Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease

Abstract

Oncogenic ras alleles are among the most common mutations found in patients with acute myeloid leukemia (AML). Previously, the role of oncogenic ras in cancer was assessed in model systems overexpressing oncogenic ras from heterologous promoters. However, there is increasing evidence that subtle differences in gene dosage and regulation of gene expression from endogenous promoters play critical roles in cancer pathogenesis. We characterized the role of oncogenic K-ras expressed from its endogenous promoter in the hematopoietic system using a conditional allele and IFN-inducible, Cre-mediated recombination. Mice developed a completely penetrant myeloproliferative syndrome characterized by leukocytosis with normal maturation of myeloid lineage cells; myeloid hyperplasia in bone marrow; and extramedullary hematopoiesis in the spleen and liver. Flow cytometry confirmed the myeloproliferative phenotype. Genotypic and Western blot analysis demonstrated Cre-mediated excision and expression, respectively, of the oncogenic K-ras allele. Bone marrow cells formed growth factor-independent colonies in methylcellulose cultures, but the myeloproliferative disease was not transplantable into secondary recipients. Thus, oncogenic K-ras induces a myeloproliferative disorder but not AML, indicating that additional mutations are required for AML development. This model system will be useful for assessing the contribution of cooperating mutations in AML and testing ras inhibitors in vivo.

Figure 1

Lethal myeloproliferative disease in mice expressing a conditional oncogenic K-ras allele. (a) Schematic of wild-type (top), floxed (middle), and activated (bottom) K-ras alleles. K-ras exons 0, 1, and 2 are depicted. Gene targeting to the endogenous K-ras locus generated the floxed LSL–K-ras G12D allele (38) containing a transcriptional termination codon flanked by loxP sites upstream of a mutation of glycine to aspartic acid in codon 12 in exon 1. Excision of the stop cassette by Cre recombinase allows expression of the oncogenic K-ras allele. Asterisk indicates G12D mutation in exon 1. (b) Breeding schematic of LSL–K-ras G12D and Mx1-Cre mice, with subsequent pI-pC treatment of progeny to generate KM+, KM–, K+, M+, and WT+ littermate mice. K, LSL–K-ras G12D; M, Mx1-Cre. + or – indicates presence or absence of pI-pC treatment. (c) Kaplan-Meier comparative survival analysis of KM+, KM– and negative control mice. Cumulative survival was plotted against days after treatment with pI-pC. For KM– mice, cumulative survival was plotted against days after their littermates received pI-pC treatment. KM+ (n = 25) and KM– (n = 8) mice developed a lethal myeloproliferative disease with median latencies of 35 and 58 days, respectively. K+ (n = 11), M+ (n = 8), and WT+ (n = 10) mice were healthy during an observation period of more than 200 days. (d) Splenomegaly in mice expressing oncogenic K-ras. Spleen weights (left to right): K+, 70 mg; M+, 130 mg; KM+, 560 mg; and KM+, 2,200 mg.

Figure 2

Mice expressing oncogenic K-ras develop a myeloproliferative disease. Representative histopathology (original magnifications in parentheses) from peripheral blood (×100), bone marrow (×100), spleen (×40), and liver (×20), showing expansion of predominantly mature myeloid elements, without an increase in immature/blast forms.

Figure 3

Mice expressing oncogenic K-ras develop esophageal squamous papillomas and lung adenomas. Shown are normal gastroesophageal junction from M+ mouse (×10) and esophageal squamous papilloma (×5), ear squamous papilloma (×2), and lung adenomas (×5; inset, ×40) from KM+ mice. Original magnifications in parentheses.

Figure 4

Flow cytometric analysis of spleen and bone marrow cells. (a) Spleen cells from KM+ (n = 4), KM– (n = 2), and negative control WT+, K+, and M+ mice (n = 2 each) were stained with a combination of antibodies to Gr-1, Mac-1, CD14, CD45, Ter-119, Thy-1, and CD19. Dot plots were gated for live cells based on forward and side scatter profiles. Representative data are shown. (b) Bone marrow cells from KM+ (n = 4) and negative control mice (n = 2 each) were stained with a combination of antibodies to Gr-1, Mac-1, c-Kit, CD4, CD8, Ter-119, and CD45. Dot plots were gated for live cells based on forward and side scatter profiles. Representative data are shown. The percentages of cells in quadrants of interest are indicated.

Figure 5

Analysis of myeloid progenitors in bone marrow and spleen of K+, M+, and KM+ mice. Percentages of myeloid progenitors (IL-7Rα^–Lin^–Sca1^–c-Kit⁺ cells) and percentages of CMPs (FcγR^loCD34⁺), GMPs (FcγR^hiCD34⁺), and MEPs (FcγR^loCD34^–) relative to whole bone marrow and spleen are indicated. Quadrants represent the respective gated populations of CMPs, GMPs, and MEPs.

Figure 6

Cre-mediated activation of the oncogenic K-ras allele and expression of oncogenic K-ras protein in diseased KM+ and KM– tissues. (a) PCR for WT and activated (Δ) K-ras alleles demonstrates the presence of the activated K-ras allele in KM+ and KM– but not K+, M+, or WT+ tissues. B, bone marrow; L, liver; S, spleen; MW, molecular weight marker; c+, positive control DNA from an individual KM+ methycellulose colony; c–, negative control DNA from an individual K+ methylcellulose colony. (b) Oncogenic K-ras expression in diseased KM+ and KM– tissues. Tissues extracts were immunoprecipitated with a pan-ras antibody, followed by immunoblotting with polyclonal antibodies specific to wild-type ras (α-ras G12) and oncogenic ras G12D (α-ras D12). The WT ras doublet corresnomas; WB, Western blot; IP, immunoprecipitation.

Figure 7

Methylcellulose cultures with KM+, K+, and M+ bone marrow in the presence and absence of growth factors. (a) Number of colonies generated after methylcellulose culture of 100,000 bone marrow cells in the presence (plus) and absence (minus) of growth factors (GF: SCF, IL-3, and IL-6). Growth factor–independent colony-forming activity of KM+ bone marrow cells was demonstrated in two independent experiments. The values shown are the mean of duplicate cultures from one representative experiment. (b) Number of secondary, tertiary, and quaternary colonies generated in serial methylcellulose cultures using 10⁴ input bone marrow cells in the presence of growth factors. Serial replating assays were performed in two independent experiments. The values shown are the means of duplicate cultures from one representative experiment. (c) PCR for WT and activated (Δ) K-ras alleles demonstrates presence of activated K-ras allele in individual methylcellulose colonies derived from KM+ bone marrow in the presence and absence of growth factors, but not from K+ or M+ colonies. The intensity of the PCR products generated from KM+ growth factor–independent colonies was less robust than that from KM+ growth factor–dependent colonies, corresponding to lesser amounts of input template genomic DNA purified from the smaller individual growth factor–independent colonies. (d) Cytospins (Wright-Giemsa stain) of individual methylcellulose colonies show GM-CFUs derived from K+, M+, and KM+ bone marrow cultured in the presence of growth factors, and M-CFUs from KM+ bone marrow cultured in the absence of growth factors. (e) Quantitation of M-CFU (M), GM-CFU (GM), and GEMM-CFU (GEMM) colonies from K+, M+, and KM+ bone marrow cultured in the presence of growth factors and KM+ bone marrow cultured in the absence of growth factors.