Somatic activation of a conditional KrasG12D allele causes ineffective erythropoiesis in vivo

Abstract

Somatic activation of a conditional targeted Kras(G12D) allele induces a fatal myeloproliferative disease in mice that closely models juvenile and chronic myelomonocytic leukemia. These mice consistently develop severe and progressive anemia despite adequate numbers of clonogenic erythroid progenitors in the bone marrow and expanded splenic hematopoiesis. Ineffective erythropoiesis is characterized by impaired differentiation. These results demonstrate that endogenous levels of oncogenic Ras have cell lineage-specific effects and support efforts to modulate Ras signaling for therapy of anemia in patients with myelodysplastic syndromes and myeloproliferative disorders.

Figure 1.

Serum EPO and erythroid progenitors in Mx1-Cre, Kras^G12D mice. (A) Serum EPO and hemoglobin (Hb) concentrations in Mx1-Cre, Kras^G12D mice (open symbols) and wild-type (wt) littermates (closed symbols); data are pooled from F1 (n = 18; circles) and C57BL/6 (n = 9; squares) mice. (B) Total CFU-Es recovered from bone marrow (2 femurs and 2 tibias) and spleens of Mx1-Cre, Kras^G12D mice (□) and wild-type littermates (▪) shown as mean ± SEM. (C) Total BFU-Es are enumerated as in panel B. (D) EPO dose-response of BFU-Es derived from C57BL/6 bone marrow, showing mean ± SEM from 3 independent experiments. (E,F) Photomicrographs of typical BFU-Es from bone marrow cultured with 50 ng/mL EPO for 7 days (bar represents 500 μm).

Figure 2.

Differentiation and proliferation of erythroid progenitors. (A) Flow cytometry analysis of CD71 and TER119 expression of freshly harvested hematopoietic cells, with regions of maturing erythroid cells indicated as described. Table S1 presents the quantification. (B) CD71 and TER119 expression of E14.5 fetal liver cells after in vitro induction of Kras^G12D for 4 days (day 0), then after 1 day of EPO stimulation (day +1), or after 2 days of EPO stimulation and 1 day of EPO withdrawal (day +3). (C) BrdU incorporation in vivo by CD71⁺TER119⁺ bone marrow cells. Note that the CD71/TER119 staining profile reflects lysis of erythrocytes and fixation required for BrdU staining. Mean percentage BrdU⁺ cells ± SEM is shown.