Dimerization of MLL fusion proteins and FLT3 activation synergize to induce multiple-lineage leukemogenesis
J. Clin. Invest. Ryoichi Ono, et al. 115:919 doi:10.1172/JCI22725 [
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Figure 1Immortalization 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. β
2m 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.
