HOXB6 overexpression results in delayed AML. (A) HOXB6 primary transplant chimeras developed fatal acute myeloid leukemia with a latency of approximately 8 months (median, 223 days; range, 83-261 days). Leukemias were transplantable with a rapid disease onset. (B) Leukemic blasts had immature morphologic appearance on Wright-Giemsa–stained cytospins; however, there was a variable expression of monocytic features such as increased cytoplasm, cytoplasmic vacuolization, and nuclear indentation. Leukemic blasts infiltrated all tissues examined including spleen, thymus, liver, kidneys, and lung (data not shown). Image acquisition was performed as described for Figure 1. (C) FACS analysis of leukemic cells, with EGFP expression on the x-axis and lineage markers on the y-axis, demonstrates the blasts express myeloid markers Gr-1 and Mac-1 as well as c-kit. The blasts do not stain for lymphoid antigens B220 or CD8. (D) HOXB6 protein was easily detected in bone marrow (M) and spleen (S) lysates from both primary and secondary recipients. Wild-type (wt), unfractionated BM was used as a negative control, and HOXB6-transfected 293-T packaging cells were used as positive control (pos). (E) Southern blot analysis of 2 HOXB6-induced primary leukemias and secondary recipients. XhoI/ClaI double digest confirms proviral integration in all leukemias (left). BamHI single digest indicates the primary leukemias were clonal (#1) or oligoclonal versus clonal with 2 proviral integrations (#2) and were transmitted to secondary recipients.