Leukemogenesis in ENU-treated URC mice. (A) Strategy for testing leukemogenesis. Mice treated with poly (IC) and 1 wk later with ENU (100 mg/kg) were monitored until they were moribund or up to 1.5 y after receiving poly (IC). Splenic or bone marrow cells from these mice without frank AML were transplanted into sublethally irradiated WT recipient mice. Three to five recipient mice were used for each transplantation and were monitored for up to 1 y for the development of AML. (B) Summary of AML incidence. Mice with the control genotype lackwd either U2af1(S34F) or Runx1 deletions and were not used for transplant. NA, not available. Mice with T cell lymphoma or thymoma were censored from the study cohort. In total, 3 of 16 URC mice developed AML (P value = 0.13, based on four separate genotypes as shown; P value = 0.03 based on URC mice vs. all other genotypes, by Fisher’s exact test). (C) A case of primary AML in 1 of 16 poly (IC)- and ENU-treated URC mice (case 1). (Left) A high percentage of c-Kit+ cells was present in the peripheral blood of this mouse by flow cytometry, which coincided with infiltration of morphologically defined myeloblasts (arrowheads) in a Wright–Giemsa–stained blood smear (Center) and an H&E-stained section of the bone marrow (Right). (D) A case of late AML. Representative results are shown for the case 3 donor (Upper Row) and one of transplant recipients when it was moribund (Lower Row). (Left) Percentages of c-Kit+ cells were markedly increased in the peripheral blood of the recipient mouse compared with that of the donor. (Center) Abnormally nucleated RBCs (arrows) were observed in the blood smears from both donor and recipient, while myeloblasts (arrowheads) were observed only in blood from the recipient. (Right) Blast cells also filled the bone marrow of the recipient mouse. (Scale bars in C and D: 50 µm.) (E) Survival curves of sublethally irradiated mice after transplantations of bone marrow (black trace) or spleen (blue trace) cells from the primary donor mice of cases 1–3. The day count started with the day of transplantation. See SI Appendix, Fig. S9 for additional characterization of these AML cases.