Richard Koller

A Moloney murine leukemia virus-based retrovirus with 4070A long terminal repeat sequences induces a high incidence of myeloid as well as lymphoid neoplasms.

ABSTRACT Retroviruses can be used to accelerate hematopoietic cancers predisposed to neoplastic disease by prior genetic manipulations such as in transgenic or knockout mice. The virus imparts a second neoplastic “hit,” providing evidence that the initial hit is transforming. In the present study, a unique retrovirus was developed that can induce a high incidence of myeloid disease and has a broad host range. This agent is a Moloney murine leukemia virus (Mo-MuLV)-based virus that has most of the U3 region of the long terminal repeat (LTR) replaced with that of retrovirus 4070A. Like Mo-MuLV, this virus, called MOL4070LTR, is NB-tropic and not restricted by Fv1 allelles. MOL4070LTR causes myeloid leukemias in ca. 50% of mice, a finding in contrast to Mo-MuLV, which induces almost exclusively lymphoid disease. The data suggest that the LTR of the 4070A virus expands the tissue tropism of the disease to the myeloid lineage. Interesting, MCF recombinant envelope was expressed in the lymphoid but not the myeloid neoplasms of BALB/c mice. This retrovirus has the potential for accelerating myeloid disease in genetically engineered mice.

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

The Ink4b gene (Cdkn2b) encodes p15Ink4b, 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. p15Ink4b 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.

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.

Identification of Zfp521 / ZNF521 as a cooperative gene for E2A-HLF to develop acute B-lineage leukemia

E2A-hepatic leukemia factor (HLF) is a chimeric protein found in B-lineage acute lymphoblastic leukemia (ALL) with t(17;19). To analyze the leukemogenic process and to create model mice for t(17;19)-positive leukemia, we generated inducible knock-in (iKI) mice for E2A-HLF. Despite the induced expression of E2A-HLF in the hematopoietic tissues, no disease was developed during the long observation period, indicating that additional gene alterations are required to develop leukemia. To elucidate this process, E2A-HLF iKI and control littermates were subjected to retroviral insertional mutagenesis. Virus infection induced acute leukemias in E2A-HLF iKI mice with higher morbidity and mortality than in control mice. Inverse PCR detected three common integration sites specific for E2A-HLF iKI leukemic mice, which induced overexpression of zinc-finger transcription factors: growth factor independent 1 (Gfi1), zinc-finger protein subfamily 1A1 isoform a (Zfp1a1, also known as Ikaros) and zinc-finger protein 521 (Zfp521). Interestingly, tumors with Zfp521 integration exclusively showed B-lineage ALL, which corresponds to the phenotype of human t(17;19)-positive leukemia. In addition, ZNF521 (human counterpart of Zfp521) was found to be overexpressed in human leukemic cell lines harboring t(17;19). Moreover, both iKI for E2A-HLF and transgenic for Zfp521 mice frequently developed B-lineage ALL. These results indicate that a set of transcription factors promote leukemic transformation of E2A-HLF-expressing hematopoietic progenitors and suggest that aberrant expression of Zfp521/ZNF521 may be clinically relevant to t(17;19)-positive B-lineage ALL.

Overexpression/enhanced kinase activity of BCR/ABL and altered expression of Notch1 induced acute leukemia in p210BCR/ABL transgenic mice

Chronic myelogenous leukemia (CML) is a hematopoietic disorder, which begins as indolent chronic phase but inevitably progresses to fatal blast crisis. p210BCR/ABL, a constitutively active tyrosine kinase, is responsible for disease initiation but molecular mechanism(s) underlying disease evolution remains largely unknown. To explore this process, we employed retroviral insertional mutagenesis to CML-exhibiting p210BCR/ABL transgenic mice (Tg). Virus infection induced acute lymphoblastic leukemia (ALL) in p210BCR/ABL Tg with a higher frequency and in a shorter latency than wild-type littermates, and inverse PCR detected two retrovirus common integration sites (CISs) in p210BCR/ABL Tg tumors. Interestingly, one CIS was the transgene itself, where retrovirus integrations induced upregulation of p210BCR/ABL and production of truncated BCR/ABL with an enhanced kinase activity. Another CIS was Notch1 gene, where retrovirus integrations resulted in overexpression of Notch1 and generation of Notch1 lacking the C-terminal region (Notch1ΔC) associated with stable expression of its activated product, C-terminal-truncated Notch intracellular domain (NICDΔC). In addition, generation of Tg for both p210BCR/ABL and Notch1ΔC developed ALL in a shortened period with Stat5 activation, demonstrating the cooperative oncogenicity of Notch1ΔC/NICDΔC with p210BCR/ABL involving Stat5-mediated pathway. These results demonstrated that overexpression/enhanced kinase activity of BCR/ABL and altered expression of Notch1 induces acute leukemia in a transgenic model for CML.

Deregulated c-Myb expression in murine myeloid leukemias prevents the up-regulation of p15 INK4b normally associated with differentiation

Deregulated expression of the proto-oncogene c-myb, which results from provirus integration, is thought to be responsible for transformation in a set of murine leukemia virus (MuLV)-induced myeloid leukemias (MML). We reported recently that this transcription factor promotes proliferation by directly transactivating c-myc and inhibits cell death through its up-regulation of Bcl-2 (Schmidt et al., 2000). To understand more about how these cells become transformed we looked at how they deal with cellular pathways inducing growth arrest. Specifically, we were interested in the expression of the tumor suppressor gene Cdkn2b (p15INK4b) in MML because this gene is expressed during myeloid differentiation and its inactivation by methylation has been shown to be important for the development of human acute myeloid leukemia. mRNA levels for p15INK4b and another INK4 gene p16INK4a were examined in monocytic Myb tumors and were compared with expression of the same genes in c-myc transformed monocytic tumors that do not express c-Myb. The Cdkn2a (p16INK4a) gene was generally not expressed in either tumor type, an observation explained by methylation or deletion in the promoter region. Although Cdkn2b (p15INK4b) mRNA was expressed in the Myc tumors, many transcripts were aberrant in size and contained only exon 1. Surprisingly, in the majority of the Myb tumors there was no p15INK4b transcription and neither deletion nor methylation could explain this result. Additional experiments demonstrated that, in the presence of constitutive c-Myb expression, the induction of p15INK4b mRNA that accompanies differentiation of M1 cells to monocytes does not occur. Therefore, the transcriptional regulator c-Myb appears to prevent activation of a growth arrest pathway that normally accompanies monocyte maturation.

CIZ1, a p21Cip1/Waf1-interacting protein, functions as a tumor suppressor in vivo

CIZ1 is a nuclear protein involved in DNA replication and is also implicated in human diseases including cancers. To gain an insight into its function in vivo, we generated mice lacking Ciz1. Ciz1‐deficient (Ciz1 −/−) mice grew without any obvious abnormalities, and Ciz1 −/− mouse embryonic fibroblasts (MEFs) did not show any defects in cell cycle status, cell growth, and DNA damage response. However, Ciz1 −/− MEFs were sensitive to hydroxyurea‐mediated replication stress and susceptible to oncogene‐induced cellular transformation. In addition, Ciz1 −/− mice developed various types of leukemias by retroviral insertional mutagenesis. These results indicate that CIZ1 functions as a tumor suppressor in vivo.

Linkage on chromosome 10 of several murine retroviral integration loci associated with leukaemia.

Mml loci have been identified as provirus integration sites among a subset of monocytic tumours induced by murine leukaemia virus (MuLV) infection of BALB/c and DBA/2 mice. These myeloid leukaemias contain a retrovirus integrated on chromosome 10 in proximity to the c-myb locus; however, c-myb expression was not altered. Detailed physical mapping enabled placement of the retroviral integration sites approximately 25 kb (Mml1), approximately 51 kb (Mml2), and approximately 70 kb (Mml3) upstream of the c-myb locus. Furthermore, the Fti1 (fit-1) locus, a common integration site in feline leukaemia virus-induced T cell lymphomas, was mapped upstream of Mml3. Sequence analysis of Mml1, Mml2 and Mml3 loci (39.6, 16.4 and 5.9 kb, respectively) in conjunction with the BLAST (basic local alignment search tool) homology searches against the expressed sequence tag (EST) database and the use of gene/exon prediction programs revealed potential coding sequences that were not confirmed by Northern analysis or RT-PCR. The sequences between c-myb and Fti1, which were shown to include two potential scaffold/matrix attachment regions (S/MARs), are most likely regulatory in nature. An extended search for transcribed sequences far upstream of Mml3 revealed five genes, four of which were expressed in multiple tissues in mice. These genes could not be linked to tumour formation by the virus but their homologous sequences were found on human chromosome 6, thus allowing extension of the syntenic region on mouse chromosome 10 to approximately 250 kb.

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.