Mice expressing KrasG12D in hematopoietic multipotent progenitor cells develop neonatal myeloid leukemia

Abstract

Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that bears distinct characteristics associated with abnormal fetal development. JMML has been extensively modeled in mice expressing the oncogenic KrasG12D mutation. However, these models have struggled to recapitulate the defining features of JMML due to in utero lethality, nonhematopoietic expression, and the pervasive emergence of T cell acute lymphoblastic leukemia. Here, we have developed a model of JMML using mice that express KrasG12D in multipotent progenitor cells (Flt3Cre+ KrasG12D mice). These mice express KrasG12D in utero, are born at normal Mendelian ratios, develop hepatosplenomegaly, anemia, and thrombocytopenia, and succumb to a rapidly progressing and fully penetrant neonatal myeloid disease. Mutant mice have altered hematopoietic stem and progenitor cell populations in the BM and spleen that are hypersensitive to granulocyte macrophage-CSF due to hyperactive RAS/ERK signaling. Biased differentiation in these progenitors results in an expansion of neutrophils and DCs and a concomitant decrease in T lymphocytes. Flt3Cre+ KrasG12D fetal liver hematopoietic progenitors give rise to a myeloid disease upon transplantation. In summary, we describe a KrasG12D mouse model that reproducibly develops JMML-like disease. This model will prove useful for preclinical drug studies and for elucidating the developmental origins of pediatric neoplasms.

Figure 1. Flt3Cre⁺ Kras^G12D mice develop a JMML-like disease.

(A) Weight gain from birth (n = 12 mutants and 19 controls). (B) Overall survival (statistical analysis by Mantel-Cox test). (C) Peripheral blood smear (n = 5). Scale bar: 100 μm. (D) Flow cytometric quantification of tissue leukocytes. (E) Seven-day BM colony formation with 100 nM PD0325901 or 0.1% DMSO (n = 3 biological replicates/group). All analyses were performed on 3- to 4-week-old moribund Flt3Cre⁺ Kras^G12D mice and age-matched littermates. *P < 0.05, ^#P < 0.01, and ^§P < 0.001, by unpaired, 2-tailed Student’s t test (A, D, and E).

Figure 2. Fetal Flt3Cre⁺ Kras^G12D progenitors initiate a JMML-like disease upon transplantation.

(A) Schematic of FL transplants (n = 12 mutant and 8 control recipients). Analysis of donor cell (B) engraftment and (C) myeloid contribution in peripheral blood. (D and E) Flow cytometric quantification of donor leukocytes in BM and spleens of moribund mutant recipients and control recipients 16 weeks after transplantation. max, maximum. (F) Overall survival following transplantation (statistical analysis by Mantel-Cox test). (G–I) Normalized tissue weights of analyzed animals. *P < 0.05, ^#P < 0.01, and ^§P < 0.001, by unpaired, 2-tailed Student’s t test (B–E and G–I).

Figure 3. Analysis of Flt3Cre⁺ Kras^G12D progenitor frequency and differentiation.

(A) Frequency of HSCs and MPPs in the BM and spleens of mutants and littermates. (B) Cell-cycle analysis of HSCs and MPPs from 1-day-old liver (n = 3 WT and 4 mutants) and 21-day-old BM (n = 2/group); representative gating is shown in Supplemental Figure 9. (C) Flow cytometric quantification of tissue DCs. (D–F) Flow cytometric quantification and representative gating of thymic cells. Error bars represent the SEM. Cell-cycle statistical analyses were performed using a χ² test, and other analyses were performed using an unpaired, 2-tailed Student’s t test. *P < 0.05, ^#P < 0.01, and ^§P < 0.001.