Gene-expression profiling allows a comprehensive classification of AML that includes previously identified genetically defined subgroups and a novel cluster with an adverse prognosis.

Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.

MK enables (in addition to CN and CBF) the prognostic classification of two new aggregates of cytogenetically abnormal AML, the unfavorable risk MK-negative category (4-year OS, 26% +/- 2%) and the highly unfavorable risk MK-positive category (4-year OS, 4% +/- 1%).

The use of Cre͞loxP recombination in mammalian cells has expanded rapidly. We describe here that Cre expression in cultured mammalian cells may result in a markedly reduced proliferation and that this effect is dependent on the endonuclease activity of Cre. Chromosome analysis after Cre expression revealed numerous chromosomal aberrations and an increased number of sister chromatid exchanges. Titration experiments in mouse embryo fibroblasts with a ligand-regulatable Cre-ER T show that toxicity is dependent on the level of Cre activity. Prolonged, low levels of Cre activity permit recombination without concomitant toxicity. This urges for a careful titration of Cre activity in conditional gene modification in mammalian cells.genotoxicity ͉ conditional knockout

Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (␥-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced ␥-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, ␥-H2AX foci were also induced in Ercc1 MMC-induced ␥-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1؊/؊ and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.Interstrand cross-links (ICLs) comprise a unique class of DNA lesions that have a potent biological effect. By definition, ICLs involve covalent modification of both strands of DNA. Therefore, these adducts prevent DNA strand separation and block DNA metabolism, such as transcription and replication (31). DNA-damaging agents that cause ICLs are extremely cytotoxic, and their utility as anticancer chemotherapeutics likely stems from their selective toxicity to proliferating cells. ICLs occur via a two-step reaction mechanism in which first a monoadduct involving one strand of DNA is formed (24). Although cross-linking agents induce a variety of DNA adducts, the relative cytotoxicity of each agent correlates with its ability to form ICLs (43, 44).The repair of DNA ICLs presents a unique challenge to cells. Since both strands of DNA are covalently modified, simple excision of the lesion followed by template-driven DNA resynthesis is precluded. In Escherichia coli, two solutions to this problem have been identified (reviewed in reference 19). In both these repair mechanisms, the ICL is excised from one strand. In error-free repair, an undamaged chromosome is then utilized as a template for gap-filling DNA polymerization (55). ICL repair also occurs in recombination-deficient E. coli, likely via translesional DNA polymerization of the second damaged strand (5). Similarly, genetic analysis of Saccharomyces cerevisiae (23) and mammalian DNA repair mutants (reviewed in reference 19) indicates the involvement of proteins from multiple DNA repair pathways in ICL repair: nucleotide excision repair (NER), homologous recombination, and postreplication...