Michael L. Cleary

Decreased lesion formation in CCR2-/- mice reveals a role for chemokines in the initiation of atherosclerosis

Chemokines are proinflammatory cytokines that function in leukocyte chemoattraction and activation and have recently been shown to block the HIV-1 infection of target cells through interactions with chemokine receptors,. In addition to their function in viral disease, chemokines have been implicated in the pathogenesis of atherosclerosis. Expression of the CC chemokine monocyte chemoattractant protein-1 (MCP-1) is upregulated in human atherosclerotic plaques,, in arteries of primates on a hypercholesterolaemic diet and in vascular endothelial and smooth muscle cells exposed to minimally modified lipids,. To determine whether MCP-1 is causally related to the development of atherosclerosis, we generated mice that lack CCR2, the receptor for MCP-1 (ref. 7), and crossed them with apolipoprotein (apo) E-null mice which develop severe atherosclerosis. Here we show that the selective absence of CCR2 decreases lesion formation markedly in apoE−/− mice but has no effect on plasma lipid or lipoprotein concentrations. These data reveal a role for MCP-1 in the development of early atherosclerotic lesions and suggest that upregulation of this chemokine by minimally oxidized lipids is an important link between hyperlipidaemia and fatty streak formation.

Rb targets histone H3 methylation and HP1 to promoters

In eukaryotic cells the histone methylase SUV39H1 and the methyl-lysine binding protein HP1 functionally interact to repress transcription at heterochromatic sites. Lysine 9 of histone H3 is methylated by SUV39H1 (ref. 2), creating a binding site for the chromo domain of HP1 (refs 3, 4). Here we show that SUV39H1 and HP1 are both involved in the repressive functions of the retinoblastoma (Rb) protein. Rb associates with SUV39H1 and HP1 in vivo by means of its pocket domain. SUV39H1 cooperates with Rb to repress the cyclin E promoter, and in fibroblasts that are disrupted for SUV39, the activity of the cyclin E and cyclin A2 genes are specifically elevated. Chromatin immunoprecipitations show that Rb is necessary to direct methylation of histone H3, and is necessary for binding of HP1 to the cyclin E promoter. These results indicate that the SUV39H1–HP1 complex is not only involved in heterochromatic silencing but also has a role in repression of euchromatic genes by Rb and perhaps other co-repressor proteins.

Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factor

The gene (E2A) for enhancer binding transcription factors E12 and E47 maps to the t(1;19) chromosomal translocation breakpoint in pre-B cell leukemias. Altered E2A transcripts lacking sequences coding for the helix-loop-helix DNA binding motif were detected in several t(1;19)-carrying cell lines. Fusion cDNAs that crossed the t(1;19) breakpoint were cloned and shown to code for an 85 kd protein consisting of the amino-terminal two-thirds of E2A fused to a chromosome 1-derived protein. The fusion protein has the features of a chimeric transcription factor in which the DNA binding domain of E2A is replaced by the putative DNA binding domain of a homeoprotein from chromosome 1 for which the name Prl (pre-B cell leukemia) is proposed. Identical E2A-prl mRNA junctions were detected by PCR in three t(1;19)-carrying cell lines, indicating that the fusion transcripts and predicted chimeric protein are a consistent feature of this translocation.

Molecular Analysis of the T(14;18) Chromosomal Translocation in Malignant Lymphomas

One of the most common karyotypic abnormalities is the t(14;18) translocation, which is found in many lymphomas that have a characteristic follicular morphology. Recent molecular studies have shown that this chromosomal translocation results in the juxtaposition of the candidate proto-oncogene bcl-2 (B-cell leukemia-lymphoma) on chromosome 18 with the immunoglobulin heavy-chain locus on chromosome 14. However, because performing accurate cytogenetic studies in solid hematolymphoid neoplasms is difficult, knowledge of the prevalence of the t(14;18) translocation and, by association, the extent of bcl-2 involvement in human lymphomas is limited. We used a number of chromosome-18 DNA probes to analyze various subtypes of Hodgkins and non-Hodgkins lymphomas and test for structural abnormalities near or within the bcl-2 gene. Molecular features of the t(14;18) translocation were found in virtually all follicular neoplasms and about 28 percent of diffuse large-cell lymphomas. No changes in bcl-2 were found in several other subtypes of Hodgkins and non-Hodgkins lymphomas, including those previously suggested to originate from follicular-center cells and those about which cytogenetic data have been difficult to obtain. Our findings suggest a close pathogenetic relation between bcl-2 and a large group of non-Hodgkins lymphomas, both with and without a follicular morphology. The methods employed in this study may be useful in improving the accuracy of diagnosis and subclassification of malignant lymphomas.

Leukemia Proto-Oncoprotein MLL Forms a SET1-Like Histone Methyltransferase Complex with Menin To Regulate Hox Gene Expression

ABSTRACT MLL (for mixed-lineage leukemia) is a proto-oncogene that is mutated in a variety of human leukemias. Its product, a homolog of Drosophila melanogaster trithorax, displays intrinsic histone methyltransferase activity and functions genetically to maintain embryonic Hox gene expression. Here we report the biochemical purification of MLL and demonstrate that it associates with a cohort of proteins shared with the yeast and human SET1 histone methyltransferase complexes, including a homolog of Ash2, another Trx-G group protein. Two other members of the novel MLL complex identified here are host cell factor 1 (HCF-1), a transcriptional coregulator, and the related HCF-2, both of which specifically interact with a conserved binding motif in the MLLN (p300) subunit of MLL and provide a potential mechanism for regulating its antagonistic transcriptional properties. Menin, a product of the MEN1 tumor suppressor gene, is also a component of the 1-MDa MLL complex. Abrogation of menin expression phenocopies loss of MLL and reveals a critical role for menin in the maintenance of Hox gene expression. Oncogenic mutant forms of MLL retain an ability to interact with menin but not other identified complex components. These studies link the menin tumor suppressor protein with the MLL histone methyltransferase machinery, with implications for Hox gene expression in development and leukemia pathogenesis.

Molecular mechanisms of leukemogenesis mediated by MLL fusion proteins

The MLL (Mixed Lineage Leukemia) gene is a common target for chromosomal translocations associated with human acute leukemias. These translocations result in a gain of MLL function by generating novel chimeric proteins containing the amino-terminus of MLL fused in-frame with one of 30 distinct partner proteins. Structure/function studies using an in vitro myeloid progenitor immortalization assay have revealed that at least four nuclear partner proteins contribute transcriptional effector properties to MLL to produce a range of chimeric transcription factors with leukemogenic potential. Mouse models suggest that expression of an MLL fusion protein is necessary but not sufficient for leukemogenesis. Interestingly, whilst all MLL fusion proteins tested so far phenocopy each other with respect to in vitro immortalization, the latency period required for the onset of acute leukemia in vivo is variable and partner protein dependent. We discuss potential mechanisms that may account for the ability of distinct MLL fusion proteins to promote short or long latency leukemogenesis.

Immortalization and leukemic transformation of a myelomonocytic precursor by retrovirally transduced HRX-ENL

A subset of chromosomal translocations in acute leukemias results in the fusion of the trithorax‐related protein HRX with a variety of heterologous proteins. In particular, leukemias with the t(11;19)(q23;p13.3) translocation express HRX–ENL fusion proteins and display features which suggest the malignant transformation of myeloid and/or lymphoid progenitor(s). To characterize directly the potential transforming effects of HRX–ENL on primitive hematopoietic precursors, the fusion cDNA was transduced by retroviral gene transfer into cell populations enriched in hematopoietic stem cells. The infected cells had a dramatically enhanced potential to generate myeloid colonies with primitive morphology in vitro. Primary colonies could be replated for at least three generations in vitro and established primitive myelomonocytic cell lines upon transfer into suspension cultures supplemented with interleukin‐3 and stem cell factor. Immortalized cells contained structurally intact HRX–ENL proviral DNA and expressed a low‐level of HRX–ENL mRNA. In contrast, wild‐type ENL or a deletion mutant of HRX–ENL lacking the ENL component did not demonstrate in vitro transforming capabilities. Immortalized cells or enriched primary hematopoietic stem cells transduced with HRX–ENL induced myeloid leukemias in syngeneic and SCID recipients. These studies demonstrate a direct role for HRX–ENL in the immortalization and leukemic transformation of a myeloid progenitor and support a gain‐of‐function mechanism for HRX–ENL‐mediated leukemogenesis.

The Menin Tumor Suppressor Protein Is an Essential Oncogenic Cofactor for MLL-Associated Leukemogenesis

The Mixed-Lineage Leukemia (MLL) protein is a histone methyltransferase that is mutated in clinically and biologically distinctive subsets of acute leukemia. MLL normally associates with a cohort of highly conserved cofactors to form a macromolecular complex that includes menin, a product of the MEN1 tumor suppressor gene, which is mutated in heritable and sporadic endocrine tumors. We demonstrate here that oncogenic MLL fusion proteins retain an ability to stably associate with menin through a high-affinity, amino-terminal, conserved binding motif and that this interaction is required for the initiation of MLL-mediated leukemogenesis. Furthermore, menin is essential for maintenance of MLL-associated but not other oncogene induced myeloid transformation. Acute genetic ablation of menin reverses aberrant Hox gene expression mediated by MLL-menin promoter-associated complexes, and specifically abrogates the differentiation arrest and oncogenic properties of MLL-transformed leukemic blasts. These results demonstrate that a human oncoprotein is critically dependent on direct physical interaction with a tumor suppressor protein for its oncogenic activity, validate a potential target for molecular therapy, and suggest central roles for menin in altered epigenetic functions underlying the pathogenesis of hematopoietic cancers.

Clonal rearrangements of T-cell receptor genes in mycosis fungoides and dermatopathic lymphadenopathy.

Histologic diagnosis of mycosis fungoides may be difficult, especially in lymph nodes that show changes frequently associated with chronic skin disease. As an alternative approach to diagnosis, we have analyzed the configuration of DNA for the beta T-cell receptor genes in biopsy tissues from 14 patients with mycosis fungoides. Clonal rearrangements of these genes were found in each specimen tht contained histologically unambiguous mycosis fungoides. Clonal rearrangements were also found in seven of nine lymph nodes removed from patients with mycosis fungoides and considered histologically to contain only benign lymphadenopathy. Matching rearrangements of beta T-cell receptor genes were detected in benign lymph nodes and histologically involved tissues when paired specimens were available from the same cases. Our findings provide molecular evidence for the clonal T-cell origin of mycosis fungoides and indicate the high incidence of extracutaneous disease in patients with palpable lymphadenopathy. In addition, this study demonstrates that the detection of rearranged T-cell receptor genes can be a sensitive and practical method for the diagnosis and characterization of T-cell neoplasms.

Menin Critically Links MLL Proteins with LEDGF on Cancer-Associated Target Genes

Menin displays the unique ability to either promote oncogenic function in the hematopoietic lineage or suppress tumorigenesis in the endocrine lineage; however, its molecular mechanism of action has not been defined. We demonstrate here that these discordant functions are unified by menins ability to serve as a molecular adaptor that physically links the MLL (mixed-lineage leukemia) histone methyltransferase with LEDGF (lens epithelium-derived growth factor), a chromatin-associated protein previously implicated in leukemia, autoimmunity, and HIV-1 pathogenesis. LEDGF is required for both MLL-dependent transcription and leukemic transformation. Conversely, a subset of menin mutations in multiple endocrine neoplasia type 1 patients abrogate interaction with LEDGF while preserving MLL interaction but nevertheless compromise MLL/menin-dependent functions. Thus, LEDGF critically associates with MLL and menin at the nexus of transcriptional pathways that are recurrently targeted in diverse diseases.