KRAS (G12D) Cooperates with AML1/ETO to Initiate a Mouse Model Mimicking Human Acute Myeloid Leukemia
Cellular Physiology and Biochemistry
This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only. These authors contributed equally to this work ( d Abstract Background/Aims: It has been demonstrated that KRAS mutations represent about 90% of cancer-associated mutations, and that KRAS mutations play an essential role
... an essential role in neoplastic transformation. Cancer-associated RAS mutations occur frequently in acute myeloid leukemia (AML), suggesting a functional role for Ras in leukemogenesis. Methods: We successfully established a mouse model of human leukemia by transplanting bone marrow cells cotransfected with the K-ras (G12D) mutation and AML1/ETO fusion protein. Results: Mice transplanted with AML/ETO+KRAS co-transduced cells had the highest mortality rate than mice transplanted with AML/ETO-or KRAS-transduced cells (115d vs. 150d). Upon reaching a terminal disease stage, EGFP-positive cells dominated their spleen, lymph nodes, peripheral blood and central nervous system tissue. Immunophenotyping, cytologic analyses revealed that AML/ETO+KRAS leukemias predominantly contained immature myeloid precursors (EGFP + /c-Kit + /Mac-1 /Gr-1 were closely packed in dense sheets that effaced the normal architecture of spleen and thymus in mice transplanted with AML1/ETO + KRAS co-transduced cells. K-ras mRNA and protein expression were upregulated in bone marrow cells of the K-ras group and AML1/ ETO + Kras group. The similar results of the AML1/ETO + Nras group were consistent with those reported previously. Conclusion: Co-transduction of Kras G12D and AML1/ETO induces acute monoblastic leukemia. leukemia, this model may be useful for investigating the multi-step leukemogenesis model of human leukemia. Tris (pH 7.4), 0.1% Tween-20, and 100 mM NaCl, incubated for 1 h with secondary antibodies conjugated to horseradish peroxidase and washed three times. Blots were developed using the ECL detection system (Pierce, Rockford, IL, USA). were cultured in the presence or absence of polybrene and FBS. The viral titers generated by transfection of the inducible packaging plasmids were 1.125 × 10 8 TU/mL for pCDH-KRAS G12D , 1.1 × 10 8 TU/mL for pCDH-NRAS G12D and 1.0 × 10 8 TU/mL for pCDH-AML1/ETO. As shown in Figure 1B , when the pCDH-KRAS G12D vector or pCDH-NRAS G12D vector were transduced into mouse bone marrow cells, more than 90% of the resulting cell populations Co-transduction of K-ras G12D and AML1/ETO induces acute monoblastic leukemia Mouse bone marrow cells were isolated, transformed with the viral vectors, transplanted into irradiated mice, and the effect of the RAS mutations and AML1/ETO translocation on the survival rate of the mice was investigated. No mortalities were observed in the mice transplanted with NRAS-transformed cells or KRAS-transformed cells up to 209 days, while the mice that were transplanted with AML1/ETO + NRAS co-transduced bone marrow cells had the highest mortality rate (95%). Mice transplanted with AML1/ETO-transduced cells or KRAS-transduced cells had a lower mortality rate than mice transplanted with AML1/ ETO + KRAS co-transduced cells (median survival, AML1/ETO + KRAS vs. KRAS, 115 vs. 150 d, P < 0.0001; AML1/ETO + KRAS vs. AML1/ETO, 115 vs. 150 d, P < 0.0001; Fig. 2A). Fig. 3. Characterization of primary leukemias in AML1/ETO+KRAS G12D mice. (A) Peripheral blood cells and (B) bone marrow cells of representative moribund AML1/ETO+KRAS G12D / AML1/ETO+NRAS G12D mice observed by Wright-Giemsa staining. (C) Imunophenotyping analysis of bone marrow cells in mice transduced with the indicated retroviral constructs. (D) Hematoxylin-eosin-stained spleen and thymus sections show-G12D / AML1/ETO+NRAS G12D mice. Bars, 50 µm. In human patients, AML is characterized by the appearance of malignant myeloid extramedullary tissues . When the mice transplanted with bone marrow cells transduced with AML1/ETO + KRAS or AML1/ETO + NRAS reached terminal disease stage, EGFP-positive cells dominated the spleen (59.79% for AML1/ETO + KRAS, 72.38% for AML1/ETO + NRAS), lymph nodes (52.80% for AML1/ETO + KRAS, 43.05% for AML1/ETO + NRAS), peripheral blood (97.23% for AML1/ETO + KRAS, 98.08% for AML1/ETO + NRAS) and central nervous system tissue (CNS; 5.78% for AML1/ETO + KRAS, 10.61% for AML1/ETO + NRAS; Fig. 2B ). We also investigated the composition of the bone marrow cells in the indicated transplanted mice by FACS. The results showed that the bone marrow cells transduced with K-ras G12D + AML1/ETO predominantly contained immature myeloid precursors (EGFP + /c-Kit + /Mac-1 /Gr-1 cells; Fig. 3C ). Wright-Giemsa staining of the peripheral blood cells and bone marrow cells of mice from the AML1/ETO + KRAS group and AML1/ETO + NRAS by progressive anemia, leukocytosis, abnormal nuclei and cytoplasmic vacuoles, in the peripheral blood and bone marrow ( Fig. 3A and B) . Histologic analyses revealed that in mice transplanted with AML1/ETO + KRAS or AML1/ETO + NRAS co-transduced cells, architecture of spleen and thymus in mice (Fig. 3D) . We then examined the mRNA and protein expression of KRAS/K-ras, NRAS/N-ras and AML1/ETO in the bone marrow cells of the transplanted mice. The levels of KRAS mRNA increased in the bone marrow cells of the K-ras group and AML1/ETO + K-ras group (Fig. 4A ). K-ras protein expression was only upregulated in the bone marrow cells of the K-ras group and AML1/ETO + K-ras group (Fig. 4B) . A similar trend in the expression of N-ras Fig. 4. Expression analysis of retroviral oncogenes in the bone marrow cells of the transplanted mice. (A) Qualitative and quantitative PCR analysis of NRAS, KRAS and AML1/ETO transcript expression in the bone marrow cells of mice transduced with the indicated retroviral constructs. (B) Western blotting of N-ras, Kof mice transduced with the indicated retroviral constructs.