Fig. 2: Development of cytogenetically normal AML induced by ITD/ITD and p53 haploinsufficiency or loss.

a Representative Pappenheim-stained blood smears and bone marrow (BM) cytospins, and hematoxylin and eosin (H & E)-stained bone marrow, and liver from p53^+/−, ITD/ITD, ITD/ITD; p53^+/− mice. Blood smear: there were no blasts and no increased monocytes in mouse #1381 (p53^+/−). Monocytes were increased in mouse #1436 (ITD/ITD). Myeloblasts were strongly increased in mouse #1376 (ITD/ITD; p53^+/−), while only few blasts were observed in mouse #1348 (ITD/ITD; p53^+/−) with biclonal disease. BM cytospins: mouse #1381 showed a normal cellular component. Monocytes were strongly increased in mouse #1436. Myeloblats made up ~60 and 30% of all nucleated cells in mice #1376 and #1348, respectively. BM section: BM histology was consistent with the cellular constituents observed in cytospins. Note the presence of megakaryocytes in mouse #1381 and the reduction/absence of megakaryocytes in other animals with leukemia. Spleen section: white pulp and red pulp were present in mouse #1381, while the structures of the spleen were destroyed in the other 3 mice with leukemia, particularly in #1376 and #1348. Liver section: while mouse #1381 showed a normal liver structure, infiltration of leukemic cells was observed in the other 3 animals, particularly in #1376. Mouse #1381 developed adenocarcinoma and osteosarcoma (Supplementary Fig. 4). However, mouse #1381 demonstrated similar cytology and histology (PB, BM and spleen) as WT mice (data not shown), except for increased granulocytes in blood smear. Data from ITD/ITD; p53^−/− mice were presented in Supplementary Fig. 5. b Cytospin from mouse #1348 showing lymphoblastic cells in thymus (left panel). Histology showing strong infiltration of lymphoblastic cells in thymus from mouse #1348 (right panel). c Macroscopic characterization of ITD/ITD; p53^+/− mouse #1454. Note strong hepatosplenomegaly of mouse #1454 (upper panel) compared with a healthy control (lower panel). d Flow cytometric analysis of BM samples demonstrated a population of myeloblast/immature cells with lower side scatter (SSC) and CD45^dim expression in mice #1376 and #1348, which were positive for CD11b, c-Kit, and CD34 (Supplementary Fig. 6). Moreover, T-lymphoblastic lymphoma cells from the thymus of mouse #1348 showed lower SSC but high CD45 expression [42]. Tumor cells were positive for CD4, CD8 and CD3. Notably, there was no infiltration of T-ALL cells in the BM of mouse #1348. Thus, there were two separate clones present in mouse #1348 (AML in the BM, T-ALL in the thymus). e mFISH analysis of a representative FLT3-ITD; p53^+/− BM sample from mouse #1348 showing a normal karyotype (40, XY). Table S1 summarizes the karyotypes found in mice with AML (n = 12). f The replating colony assay demonstrated no increased self-renewal activity in vitro by double mutations. Cells from one ITD/ITD; p53^+/− mouse did not form colonies in the 1^st plating and cells from another 2 ITD/ITD; p53^+/− mice formed <6 colonies in the 2^nd replating, while cells from all animals from ITD/ITD and p53^+/− groups formed colonies in the 1^st plating and >14 colonies in the 2^nd replating (except for one mouse in the 2^nd replating). Consistent with early reports, p53^+/− increased self-renewal activity compared with WT p53 (the only group in which many colonies continued to be observed after the 5th replating), which was abrogated in the presence of ITD/ITD. Results presented are the min to max of colony numbers from each replating. ITD/ITD, n = 5 mice; p53^+/−, n = 5; ITD/ITD; p53^+/−, n = 7; ITD/ITD; p53^−/−, n = 3; p53^−/−, n = 5; WT = 4.