Targeting recruitment of disruptor of telomeric silencing 1-like (DOT1L): characterizing the interactions between DOT1L and mixed lineage leukemia (MLL) fusion proteins

Abstract

The MLL fusion proteins, AF9 and ENL, activate target genes in part via recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like). Here we report biochemical, biophysical, and functional characterization of the interaction between DOT1L and MLL fusion proteins, AF9/ENL. The AF9/ENL-binding site in human DOT1L was mapped, and the interaction site was identified to a 10-amino acid region (DOT1L865-874). This region is highly conserved in DOT1L from a variety of species. Alanine scanning mutagenesis analysis shows that four conserved hydrophobic residues from the identified binding motif are essential for the interactions with AF9/ENL. Binding studies demonstrate that the entire intact C-terminal domain of AF9/ENL is required for optimal interaction with DOT1L. Functional studies show that the mapped AF9/ENL interacting site is essential for immortalization by MLL-AF9, indicating that DOT1L interaction with MLL-AF9 and its recruitment are required for transformation by MLL-AF9. These results strongly suggest that disruption of interaction between DOT1L and AF9/ENL is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL fusion protein-associated leukemia.

Keywords: AF9; DOT1L; ENL; Fusion Protein; Histone Methylation; Mixed Lineage Leukemia; Peptides; Protein-Protein Interactions; Surface Plasmon Resonance (SPR).

FIGURE 1.

Interaction between DOT1L and MLL fusion proteins, AF9 and ENL, measured by surface plasmon resonance. A, schematic presentation of AF9/ENL and DOT1L proteins used for the binding studies. B and C, sensorgrams representing the concentration-dependent binding of the C-terminal domain of AF9 and ENL tested in concentration range from 0.01 to 3 μm, with full-length FLAG-DOT1L (B) and Mocr-DOT1L (826–1095) (C), both immobilized on a CM5 sensor chip. The k_on, k_off, and K_D were calculated by simultaneous nonlinear regression using 1:1 binding model and BIAevaluation 3.1 software. The experimental data are shown in black, whereas the global fit analyses are shown in red. RU, response units.

FIGURE 2.

Mapping the AF9/ENL-binding site in DOT1L protein. A, alignment of human DOT1L (865–880) with human AF4 (761–774). The conserved residues are marked with asterisks, and the similar amino acids are marked with dots. B, alignment of the identified AF9/ENL-binding site in DOT1L protein from different species. C, SPR competitive binding curves of DOT1L 16-, 10-, and 7-mer and AF4 14-mer peptides against AF9 (497–568) and ENL (489–559) proteins running different tested concentrations over CM5 chip with immobilized DOT1L (826–1095). D, binding affinity of fluorescent-labeled DOT1L 10-mer peptide against AF9 and ENL. E, ITC of MBP-ENL (489–559) (80 μm) with a solution of DOT1L 16-mer (400 μm) and 10-mer (400 μm). For DOT1L 7-mer peptide (500 μm), 110 μm MBP-ENL was used. For all titrations, the raw data are shown in the upper panels, and the integrated heat data are shown in the lower panels. No binding was observed when the DOT1L peptides were tested against the MBP tag only. F, FP competitive binding curves of AF4 (749–775) recombinant protein and AF4 14-mer peptide using fluorescein-labeled DOT1L 10-mer peptide. mP, milli-polarization units. RU, response units.

FIGURE 3.

Characterizing the DOT1L 10-mer peptide and its interactions with MLL fusion proteins. A, binding affinities of alanine mutated DOT1L 10-mer peptides to AF9 and ENL in comparison to the wild type DOT1L 10-mer peptide. B, circular dichroism spectra of DOT1L 16- and 10-mer peptides.

FIGURE 4.

Analyzing the C-terminal domain in ENL protein and its binding to DOT1L. A, alignment of ENL and AF9 C-terminal domain. The three α-helices are indicated, and conserved residues are marked with asterisks. B, binding sensorgrams of immobilized Mocr-DOT1L (826–1095) with ENL (489–544) and ENL (523–559), tested in concentration ranges from 1 to 16 μm and from 5 to 35 μm, respectively. The k_on, k_off, and K_D were calculated by simultaneous nonlinear regression using 1:1 binding model and BIAevaluation 3.1 software. The experimental data are shown in black, whereas the global fit analyses are shown in red. RU, response units.

FIGURE 5.

Homology modeling of the ENL-DOT1L complex. A, molecular surface representation of the ENL-DOT1L complex. ENL surface is colored according to electrostatic potential: charged residues (dark blue), polar residues (light blue), and hydrophobic (white). The Cα trace of DOT1L peptide, LPISIPL, is shown as a green ribbon together with the side chains of the residues. B, five hydrogen bonds can be found between the complex of ENL-DOT1L shown with red dashed lines. DOT1L peptide is presented as a ball and stick model.

FIGURE 6.

DOT1L 10-mer peptide binds cellular MLL-AF9 fusion protein and disrupts its interaction with DOT1L. A, DOT1L 10-mer peptide disrupts the interaction between DOT1L and MLL-AF9 in cells. FLAG-DOT1L and Myc-CXXC-AF9 were co-transfected in HEK293 cells, and co-immunoprecipitation (IP) was performed. B, pulldown assay using biotin-labeled DOT1L 10-mer peptide. MW, molecular mass.

FIGURE 7.

AF9 binding site in DOT1L is essential for MLL-AF9 leukemic transformation. A, schematic presentation of the CFU assay. B, genotyping of transduced bone marrow cells. PCR showed high excision efficiency of endogenous DOT1L with 4-hydroxytamoxifen (4-OHT) treatment of all cells. C, quantitative PCR of exogenous DOT1L expression. All constructs showed expression compared with Neo vector alone. D, Western blot of H3K79me2 global level. Histone 3 blot was used as loading control. E and F, colony formation on methocult plates. Pictures of iodonitrotetrazolium chloride staining (E) and bar graph of colony counts (F) after second round.

FIGURE 8.

Schematic of proposed model for targeting DOT1L and MLL fusion protein-protein interactions. A, AF9/ENL-binding sites mapped in DOT1L and AF4 proteins. B, small molecule inhibitor (SMI) that binds to C-terminal domain of AF9/ENL will disrupt the MLL fusion protein complexes involved in mixed lineage leukemia, the DOT1L, and AEP complex.