The cytoplasmic NPM mutant induces myeloproliferation in a transgenic mouse model

Abstract

Although NPM1 gene mutations leading to aberrant cytoplasmic expression of nucleophosmin (NPMc(+)) are the most frequent genetic lesions in acute myeloid leukemia, there is yet no experimental model demonstrating their oncogenicity in vivo. We report the generation and characterization of a transgenic mouse model expressing the most frequent human NPMc(+) mutation driven by the myeloid-specific human MRP8 promoter (hMRP8-NPMc(+)). In parallel, we generated a similar wild-type NPM trans-genic model (hMRP8-NPM). Interestingly, hMRP8-NPMc(+) transgenic mice developed myeloproliferation in bone marrow and spleen, whereas nontransgenic littermates and hMRP8-NPM transgenic mice remained disease free. These findings provide the first in vivo evidence indicating that NPMc(+) confers a proliferative advantage in the myeloid lineage. No spontaneous acute myeloid leukemia was found in hMPR8-NPMc(+) or hMRP8-NPM mice. This model will also aid in the development of therapeutic regimens that specifically target NPMc(+).

Figure 1

Expression of NPMc⁺ and NPM in hMRP8-NPMc⁺ and hMRP8-NPM transgenic mice. (A) Schematics of hMRP8-NPMc⁺ and hMRP8-NPM constructs used to generate NPM transgenic lines. (B) Southern blot analysis using the 3′ probe of BamHI-digested tail DNA from hMRP8-NPMc⁺ founders. Positive founders were confirmed by PCR as illustrated below the Southern blot panel. (C) Semiquantitative RT-PCR analysis of NPMc⁺ and NPM mRNA levels from whole BM cells of different founders. Red arrows represent the transgenic lines used for detailed analysis. NT indicates nontransgenic control. (D) Semiquantitative RT-PCR analysis of NPMc⁺ mRNA levels from sorted populations. Myeloid: Mac-1⁺Gr-1⁺ BM cells; B-cell: B220⁺CD19⁺ splenocytes; T-cell: CD4⁺ splenocytes. (E) Western blot of NPMc⁺ and NPM expression in whole BM cells of NPMc⁺ 28 (left panels) and NPM 65 (right panels) animals. A monoclonal Flag antibody (top left) and a polyclonal NPMc⁺-specific antibody (bottom left) were used to detect NPMc⁺ levels. *Nonspecific band. A polyclonal Flag antibody (top right) and a monoclonal NPM antibody were used to detect both overexpressed human NPM and endogenous mouse Npm levels. C indicates control lysates from HEK293 cells transfected with an NPMc⁺ expression vector; NPM 69, a negative control (an NPM transgenic mouse that was not found to overexpress Npm at the level of RNA or protein). (F) Western blot analysis of NPMc⁺ and NPM expression on purified nuclear and cytoplasmic fractions of Mac-1⁺Gr-1⁺ sorted granulocytes from BM of NT, NPM 65, and NPMc⁺ 28 animals. A polyclonal Flag antibody was used to specifically recognize the transgenic wild-type NPM or NPMc⁺ mutant. A monoclonal NPM antibody was used to detect the endogenous mouse Npm levels. Anti-LaminB1 and anti-Hsp90 assess the purity of the nuclear (N) versus the cytoplasmic (C) fraction, respectively.

Figure 2

hMRP8-NPMc⁺ transgenic mice develop a nonreactive myeloproliferation with expansion of mature granulocytes/monocytes. (A) Table showing the total number of mice analyzed from different transgenic lines, their age range, and myeloproliferation (MP) penetrance involving either BM or spleen. (B) Composite data from age-matched littermates of indicated genotypes demonstrating splenomegaly in NPMc⁺ transgenic mice (mean ± SEM; NT, n = 5; NPMc⁺ 28, n = 5). **P < .01. Spleen masses are 160 ± 60.8 mg for NT (n = 5) versus 567 ± 284 mg for NPMc⁺ 28 (n = 5; P = .014). (C) Histopathologic sections of BM and spleen from representative NT and NPMc⁺ transgenic mice. (i) NT BM (hematoxylin and eosin, original magnification, ×1000). (ii) NT BM cytospin (original magnification, ×1000). represents an erythroblast; , granulocytes. (iii) NPMc⁺ BM (hematoxylin and eosin, original magnification, ×1000). (iv) NPMc⁺ BM cytospin (original magnification, ×1000). represent granulocytes. (v) NT spleen (hematoxylin and eosin, original magnification, ×40). (vi) NT spleen (hematoxylin and eosin, original magnification, ×400). (vii) NPMc⁺ spleen (hematoxylin and eosin, original magnification, ×40). represents areas of expanded red pulp with extramedullary hematopoiesis. (viii) NPMc⁺ spleen (hematoxylin and eosin, original magnification, ×400). represents extramedullary hematopoiesis. (D) Flow cytometric analysis of single-cell suspensions of BM and spleen from representative NT and NPMc⁺ transgenic mice demonstrates an increase in granulocytic/monocytic (Mac-1⁺;Gr-1⁺) and mature myeloid (Mac-1⁺;cKit⁻) cells with a corresponding decrease in the amount of B (B220⁺) and T (CD3⁺) lymphoid cell populations. (E) Quantification of granulocytic/monocytic (Mac-1⁺;Gr-1⁺), mature myeloid (Mac-1⁺/cKit⁺), B cells (B2200⁺), and T cells (CD3⁺) in BM and spleen of age-matched NT and NPMc⁺ transgenic mice analyzed as in panel D (mean ± SEM; NT, n = 6; NPMc⁺ 28, n = 6). *P < .05. **P < .01. ***P < .001. n.s. indicates not statistically significant.