Arndt Borkhardt

High expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma.

In a recent issue of Genes, Chromosomes & Cancer, van den Berg et al. (2003) reported on the high expression of human BIC RNA in Hodgkin lymphoma by means of the serial analysis of gene expression (SAGE) technique. In addition, using a combination of RNA in situ hybridization and immunostaining, they found that the expression of BIC is specific for Reed–Sternberg (RS) cells and that the BIC transcripts were in the nuclei of the RS cells. In contrast to their findings in Hodgkin lymphoma, an analysis of 43 cases of non-Hodgkin lymphoma (NHL; 15 follicular lymphomas, 7 diffuse large B-cell lymphomas, 9 Burkitt lymphomas, 7 anaplastic large cell lymphomas, and 5 T-cell-rich B-cell lymphomas) did not reveal any remarkable up-regulation of BIC expression, although one case of Burkitt lymphoma (BL) showed weak expression of BIC in a minority of its tumor cells. The BIC locus was originally identified as a common retroviral integration site in avian-leukosis virus–induced B–cell lymphomas (Clurman and Hayward, 1989; Tam et al., 1997). It should be stressed that the human BIC gene encodes a microRNA, miR-155. This microRNA is encoded by nucleotides 241–262 of BIC, which spans 1,421 bp in total and is on chromosome 21 (GenBank accession number: AF402776). MicroRNAs (miRNAs) are an abundant class of noncoding RNAs that interact with coding mRNA and trigger either translation repression or direct RNA cleavage via RNA interference, depending on the degree of complementarity with the specific target mRNA (Ambros, 2001; Lagos-Quintana et al., 2001; Ruvkun, 2001; Lai, 2002; Pasquinelli, 2002; Ambros et al., 2003; Lagos-Quintana et al., 2003). Mature miRNAs are 21–23 nt long and are excised from an approximately 60to 80-nt double-stranded RNA hairpin by Dicer RNase III (Hutvagner et al., 2001). In the last 2 years, more than 200 human microRNA genes have been described, but the prediction and validation of their target mRNAs by computerized means and experimental approaches is a tantalizing and still unresolved task (Ambros et al., 2003). Apart from this open question, it was recently hypothesized that microRNA genes might play an important role in oncogenesis (McManus, 2003). We support the idea advanced by van den Berg et al. that BIC might play a role in the selection of B cells, but here we also extend their data by demonstrating that miR-155/BIC is highly expressed in pediatric BL but not in other hematologic malignancies, for example, pre-B/common or T-cell leukemia. The conditio sine qua non for the development of BL is activation of the MYC oncogene, mostly by chromosomal translocations in which MYC is juxtaposed to an immunoglobulin enhancer. On the other hand, there is a body of evidence that the activation of MYC alone is not sufficient for full malignancy. Providing support for this contention was the demonstration that MYC cooperates with BMI1, PIM1, RAF, BCL2, or, as shown in a very recent report, with the Werner syndrome protein WRN (Grandori et al., 2003) We analyzed tumor cells from 21 children (ages 2–13 years, with a median age of 6 years) with BL (n 11), common/pre-B acute lymphoblastic leukemia (ALL; n 6), or T-cell ALL (n 4). In all cases selected, the proportion of tumor cells was 80% or greater. The presence of an IGH/MYC rearrangement was demonstrated by long-distance polymerase chain reaction (PCR) in all BL cases, whereas neither the common/pre-B nor T-cell ALLs had such a recombination (Busch et al., unpublished data). All patients were treated according to the NHL-BFM 90, 95, or the ALL-BFM 90, 95 multicenter therapy study (Reiter et al., 1999; Schrappe et al., 2000). Immunophenotyping was done according to standard procedures (Ludwig et al., 1994). Informed parental consent was obtained in all cases. RNA isolated from peripheral blood from 11 healthy volunteers served as an additional control.

Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi)

Short 21-mer double-stranded RNA (dsRNA) molecules have recently been employed for the sequence-specific silencing of endogenous human genes. This mechanism, called RNA interference (RNAi), is extremely potent and requires only a few dsRNA molecules per cell to silence homologous gene mRNA expression. We used dsRNA targeting the M-BCR/ABL fusion site to kill leukemic cells with such a rearrangement. Transfection of dsRNA specific for the M-BCR/ABL fusion mRNA into K562 cells depleted the corresponding mRNA and the M-BCR/ABL oncoprotein. This was demonstrated by real-time quantitative PCR and Western blots. The BCR/ABL knockdown was accompanied by strong induction of apoptotic cell death. Leukemic cells without BCR/ABL rearrangement were not killed by M-BCR/ABL-dsRNA. In addition, to corroborate the extraordinary sequence specificity of RNAi, we designed another RNA oligo matching the M-BCR/ABL fusion site but having two point mutations within its central region. We show that these two point mutations abolished both p210 reduction and induction of apoptosis in K562 cells. Finally, we compared leukemic cell killing by RNAi to that caused by the ABL kinase tyrosine inhibitor, STI 571, Imatinib. For full induction of apoptosis, dsRNA targeting M-BCR/ABL required 24 h more than Imatinib. This may be caused by the relatively long half-life of the BCR/ABL oncoprotein, which is not targeted by the RNAi mechanism, but is affected by STI 571. When we applied ds M-BCR/ABL RNA and STI 571 in combination, we did not observe a further increase in the induction of apoptosis. Nevertheless, these data may open a field for further studies towards gene-therapeutic approaches using RNA interference to kill tumor cells with specific genetic abnormalities.

Mutations of JAK2 in acute lymphoblastic leukaemias associated with Down's syndrome

BACKGROUND Children with Downs syndrome have a greatly increased risk of acute megakaryoblastic and acute lymphoblastic leukaemias. Acute megakaryoblastic leukaemia in Downs syndrome is characterised by a somatic mutation in GATA1. Constitutive activation of the JAK/STAT (Janus kinase and signal transducer and activator of transcription) pathway occurs in several haematopoietic malignant diseases. We tested the hypothesis that mutations in JAK2 might be a common molecular event in acute lymphoblastic leukaemia associated with Downs syndrome. METHODS JAK2 DNA mutational analysis was done on diagnostic bone marrow samples obtained from 88 patients with Downs syndrome-associated acute lymphoblastic leukaemia; and 216 patients with sporadic acute lymphoblastic leukaemia, Downs syndrome-associated acute megakaryoblastic leukaemia, and essential thrombocythaemia. Functional consequences of identified mutations were studied in mouse haematopoietic progenitor cells. FINDINGS Somatically acquired JAK2 mutations were identified in 16 (18%) patients with Downs syndrome-associated acute lymphoblastic leukaemia. The only patient with non-Downs syndrome-associated leukaemia but with a JAK2 mutation had an isochromosome 21q. Children with a JAK2 mutation were younger (mean [SE] age 4.5 years [0.86] vs 8.6 years [0.59], p<0.0001) at diagnosis. Five mutant alleles were identified, each affecting a highly conserved arginine residue (R683). These mutations immortalised primary mouse haematopoietic progenitor cells in vitro, and caused constitutive Jak/Stat activation and cytokine-independent growth of BaF3 cells, which was sensitive to pharmacological inhibition with JAK inhibitor I. In modelling studies of the JAK2 pseudokinase domain, R683 was situated in an exposed conserved region separated from the one implicated in myeloproliferative disorders. INTERPRETATION A specific genotype-phenotype association exists between the type of somatic mutation within the JAK2 pseudokinase domain and the development of B-lymphoid or myeloid neoplasms. Somatically acquired R683 JAK2 mutations define a distinct acute lymphoblastic leukaemia subgroup that is uniquely associated with trisomy 21. JAK2 inhibitors could be useful for treatment of this leukaemia. FUNDING Israel Trade Ministry, Israel Science Ministry, Jewish National Fund UK, Sam Waxman Cancer Research Foundation, Israel Science Foundation, Israel Cancer Association, Curtis Katz, Constantiner Institute for Molecular Genetics, German-Israel Foundation, and European Commission FP6 Integrated Project EUROHEAR.

Hoxa9 and Meis1 Are Key Targets for MLL-ENL-Mediated Cellular Immortalization

ABSTRACT MLL fusion proteins are oncogenic transcription factors that are associated with aggressive lymphoid and myeloid leukemias. We constructed an inducible MLL fusion, MLL-ENL-ERtm, that rendered the transcriptional and transforming properties of MLL-ENL strictly dependent on the presence of 4-hydroxy-tamoxifen. MLL-ENL-ERtm-immortalized hematopoietic cells required 4-hydroxy-tamoxifen for continuous growth and differentiated terminally upon tamoxifen withdrawal. Microarray analysis performed on these conditionally transformed cells revealed Hoxa9 and Hoxa7 as well as the Hox coregulators Meis1 and Pbx3 among the targets upregulated by MLL-ENL-ERtm. Overexpression of the Hox repressor Bmi-1 inhibited the growth-transforming activity of MLL-ENL. Moreover, the enforced expression of Hoxa9 in combination with Meis1 was sufficient to substitute for MLL-ENL-ERtm function and to maintain a state of continuous proliferation and differentiation arrest. These results suggest that MLL fusion proteins impose a reversible block on myeloid differentiation through aberrant activation of a limited set of homeobox genes and Hox coregulators that are consistently expressed in MLL-associated leukemias.

Down syndrome acute lymphoblastic leukemia, a highly heterogeneous disease in which aberrant expression of CRLF2 is associated with mutated JAK2: a report from the International BFM Study Group.

We report gene expression and other analyses to elucidate the molecular characteristics of acute lymphoblastic leukemia (ALL) in children with Down syndrome (DS). We find that by gene expression DS-ALL is a highly heterogeneous disease not definable as a unique entity. Nevertheless, 62% (33/53) of the DS-ALL samples analyzed were characterized by high expression of the type I cytokine receptor CRLF2 caused by either immunoglobulin heavy locus (IgH@) translocations or by interstitial deletions creating chimeric transcripts P2RY8-CRLF2. In 3 of these 33 patients, a novel activating somatic mutation, F232C in CRLF2, was identified. Consistent with our previous research, mutations in R683 of JAK2 were identified in 10 specimens (19% of the patients) and, interestingly, all 10 had high CRLF2 expression. Cytokine receptor-like factor 2 (CRLF2) and mutated Janus kinase 2 (Jak2) cooperated in conferring cytokine-independent growth to BaF3 pro-B cells. Intriguingly, the gene expression signature of DS-ALL is enriched with DNA damage and BCL6 responsive genes, suggesting the possibility of B-cell lymphocytic genomic instability. Thus, DS confers increased risk for genetically highly diverse ALLs with frequent overexpression of CRLF2, associated with activating mutations in the receptor itself or in JAK2. Our data also suggest that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathways.

Cloning and characterization of AFX, the gene that fuses to MLL in acute leukemias with a t(X;11)(q13;q23).

We report the cloning and characterization of the entire AFX gene which fuses to MLL in acute leukemias with a t(X;11)(q13;q23). AFX consists of two exons and encodes for a protein of 501 amino acids. We found that normal B- and T-cells contain similar levels of AFX mRNA and that both the MLL/AFX as well as the AFX/MLL fusion transcripts are present in the cell line and the ANLL sample with a t(X;11)(q13;q23). The single intron of the AFX gene consists of 3706 nucleotides. It contains five simple sequence repeats with lengths of at least 12 bps, a chi-like octamer sequence (GCA/TGGA/TGG) and several immunoglobulin heptamer-like sequences (GATAGTG) that are distributed throughout the entire AFX intron sequence. In the KARPAS 45 cell line the breakpoints occur at nucleotides 2913/2914 of the AFX intron and at nucleotides 4900/4901 of the breakpoint cluster region of the MLL gene. The AFX protein belongs to the forkhead protein family. It is highly homologous to the human FKHR protein, the gene of which is disrupted by the t(2;13)(q35;q14), a chromosome rearrangement characteristic of alveolar rhabdomyosarcomas. It is noteworthy that the t(X;11)(q13;q23) in the KARPAS 45 cell line and in one acute nonlymphoblastic leukemia (ANLL) disrupts the forkhead domain of the AFX protein exactly at the same amino acids as does the t(2;13)(q35;q14) in case of the FKHR protein. In addition, the 5′-part of the AFX protein contains a conserved hexapeptide motif (QIYEWM) that is homologous to the functionally important conserved hexapeptide QIYPWM upstream of the homeobox domain in Hox proteins. This motif mediates the co-operative DNA binding of Pbx family members and Hox proteins and, therefore, plays an important role in physiologic and oncogenic processes. In acute leukemias with a t(X;11)(q13;q23), this hexapeptide motif is separated from the remaining forkhead domain within the AFX protein. The predicted amino acid sequence of AFX differs significantly from the partial AFX protein sequence published previously (Genes, Chromosomes and Cancer, 1994, 11, 79 – 84). This discrepancy can be explained by the occurrence of two sequencing errors in the earlier work at nucleotide number 783 and 844 (loss of a cytosine residue or guanosine residue, respectively) that lead to two reading frame shifts.

Prognostic Value of Minimal Residual Disease Quantification Before Allogeneic Stem-Cell Transplantation in Relapsed Childhood Acute Lymphoblastic Leukemia: The ALL-REZ BFM Study Group

PURPOSE Minimal residual disease (MRD) before allogeneic stem-cell transplantation was shown to predict outcome in children with relapsed acute lymphoblastic leukemia (ALL) in retrospective analysis. To verify this, the Acute Lymphoblastic Leukemia Relapse Berlin-Frankfurt-Münster (ALL-REZ BFM) Study Group conducted a prospective trial. PATIENTS AND METHODS Between March 1999 and July 2005, 91 children with relapsed ALL treated according to the ALL-REZ BFM 96 or 2002 protocols and receiving stem-cell transplantation in >or= second remission were enrolled. MRD quantification was performed by real-time polymerase chain reaction using T-cell receptor and immunoglobulin gene rearrangements. RESULTS Probability of event-free survival (pEFS) and cumulative incidence of relapse (CIR) in 45 patients with MRD >or= 10(-4) leukemic cells was 0.27 and 0.57 compared with 0.60 and 0.13 in 46 patients with MRD less than 10(-4) leukemic cells (EFS, P = .004; CIR, P < .001). Intermediate-risk patients (strategic group S1) with MRD >or= 10(-4) leukemic cells (n = 14) had a pEFS of 0.20 and CIR of 0.73, whereas patients with MRD less than 10(-4) leukemic cells (n = 21) had a pEFS of 0.68 and CIR of 0.09 (EFS, P = .020; CIR, P < .001). High-risk patients (S3/4, third complete remission) who received transplantation with an MRD load of less than 10(-4) leukemic cells (n = 25) showed a pEFS and CRI of 0.53 and 0.18, respectively. In contrast, pEFS and CRI were 0.30 and 0.50 in patients who received transplantation with an MRD load of >or= 10(-4) leukemic cells. Multivariate Cox regression analysis revealed MRD as the only independent parameter predictive for EFS (P = .006). CONCLUSION MRD is an important predictor for post-transplantation outcome. As a result, new strategies with modified stem-cell transplantation procedures will be evaluated in ALL-BFM trials.

Recurrent mutation of the ID3 gene in Burkitt lymphoma identified by integrated genome, exome and transcriptome sequencing

Burkitt lymphoma is a mature aggressive B-cell lymphoma derived from germinal center B cells. Its cytogenetic hallmark is the Burkitt translocation t(8;14)(q24;q32) and its variants, which juxtapose the MYC oncogene with one of the three immunoglobulin loci. Consequently, MYC is deregulated, resulting in massive perturbation of gene expression. Nevertheless, MYC deregulation alone seems not to be sufficient to drive Burkitt lymphomagenesis. By whole-genome, whole-exome and transcriptome sequencing of four prototypical Burkitt lymphomas with immunoglobulin gene (IG)-MYC translocation, we identified seven recurrently mutated genes. One of these genes, ID3, mapped to a region of focal homozygous loss in Burkitt lymphoma. In an extended cohort, 36 of 53 molecularly defined Burkitt lymphomas (68%) carried potentially damaging mutations of ID3. These were strongly enriched at somatic hypermutation motifs. Only 6 of 47 other B-cell lymphomas with the IG-MYC translocation (13%) carried ID3 mutations. These findings suggest that cooperation between ID3 inactivation and IG-MYC translocation is a hallmark of Burkitt lymphomagenesis.

RNA targets of wild-type and mutant FET family proteins

FUS, EWSR1 and TAF15, constituting the FET protein family, are abundant, highly conserved RNA-binding proteins with important roles in oncogenesis and neuronal disease, yet their RNA targets and recognition elements are unknown. Using PAR-CLIP, we defined global RNA targets for all human FET proteins and two ALS-causing human FUS mutants. FET members showed similar binding profiles, whereas FUS mutants showed a drastically altered binding pattern, consistent with changes in subcellular localization.

Decoding the regulatory landscape of medulloblastoma using DNA methylation sequencing

Epigenetic alterations, that is, disruption of DNA methylation and chromatin architecture, are now acknowledged as a universal feature of tumorigenesis. Medulloblastoma, a clinically challenging, malignant childhood brain tumour, is no exception. Despite much progress from recent genomics studies, with recurrent changes identified in each of the four distinct tumour subgroups (WNT-pathway-activated, SHH-pathway-activated, and the less-well-characterized Group 3 and Group 4), many cases still lack an obvious genetic driver. Here we present whole-genome bisulphite-sequencing data from thirty-four human and five murine tumours plus eight human and three murine normal controls, augmented with matched whole-genome, RNA and chromatin immunoprecipitation sequencing data. This comprehensive data set allowed us to decipher several features underlying the interplay between the genome, epigenome and transcriptome, and its effects on medulloblastoma pathophysiology. Most notable were highly prevalent regions of hypomethylation correlating with increased gene expression, extending tens of kilobases downstream of transcription start sites. Focal regions of low methylation linked to transcription-factor-binding sites shed light on differential transcriptional networks between subgroups, whereas increased methylation due to re-normalization of repressed chromatin in DNA methylation valleys was positively correlated with gene expression. Large, partially methylated domains affecting up to one-third of the genome showed increased mutation rates and gene silencing in a subgroup-specific fashion. Epigenetic alterations also affected novel medulloblastoma candidate genes (for example, LIN28B), resulting in alternative promoter usage and/or differential messenger RNA/microRNA expression. Analysis of mouse medulloblastoma and precursor-cell methylation demonstrated a somatic origin for many alterations. Our data provide insights into the epigenetic regulation of transcription and genome organization in medulloblastoma pathogenesis, which are probably also of importance in a wider developmental and disease context.