Leukemogenic rearrangements at the mixed lineage leukemia gene (MLL)-multiple rather than a single mechanism

Abstract

Despite manifold efforts to achieve reduced-intensity and -toxicity regimens, secondary leukemia has remained the most severe side effect of chemotherapeutic cancer treatment. Rearrangements involving a short telomeric <1 kb region of the mixed lineage leukemia (MLL) gene are the most frequently observed molecular changes in secondary as well as infant acute leukemia. Due to the mode-of-action of epipodophyllotoxins and anthracyclines, which have widely been used in cancer therapy, and support from in vitro experiments, cleavage of this MLL breakpoint cluster hotspot by poisoned topoisomerase II was proposed to trigger the molecular events leading to malignant transformation. Later on, clinical patient data and cell-based studies addressing a wider spectrum of stimuli identified cellular stress signaling pathways, which create secondary DNA structures, provide chromatin accessibility, and activate nucleases other than topoisomerase II at the MLL. The MLL destabilizing signaling pathways under discussion, namely early apoptotic DNA fragmentation, transcription stalling, and replication stalling, may all act in concert upon infection-, transplantation-, or therapy-induced cell cycle entry of hematopoietic stem and progenitor cells (HSPCs), to permit misguided cleavage and error-prone DNA repair in the cell-of-leukemia-origin.

Keywords: MLL breakpoint cluster region; apoptosis; error-prone DNA repair; infant acute leukemia; replication stress; therapy-related leukemia.

Figure 1

Position, structure, and regulatory features of MLL breakpoint cluster region. (A–C) Selected features of the KMT2A/MLL gene based on current issue of Ensembl data (release 79, March 2015; http://www.ensembl.org/index.html; Cunningham et al., 2015). (A) KMT2A/MLL position on chromosome 11, exon structure according to the current reference sequence (Human RefSeq), transcripts (protein coding and non-protein coding), regulatory features and open chromatin (DNase I sensitive) sites. Some transcripts harbor exons that are not acknowledged by the reference sequence (for example compare Human RefSeq, KMT2A-001 and KMT2A-002). Open chromatin sites are detectable in promoter-exon 1, exon 12 and exon 37. The position of the breakpoint cluster region (MLLbcr) is marked by dashed lines. (B) Enhanced view of the MLLbcr with exon structure of the reference sequence and transcripts, regulatory features and open chromatin (DNase I) sites. The position of the therapy-related hotspot is marked by dashed lines. (C) Enhanced view of a therapy-related hotspot flanking a major therapy-related translocation breakpoint described by Mirault et al. (2006), with exon structure, sequence, regulatory features and open chromatin (DNase I) sites. The major therapy-related breakpoint cluster (marked by red triangle) lies at the 5′ end of intron 11, just 3′ of the DNase I and CTCF-binding site found in 17/18 and 18/18 cell lines used in Ensembl, respectively. The indicated 399 bp segment was used in reporter studies with exogenous MLLbcr fragment (Boehden et al., ; Gole et al., ; Ireno et al., 2014). (D) Secondary structure of the 399 bp therapy-related hotspot. The structure was calculated using programs performing searches based on sequence, namely Quadruplex forming G-Rich Sequences (QGRS) Mapper (http://bioinformatics.ramapo.edu/QGRS/index.php) and M-Fold for hairpins (http://mfold.rna.albany.edu/?q=mfold).

Figure 2

Comprehensive overview of the pathways leading to MLLbcr rearrangements. Exposure of the cells to genotoxic agents can directly or indirectly cause stalling of DNA replication, e.g., by antimetabolite treatment, and by formation of DNA adducts and downstream repair intermediates (e.g., by treatment with alkylating agents and downstream excision repair) or transcription stalling, respectively. Excessive proliferation of the hematopoietic stem/progenitor cells (cells-of-leukemia-origin) due to enforced self-renewal after bone marrow transplantation or genotoxic insults also results in replication stress. MLLbcr in turn is cleaved either as part of the attempt to rescue stalled forks or as part of DNA damage-induced early apoptotic high order DNA fragmentation. DSBs at the MLLbcr can be repaired through NHEJ, MMEJ, or homology-directed repair which can lead to leukemogenic rearrangements preventing further oligomeric DNA fragmentation and cell death.