Subject(s)
Leukemia, Myeloid, Acute/drug therapy , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/genetics , Amino Acid Sequence , Animals , Cell Proliferation , Dose-Response Relationship, Drug , Humans , In Vitro Techniques , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred BALB C , MutationABSTRACT
The t(10;11)(p12;q23) translocation and the t(10;11)(p12;q14) translocation, which encode the MLL (mixed lineage leukemia)-AF10 and CALM (clathrin assembly lymphoid myeloid leukemia)-AF10 fusion oncoproteins, respectively, are two recurrent chromosomal rearrangements observed in patients with acute myeloid leukemia and acute lymphoblastic leukemia. Here, we demonstrate that MLL-AF10 and CALM-AF10-mediated transformation is dependent on the H3K79 methyltransferase Dot1l using genetic and pharmacological approaches in mouse models. Targeted disruption of Dot1l using a conditional knockout mouse model abolished in vitro transformation of murine bone marrow cells and in vivo initiation and maintenance of MLL-AF10 or CALM-AF10 leukemia. The treatment of MLL-AF10 and CALM-AF10 transformed cells with EPZ004777, a specific small-molecule inhibitor of Dot1l, suppressed expression of leukemogenic genes such as Hoxa cluster genes and Meis1, and selectively impaired proliferation of MLL-AF10 and CALM-AF10 transformed cells. Pretreatment with EPZ004777 profoundly decreased the in vivo spleen-colony-forming ability of MLL-AF10 or CALM-AF10 transformed bone marrow cells. These results show that patients with leukemia-bearing chromosomal translocations that involve the AF10 gene may benefit from small-molecule therapeutics that inhibit H3K79 methylation.
Subject(s)
Enzyme Inhibitors/pharmacology , Gene Silencing , Methyltransferases/antagonists & inhibitors , Myeloid-Lymphoid Leukemia Protein/genetics , Oncogene Proteins, Fusion/genetics , Animals , Apoptosis , Blotting, Western , Cell Cycle , Cell Proliferation , Fluorescent Antibody Technique , Humans , Mice , Mice, Mutant Strains , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The t(10;11)(p12;q14) is a recurring chromosomal translocation that gives rise to the CALM/AF10 fusion gene, which is found in acute myeloid leukemia, acute lymphoblastic leukemia and malignant lymphoma. We analyzed the fusion transcripts in 20 new cases of CALM/AF10-positive leukemias, and compared the gene expression profile of 10 of these to 125 patients with other types of leukemia and 10 normal bone marrow samples. Based on gene set enrichment analyses, the CALM/AF10-positive samples showed significant upregulation of genes involved in chromatin assembly and maintenance and DNA repair process, and downregulation of angiogenesis and cell communication genes. Interestingly, we observed a striking upregulation of four genes located immediately centromeric to the break point of the t(10;11)(p12;q14) on 10p12 (COMMD3 (COMM domain containing 3), BMI1 (B lymphoma Mo-MLV insertion region 1 homolog), DNAJC1 (DnaJ (Hsp40) homolog subfamily C member 1) and SPAG6 (sperm associated antigen 6)). We also conducted semiquantitative reverse transcriptase-PCR analysis on leukemic blasts from a murine CALM/AF10 transplantation model that does not have the translocation. Commd3, Bmi1 and Dnajc1, but not Spag6 were upregulated in these samples. These results strongly indicate that the differential regulation of these three genes is not due to the break point effect but as a consequence of the CALM/AF10 fusion gene expression, though the mechanism of regulation is not well understood.
Subject(s)
Chromatin Assembly and Disassembly/genetics , Chromosome Fragile Sites , Chromosomes, Human, Pair 10 , DNA Repair/genetics , Leukemia/genetics , Monomeric Clathrin Assembly Proteins/genetics , Transcription Factors/genetics , Up-Regulation , Animals , Humans , Mice , Reverse Transcriptase Polymerase Chain Reaction , Translocation, GeneticABSTRACT
The t(10;11)(p13-14;q14-21) translocation, giving rise to the CALM-AF10 fusion gene, is a recurrent chromosomal rearrangement observed in patients with poor prognosis acute myeloid leukemia (AML). Although splicing of the CALM-AF10 fusion transcripts has been described in AML patients, the contribution of different CALM and AF10 domains to in vivo leukemogenesis remains to be defined. We therefore performed detailed structure-function studies of the CALM-AF10 fusion protein. We demonstrate that fusion of the C-terminal 248 amino acids of CALM, which include the clathrin-binding domain, to the octapeptide motif-leucine-zipper (OM-LZ) domain of AF10 generated a fusion protein (termed CALM-AF10 minimal fusion (MF)), with strikingly enhanced transformation capabilities in colony assays, providing an efficient system for the expeditious assessment of CALM-AF10-mediated transformation. Leukemias induced by the CALM-AF10 (MF) mutant recapitulated multiple aspects of full-length CALM-AF10-induced leukemia, including aberrant Hoxa cluster upregulation, a characteristic molecular lesion of CALM-AF10 leukemias. In summary, this study indicates that collaboration of the clathrin-binding and the OM-LZ domains of CALM-AF10 is sufficient to induce AML. These findings further suggest that future approaches to antagonize CALM-AF10-induced transformation should incorporate strategies, which aim at blocking these key domains.
Subject(s)
Clathrin/metabolism , Leukemia, Myeloid, Acute/genetics , Monomeric Clathrin Assembly Proteins/metabolism , Transcription Factors/metabolism , Animals , Blotting, Western , Cells, Cultured , Gene Expression Profiling , Humans , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Microscopy, Fluorescence , Monomeric Clathrin Assembly Proteins/chemistry , Transcription Factors/chemistryABSTRACT
Diffraction gratings affect the absolute phase of light in a way that is not obvious from the usual derivation of optical paths using the grating equation. For example, consider light which encounters first one and then the second of two parallel gratings. If one grating is moved parallel to its surface, the phase of the light diffracted from the grating pair is shifted by 2pi each time the grating is moved by one grating constant, even though the geometric path length is not altered by the motion. This additional phase shift must be included when incorporating diffraction gratings in interferometers.
