cells by increasing their self-renewal/proliferative capacity. The availability of lines with repopulating potential and capacity for leukemic conversion should open new avenues for understanding progression of Hox-mediated acute myeloid leukemia.

NOD/SCID mice engineered to express human IL-3, GM-CSF and Steel factor constitutively mobilize engrafted human progenitors and compromise human stem cell regeneration.

Transplantation of immunodeficient mice with human hematopoietic cells has greatly facilitated studies of the earliest stages of human hematopoiesis. These include demonstration of the ability of injected 'human-specific' hematopoietic growth factors to enhance the production of human cells at multiple levels of differentiation. In contrast, the effects of continuous exposure to such molecules have not been well investigated. Here, we show that nonobese diabetic severe combined immunodeficiency mice genetically engineered to produce ng/ml serum levels of human interleukin-3 (IL-3), granulocyte/macrophage-stimulating factor (GM-CSF) and Steel factor (SF) display a complex phenotype when transplanted with primitive human bone marrow (BM) or fetal liver cells. This phenotype is characterized by an enhancement of terminal human myelopoiesis and a matched suppression of terminal human erythropoiesis, with a slight reduction in human B-lymphopoiesis in the BM of the engrafted mice. Human clonogenic progenitors are more prevalent in the blood of the transplanted growth factor-producing mice and this is accompanied by a very marked reduction of more primitive human cells in the BM. Our findings suggest that long-term exposure of primitive human hematopoietic cells to elevated levels of human IL-3, GM-CSF and SF in vivo may deleteriously affect the stem cell compartment, while expanding terminal myelopoiesis.

Improved engraftment of human acute myeloid leukemia progenitor cells in beta 2-microglobulin-deficient NOD/SCID mice and in NOD/SCID mice transgenic for human growth factors.

Primitive malignant progenitors defined as nonobese diabetic/severe combined immunodeficient (NOD/SCID) leukemia-initiating cells or NOD/SL-IC from patients with acute myeloid leukemia (AML) can be detected and quantitated in sublethally irradiated NOD/SCID mice. However, there is variability in the levels of bone marrow (BM) engraftment obtained after intravenous injection of cells from different AML samples. In the current study, AML cell engraftment in standard NOD/SCID mice was compared to that obtained with NOD/SCID mice transgenic for the human growth factor genes Steel factor (SF), interleukin-3 (IL-3) and granulocyte macrophage-colony-stimulating factor (GM-CSF) (N/S-S/GM/3) as well as beta 2 microglobulin-null NOD/SCID (N/S-beta 2m(-/-)) mice. Three of the eight AML samples that failed to engraft in standard NOD/SCID animals showed easily detectable and up to 70-fold increased in the number of leukemic cells in BM 8-12 weeks post-transplantation in each of the N/S-beta 2m(-/-) and N/S-S/GM/3 mouse strains. In two of the four AML samples studied at limiting dilution, the frequency of NOD/SL-IC detected was increased six- and seven-fold. Thus, in these novel mouse strains a broader spectrum of AML patient samples can be evaluated for their progenitor content and potentially studied for their response to innovative therapeutics in vivo.