Knock-in of an internal tandem duplication mutation into murine FLT3 confers myeloproliferative disease in a mouse model

Abstract

Constitutive activation of FMS-like tyrosine kinase 3 (FLT3) by internal tandem duplication (ITD) mutations is one of the most common molecular alterations known in acute myeloid leukemia (AML). To investigate the role FLT3/ITD mutations play in the development of leukemia, we generated a FLT3/ITD knock-in mouse model by inserting an ITD mutation into the juxtamembrane domain of murine Flt3. FLT3wt/ITD mice developed myeloproliferative disease, characterized by splenomegaly, leukocytosis, and myeloid hypercellularity, which progressed to mortality by 6 to 20 months. Bone marrow (BM) and spleen from FLT3wt/ITD mice had an increased fraction of granulocytes/monocytes and dendritic cells, and a decreased fraction of B-lymphocytes. No sign of acute leukemia was observed over the lifetime of these mice. BM from FLT3wt/ITD mice showed enhanced potential to generate myeloid colonies in vitro. BM from FLT3wt/ITD mice also produced more spleen colonies in the in vivo colony-forming unit (CFU)-spleen assay. In the long-term competitive repopulation assay, BM cells from FLT3wt/ITD mice outgrew the wild-type competitor cells and showed increased myeloid and reduced lymphoid expansion activity. In summary, our data indicate that expression of FLT3/ITD mutations alone is capable of conferring normal hematopoietic stem/progenitor cells (HSPCs) with enhanced myeloid expansion. It also appears to suppress B lymphoid maturation. Additional cooperative events appear to be required to progress to acute leukemia.

Figure 1

Generation of a mouse model with FLT3^wt/ITD mutation. (A) Strategy for targeted insertion of an ITD mutation into murine Flt3 genomic DNA. The targeting vector was a 12-kb fragment flanking exons 10 to 16 of murine FLT3 genomic DNA. An 18-bp ITD mutation was inserted into exon 14. MC1-TK cassette and LoxP-flanked PGK-Neo cassette were inserted as indicated into the targeting vector, acting as negative and positive selection markers, respectively. The sites of diagnostic restriction enzyme KpnI (K) and ProbeA used for Southern Blotting analysis are shown; ▵ indicates LoxP. (B) Homologous recombination and presence of ITD mutation in FLT3^wt/ITD mice was confirmed by Southern blotting analysis (i) and PCR analysis (ii). (Bi) Southern blotting analysis confirmed the presence of homologous recombination on one allele of Flt3 genomic DNA. (Bii) PCR analysis detected the presence of homologously recombined allele (top panel) and ITD mutation (bottom panel). (C) PIPC-treated FLT3^wt/ITD mice showed enhanced FLT3 expression. (Ci) RT-PCR detected transcription of both ITD and wild-type FLT3 in BM cells from FLT3^wt/ITD mice; numbers shown below each lane in the top panel are the mean ratio of the ITD (top band) versus wild-type (bottom band) FLT3 expression determined by densitometry analysis. (Cii) Quantitative RT-PCR showed increased expression of FLT3 in FLT3^wt/ITD mice. The level of total FLT3 expression was normalized based on that of mS16. Data are expressed as means (bars) plus or minus SEM (error bars). (Di) Western blotting analysis showed enhanced FLT3 activity and expression in FLT3^wt/ITD mice. Lysed spleen cells were immunoprecipitated with EB-10 antibody and immunoblotted using 4G10 (top panel) or anti-murine FLT3 antibodies (bottom panel). (Dii) Flow cytometric analysis shows elevated FLT3 expression in the Lin^−/low population of the BM from FLT3^wt/ITD mice. wt indicates wild-type.

Figure 2

Myeloproliferative disease develops in mice with FLT3^wt/ITD mutation. (A) Kaplan-Meier plot of survival of FLT3^wt/ITD and wild-type control mice (n = 12). (B) FLT3^wt/ITD mice manifested splenomegaly. Peripheral WBC count (C) and WBC differential (D) in 12-month-old FLT3^wt/ITD and wild-type mice (n = 10) are shown. wt indicates wild-type. *P < .05. Data in panel C are expressed as means (bars) plus or minus SEM (error bars).

Figure 3

Spleen and BM from FLT3^wt/ITD display MPD. (A-F) Spleen sections from 2-month-old wild-type (A) and FLT3^wt/ITD (B) mice and from 12-month-old wild-type (C,E) and FLT3^wt/ITD (D,F) mice. (G-J) BM sections from 12-month-old wild-type (G,I) and FLT3^wt/ITD mice (H,J). (K,L) Cytospins of BM from 12-month-old wild-type (K) and FLT3^wt/ITD (L) mice. (A-J) H&E stain. (K,L) Wright & Giemsa stain. Scale bars: 100 μm (A,B); 50 μm (C,D,G,H); and 10 μm (E,F,I,J). Image acquisition: Images A-J were acquired at room temperature using a Zeiss Axioskop upright microscope system (Zeiss, Thornwood, NY) with Achroplan 5×/0.16 NA, 10×/0.3 NA, and 40×/0.6 NA objectives. The images were photographed with an AxioCam camera (Zeiss) and Axiovision 4.0 software (Zeiss). Images K and L were acquired at room temperature using a Nikon Eclipse E600 microscope system (Nikon, Tokyo, Japan) with a Nikon 100×/0.90 NA oil objective (magnification: ×1000) and were photographed with a Nikon DMX 1200 digital camera with ACT-1 2.0 software (Nikon).

Figure 4

Immunophenotype of BM and spleen cells from FLT3^wt/ITD mice shows expansion of granulocytes /monocytes and DCs and inhibition of B-lymphoid development. (A,E) Increased Lin^−/low and c-KIT⁺ population. (B,F) Increased granulocytic/monocytic and reduced erythroid populations. (C,G,I) Reduced B-cell populations; analyses of total BM/spleen cells (top panels) and B220⁺CD43⁻ BM/spleen cells (bottom panels) are shown. (D,H) Increased DC production. Flow cytometric analysis of spleen and BM cells from representative 1-, 2-, and 12-month-old wild-type and FLT3^wt/ITD mice is shown. Numbers indicate the percentage of cells in each quadrant.

Figure 5

BM from FLT3^wt/ITD mice demonstrates enhanced myeloid colony-forming activity and can be immortalized in vitro. Methylcellulose-based in vitro colony-forming assay of total BM cells (A) and Lin^−/low BM cells (B) from 2-month-old mice; (C) Secondary and tertiary colony-forming assay; (D) Flow cytometic analysis of cells collected from colonies 11 days after the first plating and from the long-term liquid culture (6 months). (E) Cells derived from FLT3^wt/ITD BM could be serially propagated in RPMI 1640 medium containing SCF and IL-3. *P < .05. (F) BM cells from FLT3^wt/ITD mice have a lower cytokine requirement to form CFU-GM. Data are results from 3 independent experiments. Data are expressed as means (bars) plus or minus SEM (error bars). (G) Representative CFU-GM colonies generated from FLT3^wt/ITD or wild-type BM in the presence of 1 ng/mL GM-CSF. Original magnification: ×40. Images were acquired at room temperature using a Nikon Eclipse TE300 inverted microscope system with a Nikon Plan Fluor 4×/0.13 NA objective. The images were photographed using a Diagnostics Instruments Spot camera with SPOT 3.3.2 software (Diagnostic Instruments, Sterling Heights, MI).

Figure 6

FLT3^wt/ITD mice possess enhanced myeloid expansion activity in vivo. (A) In vivo CFU-spleen assay. CFU-S 1, CFU-S 2, and CFU-S 3 indicate primary, secondary and tertiary CFU-spleen assays, respectively. Data are expressed as means (bars) plus or minus SEM (error bars). (B) In vivo competitive repopulation assay. A representative from 3 independent experiments is shown. *P < .05; **P < .001. (C) BM cells from the FLT3^wt/ITD donors demonstrated increased myeloid and reduced lymphoid expansion activity compared with the wild-type competitor cells in the same recipients. Flow cytometry analysis of BM cells from representative recipients in the FLT3^wt/ITD and wild-type groups is shown. Numbers indicate the percentage of cells in each quadrant. wt indicates wild-type.