Mutation in the c-KIT gene that result in constitutive tyrosine kinase activity is associated with mastocytosis/mast cell leukemia, gastrointestinal stromal tumors (GIST), and acute myeloid leukemia (AML). The analogous mutation in murine c-Kit (D814V) renders Ba/F3 cells factor-independent and when expressed in murine bone marrow causes lymphocytic leukemia. An animal model using the human allele has not been described. We generated retroviral expression constructs that express either wild-type, or mutated human or murine c-Kit and all proteins were well expressed in human-derived 293T cells. However, upon retroviral infection of murine-derived Ba/F3 cells, the human KIT D816V was expressed at a significantly lower level, and downstream signaling pathways, including STAT3, STAT5 and AKT were activated to a lesser extent than cells expressing the murine D814V. Surprisingly, without prior antibiotic selection, the human D816V was not able to transform Ba/F3 cells in short term assays for factor independent growth and mice transplanted with bone marrow cells transduced with human D816V did not succumb to disease, even after 6 months of observation. In contrast, 100% of mice receiving the murine D814V transduced marrow succumbed to disease; 80% died with short latency (46 ± 17 days) acute leukemia with immature B lymphocytes (B220+CD43+IgM-); and 20% of D814V recipients died from myeloid proliferative disease (MPD) with longer latency (68 ± 22 days). We then created a chimeric cDNA that fuses extracellular and transmembrane domain of mouse c-Kit to the human c-KIT intracellular domain (Hybrid KIT). Both Hybrid KIT wild type (WT) and Hybrid D816V were well expressed in Ba/F3 cells. Hybrid D816V, but not Hybrid KIT WT rapidly stimulated BaF3 cells to proliferate without IL3, and all recipient mice transplanted with bone marrow cells transduced with Hybrid D816V mutation, but not Hybrid KIT-WT, developed fatal MPD. We conclude that the extracellular domain of human c-KIT prohibits efficient expression in murine cells. Our model, using a mouse-human hybrid c-Kit, provides a useful animal model to test potential small molecule inhibitors.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]