The Activated K-RAS Protein Accelerates Human Derived-MLL/AF4 Induced Leukemo-Lymphomogenicity In Transgenic Mice Model

Abstract 61

Acute lymphoblastic leukemia (ALL) with MLL/AF4 fusion is a common infant leukemia associated with a poor prognosis. Research into the molecular mechanisms underlying ALL with MLL/AF4 has been hampered by the absence of a good animal model: there is a large discrepancy between the time to disease onset in infants and the long latency seen in previously established murine models (human derived-AF4 knock-in and inverter model). We speculated that the absence of associated K-RAS mutations could explain the long latency seen in murine models of ALL with MLL/AF4. To establish a model combining MLL/AF4 fusion and activated K-RAS, we first established MLL/AF4+ transgenic (Tg) mice through overexpression of human MLL/AF4 fusion protein. These MLL/AF4+ Tg mice exhibited splenomegaly and lung tumors at 8–12 months of age (median, 12 months), and histopathological analysis showed disruption of the spleen structure and infiltration of the liver, lung and spleen by lymphoma cells consisted of pro B-cells (CD45R/B220+CD43+). Western blot analysis showed expression of both MLL/AF4 and HoxA9 was upregulated in all of the tissues tested. Infiltration of tumor cells into the peripheral blood was observed after a long latency period in MLL/AF4+ Tg mice (median, 14 months), at which time abnormal lymphocytes accounted for 7.2±1.2% (n=5) of white blood cells. FACS analysis showed these abnormal lymphocytes to be CD45R/B220+CD43+CD19+ cells, which is the same phenotype as the lymphoma cells. To develop a more aggressive oncogenic model mouse, we generated MLL/AF4+K-RAS mutation+ Tg mice by crossing MLL/AF4+ Tg mice and K-RAS mutation (G12D)+ Tg mice. The resultant mice developed lymphoma significantly earlier (median, 6 months) than MLL/AF4+ Tg mice (median LFS: 5.5 months vs. 12 months, P=0.001). Infiltration of tumor cells into the peripheral blood was observed after a short latency period in MLL/AF4+K-RAS mutation+ Tg mice (median age, 6 months), at which time leukemia cells accounted for 3.2±0.9% of WBCs, suggesting the collaborative acceleration of leukemo-lymphomogenicity through overexpression of MLL/AF4 and mutant K-RAS. We anticipate that the MLL/AF4+K-RAS mutation+ Tg mouse model will be useful for studying the molecular mechanisms of MLL/AF4 leukemogenesis and for developing new therapies against MLL-related malignancies.