The methyltransferase G9a regulates HoxA9-dependent transcription in AML.

Chromatin modulators are emerging as attractive drug targets, given their widespread implication in human cancers and susceptibility to pharmacological inhibition. Here we establish the histone methyltransferase G9a/EHMT2 as a selective regulator of fast proliferating myeloid progenitors with no discernible function in hematopoietic stem cells (HSCs). In mouse models of acute myeloid leukemia (AML), loss of G9a significantly delays disease progression and reduces leukemia stem cell (LSC) frequency. We connect this function of G9a to its methyltransferase activity and its interaction with the leukemogenic transcription factor HoxA9 and provide evidence that primary human AML cells are sensitive to G9A inhibition. Our results highlight a clinical potential of G9A inhibition as a means to counteract the proliferation and self-renewal of AML cells by attenuating HoxA9-dependent transcription.

A transgenic mouse model demonstrating the oncogenic role of mutations in the polycomb-group gene EZH2 in lymphomagenesis.

The histone methyltransferase EZH2 is frequently mutated in germinal center-derived diffuse large B-cell lymphoma and follicular lymphoma. To further characterize these EZH2 mutations in lymphomagenesis, we generated a mouse line where EZH2(Y641F) is expressed from a lymphocyte-specific promoter. Spleen cells isolated from the transgenic mice displayed a global increase in trimethylated H3K27, but the mice did not show an increased tendency to develop lymphoma. As EZH2 mutations often coincide with other mutations in lymphoma, we combined the expression of EZH2(Y641F) by crossing these transgenic mice with Eµ-Myc transgenic mice. We observed a dramatic acceleration of lymphoma development in this combination model of Myc and EZH2(Y641F). The lymphomas show histologic features of high-grade disease with a shift toward a more mature B-cell phenotype, increased cycling and gene expression, and epigenetic changes involving important pathways in B-cell regulation and function. Furthermore, they initiate disease in secondary recipients. In summary, EZH2(Y641F) can collaborate with Myc to accelerate lymphomagenesis demonstrating a cooperative role of EZH2 mutations in oncogenesis. This murine lymphoma model provides a new tool to study global changes in the epigenome caused by this frequent mutation and a promising model system for testing novel treatments.

Ontogeny stage-independent and high-level clonal expansion in vitro of mouse hematopoietic stem cells stimulated by an engineered NUP98-HOX fusion transcription factor.

Achieving high-level expansion of hematopoietic stem cells (HSCs) in vitro will have an important clinical impact in addition to enabling elucidation of their regulation. Here, we couple the ability of engineered NUP98-HOXA10hd expression to stimulate > 1000-fold net expansions of murine HSCs in 10-day cultures initiated with bulk lin(-)Sca-1(+)c-kit(+) cells, with strategies to purify fetal and adult HSCs and analyze their expansion clonally. We find that NUP98-HOXA10hd stimulates comparable expansions of HSCs from both sources at ∼ 60% to 90% unit efficiency in cultures initiated with single cells. Clonally expanded HSCs consistently show balanced long-term contributions to the lymphoid and myeloid lineages without evidence of leukemogenic activity. Although effects on fetal and adult HSCs were indistinguishable, NUP98-HOXA10hd-transduced adult HSCs did not thereby gain a competitive advantage in vivo over freshly isolated fetal HSCs. Live-cell image tracking of single transduced HSCs cultured in a microfluidic device indicates that NUP98-HOXA10hd does not affect their proliferation kinetics, and flow cytometry confirmed the phenotype of normal proliferating HSCs and allowed reisolation of large numbers of expanded HSCs at a purity of 25%. These findings point to the effects of NUP98-HOXA10hd on HSCs in vitro being mediated by promoting self-renewal and set the stage for further dissection of this process.

Linkage of the potent leukemogenic activity of Meis1 to cell-cycle entry and transcriptional regulation of cyclin D3.

MEIS1 is a three-amino acid loop extension class homeodomain-containing homeobox (HOX) cofactor that plays key roles in normal hematopoiesis and leukemogenesis. Expression of Meis1 is rate-limiting in MLL-associated leukemias and potently interacts with Hox and NUP98-HOX genes in leukemic transformation to promote self-renewal and proliferation of hematopoietic progenitors. The oncogenicity of MEIS1 has been linked to its transcriptional activation properties. To further reveal the pathways triggered by Meis1, we assessed the function of a novel engineered fusion form of Meis1, M33-MEIS1, designed to confer transcriptional repression to Meis1 target genes that are otherwise up-regulated in normal and malignant hematopoiesis. Retroviral overexpression of M33-Meis1 resulted in the rapid and complete eradication of M33-Meis1-transduced normal and leukemic cells in vivo. Cell-cycle analysis showed that M33-Meis1 impeded the progression of cells from G(1)-to-S phase, which correlated with significant reduction of cyclin D3 levels and the inhibition of retinoblastoma (pRb) hyperphosphorylation. We identified cyclin D3 as a direct downstream target of MEIS1 and M33-MEIS1 and showed that the G(1)-phase accumulation and growth suppression induced by M33-Meis1 was partially relieved by overexpression of cyclin D3. This study provides strong evidence linking the growth-promoting activities of Meis1 to the cyclin D-pRb cell-cycle control pathway.

Brief report: ectopic expression of NUP98-HOXA10 augments erythroid differentiation of human embryonic stem cells.

Hox genes encode highly conserved transcription factors that have been implicated in hematopoietic development and self-renewal of hematopoietic stem cells (HSCs) and hematopoietic development. The potency of NUP98-HOXA10hd (NA10) on adult murine bone marrow HSC self-renewal prompted us to examine its effect on specification and proliferation of hematopoietic cells derived from human embryonic stem cells (hESCs). Here, we demonstrate that expression of NA10 in hESCs influences the hematopoietic differentiation program. The specific effect of NA10 is dependent on the developmental stage of hematopoietic emergence from hESCs. Overexpression of NA10 in either undifferentiated hESCs or early hemogenic precursors augmented the frequency of CD45(-) GlycophorinA(+) cells and erythroid progenitors (blast-forming unit-erythrocyte). In contrast, targeted NA10 expression in primitive CD34+ cells committed to the hematopoietic lineage had no effect on erythropoietic capacity but instead increased hematopoietic progenitor proliferation. Our study reveals a novel neomorphic effect of NA10 in early human erythroid development from pluripotent stem cells.

Retroviral integration site analysis identifies ICSBP as a collaborating tumor suppressor gene in NUP98-TOP1-induced leukemia.

INTRODUCTION: The NUP98-TOP1 fusion gene is one of 18 distinct translocations identified in acute myeloid leukemia involving the N-terminal portion of the nucleoporin NUP98. We previously reported that expression of NUP98-TOP in murine bone marrow induces a lethal, transplantable leukemia. However, the long latency suggests the in vivo acquisition of additional mutations and/or time required for clonal outgrowth of rare transformed cells arising from the collaboration of NUP98-TOP1 and a cooperating event. The aim of this study was to test whether retroviral insertional mutagenesis contributes to disease onset and whether integration site analysis can identify collaborating genes. METHODS: The genomic sites of retroviral integration in NUP98-TOP1-induced leukemic mice were analyzed. This screen identified a proviral integration that disrupts expression of the Interferon consensus sequence binding protein (ICSBP) tumor suppressor gene. Intriguingly, an ICSBP deficiency induces a chronic myeloid leukemia-like disease in mice and its reduced expression has been observed in several human leukemias. To ascertain whether an ISCBP deficiency collaborates with NUP98-TOP1 in leukemogenesis, we expressed NUP98-TOP1 in ICSBP(-/-) bone marrow. RESULTS: The in vivo myeloproliferation induced by NUP98-TOP1 was markedly exaggerated with the ICSBP(-/-) deficiency. Moreover, NUP98-TOP1/ICSBP(-/-) mice had a reduced survival compared with NUP98-TOP1/ICSBP(+/+) mice. CONCLUSION: These results reveal the novel finding of collaboration between the ICSBP tumor suppressor gene and NUP98-TOP1 in leukemogenesis. Moreover they further illustrate the power of retroviral integration site analysis for identifying novel cooperating tumor suppressor genes.

Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion.

Meis1 is recognized as an important transcriptional regulator in hematopoietic development and is strongly implicated in the pathogenesis of leukemia, both as a Hox transcription factor co-factor and independently. Despite the emerging recognition of Meis1's importance in the context of both normal and leukemic hematopoiesis, there is not yet a full understanding of Meis1's functions and the relevant pathways and genes mediating its functions. Recently, several conditional mouse models for Meis1 have been established. These models highlight a critical role for Meis1 in adult mouse hematopoietic stem cells (HSCs) and implicate reactive oxygen species (ROS) as a mediator of Meis1 function in this compartment. There are, however, several reported differences between these studies in terms of downstream progenitor populations impacted and effectors of function. In this study, we describe further characterization of a conditional knockout model based on mice carrying a loxP-flanked exon 8 of Meis1 which we crossed onto the inducible Cre localization/expression strains, B6;129-Gt(ROSA)26Sor(tm1(Cre/ERT)Nat)/J or B6.Cg-Tg(Mx1-Cre)1Cgn/J. Findings obtained from these two inducible Meis1 knockout models confirm and extend previous reports of the essential role of Meis1 in adult HSC maintenance and expansion and provide new evidence that highlights key roles of Meis1 in both megakaryopoiesis and erythropoiesis. Gene expression analyses point to a number of candidate genes involved in Meis1's role in hematopoiesis. Our data additionally support recent evidence of a role of Meis1 in ROS regulation.

Aldehyde dehydrogenases are regulators of hematopoietic stem cell numbers and B-cell development.

High levels of the aldehyde dehydrogenase isoform ALDH1A1 are expressed in hematopoietic stem cells (HSCs); however, its importance in these cells remains unclear. Consistent with an earlier report, we find that loss of ALDH1A1 does not affect HSCs. Intriguingly, however, we find that ALDH1A1 deficiency is associated with increased expression of the ALDH3A1 isoform, suggesting its potential to compensate for ALDH1A1. Mice deficient in ALDH3A1 have a block in B-cell development as well as abnormalities in cell cycling, intracellular signaling, and gene expression. Early B cells from these mice exhibit excess reactive oxygen species and reduced metabolism of reactive aldehydes. Mice deficient in both ALDH3A1 and ALDH1A1 have reduced numbers of HSCs as well as aberrant cell cycle distribution, increased reactive oxygen species levels, p38 mitogen-activated protein kinase activity and sensitivity to DNA damage. These findings demonstrate that ALDH3A1 can compensate for ALDH1A1 in bone marrow and is important in B-cell development, both ALDH1A1 and 3A1 are important in HSC biology; and these effects may be due, in part, to changes in metabolism of reactive oxygen species and reactive aldehydes.

Induction of acute myeloid leukemia in mice by the human leukemia-specific fusion gene NUP98-HOXD13 in concert with Meis1.

HOX genes, notably members of the HOXA cluster, and HOX cofactors have increasingly been linked to human leukemia. Intriguingly, HOXD13, a member of the HOXD cluster not normally expressed in hematopoietic cells, was recently identified as a partner of NUP98 in a t(2;11) translocation associated with t-AML/MDS. We have now tested directly the leukemogenic potential of the NUP98-HOXD13 t(2; 11) fusion gene in the murine hematopoietic model. NUP98-HOXD13 strongly promoted growth and impaired differentiation of early hematopoietic progenitor cells in vitro; this effect was dependent on the NUP98 portion and an intact HOXD13 homeodomain. Expression of the NUP98-HOXD13 fusion gene in vivo resulted in a partial impairment of lymphopoiesis but did not induce evident hematologic disease until late after transplantation (more than 5 months), when some mice developed a myeloproliferative-like disease. In contrast, mice transplanted with bone marrow (BM) cells cotransduced with NUP98-HOXD13 and the HOX cofactor Meis1 rapidly developed lethal and transplantable acute myeloid leukemia (AML), with a median disease onset of 75 days. In summary, this study demonstrates that NUP98-HOXD13 can be directly implicated in the molecular process leading to leukemic transformation, and it supports a model in which the transforming properties of NUP98-HOXD13 are mediated through HOX-dependent pathways.