The chromosomal translocation t(4;11) marks infant acute lymphoblastic leukemia associated with a particularly dismal prognosis. The leukemogenic role of the corresponding fusion gene MLL-AF4 is not well understood. We show that transient inhibition of MLL-AF4 expression with small interfering RNAs impairs the proliferation and clonogenicity of the t(4; 11 IntroductionChromosomal aberrations giving rise to fusion genes are observed for many different leukemias. 1 Such tumor-specific oncogenes would be promising targets for new therapeutic approaches with improved specificity if these oncogenes were important for maintaining the leukemic phenotype. However, in contrast to the development of leukemia, a central role for leukemic persistence has only been established for a minority of fusion genes.The mixed-lineage leukemia (MLL) gene located on chromosome 11 band q23 is involved in numerous chromosomal aberrations associated with human leukemia. 2 The most prevalent among those is the translocation t(4;11)(q21;q23), which fuses MLL with the AF4 gene located on chromosome 4 band q21. [3][4][5] This translocation is the hallmark of a high-risk acute lymphoblastic leukemia (ALL) with a particularly poor prognosis in infants. 6 The wild-type MLL gene is a member of the trithorax family and encodes for a 430-kDa protein, which is proteolytically processed into 2 fragments of 300 and 180 kDa heterodimerizing with each other. [7][8][9][10] The MLL protein has a complex structure that includes an AT hook domain for A-T base-pair-rich DNA binding, a metallothionein domain showing homology to DNA methyltransferase, and methyl-binding domain protein 1 (MBD1), a plant homeodomain (PHD) containing zinc fingers and a Su(var)3-9, enhancer of zeste, trithorax (SET) histone methyl transferase domain. 2 MLL and at least some of its leukemic derivatives are involved in mechanisms controlling HOX gene transcription. [11][12][13][14] Moreover, the HOX genes HOXA7 and HOXA9 in combination with the homeotic gene MEIS1 are necessary for the transformation induced by several different MLL fusion genes. 15-17 Such a crucial role has not yet been reported for MLL-AF4. Nevertheless, expression levels of several HOX genes and of MEIS1 are raised in both primary t(4;11) ALL and t(4;11) leukemic cell lines. [18][19][20][21] The AF4 gene encodes a serine/proline-rich protein containing a nuclear localization signal and a guanosine triphosphate (GTP)-binding domain. It localizes to the nucleus 22 and is probably involved in the control of gene transcription. Whereas homozygous inactivation of MLL is embryonally lethal, 23 AF4-deficient mice exhibit imperfect T-cell development and modest alterations in B-cell development. 24 The t(4;11) translocation generates 2 fusion genes, AF4MLL and MLL-AF4. The significance of either fusion gene for leukemogenesis is currently not completely understood. AF4MLL has recently been shown to interfere with ubiquitin-mediated ALL-1 fused gene from chromosome 4 (AF4) degradation and to transform murine embryonic fi...

ObjectivesTo investigate whether monocytes contribute to matrix deposition in systemic sclerosis (SSc) by production of tissue-inhibitor of metalloproteinase-1 (TIMP-1).MethodsMatrix metalloproteinase-1 (MMP-1) and TIMP-1 expression and secretion were measured by qRT-PCR and ELISA in circulating monocytes from patients with SSc, patients with rheumatoid arthritis (RA) and healthy controls (HC) and in healthy monocytes cultured in the presence of SSc or HC serum samples. Production of TIMP-1 was determined in response to a panel of Toll-like receptor (TLR) agonists and MyD88 inhibitory peptide. The functional effect of conditioned media from SSc and HC serum samples or TLR8-stimulated monocytes was studied in an MMP-1 activity assay.ResultsTIMP-1 production by monocytes was upregulated in patients with SSc compared with patients with RA and HC. Incubation of HC monocytes with SSc serum samples resulted in functionally active TIMP-1 production. However, pretreatment with MyD88 inhibitor, but not control peptide, decreased TIMP-1 secretion. TIMP-1 production was significantly stronger when SSc and HC monocytes were stimulated with TLR8 (ssRNA) agonist, but the response was more pronounced in SSc monocytes. TIMP-1 production after TLR stimulation was also strongly reduced in the presence of MyD88 inhibitory peptide or in the monocytes isolated from a patient with a genetic TLR signalling defect. MMP-1 activity was significantly inhibited in media from serum samples or TLR8-stimulated monocytes indicative of functional TIMP activity.ConclusionsThis study demonstrates profibrotic properties of circulating monocytes from patients with SSc and a key role for TLR signalling, particularly TLR8, in TIMP-1 secretion and matrix remodelling.

MLL/AF4 and AML/MTG8 represent two leukemic fusion genes, which are most frequently found in infant acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), respectively. We examined the influence of MLL/AF4 and AML1/MTG8 fusion genes on the expression of TERT coding for the telomerase protein subunit, and subsequently telomerase activity in t(4;11)-positive ALL and t(8;21)-positive cell lines, respectively. MLL/AF4 suppression diminished telomerase activity and expression of TERT. Blocking pro-apoptotic caspase activation in conjunction with MLL/AF4 knockdown enhanced the inhibition of TERT gene expression, which suggests that MLL/AF4 depletion does not reduce TERT expression levels by inducing apoptosis. Knockdown of HOXA7, a direct transcriptional target of MLL/AF4 fusion gene, caused a reduction of telomerase and TERT to an extent similar to that observed with MLL/AF4 suppression. Chromatin immunoprecipitation of SEM cells, using ectopically expressed FLAG-tagged Hoxa7, indicates HOXA7 binding site in the TERT promoter region. Furthermore, suppression of the AML1/MTG8 fusion gene was associated with severely reduced clonogenicity, induction of replicative senescence, impaired TERT expression and accelerated telomere shortening. We thus present findings that show a mechanistic link between leukemic fusion proteins, essential for development and maintenance of leukemia, and telomerase, a key element of both normal and malignant self-renewal.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu) AML1/ETO and POU4F1 synergy drives B-lymphoid gene expression typical of t(8;21) acute myeloid leukemia Leukemia (2012Leukemia ( ) 26, 1131Leukemia ( --1135 doi:10.1038/leu.2011; published online 8 November 2011Cells of the acute myeloid leukemia (AML) t(8;21) subtype express the B-lymphoid lineage marker Paired Box 5 (PAX5), and frequently, but not always, express the characteristically B-lymphoid surface markers CD19 and CD79a. 1 --3 However, mechanisms for activation of this 'B-lineage' program and its potential contribution to the transformation process are unclear.Comprehensive studies of the t(8;21) translocation product AML1/ETO (also known as RUNX1/RUNX1T1 or AML1/MTG8) have identified its sufficiency in inhibition of myeloid gene function and immortalisation of both human and murine progenitors, however AML1/ETO alone is insufficient for leukemogenesis, suggesting it requires co-operating events to drive disease. 4 The POU homeobox gene POU4F1 (also known as BRN3A) is highly expressed in t(8;21) samples, with AML1/ETO appearing both to promote some BRN3A expression and co-operate with its protein product Brn3a to restrict myeloid gene expression. 5,6 An ability of Brn3a shRNA to impair AML1/ETO-dependent immortalisation of murine haematopoietic progenitor cells in vitro suggests an important role for BRN3A in the human disease. 6

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