Patty Rosten

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.

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.

Modeling de novo leukemogenesis from human cord blood with MN1 and NUP98HOXD13

Leukemic transformation of human cells is a complex process. Here we show that forced expression of MN1 in primitive human cord blood cells maintained on stromal cells in vitro induces a transient, but not serially transplantable, myeloproliferation in engrafted mice. However, cotransduction of an activated HOX gene (NUP98HOXD13) with MN1 induces a serially transplantable acute myeloid leukemia (AML). Further characterization of the leukemic cells generated from the dually transduced cells showed the activation of stem cell gene expression signatures also found in primary human AML. These findings show a new forward genetic model of human leukemogenesis and further highlight the relevance of homeobox transcription factors in the transformation process.

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.

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)26Sortm1(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.

Delineating MEIS1 cis-regulatory elements active in hematopoietic cells.

MEIS1 (Myeloid ecotropic viral integration site 1) is a homeodomain transcription factor that cooperates with Hox family proteins in both normal and leukemic hematopoiesis.1 MEIS1 is highly expressed in hematopoietic stem cells (HSCs), and its level decreases during differentiation but with a relatively higher expression in the megakaryocytic lineage.2, 3 MEIS1 expression is often elevated in leukemia,4 and it is one of the leukemic stem cell (LSC)-related signature genes whose higher expression is correlated with worse prognosis.5 Previous work, including our own, has shown that MEIS1 acts as an important driver for leukemogenesis.1 Elevated expression of MEIS1 with concurrent high expression of HOX can induce rapid-onset AML in murine models.6 Decreased MEIS1 expression via short hairpin RNA-mediated knockdown has been shown to significantly reduce LSC potential.7, 8 Together, these findings point to the importance of tightly regulated expression of MEIS1 for normal hematopoiesis and reveal how its deregulated expression can be a major factor in leukemogenesis.

HOXA10-Overexpression causes severe perturbations in human hematopoiesis

Abstract Normal hematopoiesis is linked to the differential expression of multiple members of the Hox transcription factor family. Moreover, studies in murine transplantation models have shown that overexpression of A cluster HOX genes (i.e. HOXA10, HOXA9) is leukemogenic. We have now tested directly the impact of dysregulated HOXA10 expression on human hematopoietic cells in vitro and in vivo using highly purified CD34+/GFP+ cells transduced with a MSCV vector carrying a HOXA10-IRES-GFP cassette or a GFP cassette only (control). HOXA10 altered lineage differentiation with a 85% reduction in BFU-E colony formation (P = 0.017) and increased the self-renewal of clonogenic progenitor cells with an over 120 fold increase in secondary colony formation which were predominantly of blast morphology compared to control. The in vitro data were extended by in vivo analysis of NOD/SCID mice fulfilling the criteria of lymphomyeloid engraftment with transduced human cells. 12 wks post transplantation there was a 2 fold increase in the total number of clonogenic progenitors per 10 6 engrafted CD34+ human cells in the HOXA10- transduced compartment (P = 0.04). Furthermore, ex vivo HOXA10 overexpressing progenitors formed a 19 fold increased number of blast colonies. HOXA10 also markedly impaired in vivo differentiation into B cells (70% drop in absolute number). Imbalanced HOXA10 expression thus initiates profound perturbations in human hematopoiesis pointing to a potential role of abnormally increased self-renewal and altered lineage differentiation in HOX gene-associated leukemogenesis.