UTX maintains the functional integrity of the murine hematopoietic system by globally regulating aging-associated genes.

Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in hematopoietic stem cells declined with age, and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.

Acquired expression of CblQ367P in mice induces dysplastic myelopoiesis mimicking chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a hematological malignancy characterized by uncontrolled proliferation of dysplastic myelomonocytes and frequent progression to acute myeloid leukemia (AML). We identified mutations in the Cbl gene, which encodes a negative regulator of cytokine signaling, in a subset of CMML patients. To investigate the contribution of mutant Cbl in CMML pathogenesis, we generated conditional knockin mice for Cbl that express wild-type Cbl in a steady state and inducibly express CblQ367P , a CMML-associated Cbl mutant. CblQ367P mice exhibited sustained proliferation of myelomonocytes, multilineage dysplasia, and splenomegaly, which are the hallmarks of CMML. The phosphatidylinositol 3-kinase (PI3K)-AKT and JAK-STAT pathways were constitutively activated in CblQ367P hematopoietic stem cells, which promoted cell cycle progression and enhanced chemokine-chemokine receptor activity. Gem, a gene encoding a GTPase that is upregulated by CblQ367P , enhanced hematopoietic stem cell activity and induced myeloid cell proliferation. In addition, Evi1, a gene encoding a transcription factor, was found to cooperate with CblQ367P and progress CMML to AML. Furthermore, targeted inhibition for the PI3K-AKT and JAK-STAT pathways efficiently suppressed the proliferative activity of CblQ367P -bearing CMML cells. Our findings provide insights into the molecular mechanisms underlying mutant Cbl-induced CMML and propose a possible molecular targeting therapy for mutant Cbl-carrying CMML patients.

Propagation of trimethylated H3K27 regulated by polycomb protein EED is required for embryogenesis, hematopoietic maintenance, and tumor suppression.

Polycomb repressive complex 2 (PRC2) catalyzes the monomethylation, dimethylation, and trimethylation of histone H3 Lys27 (H3K27) and acts as a central epigenetic regulator that marks the repressive chromatin domain. Embryonic ectoderm development (EED), an essential component of PRC2, interacts with trimethylated H3K27 (H3K27me3) through the aromatic cage structure composed of its three aromatic amino acids, Phe97, Trp364, and Tyr365. This interaction allosterically activates the histone methyltransferase activity of PRC2 and thereby propagates repressive histone marks. In this study, we report the analysis of knock-in mice harboring the myeloid disorder-associated EED Ile363Met (I363M) mutation, analogous to the EED aromatic cage mutants. The I363M homozygotes displayed a remarkable and preferential reduction of H3K27me3 and died at midgestation. The heterozygotes increased the clonogenic capacity and bone marrow repopulating activity of hematopoietic stem/progenitor cells (HSPCs) and were susceptible to leukemia. Lgals3, a PRC2 target gene encoding a multifunctional galactose-binding lectin, was derepressed in I363M heterozygotes, which enhanced the stemness of HSPCs. Thus, our work provides in vivo evidence that the structural integrity of EED to H3K27me3 propagation is critical, especially for embryonic development and hematopoietic homeostasis, and that its perturbation increases the predisposition to hematologic malignancies.

Fbxl10 overexpression in murine hematopoietic stem cells induces leukemia involving metabolic activation and upregulation of Nsg2.

We previously reported that deficiency for Samd9L, which was cloned as a candidate gene for -7/7q- syndrome, accelerated leukemia cooperatively with enhanced expression of a histone demethylase: F-box and leucine-rich repeat protein 10 (Fbxl10, also known as Jhdm1b, Kdm2b, and Ndy1). To further investigate the role of Fbxl10 in leukemogenesis, we generated transgenic (Tg) mice that overexpress Fbxl10 in hematopoietic stem cells (HSCs). Interestingly, Fbxl10 Tg mice developed myeloid or B-lymphoid leukemia with complete penetrance. HSCs from the Tg mice exhibited an accelerated G0/G1-to-S transition with a normal G0 to G1 entry, resulting in pleiotropic progenitor cell expansion. Fbxl10 Tg HSCs displayed enhanced expression of neuron-specific gene family member 2 (Nsg2), and forced expression of Nsg2 in primary bone marrow cells resulted in expansion of immature cells. In addition, the genes involved in mitochondrial oxidative phosphorylation were markedly enriched in Fbxl10 Tg HSCs, coupled with increased cellular adenosine 5'-triphosphate levels. Moreover, chromatin immunoprecipitation followed by sequencing analysis demonstrated that Fbxl10 directly binds to the regulatory regions of Nsg2 and oxidative phosphorylation genes. These findings define Fbxl10 as a bona fide oncogene, whose deregulated expression contributes to the development of leukemia involving metabolic proliferative advantage and Nsg2-mediated impaired differentiation.

Identification of cooperative genes for E2A-PBX1 to develop acute lymphoblastic leukemia.

E2A-PBX1 is a chimeric gene product detected in t(1;19)-bearing acute lymphoblastic leukemia (ALL) with B-cell lineage. To investigate the leukemogenic process, we generated conditional knock-in (cKI) mice for E2A-PBX1, in which E2A-PBX1 is inducibly expressed under the control of the endogenous E2A promoter. Despite the induced expression of E2A-PBX1, no hematopoietic disease was observed, strongly suggesting that additional genetic alterations are required to develop leukemia. To address this possibility, retroviral insertional mutagenesis was used. Virus infection efficiently induced T-cell, B-cell, and biphenotypic ALL in E2A-PBX1 cKI mice. Inverse PCR identified eight retroviral common integration sites, in which enhanced expression was observed in the Gfi1, Mycn, and Pim1 genes. In addition, it is of note that viral integration and overexpression of the Zfp521 gene was detected in one tumor with B-cell lineage; we previously identified Zfp521 as a cooperative gene with E2A-HLF, another E2A-involving fusion gene with B-lineage ALL. The cooperative oncogenicity of E2A-PBX1 with overexpressed Zfp521 in B-cell tumorigenesis was indicated by the finding that E2A-PBX1 cKI, Zfp521 transgenic compound mice developed B-lineage ALL. Moreover, upregulation of ZNF521, the human counterpart of Zfp521, was found in several human leukemic cell lines bearing t(1;19). These results indicate that E2A-PBX1 cooperates with additional gene alterations to develop ALL. Among them, enhanced expression of ZNF521 may play a clinically relevant role in E2A fusion genes to develop B-lineage ALL.

Brief report: Loss of p15Ink4b accelerates development of myeloid neoplasms in Nup98-HoxD13 transgenic mice.

Homeostasis of hematopoietic stem and progenitor cells is a tightly regulated process. The disturbance of the balance in the hematopoietic progenitor pool can result in favorable conditions for development of diseases such as myelodysplastic syndromes and leukemia. It has been shown recently that mice lacking p15Ink4b have skewed differentiation of common myeloid progenitors toward the myeloid lineage at the expense of erythroid progenitors. The lack of p15INK4B expression in human leukemic blasts has been linked to poor prognosis and increased risk of myelodysplastic syndromes transformation to acute myeloid leukemia. However, the role of p15Ink4b in disease development is just beginning to be elucidated. This study examines the collaboration of the loss of p15Ink4b with Nup98-HoxD13 translocation in the development of hematological malignancies in a mouse model. Here, we report that loss of p15Ink4b collaborates with Nup98-HoxD13 transgene in the development of predominantly myeloid neoplasms, namely acute myeloid leukemia, myeloproliferative disease, and myelodysplastic syndromes. This mouse model could be a very valuable tool for studying p15Ink4b function in tumorigenesis as well as preclinical drug testing.

Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras.

The cascade comprising Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) is a therapeutic target in human cancers with deregulated Ras signalling, which includes tumours that have inactivated the Nf1 tumour suppressor. Nf1 encodes neurofibromin, a GTPase-activating protein that terminates Ras signalling by stimulating hydrolysis of Ras-GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed because of outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4 and Mapk14, which encodes p38alpha. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance.

PIM1 gene cooperates with human BCL6 gene to promote the development of lymphomas.

Diffuse large B-cell lymphomas in humans are associated with chromosomal rearrangements (∼40%) and/or mutations disrupting autoregulation (∼16%) involving the BCL6 gene. Studies of lymphoma development in humans and mouse models have indicated that lymphomagenesis evolves through the accumulation of multiple genetic alterations. Based on our prior studies, which indicated that carcinogen-induced DNA mutations enhance the incidence of lymphomas in our mouse model expressing a human BCL6 transgene, we hypothesized that mutated genes are likely to play an important cooperative role in BCL6-associated lymphoma development. We used retroviral insertional mutagenesis in an effort to identify which genes cooperate with BCL6 in lymphomagenesis in our BCL6 transgenic mice. We identified PIM1 as the most frequently recurring cooperating gene in our murine BCL6-associated lymphomas (T- and B-cell types), and we observed elevated levels of PIM1 mRNA and protein expression in these neoplasms. Further, immunohistochemical staining, which was performed in 20 randomly selected BCL6-positive human B- and T-cell lymphomas, revealed concurrent expression of BCL6 and PIM1 in these neoplasms. As PIM1 encodes a serine/threonine kinase, PIM1 kinase inhibition may be a promising therapy for BCL6/PIM1-positive human lymphomas.

Stress-induced phosphorylation of Thr486 in c-Myb by p38 mitogen-activated protein kinases attenuates conjugation of SUMO-2/3.

c-Myb plays an essential role in regulation of properly balanced hematopoiesis through transcriptional regulation of genes directly controlling cellular processes such as proliferation, differentiation, and apoptosis. The transcriptional activity and protein levels of c-Myb are strictly controlled through post-translational modifications such as phosphorylation, acetylation, ubiquitination, and SUMOylation. Conjugation of small ubiquitin-like modifier (SUMO) proteins has been shown to suppress the transcriptional activity of c-Myb. SUMO-1 modifies c-Myb under physiological conditions, whereas SUMO-2/3 conjugation was reported in cells under stress. Because stress also activates several cellular protein kinases, we investigated whether phosphorylation of c-Myb changes in stressed cells and whether a mutual interplay exists between phosphorylation and SUMOylation of c-Myb. Here we show that several types of environmental stress induce a rapid change in c-Myb phosphorylation. Interestingly, the phosphorylation of Thr(486), located in close proximity to SUMOylation site Lys(499) of c-Myb, is detected preferentially in nonSUMOylated protein and has a negative effect on stress-induced SUMOylation of c-Myb. Stress-activated p38 MAPKs phosphorylate Thr(486) in c-Myb, attenuate its SUMOylation, and increase its proteolytic turnover. Stressed cells expressing a phosphorylation-deficient T486A mutant demonstrate decreased expression of c-Myb target genes Bcl-2 and Bcl-xL and accelerated apoptosis because of increased SUMOylation of the mutant protein. These results suggest that phosphorylation-dependent modulation of c-Myb SUMOylation may be important for proper response of cells to stress. In summary, we have identified a novel regulatory interplay between phosphorylation and SUMOylation of c-Myb that regulates its activity in stressed cells.

GFI1B, EVI5, MYB--additional genes that cooperate with the human BCL6 gene to promote the development of lymphomas.

The BCL6 gene, which is expressed in certain B- and T-cell human lymphomas, is involved with chromosomal rearrangements and mutations in a number of these neoplasms. Lymphomagenesis is believed to evolve through a multi-step accumulation of genetic alterations in these tumors. We used retroviral insertional mutagenesis in transgenic mice expressing the human BCL6 transgene in order to identify genes that cooperate with BCL6 during lymphomatous transformation. We previously reported PIM1 as the most frequently recurring cooperating gene in this model. We now report three newly identified cooperating genes-GFI1B, EVI5, and MYB-that we identified in the lymphomas of retroviral-injected BCL6 transgenic mice (but not in retroviral-injected non-transgenic controls); mRNA and protein expression of GFI1B and EVI5 were decreased in the murine tumors, whereas MYB mRNA and protein expression were increased or decreased. These findings correlated with protein expression in human lymphomas, both B- and T-cell. Improved therapy of lymphomas may necessitate the development of combinations of drugs that target the alterations specific to each neoplasm.

The tumor suppressor p15Ink4b regulates the differentiation and maturation of conventional dendritic cells.

The tumor suppressor p15Ink4b is frequently inactivated by methylation in acute myeloid leukemia and premalignant myeloid disorders. Dendritic cells (DCs) as potent APCs play critical regulatory roles in antileukemic immune responses. In the present study, we investigated whether p15Ink4b can function as modulator of DC development. The expression of p15Ink4b is induced strongly during differentiation and activation of DCs, and its loss resulted in significant quantitative and qualitative impairments of conventional DC (cDC) development. Accordingly, ex vivo-generated BM-derived DCs from p15Ink4b-knockout mice express significantly decreased levels of the antigen-presenting (MHC II) and costimulatory (CD80 and CD86) molecules and have impaired immunostimulatory functions, such as antigen uptake and T-cell stimulation. Reexpression of p15Ink4b in progenitors restored these defects, and confirmed a positive role for p15Ink4b during cDC differentiation and maturation. Furthermore, we have shown herein that p15Ink4b expression increases phosphorylation of Erk1/Erk2 kinases, which leads to an elevated activity of the PU.1 transcription factor. In conclusion, our results establish p15Ink4b as an important modulator of cDC development and implicate a novel function for this tumor suppressor in the regulation of adaptive immune responses.

Sox4 cooperates with CREB in myeloid transformation.

The cAMP response element-binding protein (CREB) is a nuclear transcription factor that is critical for normal and neoplastic hematopoiesis. Previous studies have demonstrated that CREB is a proto-oncogene whose overexpression promotes cellular proliferation in hematopoietic cells. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative disease with splenomegaly and aberrant myelopoiesis. However, CREB overexpressing mice do not spontaneously develop acute myeloid leukemia. In this study, we used retroviral insertional mutagenesis to identify genes that accelerate leukemia in CREB transgenic mice. Our mutagenesis screen identified several integration sites, including oncogenes Gfi1, Myb, and Ras. The Sox4 transcription factor was identified by our screen as a gene that cooperates with CREB in myeloid leukemogenesis. We show that the transduction of CREB transgenic mouse bone marrow cells with a Sox4 retrovirus increases survival and self-renewal of cells in vitro. Furthermore, leukemic blasts from the majority of acute myeloid leukemia patients have higher CREB, phosphorylated CREB, and Sox 4 protein expression. Sox4 transduction of mouse bone marrow cells results in increased expression of CREB target genes. We also demonstrate that CREB is a direct target of Sox4 by chromatin immunoprecipitation assays. These results indicate that Sox4 and CREB cooperate and contribute to increased proliferation of hematopoietic progenitor cells.

An insertional mutagenesis screen identifies genes that cooperate with Mll-AF9 in a murine leukemogenesis model.

Patients with a t(9;11) translocation (MLL-AF9) develop acute myeloid leukemia (AML), and while in mice the expression of this fusion oncogene also results in the development of myeloid leukemia, it is with long latency. To identify mutations that cooperate with Mll-AF9, we infected neonatal wild-type (WT) or Mll-AF9 mice with a murine leukemia virus (MuLV). MuLV-infected Mll-AF9 mice succumbed to disease significantly faster than controls presenting predominantly with myeloid leukemia while infected WT animals developed predominantly lymphoid leukemia. We identified 88 candidate cancer genes near common sites of proviral insertion. Analysis of transcript levels revealed significantly elevated expression of Mn1, and a trend toward increased expression of Bcl11a and Fosb in Mll-AF9 murine leukemia samples with proviral insertions proximal to these genes. Accordingly, FOSB and BCL11A were also overexpressed in human AML harboring MLL gene translocations. FOSB was revealed to be essential for growth in mouse and human myeloid leukemia cells using shRNA lentiviral vectors in vitro. Importantly, MN1 cooperated with Mll-AF9 in leukemogenesis in an in vivo BM viral transduction and transplantation assay. Together, our data identified genes that define transcription factor networks and important genetic pathways acting during progression of leukemia induced by MLL fusion oncogenes.

Concise review: erythroid versus myeloid lineage commitment: regulating the master regulators.

Developmental processes, like blood formation, are orchestrated by transcriptional networks. Those transcriptional networks are highly responsive to various environmental stimuli and affect common precursors resulting in increased production of cells of the erythroid lineage or myeloid lineage (granulocytes, neutrophils, and macrophages). A significant body of knowledge has accumulated describing transcription factors that drive differentiation of these two major cellular pathways, in particular the antagonistic master regulators such as GATA-1 and PU.1. However, little is known about factors that work upstream of master regulators to enhance differentiation toward one lineage. These functions become especially important under various stress conditions like sudden loss of red blood cells or pathogen infection. This review describes recent studies that begin to provide evidence for such factors. An increased understanding of factors regulating cellular commitment will advance our understanding of the etiology of diseases like anemia, cancer, and possibly other blood related disorders.

p15Ink4b Functions in determining hematopoietic cell fates: implications for its role as a tumor suppressor.

The p15Ink4b gene is frequently hypermethylated in myeloid neoplasia and has been demonstrated to be a tumor suppressor. Since it is a member of the INK4b family of cyclin-dependent kinase inhibitors, it was initially presumed that its loss in leukemic blasts caused a dysregulation of the cell cycle. However, animal model experiments over the last several years have produced a very different picture of how p15Ink4b functions in hematopoietic cells and how its loss contributes to myelodysplastic syndrome and myeloid leukemia. It is clear now, that in early hematopoietic progenitors, p15Ink4b functions outside of its canonical role as a cell cycle inhibitor. Its functions are involved in signal transduction and influence the development of erythroid, monocytic and dendritic cells.

Three murine leukemia virus integration regions within 100 kilobases upstream of c-myb are proximal to the 5' regulatory region of the gene through DNA looping.

Retroviruses integrated into genomic DNA participate in long-range gene activation from as far away as several hundred kilobases. Hypotheses have been put forth to account for these phenomena, but data have not been provided to support a physical mechanism that explains long-range activation. In murine leukemia virus-induced myeloid leukemia in mice, integrated proviruses have been found upstream of c-myb in three regions, named Mml1, Mml2, and Mml3 (25, 50, and 70 kb upstream, respectively). The transcription factor c-Myb is an oncogene whose dysregulation and/or mutation can lead to human leukemia. We hypothesized that the murine c-myb upstream region contains regulatory elements accessed by the retrovirus. To identify regulatory sites in the murine c-myb upstream region, we looked by chromatin immunoprecipitation with microarray technology (ChIP-on-chip) for histone modifications implicating gene activation in normal cells. H3K4me3, H3K4me1, and H3K9/14ac were enriched at Mml1 and/or Mml2 in the myeloblastic cell line M1, which expresses c-myb. The enrichment of all of these histone marks decreased with differentiation-induced downregulation of the gene in M1 cells but increased and spread in tumor cells containing integrated provirus. Importantly, using chromosome conformation capture (3C)-quantitative PCR assays, interactions between the 5' region, including the promoter and all Mml sites (Mml1, Mml2, and Mml3), were detected due to DNA looping in M1 cells and tumor cells with provirus in Mml1, Mml2, or Mml3. Therefore, our study provides a new mechanism of retrovirus insertional mutagenesis whereby spatial chromatin organization allows distally located provirus, with its own enhancer elements, to access the 5' regulatory region of the gene.

Hematopoiesis and leukemogenesis in mice expressing oncogenic NrasG12D from the endogenous locus.

NRAS is frequently mutated in hematologic malignancies. We generated Mx1-Cre, Lox-STOP-Lox (LSL)-Nras(G12D) mice to comprehensively analyze the phenotypic, cellular, and biochemical consequences of endogenous oncogenic Nras expression in hematopoietic cells. Here we show that Mx1-Cre, LSL-Nras(G12D) mice develop an indolent myeloproliferative disorder but ultimately die of a diverse spectrum of hematologic cancers. Expressing mutant Nras in hematopoietic tissues alters the distribution of hematopoietic stem and progenitor cell populations, and Nras mutant progenitors show distinct responses to cytokine growth factors. Injecting Mx1-Cre, LSL-Nras(G12D) mice with the MOL4070LTR retrovirus causes acute myeloid leukemia that faithfully recapitulates many aspects of human NRAS-associated leukemias, including cooperation with deregulated Evi1 expression. The disease phenotype in Mx1-Cre, LSL-Nras(G12D) mice is attenuated compared with Mx1-Cre, LSL-Kras(G12D) mice, which die of aggressive myeloproliferative disorder by 4 months of age. We found that endogenous Kras(G12D) expression results in markedly elevated Ras protein expression and Ras-GTP levels in Mac1(+) cells, whereas Mx1-Cre, LSL-Nras(G12D) mice show much lower Ras protein and Ras-GTP levels. Together, these studies establish a robust and tractable system for interrogating the differential properties of oncogenic Ras proteins in primary cells, for identifying candidate cooperating genes, and for testing novel therapeutic strategies.

Mutant Ikzf1, KrasG12D, and Notch1 cooperate in T lineage leukemogenesis and modulate responses to targeted agents.

Mice that accurately model the genetic diversity found in human cancer are valuable tools for interrogating disease mechanisms and investigating novel therapeutic strategies. We performed insertional mutagenesis with the MOL4070LTR retrovirus in Mx1-Cre, Kras(G12D) mice and generated a large cohort of T lineage acute lymphoblastic leukemias (T-ALLs). Molecular analysis infers that retroviral integration within Ikzf1 is an early event in leukemogenesis that precedes Kras(G12D) expression and later acquisition of somatic Notch1 mutations. Importantly, biochemical analysis uncovered unexpected heterogeneity, which suggests that Ras signaling networks are remodeled during multistep tumorigenesis. We tested tumor-derived cell lines to identify biomarkers of therapeutic response to targeted inhibitors. Whereas all T-ALLs tested were sensitive to a dual-specificity phosphoinosityl 3-kinase/mammalian target of rapamycin inhibitor, biochemical evidence of Notch1 activation correlated with sensitivity to gamma-secretase inhibition. In addition, Kras(G12D) T-ALLs were more responsive to a MAP/ERK kinase inhibitor in vitro and in vivo. Together, these studies identify a genetic pathway involving Ikzf1, Kras(G12D), and Notch1 in T lineage leukemogenesis, reveal unexpected diversity in Ras-regulated signaling networks, and define biomarkers of drug responses that may inform treatment strategies.

Signatures of polycomb repression and reduced H3K4 trimethylation are associated with p15INK4b DNA methylation in AML.

DNA hypermethylation of the p15INK4b tumor suppressor gene is commonly observed in acute myeloid leukemia (AML). Repressive histone modifications and their associated binding proteins have been implicated in the regulation of DNA methylation and the transcriptional repression of genes with DNA methylation. We have used high-density chromatin immunoprecipitation-on-chip to determine the histone modifications that normally regulate p15INK4b expression in AML cells and how these marks are altered in cells that have p15INK4b DNA methylation. In AML patient blasts without p15INK4b DNA methylation, a bivalent pattern of active (H3K4me3) and repressive (H3K27me3) modifications exist at the p15INK4b promoter. AML patient blasts with p15INK4b DNA methylation lose H3K4me3 at p15INK4b and become exclusively marked by H3K27me3. H3K27me3, as well as EZH2, extends throughout p14ARF and p16INK4a, indicating that polycomb repression of p15INK4b is a common feature in all AML blasts irrespective of the DNA methylation status of the gene. Reactivation of p15INK4b expression in AML cell lines and patient blasts using 5-aza-2'-deoxycytidine (decitabine) and trichostatin A increased H3K4me3 and maintained H3K27me3 enrichment at p15INK4b. These data indicate that AML cells with p15INK4b DNA methylation have an altered histone methylation pattern compared with unmethylated samples and that these changes are reversible by epigenetic drugs.

Myeloid-specific inactivation of p15Ink4b results in monocytosis and predisposition to myeloid leukemia.

Inactivation of p15INK4b, an inhibitor of cyclin-dependent kinases, through DNA methylation is one of the most common epigenetic abnormalities in myeloid leukemia. Although this suggests a key role for this protein in myeloid disease suppression, experimental evidence to support this has not been reported. To address whether this event is critical for premalignant myeloid disorders and leukemia development, mice were generated that have loss of p15Ink4b specifically in myeloid cells. The p15Ink4b(fl/fl)-LysMcre mice develop nonreactive monocytosis in the peripheral blood accompanied by increased numbers of myeloid and monocytic cells in the bone marrow resembling the myeloproliferative form of chronic myelomonocytic leukemia. Spontaneous progression from chronic disease to acute leukemia was not observed. Nevertheless, MOL4070LTR retrovirus integrations provided cooperative genetic mutations resulting in a high frequency of myeloid leukemia in knockout mice. Two common retrovirus insertion sites near c-myb and Sox4 genes were identified, and their transcript up-regulated in leukemia, suggesting a collaborative role of their protein products with p15Ink4b-deficiency in promoting malignant disease. This new animal model demonstrates experimentally that p15Ink4b is a tumor suppressor for myeloid leukemia, and its loss may play an active role in the establishment of preleukemic conditions.