No abstract

Expression of the AF4-MLL fusion protein in murine hematopoietic progenitor/stem cells results in the development of proB acute lymphoblastic leukemia. In this study, we affinity purified the AF4-MLL and AF4 protein complexes to elucidate their function. We observed that the AF4 complex consists of 11 binding partners and exhibits positive transcription elongation factor b (P-TEFb)-mediated activation of promoter-arrested RNA polymerase (pol) II in conjunction with several chromatin-modifying activities. In contrast, the AF4-MLL complex consists of at least 16 constituents including P-TEFb kinase, H3K4 me3 and H3K79 me3 histone methyltransferases (HMT), a protein arginine N-methyltransferase and a histone acetyltransferase. These findings suggest that the AF4-MLL protein disturbs the fine-tuned activation cycle of promoter-arrested RNA Pol II and causes altered histone methylation signatures. Thus, we propose that these two processes are key to trigger cellular reprogramming that leads to the onset of acute leukemia.

The human AF4 (ALL-1 fused gene on chromosome 4) gene (4q11) is recurrently involved in reciprocal translocations to the MLL (mixed lineage leukemia) gene (11q23), correlated with high-risk acute lymphoblastic leukemia (ALL) in infants and early childhood. The t(4;11) translocation is one of the most frequent MLL translocations known today. In general, MLL translocations are the result of an illegitimate recombination process leading to reciprocal fusions of unrelated translocation partner (TP) genes with the MLL gene. Owing to the constant presence of the derivative (11) product, it was hypothesised that only MLL . TP fusion genes are responsible for the leukemogenic process. This concept has been successfully tested for some known MLL fusions, while other MLL fusions failed. Here, we demonstrate growthtransforming potential of AF4 wild-type and the AF4 . MLL fusion protein. The underlying oncogenic mechanism involves the two E3 ubiquitin ligases SIAH1 and SIAH2, the N-terminal portion of AF4 and the protection of the AF4 . MLL fusion protein against proteosomal degradation.

The MLL gene is frequently involved in chromosomal translocations associated with high-risk acute leukaemia. Infant and therapy-related acute leukaemia patients display chromosomal breakpoints preferentially clustered in the telomeric portion of the MLL breakpoint cluster region (SCII). Here, we demonstrate that SCII colocalizes with a gene-internal promoter element in the mouse and human MLL gene, respectively. The mRNA generated encodes an N-terminally truncated version of MLL that still exhibits many functional regions, including the C-terminal SET-domain. Etoposide-induced DNA doublestrand breaks colocalize with the binding site of RNA polymerase II and the transcription initiation region, but not with a nearby Topo II consensus sequence. Thus, the observed genomic instability of the human MLL gene is presumably linked to transcriptional processes. The consequences of this novel finding for the creation of chromosomal translocations, the biology of the MLL protein and for MLL-mediated acute leukaemia are discussed.

The reciprocal chromosomal translocation t(4;11) is correlated with infant, childhood, adult and therapyrelated high-risk acute leukemia. Here, we investigated the biological effects of MLL . AF4, AF4 . MLL or the combination of both reciprocal fusion proteins in a conditional in vitro cell culture model system. Several parameters like cell growth, cell cycling capacity, apoptotic behavior and growth transformation were investigated under physiological and stress conditions. Co-transfected cells displayed the highest resistance against apoptotic triggers, cell cycling capacity and lossof-contact inhibition. These analyses were complemented by gene expression profiling experiments and specific gene signatures were established for each of the three cell lines. Interestingly, co-transfected cells strongly upregulate the homeobox gene Nanog. In combination with Oct4, the Nanog homeoprotein is steering maintenance of pluripotency and self-renewal in embryonic stem cells. Transcription of Nanog and other stem cell factors, like Oct4 and Bmi1, was verified in biopsy material of t(4;11) patient cells which express both reciprocal t(4;11) fusion genes. In conclusion, the presence of both reciprocal MLL fusion proteins confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype.