Abstract
Rearrangements in the Mixed Lineage Leukemia breakpoint cluster region (MLLbcr) are frequently involved in therapy-induced leukemia, a severe side effect of anti-cancer therapies. Previous work unraveled Endonuclease G as the critical nuclease causing initial breakage in the MLLbcr in response to different types of chemotherapeutic treatment. To identify peptides protecting against therapy-induced leukemia, we screened a hemofiltrate-derived peptide library by use of an enhanced green fluorescent protein (EGFP)-based chromosomal reporter of MLLbcr rearrangements. Chromatographic purification of one active fraction and subsequent mass spectrometry allowed to isolate a C-terminal 27-mer of fibrinogen α encompassing amino acids 603 to 629. The chemically synthesized peptide, termed Fα27, inhibited MLLbcr rearrangements in immortalized hematopoietic cells following treatment with the cytostatics etoposide or doxorubicin. We also provide evidence for protection of primary human hematopoietic stem and progenitor cells from therapy-induced MLLbcr breakage. Of note, fibrinogen has been described to activate toll-like receptor 4 (TLR4). Dissecting the Fα27 mode-of action revealed association of the peptide with TLR4 in an antagonistic fashion affecting downstream NFκB signaling and pro-inflammatory cytokine production. In conclusion, we identified a hemofiltrate-derived peptide inhibitor of the genome destabilizing events causing secondary leukemia in patients undergoing chemotherapy.
Highlights
Chromosomal translocations, inversions, amplifications and complex rearrangements at the 11q human genomic locus encoding the lysine methyl transferase 2 A gene (KMT2A), known as mixed lineage leukemia gene (MLL), are a hallmark of acute lymphoblastic leukemia (ALL) in infants and of therapy-induced acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) in Fibrinogen Peptide Mitigating Leukemic Rearrangements adults [1,2,3]
To identify a human peptide which protects against leukemic Mixed Lineage Leukemia breakpoint cluster region (MLLbcr) rearrangements during chemotherapeutic treatment, we screened a hemofiltrate-derived library from patients suffering from renal failure [33, 37]
Etoposide treatment conditions were previously established and demonstrated to induce a one order of magnitude increase of MLLbcr rearrangements in K562MLL and WTK1MLL reporter cells derived from human erythroleukemia and human lymphoblastoid cells, respectively [30]
Summary
Chromosomal translocations, inversions, amplifications and complex rearrangements at the 11q human genomic locus encoding the lysine methyl transferase 2 A gene (KMT2A), known as mixed lineage leukemia gene (MLL), are a hallmark of acute lymphoblastic leukemia (ALL) in infants and of therapy-induced acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) in Fibrinogen Peptide Mitigating Leukemic Rearrangements adults [1,2,3]. MLL abnormalities were reported to be detectable in up to 85% of infant ALL and approximately one third of therapyinduced AML cases [2, 4] Underscoring their impact, these chromosomal changes correlate with poor prognosis, resistance to treatment and reduced overall survival [5, 6]. The MLLbcr sequence has been predicted to adopt complex secondary structures with hairpins that are known to be highly enriched at translocation breakpoints in human cancer genomes [2, 16]. Such non-B DNA structures represent obstacles to the transcription and DNA replication machinery and are subject to incisions by nucleases. MLLbcr rearrangements can be explained by errorprone repair attempts in apoptosis-escaping cells and can be recapitulated ex vivo in cultured lymphocytes as well as hematopoietic stem and progenitor cells (HSPCs) after exposure to nutritional risk factors or cytostatic drugs [8, 9, 17,18,19]
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