PML/RARalpha and FLT3-ITD induce an APL-like disease in a mouse model

Abstract

Acute promyelocytic leukemia (APL) cells invariably express aberrant fusion proteins involving the retinoic acid receptor alpha (RARalpha). The most common fusion partner is promyelocytic leukemia protein (PML), which is fused to RARalpha in the balanced reciprocal chromosomal translocation, t(15;17)(q22:q11). Expression of PML/RARalpha from the cathepsin G promoter in transgenic mice causes a nonfatal myeloproliferative syndrome in all mice; about 15% go on to develop APL after a long latent period, suggesting that additional mutations are required for the development of APL. A candidate target gene for a second mutation is FLT3, because it is mutated in approximately 40% of human APL cases. Activating mutations in FLT3, including internal tandem duplication (ITD) in the juxtamembrane domain, transform hematopoietic cell lines to factor independent growth. FLT3-ITDs also induce a myeloproliferative disease in a murine bone marrow transplant model, but are not sufficient to cause AML. Here, we test the hypothesis that PML/RARalpha can cooperate with FLT3-ITD to induce an APL-like disease in the mouse. Retroviral transduction of FLT3-ITD into bone marrow cells obtained from PML/RARalpha transgenic mice results in a short latency APL-like disease with complete penetrance. This disease resembles the APL-like disease that occurs with long latency in the PML/RARalpha transgenics, suggesting that activating mutations in FLT3 can functionally substitute for the additional mutations that occur during mouse APL progression. The leukemia is transplantable to secondary recipients and is ATRA responsive. These observations document cooperation between PML/RARalpha and FLT3-ITD in development of the murine APL phenotype.

Figure 1

Kaplan–Meier analysis for leukemia-free survival. The percentage of surviving mice (y axis) is plotted with respect to time in days (x axis). The number of animals per group is indicated. (A) The disease-free survival of BALB/c animals receiving 10⁶ cells transduced with FLT3-ITD compared with the survival of (B6×C3H)F₁ animals receiving 10⁶ cells transduced with FLT3-ITD. This plot presents data from four independent experiments. (B) The leukemia-free survival of (B6×C3H)F₁ animals receiving PML/RARα transgenic cells (PR) transduced with either EGFP or FLT3-ITD. This plot presents data from two independent experiments.

Figure 2

Histopathology of the spleen. Sections of hematoxylin and eosin-stained spleen (×500 magnification) are shown. (A) Wild-type healthy (B6×C3H)F₁ mouse spleen transduced with EGFP. (B) Diseased mouse spleen expressing FLT3-ITD on wild-type background, with an Inset showing a section of the lymphoid tumor. (C) Diseased mouse spleen with PML/RARα alone, which developed APL after long latency. (D) Diseased mouse spleen expressing FLT3-ITD on the PML/RARα transgenic background.

Figure 3

Immunophenotype of spleen cells. The immunophenotypes for normal (B6×C3H)F₁ control spleen cells, FLT3-ITD-transduced control spleen cells, FLT3-ITD-transduced PML/RARα transgenic spleen cells, and EGFP-transduced PML/RARα transgenic cells are shown. Spleen cells were stained with allophycocyanin (APC)-anti-Gr-1 and phycoerythrin (PE)-conjugated Mac-1, APC-Mac-1, and PE-c-Kit or APC-CD4 and PE-CD8. The dot plots are gated for live cells based on forward- and side-scatter profiles. The percentages of cells in quadrants of interest are shown.

Figure 4

The FLT3-ITD, PML/RARα-induced disease is transplantable. The c-Kit (allophycocyanin conjugated), Mac-1 (phycoerythrin conjugated) staining profile of spleen cells from primary, secondary, and tertiary recipients of FLT3 and PML/RARα (F3+PR) cells is shown. The percentages of cells in each quadrant is indicated. Below each FACS profile is a histological section of the spleen from a primary, secondary, or tertiary animal, stained with hematoxylin and eosin (×500 magnification). Southern analysis of cells from primary (lanes 1–6) and secondary (lanes 7–9) recipients is shown; each lane represents an individual mouse. The animal represented in lane 6 was the source of primary cells used for secondary transplantation (lanes 7–9), hybridized with EGFP. These data were generated on a single blot. The blot was rehybridized with cathepsin G as a control for DNA loading.

Figure 5

ATRA responsiveness of FLT3-ITD, PML/RARα APL cells. Colony assays with PML/RARα APL cells and FLT3-ITD + PML/RARα APL cells were performed in clotted plasma in the absence or presence of ATRA, dried, and stained with Wright and Giemsa. In each case the colonies are shown at ×100 magnification with an Inset showing the cells at ×500 magnification. (A) PML/RARα cells without ATRA. (B) PML/RARα cells with ATRA 10⁻⁶ M. (C) FLT3-ITD + PML/RARα cells without ATRA. (D) FLT3-ITD + PML/RARα cells with ATRA 10⁻⁶ M.