FTY720 Has Potent Anti-Leukemic Effects on Acute Lymphoblastic Leukemia Cells and Results In Caspase Independent Cell Death

Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer, which usually responds to chemotherapy. Long-term survival in adults is poor with most developing disease relapse, whilst Ph⁺ ALL has a particularly poor prognosis. FTY720 is an immunosuppressive drug that has recently demonstrated efficacy in phase 3 trials of relapsing/remitting multiple sclerosis. FTY720 also appears promising in a number of malignancies with the proposed mechanism being the reactivation of PP2A, a protein serine/threonine phosphatase whose activity may be reduced in malignant cells. Here we report findings of in vitro testing of FTY720 on Ph⁺ and negative ALL cell lines and primary patient samples, describing mechanisms of cell death.

ALL cell lines and primary patient samples were treated with 1 nM - 100 μM FTY720 for 24 hours. Viability was measured by flow cytometry using propidium iodide and annexin V staining. Cellular proliferation was measured by ³H-thymidine incorporation. Flow cytometry and western blotting were used to measure caspase 3 activation whilst western blotting was used to assess caspase 3, PARP cleavage and LC3II formation. Electron microscopy permitted a detailed examination of cell ultra-structure and confocal microscopy with lysosensor blue staining enabled visualisation of acidic vacuoles. Reactive oxygen species generation was assessed by flow cytometry using the cell permeable dye carboxy-H₂DCFDA.

FTY720 produced a profound reduction in proliferation and viability of Ph⁺ (ALL1 cells) and Ph⁻ (REH, NALM6 and LK63 cells) cell lines and patient samples (n=7) in the low micromolar range. IC₅₀ values for loss of viability at 24 hours ranged from 5.3 μM for ALL1 to 7.9 μM for LK63. The IC₅₀ values for proliferation at 24 hours were 1.4 μM for ALL1 and 3.5 μM for REH. Caspase 3 activation was observed only at very low levels by flow cytometry whilst both caspase 3 and PARP cleavage were not detected by western blotting. Inhibition of caspases by ZVAD-FMK failed to rescue ALL cells from FTY720 induced cell death, demonstrating a caspase independent cell death mechanism. Light microscopy revealed prominent cytoplasmic vacuolation, and electron microscopy showed features consistent with autophagy and necrosis. Western blotting demonstrated strong LC3II bands and confocal microscopy, using lysosensor blue, revealed prominent acidic vacuolation, all confirming the induction of autophagy. Reactive oxygen species were generated in response to FTY720 treatment and partial reversal of this by N-acetyl-cysteine produced a concomitant increase in cell viability. PP2A inhibition with okadaic acid failed to rescue cells from FTY720-induced cell death.

FTY720 is a highly active drug in vitro in ALL cell lines and patient samples. Evidence supports a caspase independent mechanism of cell death with the occurrence of autophagy and necrosis. PP2A activation is not solely responsible for leukemic cell death. Data on the in vivo effects of FTY720 on ALL cells in NOD-SCID mice will be presented.

Bendall:Genzyme: Honoraria.