MLL-AFX requires the transcriptional effector domains of AFX to transform myeloid progenitors and transdominantly interfere with forkhead protein function

Abstract

MLL-AFX is a fusion gene created by t(X;11) chromosomal translocations in a subset of acute leukemias of either myeloid or lymphoid derivation. It codes for a chimeric protein consisting of MLL fused to AFX, a forkhead transcription factor that normally regulates genes involved in apoptosis and cell cycle progression. We demonstrate here that forced expression of MLL-AFX enhances the self-renewal of hematopoietic progenitors in vitro and induces acute myeloid leukemias after long latencies in syngeneic recipient mice. MLL-AFX interacts with the transcriptional coactivator CBP, which is also a fusion partner for MLL in human leukemias. A potent minimal transactivation domain (CR3) at the C terminus of AFX mediates interactions with the KIX domain of CBP and is necessary for transformation of myeloid progenitors by MLL-AFX. However, CR3 alone is not sufficient, suggesting that simple acquisition of a transactivation domain per se does not activate the oncogenic potential of MLL. Rather, two conserved transcriptional effector domains (CR2 and CR3) of AFX are required for full oncogenicity of MLL-AFX and also endow it with the potential to competitively interfere with transcription and apoptosis mediated by wild-type forkhead proteins. Furthermore, a dominant-negative mutant of AFX containing CR2 and CR3 enhances the growth of myeloid progenitors in vitro, although considerably less effectively than does MLL-AFX. Taken together, these data suggest that recruitment of transcriptional cofactors utilized by forkhead proteins is a critical requirement for oncogenic action of MLL-AFX, which may impact both MLL- and forkhead-dependent transcriptional pathways.

FIG. 1.

Transformation of myeloid progenitors by MLL-AFX. (A) Schematic diagram of MLL-AFX and the retroviral constructs used in hematopoietic progenitor transformation assays (left). MTase, DNA methyltransferase homology region; AT hook, AT hook DBD; FK, forkhead DBD; S, consensus serine phosphorylation sites; PFK, post-forkhead homology region (1); CR2, conserved region 2, which contains three alpha helices shown as thin vertical bars; CR3, conserved region 3. The bar graph (right) represents corresponding numbers of colonies after each round of plating in methylcellulose (average of three independent assays). (B) Typical morphology of methylcellulose colonies generated from bone marrow cells transduced with retroviruses expressing the indicated constructs. (C) Phenotypic analysis of cells transduced by MLL-AFX. Red lines represent FACS staining obtained with antibodies specific for the indicated cell surface antigens. Blue lines represent staining obtained with isotype control antibodies.

FIG. 2.

MLL-AFX transformed cells induce acute myeloid leukemias. (A) Representative histology is shown for control and MLL-AFX mice. Paraffin sections were stained with hematoxylin and eosin; blood smears were stained with MGG. In MLL-AFX mice, the spleen and liver were infiltrated with leukemic blasts. Bone marrow was densely packed with a homogeneous population of blasts. Leukemic cells are present in the peripheral blood. (B) Survival curves are shown for cohorts (n = 10) of sublethally irradiated C57BL/6 mice that were injected with MLL-AFX immortalized cells (MLL-AFX), AFX-3′ immortalized cells, or mock injected (control).

FIG. 3.

Mapping of transcriptional activation domains in AFX. Schematic diagram (top left) illustrates the conserved domains of AFX. The arrow indicates the fusion site with MLL in human leukemias. Thick horizontal black lines with amino acid numbers represent the AFX fragments fused to the Gal4-DBD for transactivation assays. ENL-C (477 to 559 aa) and ELL-C (496 to 621 aa) correspond to the minimal regions sufficient for immortalization of myeloid progenitors by MLL-ENL and MLL-ELL fusion proteins, respectively (15, 50). VP16-MTD encodes the minimal transcriptional activation domain (aa 413 to 453) of herpes simplex virus VP-16. Expression constructs were cotransfected with pcDNA-LacZ into 293 cells with a luciferase reporter gene under the control of the indicated promoters. TK, herpes simplex virus TK promoter; E1b, adenovirus E1b promoter; Myelomono, myelomonocytic growth factor promoter (described in Materials and Methods). Luciferase values were normalized for β-galactosidase expression from the internal LacZ control construct. Similar expression level of the Gal4 constructs was confirmed by Western blot (data not shown).

FIG. 4.

The AFX CR3 interacts in vitro with the KIX domain of CBP. (A) GST pull-down experiments were conducted with in vitro translated [³⁵S]methionine-labeled proteins indicated on the left. GST constructs are indicated at the top of gel lanes. GST-CBP constructs have been previously described (58). (B) Mapping of KIX interacting domain to CR3 of AFX. Various AFX fragments (indicated by horizontal bars on left) were in vitro translated with [³⁵S]methionine and incubated with GST (lane 2) or GST-KIX (lane 3) fusion proteins. Bound proteins were washed, eluted, and subjected to SDS-PAGE after autoradiography. Protein inputs are shown in lane 1. (C) In vivo interaction between CBP and AFX proteins. Constructs used in cell transfection are shown at the top of each lane and their expression was confirmed by Western blots (bottom panel). Cell lysates were precipitated with antibodies to MLL or Gal4 DBD and then blotted with anti-CBP antibody. Specific 265-kDa CBP bands are indicated in the top panel.

FIG. 5.

CR2 and CR3 are both necessary for MLL-AFX-mediated transformation. Schematic diagram (left column) shows the deletion mutants of MLL-AFX used for hematopoietic transformation assays. A synthetic MLL-VP16 (MTD) construct was also included. Transformation abilities, transactivation properties, and in vitro binding to the CBP KIX domain are summarized in the middle. Bar graph (right) represents the number of colonies generated by the respective constructs in replating assays (mean ± the standard derivation, n = 3).

FIG. 6.

Potential dominant-negative effects of MLL-AFX fusion proteins. (A) MLL-AFX is unable to bind consensus forkhead DNA-binding sites. In vitro-translated proteins (indicated at tops of gel lanes) were incubated with ³²P-labeled oligonucleotides encompassing the Fas ligand site corresponding to IGFBP1 IRS (Fas ligand promoter) or a mutated IRS (Am2Bm2) of the IGFBP-1. Reticulocyte lysate (lane 1) was used as a control for nonspecific binding. The arrow indicates specific DNA-binding complexes. (B) MLL-AFX antagonizes FKHRL1-mediated transcriptional activation. An expression construct encoding FKHRL1 was cotransfected into 293 cells with FKHRL1, MLL-AFX, or AFX expression constructs and a reporter gene driven by the Fas ligand promoter (FHRE promoter) or the p27^kip-1 promoter (p27kip promoter). The fold induction was corrected for β-galactosidase activity from an internal lacZ control construct in each transfection. (C) Comparable expression levels of MLL-AFX in leukemic (lane 2) and transfected (lane 3) cells. The control (lane 1) consisted of the REH pre-B cell line, which expressed wild-type MLL but not MLL-AFX. Specific bands corresponding to wild-type MLL, MLL-AFX, and FKHRL1 are indicated to the left.

FIG. 7.

MLL-AFX suppresses FKHRL1-mediated apoptosis in Ba/F3 cells. (A) Ba/F3 cells stably expressing FKHRL1 (A3): ER∗ were cotransfected with EGFP and various expression constructs (left). Cells were treated with or without 0.1 μM 4-OHT for 24 h before analysis of apoptosis by Annexin V staining. EGFP was used as a marker to identify transfected cells for apoptosis analysis. Bars (right) represent the percentage change in apoptosis after the induction of FKHRL1 (A3) expression in the absence or presence of various DNA constructs. (B) FACS data of Ba/F3 cells from a representative transfection experiment. The results are shown for EGFP-positive, PI-negative gated cells. Black lines represent controls transfected with EGFP only and treated with 4-OHT for 24 h. Shadow profiles represent either the control without 4-OHT treatment or cells transfected with various constructs and treated with 4-OHT. Expression of FKHRL1 (A3)∗ER was detected by Western blot, as shown by the small insert in the left top panel. (C) MLL-AFX- or MSCV-transfected cells were sorted into GFP-positive and -negative populations. Cell lysates from 10⁵ cells were analyzed by Western blotting with antibodies specific for p27^kip-1 or actin.