Therapeutic Efficacy of FTY720 In a Rat Model of LGL Leukemia

Abstract 776

Natural kill cell large granular lymphocytic (NK-LGL) leukemia is a fatal disorder with death occurring in days to weeks following diagnosis. There is no known curative therapy for this disease. Therefore, there is an urgent unmet need for development of new therapeutics for this deadly leukemia. Fischer F344 rat LGL leukemia model has been established as an important experimental model for the study of NK-LGL leukemia progression and closely resembles human aggressive NK-LGL leukemia exhibiting clonal expansion of CD3-CD8a+ lymphocytes. FTY720, a new immunosuppressant, has been studied for its putative anti-cancer properties in the recent years. At four weeks after transplantation of leukemic LGL cell line, the rats displayed early signs of leukemia, including weight loss, rough hair coat and increased level of neutrophils. By week 5, circulating blasts, anemia, thrombocytopenia, and splenomegaly were observed. These leukemic rats were then injected intraperitoneally with 4.5mg/kg of FTY720 or PBS every day over a 4-week treatment period. Animals died within the next 1 to 2 weeks if treated with PBS. The median survival in PBS treated group was 41 days compared to 51 days in FTY720 treated group (Mantel-Cox test, p<0.0001). Importantly, 5 of 16 leukemic rats treated with FTY720 had maintenance of normal blood counts without circulating blasts suggesting achievement of complete clinical remission. The remaining eleven leukemic rats treated with FTY720 had a transient improvement as evidenced by reduction of white blood cell counts and elevated platelet counts after two weeks treatment. Subsequently, however, blast counts rose and animals died within the following one week. To further determine remission status, the five responsive animals were euthanized after cessation of the treatment. At necropsy, we found these rats had normal levels of CD3-CD8a+ LGL cells in the blood, marrow, and spleen. Of note, the eleven leukemic rats not achieving remission also displayed significant reduction of LGL cells in these tissues, to lesser extent. Examination of spleen sections from rats responsive to FTY720 showed normal splenic histology. In contrast, leukemic rats not achieving remission showed leukemic LGL infiltration of the red pulp and depletion of the white pulp. Interestingly, pro-apoptotic proteins Bax and Bak were dramatically increased while anti-apoptotic protein Mcl-1 was decreased in the spleens of the rats achieving remission; however, they remained unchanged in leukemic rats not achieving remission. These data indicate that in vivo therapeutic efficacy of FTY720 may be a consequence of modulation of anti-apoptosis signaling which led to resolution of leukemic cell infiltration. We then extended these studies to NK LGL from patients. Initial experiments demonstrated that FTY720 displayed dose- and time-dependent apoptotic cell death in PBMC from NK-LGL leukemia patients (CD3⁻CD56⁺>80%). In contrast, treatment with 10uM FTY720 did not induce significant cytotoxic effects in PBMC from normal donors, or normal NK cells. In addition, treatment of human and rat NK-LGL leukemia cells with FTY720 led to caspase-dependent apoptosis, generation of reactive oxygen species (ROS), and Mcl-1 degradation which did not occur at the transcriptional level. Of interest, inhibition of ROS rescued FTY720 induced apoptosis in leukemic NK cells. Moreover, efficient knockdown of Mcl-1 resulted in more than two fold increase in apoptotic cell death of NKL, a human NK-LGL leukemia cell line. Collectively, these results indicate efficacy of FTY720 in a rat model of NK LGL leukemia via production of ROS and decreased mcl-1 expression or signaling.