Dimerization of MLL fusion proteins and FLT3 activation synergize to induce multiple-lineage leukemogenesis

Abstract

The mechanisms by which mixed-lineage leukemia (MLL) fusion products resulting from in utero translocations in 11q23 contribute to leukemogenesis and infant acute leukemia remain elusive. It is still controversial whether the MLL fusion protein is sufficient to induce acute leukemia without additional genetic alterations, although carcinogenesis in general is known to result from more than 1 genetic disorder accumulating during a lifetime. Here we demonstrate that the fusion partner-mediated homo-oligomerization of MLL-SEPT6 is essential to immortalize hematopoietic progenitors in vitro. MLL-SEPT6 induced myeloproliferative disease with long latency in mice, but not acute leukemia, implying that secondary genotoxic events are required to develop leukemia. We developed in vitro and in vivo model systems of leukemogenesis by MLL fusion proteins, where activated FMS-like receptor tyrosine kinase 3 (FLT3) together with MLL-SEPT6 not only transformed hematopoietic progenitors in vitro but also induced acute biphenotypic or myeloid leukemia with short latency in vivo. In these systems, MLL-ENL, another type of the fusion product that seems to act as a monomer, also induced the transformation in vitro and leukemogenesis in vivo in concert with activated FLT3. These findings show direct evidence for a multistep leukemogenesis mediated by MLL fusion proteins and may be applicable to development of direct MLL fusion-targeted therapy.

Figure 1

Immortalization of murine hematopoietic progenitors by MLL-SEPT6 fusion protein via aberrant expression of Hox genes. (A) Schematic representation of the retroviral constructions used. CXXC, CXXC domain; Zn fingers, zinc fingers; CS, cleavage sites; TAD, transactivation domain; SET, SET domain. (B) Western blot analysis of proteins extracted from PlatE cells transfected with the constructs shown in A, after immunoprecipitation using the anti-MLL Ab (lanes 1–9). Each lysate was blotted with the anti-FLAG Ab (lanes 1–9) or anti-SEPT6 Ab (lanes 10 and 11). Endogenous expression of SEPT6 was detected in lane 10. Lane 1, mock; lane 2, 5′-MLL; lane 3, MLL-SEPT6; lane 4, MLL-SEPT6^Δcoil; lane 5, MLL-SEPT6^Δcoil-ER; lane 6, MLL-SEPT6^ΔGTP; lane 7, MLL-SEPT6^ΔP-loop; lane 8, MLL-SEPT6^S56N; lane 9, MLL-ENLs; lane 10, pMXs-neo alone (endogenous SEPT6); lane 11, SEPT6. (C) Experimental strategy for myeloid immortalization assay. (D) Myeloid immortalization assay using the constructs shown in A. Lanes for MLL-SEPT6^Δcoil-ER indicate the presence (+) or absence (–) of 4-OHT. The bar graph shows numbers of colonies obtained after each round of replating in methylcellulose (average ± SD). (E and F) Typical morphology of the colonies generated by MLL-SEPT6 (E), and the cells constituting these colonies (F). Original magnification, ×40 (E), ×400 (F). (G) Expression of Hox a7, Hox a9, and Meis1 by RT-PCR in the cells from third-round cultures. β₂m was used as an internal standard. M, 100-bp DNA ladder (New England Biolabs Inc.); lane 1, control (Ba/F3 with IL-3) cells; lane 2, mock; lane 3, MLL-SEPT6; lane 4, MLL-ENLs; lane 5, negative control.

Figure 2

Characterization of the cells immortalized by MLL-SEPT6. (A) Schematic representation of pMXs-neo-MLL-SEPT6. The restriction endonuclease sites and the Neo probe used in the Southern blot analysis are indicated by vertical arrows and a thick horizontal line, respectively. The primers used are indicated by horizontal arrows. LTR, long-terminal repeat. (B) Immunophenotype of the cells immortalized by MLL-SEPT6. Shadow profiles represent FACS staining obtained with Abs specific for the indicated cell surface antigens. Green lines represent staining obtained with isotype control Abs. (C) Southern blot analysis to detect clonality (lane 1) and integration (lane 2). Genomic DNA extracted from the cells immortalized by MLL-SEPT6 was digested with BamHI (lane 1) or NheI (lane 2), and hybridized with the Neo probe. Oligoclonal bands of proviral integration sites and a single band of the intact proviral DNA are indicated by arrows and an arrowhead, respectively. (D) Expression of MLL-SEPT6 fusion transcripts by RT-PCR. M, 1-kb DNA ladder (New England Biolabs Inc.); lane 1, cells immortalized by MLL-SEPT6; lane 2, negative control; lane 3, positive control (pMXs-neo-MLL-SEPT6).

Figure 3

Oligomerization of MLL-SEPT6 fusion protein through both its GTP-binding domain and its coiled-coil region in the nucleus. 293T cells were cotransfected with equal amounts of FLAG-tagged and HA-tagged constructs (A), and transfected with pMXs-neo-SEPT6 or pMXs-neo-MLL-SEPT6 (B). (A) Self-interaction among MLL-SEPT6 fusion proteins or MLL-SEPT6 mutants, analyzed by Western blot analysis after immunoprecipitation. Lysates of 293T cells coexpressing FLAG-tagged and HA-tagged MLL-SEPT6 or its mutants (top and middle) were immunoprecipitated by the anti-FLAG Ab, and lysates of the cells expressing HA-tagged MLL-SEPT6 or its mutants (bottom) were immunoprecipitated by the anti-HA Ab. Anti-FLAG immunoprecipitates were blotted with the anti-HA Ab (top) or the anti-FLAG Ab (middle), while anti-HA immunoprecipitates were blotted with the anti-HA Ab (bottom). Lane 1, MLL-SEPT6; lane 2, MLL-SEPT6^Δcoil; lane 3, MLL-SEPT6^ΔP-loop; lane 4, MLL-SEPT6^S56N. (B) Localization of SEPT6 and MLL-SEPT6, analyzed by immunofluorescent confocal microscopy. FITC-conjugated secondary Abs reacting with the anti-FLAG Ab in 293T cells expressing SEPT6 or MLL-SEPT6 visualized their cellular localizations (middle). Nuclei were visualized with DAPI (left), and merged images are displayed (right). Original magnification, ×400.

Figure 4

Synergistic transformation of murine hemato-poietic progenitors by MLL fusion genes and FLT3-ITD in vitro. (A) Schematic representation of the retroviral constructions expressing FLT3-ITD. (B) Transformation assay of the cells immortalized by MLL-SEPT6, after transduction with FLT3-ITD in the pMY-IRES-EGFP construct shown in A in the presence (+) or absence (–) of IL-3. (C) Western blot analysis of proteins extracted from PlatE cells transfected with the constructs shown in A and each vector alone as a control, after immunoprecipitation using the anti-FLT3 Ab (lanes 1–4). Each lysate was blotted with the anti-FLT3 Ab. Lane 1, pMY-IRES-EGFP alone; lane 2, pMY-FLT3-ITD-IRES-EGFP; lane 3, pMYpuro alone; lane 4, pMYpuro-FLT3-ITD. (D) Myeloid immortalization assay using the pMYpuro constructs shown in A. The bar graph shows numbers of colonies obtained after each round of replating in methylcellulose (average ± SD). (E) Myeloid transformation assay using the sequential transduction with FLT3-ITD or control (pMYpuro alone) after MLL-SEPT6, MLL-ENLs, or mock (pMXs-neo alone) in the presence (+) or absence (–) of IL-3.

Figure 5

Synergistic leukemogenesis induced by MLL fusion genes and FLT3-ITD in vivo. (A and B) Survival curves of mice transplanted with mock/GFP (n = 4), MLL-SEPT6/GFP (MS6/GFP; n = 6), mock/FLT3 (n = 5), and MS6/FLT3 (n = 9) (A), and with MLL-ENLs/GFP (MEs/GFP; n = 4) and MEs/FLT3 (n = 5) (B). Mice transplanted with MLL fusion genes in combination with FLT3-ITD showed significantly shorter survival time than those with corresponding MLL fusion genes in combination with GFP (P < 0.05, log-rank test). (C) Representative macroscopic and histopathologic analysis of morbid mice transplanted with MS6/FLT3 and MS6/GFP, respectively. Arrowheads show lymphadenopathy. BM cells were stained with Wright-Giemsa, and paraffin sections of liver and spleen were stained with H&E. Original magnification of BM cells, ×200; scale bars, 200 μm.

Figure 6

Immunophenotype of bone marrow cells obtained from representative mice (nos. 213 and 212) transplanted with mock/GFP, MS6/GFP, and MS6/FLT3. The dot plots show each surface antigen labeled with a corresponding PE-conjugated mAb versus expression of GFP.

Figure 7

Southern blot analysis of spleen DNA obtained from representative mice transplanted with mock/GFP, MS6/GFP, and MS6/FLT3. Genomic DNA extracted from each spleen was digested with NheI (A), BamHI (B), or EcoRI (C) and hybridized with the Neo probe (A and B, left), the GFP probe (A and B, right), or the J_H probe (C). (A) Each single band of intact proviral DNA, including MLL-SEPT6 (MS6) and FLT3-ITD (FLT3), is indicated by an arrow with each abbreviation. (B) Oligoclonal bands of proviral integration sites of MS6 or FLT3 are indicated by black or white arrowheads, respectively. (C) Germ line (G) or rearrangements (R) of IgH gene are indicated by an arrow with each abbreviation. Lane 1, mock/GFP mouse; lanes 2 and 3, MS6/GFP mice; lanes 4 and 5, MS6/FLT3 mice.