Joerg Faber

Transformation from committed progenitor to leukaemia stem cell initiated by MLL–AF9

Leukaemias and other cancers possess a rare population of cells capable of the limitless self-renewal necessary for cancer initiation and maintenance. Eradication of these cancer stem cells is probably a critical part of any successful anti-cancer therapy, and may explain why conventional cancer therapies are often effective in reducing tumour burden, but are only rarely curative. Given that both normal and cancer stem cells are capable of self-renewal, the extent to which cancer stem cells resemble normal tissue stem cells is a critical issue if targeted therapies are to be developed. However, it remains unclear whether cancer stem cells must be phenotypically similar to normal tissue stem cells or whether they can retain the identity of committed progenitors. Here we show that leukaemia stem cells (LSC) can maintain the global identity of the progenitor from which they arose while activating a limited stem-cell- or self-renewal-associated programme. We isolated LSC from leukaemias initiated in committed granulocyte macrophage progenitors through introduction of the MLL–AF9 fusion protein encoded by the t(9;11)(p22;q23). The LSC were capable of transferring leukaemia to secondary recipient mice when only four cells were transferred, and possessed an immunophenotype and global gene expression profile very similar to that of normal granulocyte macrophage progenitors. However, a subset of genes highly expressed in normal haematopoietic stem cells was re-activated in LSC. LSC can thus be generated from committed progenitors without widespread reprogramming of gene expression, and a leukaemia self-renewal-associated signature is activated in the process. Our findings define progression from normal progenitor to cancer stem cell, and suggest that targeting a self-renewal programme expressed in an abnormal context may be possible.

MLL-Rearranged Leukemia Is Dependent on Aberrant H3K79 Methylation by DOT1L

The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations of the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in an MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3, and H3K36me3 in hematopoietic progenitor and leukemia stem cells (LSCs). We found abnormal profiles only for H3K79me2 on MLL-AF9 fusion target loci in LSCs. Inactivation of Dot1l led to downregulation of direct MLL-AF9 targets and an MLL translocation-associated gene expression signature, whereas global gene expression remained largely unaffected. Suppression of MLL translocation-associated gene expression corresponded with dependence of MLL-AF9 leukemia on Dot1l in vivo. These data point to DOT1L as a potential therapeutic target in MLL-rearranged leukemia.

H3K79 methylation profiles define murine and human MLL-AF4 leukemias

We created a mouse model wherein conditional expression of an Mll-AF4 fusion oncogene induces B precursor acute lymphoblastic (ALL) or acute myeloid leukemias (AML). Gene expression profile analysis of the ALL cells demonstrated significant overlap with human MLL-rearranged ALL. ChIP-chip analysis demonstrated histone H3 lysine 79 (H3K79) methylation profiles that correlated with Mll-AF4-associated gene expression profiles in murine ALLs and in human MLL-rearranged leukemias. Human MLL-rearranged ALLs could be distinguished from other ALLs by their H3K79 profiles, and suppression of the H3K79 methyltransferase DOT1L inhibited expression of critical MLL-AF4 target genes. We thus demonstrate that ectopic H3K79 methylation is a distinguishing feature of murine and human MLL-AF4 ALLs and is important for maintenance of MLL-AF4-driven gene expression.

HOXA9 is required for survival in human MLL-rearranged acute leukemias.

Leukemias that harbor translocations involving the mixed lineage leukemia gene (MLL) possess unique biologic characteristics and often have an unfavorable prognosis. Gene expression analyses demonstrate a distinct profile for MLL-rearranged leukemias with consistent high-level expression of select Homeobox genes, including HOXA9. Here, we investigated the effects of HOXA9 suppression in MLL-rearranged and MLL-germline leukemias using RNA interference. Gene expression profiling after HOXA9 suppression demonstrated co-down-regulation of a program highly expressed in human MLL-AML and murine MLL-leukemia stem cells, including HOXA10, MEIS1, PBX3, and MEF2C. We demonstrate that HOXA9 depletion in 17 human AML/ALL cell lines (7 MLL-rearranged, 10 MLL-germline) induces proliferation arrest and apoptosis specifically in MLL-rearranged cells (P = .007). Similarly, assessment of primary AMLs demonstrated that HOXA9 suppression induces apoptosis to a greater extent in MLL-rearranged samples (P = .01). Moreover, mice transplanted with HOXA9-depleted t(4;11) SEMK2 cells revealed a significantly lower leukemia burden, thus identifying a role for HOXA9 in leukemia survival in vivo. Our data indicate an important role for HOXA9 in human MLL-rearranged leukemias and suggest that targeting HOXA9 or downstream programs may be a novel therapeutic option.

Age-Dependent Association of Human Mannose-Binding Lectin Mutations With Susceptibility to Invasive Meningococcal Disease in Childhood

Background: Mannose-binding lectin (MBL) is an important factor of the innate immune system, and MBL-initiated complement activation is an important early defense mechanism against various bacterial infections, including invasive meningococcal disease. Methods: In a pediatric cohort (ages 2–215 months) with invasive meningococcal disease, we investigated the overall and age-stratified frequency of 3 MBL exon 1 variations (C154T, G161A, G170A), previously shown to result in markedly decreased MBL plasma concentrations, by allele specific fluorescent hybridization probe real-time PCR assays and direct sequencing. Healthy age-matched volunteers with the same ethnic background and no history of meningococcal disease served as a control group. Results: The overall frequency of a MBL exon 1 variant genotype was significantly higher in patients than in controls (31.8% vs. 8.2%, P < 0.001). In the patient group with disease onset less than 24 months of age, the prevalence of MBL structural variant genotype was further increased (39.3%; P < 0.001) and most pronounced in children with disease onset less than 12 months of age (57.1%; P < 0.001) when compared with healthy controls. Analysis of clinical severity and outcome revealed no significant difference between patients with wild-type and mutant alleles. Conclusions: Our data suggest that MBL exon 1 structural variants are significantly associated with susceptibility to childhood meningococcal disease in an age-dependent manner.

The relationship of serum‐eosinophil cationic protein and eosinophil count to disease activity in children with bronchial asthma

BACKGROUND The serum-eosinophil cationic protein level (S-ECP) has been promoted as a biomarker of asthma that reflects the degree of bronchial eosinophilic inflammation. PATIENTS AND METHODS To investigate whether S-ECP is indeed a clinically useful objective parameter, especially in mild or moderate chronic childhood asthma, we studied 100 outpatient children with chronic asthma symptoms (63 boys and 37 girls, aged three to 15 years, median of age eight) and 25 controls (12 boys and 13 girls aged three to 15 years, median of age eight). Symptom scores, lung function parameters and atopy were compared with S-ECP determined by commercially available tests and eosinophils measured by an autoanalyser. RESULTS Asthma symptom scores in the patient group ranged between one and 13 (median of 8), S-ECP between 2.1 and 75.6 microg/l (median of 13.3 microg/ l), and eosinophils between 30/microl and 2002/microl (median of 314). Symptom scores and S-ECP were correlated significantly (P < 0.001) as were symptom scores and eosinophils (P = 0.001). S-ECPs were significantly higher in children with chronic asthma symptoms compared with non-asthmatic, non-atopic children (P = 0.005 for non-atopic chronic asthmatics and P < 0.001 for atopic asthmatics); similar results were found comparing eosinophils in these groups. There was no difference in S-ECP between atopic and non-atopic asthmatic children, but the 25 polysensitised asthmatic children especially with sensitisations to mite, pollen and pet allergens were found to have significantly higher S-ECP compared to 15 monosensitised children (P = 0.002). Similar results were found when correlating eosinophil numbers with atopy. Polysensitised (mite, pollen, pet) asthmatics had significantly higher eosinophil counts compared with monosensitised (pollen) asthmatics (P = 0.01); there was, however, a better discrimination between atopic and non-atopic asthmatics (P = 0.001). Non-asthmatic, non-atopic controls had significantly lower eosinophil counts compared with asthmatics (P < 0.001 for both non-atopic and atopic asthmatics). No correlation between S-ECP or eosinophils and any of the lung function parameters measured (FEV1, FEV1/FVC, MEF50, airway resistance and ITGV) was found. SUMMARY Our data thus indicate that 1) S-ECP is higher than normal in children with asthma symptoms and correlates with asthma symptom score. 2) S-ECP is better correlated to symptom score than to lung function parameters especially in children with mild and moderate asthma symptoms. 3) Raised S-ECP appears to reflect the extent of allergen sensitivity and may also reflect current allergen exposure. 4) Similar correlations were seen when measuring eosinophil number by an autoanalyser instead of S-ECP. CONCLUSIONS Although S-ECP and eosinophils are not diagnostic of asthma they are useful inflammation markers especially in the context of clinical studies. However, both methods are not yet suitable for use in daily practice because they require extensive procedures and special equipment.

A toll-like receptor 4 variant is associated with fatal outcome in children with invasive meningococcal disease

Aims: Toll‐like receptor 4 (TLR4) is the major endotoxin signalling receptor of the innate immune system and is required for efficient recognition of bacterial infections. Here, we analysed a possible association between the TLR4 variant Asp299Gly and disease outcome in children with invasive meningococcal disease.

MLL-Rearranged B Lymphoblastic Leukemias Selectively Express the Immunoregulatory Carbohydrate-Binding Protein Galectin-1

Purpose: Patients with mixed lineage leukemia (MLL)–rearranged B-lymphoblastic leukemias (B-ALL) have an unfavorable prognosis and require intensified treatment. Multiple MLL fusion partners have been identified, complicating the diagnostic evaluation of MLL rearrangements. We analyzed molecular markers of MLL rearrangement for use in rapid diagnostic assays and found the immunomodulatory protein, Galectin-1 (Gal-1), to be selectively expressed in MLL-rearranged B-ALL. Experimental Design: Transcriptional profiling of ALL subtypes revealed selective overexpression of Gal-1 in MLL-rearranged ALLs. For this reason, we analyzed Gal-1 protein expression in MLL-germline and MLL-rearranged adult and infant pediatric B-ALLs and cell lines by immunoblotting, immunohistochemistry, and intracellular flow cytometry of viable tumor cell suspensions. Because deregulated gene expression in MLL-rearranged leukemias may be related to the altered histone methyltransferase activity of the MLL fusion protein complex, we also analyzed histone H3 lysine 79 (H3K79) dimethylation in the LGALS1 promoter region using chromatin immunoprecipitation. Results: Gal-1 transcripts were significantly more abundant in MLL-rearranged B-ALLs. All 32 primary MLL-rearranged B-ALLs exhibited abundant Gal-1 immunostaining, regardless of the translocation partner, whereas only 2 of 81 germline-MLL B-ALLs expressed Gal-1. In addition, Gal-1 was selectively detected in newly diagnosed MLL-rearranged B-ALLs by intracellular flow cytometry. The LGALS1 promoter H3K79 was significantly hypermethylated in MLL-rearranged B-ALLs compared with MLL-germline B-ALLs and normal pre-B cells. Conclusion: In B-ALL, Gal-1 is a highly sensitive and specific biomarker of MLL rearrangement that is likely induced by a MLL-dependent epigenetic modification. Clin Cancer Res; 16(7); 2122–30. ©2010 AACR.

Identification of a novel compound heterozygote SCO2 mutation in cytochrome c oxidase deficient fatal infantile cardioencephalomyopathy.

Fatal infantile cardioencephalomyopathy (OMIM No. 604377) is a disorder of the mitochondrial respiratory chain and is characterised by neonatal progressive muscular hypotonia and cardiomyopathy because of severe Cytochrome c oxidase deficiency. Here we report a novel mutation in the Cytochrome c oxidase assembly gene SCO2 in an infant with fatal infantile cardioencephalomyopathy despite normal initial metabolic screening.

Rapid detection of common pathogenic Aspergillus species by a novel real-time PCR approach

Invasive aspergillosis is a major cause of morbidity and mortality in immunocompromised and critically ill patients. Standard culture based methods for the diagnosis of Aspergillus infections have limited sensitivity and specificity and are time consuming. The recent availability of novel molecular based diagnostic techniques offers the potential of rapid, highly sensitive and specific pathogen detection. In this study, we aimed to develop a diagnostic assay to detect simultaneously common pathogenic Aspergillus species including Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus in whole blood specimen. A real‐time PCR/probe set assay was designed to amplify the 18sRNA genomic region of Aspergillus. In addition to a previously probe set validated for the detection of A. fumigatus (‘Asp.fum’), two other probe sets (‘Asp.ter’; ‘Asp.nig’) were synthesised with 100% sequence homology to A. terreus or A. niger. Specificity testing demonstrated amplification of Aspergillus DNA, but not DNA from a panel of other common pathogenic fungi and bacteria and/or human DNA. Sensitivity testing in serial dilution assays revealed a lower detection limit of 1–5 CFU ml−1 whole blood. However, no single probe set was most sensitive for all Aspergillus species tested and a combination of all three probe sets was necessary to achieve maximal overall assay sensitivity. Furthermore, inclusion of a subsequent probe melting temperature (Tm) analysis demonstrated species‐specific Tm‐patterns, useful to differentiate simultaneously between the different Aspergillus species detected. We describe a highly specific and sensitive real‐time PCR approach to detect and differentiate the most common pathogenic Aspergillus species in a rapid ‘same day’ assay.