Distal regulation of c-myb expression during IL-6-induced differentiation in murine myeloid progenitor M1 cells.

The c-Myb transcription factor is a major regulator that controls differentiation and proliferation of hematopoietic progenitor cells, which is frequently deregulated in hematological diseases, such as lymphoma and leukemia. Understanding of the mechanisms regulating the transcription of c-myb gene is challenging as it lacks a typical promoter and multiple factors are involved. Our previous studies identified some distal regulatory elements in the upstream regions of c-myb gene in murine myeloid progenitor M1 cells, but the detailed mechanisms still remain unclear. In the present study, we found that a cell differentiation-related DNase1 hypersensitive site is located at a -28k region upstream of c-myb gene and that transcription factors Hoxa9, Meis1 and PU.1 bind to the -28k region. Circular chromosome conformation capture (4C) assay confirmed the interaction between the -28k region and the c-myb promoter, which is supported by the enrichment of CTCF and Cohesin. Our analysis also points to a critical role for Hoxa9 and PU.1 in distal regulation of c-myb expression in murine myeloid cells and cell differentiation. Overexpression of Hoxa9 disrupted the IL-6-induced differentiation of M1 cells and upregulated c-myb expression through binding of the -28k region. Taken together, our results provide an evidence for critical role of the -28k region in distal regulatory mechanism for c-myb gene expression during differentiation of myeloid progenitor M1 cells.

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.

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.

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.

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.

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.

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.

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.

Loss of Dnmt3b function upregulates the tumor modifier Ment and accelerates mouse lymphomagenesis.

DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b-/- lymphomas, but not in Dnmt3b-/- pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b-/- lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b+/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies.

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.

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.

Retroviral insertional mutagenesis identifies genes that collaborate with NUP98-HOXD13 during leukemic transformation.

The t(2;11)(q31;p15) chromosomal translocation results in a fusion between the NUP98 and HOXD13 genes and has been observed in patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia. We previously showed that expression of the NUP98-HOXD13 (NHD13) fusion gene in transgenic mice results in an invariably fatal MDS; approximately one third of mice die due to complications of severe pancytopenia, and about two thirds progress to a fatal acute leukemia. In the present study, we used retroviral insertional mutagenesis to identify genes that might collaborate with NHD13 as the MDS transformed to an acute leukemia. Newborn NHD13 transgenic mice and littermate controls were infected with the MOL4070LTR retrovirus. The onset of leukemia was accelerated, suggesting a synergistic effect between the NHD13 transgene and the genes neighboring retroviral insertion events. We identified numerous common insertion sites located near protein-coding genes and confirmed dysregulation of a subset of these by expression analyses. Among these genes were Meis1, a known collaborator of HOX and NUP98-HOX fusion genes, and Mn1, a transcriptional coactivator involved in human leukemia through fusion with the TEL gene. Other putative collaborators included Gata2, Erg, and Epor. Of note, we identified a common insertion site that was >100 kb from the nearest coding gene, but within 20 kb of the miR29a/miR29b1 microRNA locus. Both of these miRNA were up-regulated, demonstrating that retroviral insertional mutagenesis can target miRNA loci as well as protein-coding loci. Our data provide new insights into NHD13-mediated leukemogenesis as well as retroviral insertional mutagenesis mechanisms.

Methylation-independent silencing of the tumor suppressor INK4b (p15) by CBFbeta-SMMHC in acute myelogenous leukemia with inv(16).

The tumor suppressor gene INK4b (p15) is silenced by CpG island hypermethylation in most acute myelogenous leukemias (AML), and this epigenetic phenomenon can be reversed by treatment with hypomethylating agents. Thus far, it was not investigated whether INK4b is hypermethylated in all cytogenetic subtypes of AML. A comparison of levels of INK4b methylation in AML with the three most common cytogenetic alterations, inv(16), t(8;21), and t(15;17), revealed a strikingly low level of methylation in all leukemias with inv(16) compared with the other types. Surprisingly, the expression level of INK4b in inv(16)+ AML samples was low and comparable with that of the other subtypes. An investigation into an alternative mechanism of INK4b silencing determined that the loss of INK4b expression was caused by inv(16)-encoded core binding factor beta-smooth muscle myosin heavy chain (CBFbeta-SMMHC). The silencing was manifested in an inability to activate the normal expression of INK4b RNA as shown in vitamin D3-treated U937 cells expressing CBFbeta-SMMHC. CBFbeta-SMMHC was shown to displace RUNX1 from a newly determined CBF site in the promoter of INK4b. Importantly, this study (a) establishes that the gene encoding the tumor suppressor p15(INK4b) is a target of CBFbeta-SMMHC, a finding relevant to the leukemogenesis process, and (b) indicates that, in patients with inv(16)-containing AML, reexpression from the INK4b locus in the leukemia would not be predicted to occur using hypomethylating drugs.

Stress-induced inactivation of the c-Myb transcription factor through conjugation of SUMO-2/3 proteins.

Post-translational modifications, such as phosphorylation, acetylation, ubiquitination, and SUMOylation, play an important role in regulation of the stability and the transcriptional activity of c-Myb. Conjugation of small ubiquitin-like modifier type 1 (SUMO-1) to lysines in the negative regulatory domain strongly suppresses its transcriptional activity. Here we report conjugation of two other members of the SUMO protein family, SUMO-2 and SUMO-3, and provide evidence that this post-translational modification negatively affects transcriptional activity of c-Myb. Conjugation of SUMO-2/3 proteins is strongly enhanced by several different cellular stresses and occurs primarily on two lysines, Lys(523) and Lys(499). These lysines are in the negative regulatory domain of c-Myb and also serve as acceptor sites for SUMO-1. Stress-induced SUMO-2/3 conjugation is very rapid and independent of activation of stress-activated protein kinases of the SAPK and JNK families. PIAS-3 protein was identified as a new c-Myb-specific SUMO-E3 ligase that both catalyzes conjugation of SUMO-2/3 proteins to c-Myb and exerts a negative effect on c-Myb-induced reporter gene activation. Interestingly, co-expression of a SPRING finger mutant of PIAS-3 significantly suppresses SUMOylation of c-Myb under stress. These results argue that PIAS-3 SUMO-E3 ligase plays a critical role in stress-induced conjugation of SUMO-2/3 to c-Myb. We also detected stress-induced conjugation of SUMO-2/3 to c-Myb in hematopoietic cells at the levels of endogenously expressed proteins. Furthermore, according to the negative role of SUMO conjugation on c-Myb capacity, we have observed rapid stress-induced down-regulation of the targets genes c-myc and bcl-2 of c-Myb. Our findings demonstrate that SUMO-2/3 proteins conjugate to c-Myb and negatively regulate its activity in cells under stress.

Interferon beta increases c-Myc proteolysis in mouse monocyte/macrophage leukemia cells.

Growth inhibitory activity of interferons (IFNs) has been attributed to several events. These include rapid induction of cyclin-dependent kinase inhibitors, such as those in the Cip/Kip and Ink 4 families and down-regulation of c-myc mRNA and c-Myc transcriptional activity. Here, we report an additional mechanism, involving regulation of Myc protein levels, through which type 1 IFN may halt proliferation of cells. This was discovered using a cell line which constitutively expresses c-myc from a retrovirus vector and which was reported to have undergone deletion of genes encoding the Ink 4 tumor suppressors p15 and p16. IFNbeta caused a reduction in the steady state level of c-Myc protein by increasing degradation through the 26S proteasome. Our data, as well as that of others, indicate that multiple levels of c-Myc expression can be affected by IFN treatment and this contributes to rapid growth arrest in the G1 phase of the cell cycle.

Activation of c-myb by 5' retrovirus promoter insertion in myeloid neoplasms is dependent upon an intact alternative splice donor site (SD') in gag.

Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associated with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism.

The interferon regulatory factor ICSBP/IRF-8 in combination with PU.1 up-regulates expression of tumor suppressor p15(Ink4b) in murine myeloid cells.

CDKN2B (INK4B), which encodes the cyclin-dependent kinase inhibitor p15(INK4b), is up-regulated by many cytokines found in hematopoietic environments in vivo. In human acute myeloid leukemias (AMLs), it is inactivated with high frequency. To gain insight into the regulatory pathways leading to the normal activation of p15(Ink4b) expression, we examined interferon beta (IFNbeta)-induced transcription. Using reporter gene assays in murine myeloid cells M1, we determined that a 328-bp fragment, located 117 to 443 bp upstream of the translation initiation site, was sufficient to activate transcription. Both the interferon consensus sequence-binding protein/interferon regulatory factor 8 (ICSBP/IRF-8) and PU.1 were able to increase transcription from this region. It was determined that both ICSBP and PU.1 must bind to DNA to form a stable PU.1/ICSBP binding complex. Interestingly, introduction of the ICSBP into ICSBP-null Tot2 cells led to a significant increase in p15(Ink4b) RNA expression. This regulation of the Ink4b promoter is apparently myeloid specific because both ICSBP and PU.1 are myeloid commitment factors. Importantly, this provides a mechanism to explain in part the tumor suppressor activity of ICSBP, since ICSBP-deficient mice develop a chronic myelogenous leukemia (CML)-like disease and a high percentage of human AML and CML lack ICSBP transcripts.

A novel retrovirus provides the cooperating oncogenic event(s) required to demonstrate the tumor suppressor activity of p15Ink4b in myeloid cells in vivo.

Cancer is a multistep process resulting from an accumulation of several genetic changes. The determination of cooperating events in experimental models can help scientists decipher specific neoplastic pathways and place genes with similar functions in complementation groups. In leukemia models, retrovirus tagging is a powerful approach to determine genes that cooperate with oncogenic transgenes or tumor suppressors that have undergone targeted deletion. Experimental models for B and T cell leukemias involving transgenic c-myc were the first to show the utility of retroviral tagging. Here we review these experiments and present examples of new models of myeloid leukemia where retroviruses have collaborated with a transgene [Cbfbeta-MYH111 from Inv(16)] and with loss of a tumor suppressor (Ink4b) mice to induce disease.

Hypermethylation of the Ink4b locus in murine myeloid leukemia and increased susceptibility to leukemia in p15(Ink4b)-deficient mice.

The Ink4b gene (Cdkn2b) encodes p15(Ink4b), a cyclin-dependent kinase inhibitor. It has been implicated in playing a role in the development of acute myeloid leukemia (AML) in man, since it is hypermethylated with high frequency. We provide evidence that the gene is a tumor suppressor for myeloid leukemia in mice. The evidence is twofold: (1) retrovirus-induced myeloid leukemias of the myelomonocytic phenotype were found to have hypermethylation of the 5' CpG island of the Ink4b gene, and this could be correlated with reduced mRNA expression, as demonstrated by TaqMan real-time PCR. p15(Ink4b) mRNA expression in a leukemia cell line, with hypermethylation at the locus, was induced following treatment with 5-aza-2'-deoxycytidine. (2) Targeted deletion of one allele in mice by removal of exon 2 increases their susceptibility to retrovirus-induced myeloid leukemia. Mice deficient in both alleles were not more susceptible to myeloid disease than those deficient in one allele, raising the possibility that there are opposing forces related to the development of myeloid leukemia in Ink4b null mice.