Timed sequential therapy of acute leukemia with flavopiridol: In vitro model for a phase I clinical trial

Purpose: The survival of adults with acute leukemias remains unsatisfactory and requires new treatment approaches. Flavopiridol modulates cell cycle progression, inhibits transcription, and induces apoptosis. We designed an in vitro model of timed sequential therapy for acute leukemia to determine whether flavopiridol can: (a) trigger apoptosis in fresh acute leukemia; and (b) recruit surviving leukemic cells to a proliferative state, thereby priming such cells for the S-phase-related cytotoxicity of 1-β-D-arabinofuranosylcytosine (ara-C). Experimental Design: Bone marrow cells from 20 adults with relapsed and refractory acute leukemias were enriched for blasts by Ficoll Hypaque sedimentation. Blasts were cultured on day 0 in flavopiridol 250 nM for 24 h, removed from flavopiridol for 24 h, and then cultured in ara-C 1 μM for an additional 72 h (F₂₅₀A₁). Apoptosis and cell cycle phase distribution were estimated from cells stained with propidium iodide. Cell survival was determined after the 72 h ara-C exposure by double cytofluorescence assay with fluorescein diacetate and propidium iodide. Results: Flavopiridol induced a 4.3-fold increase in apoptosis in human leukemia samples within the first 24 h of culture. Subsequent removal of flavopiridol led to a 1.7-fold increase in the proportion of cells in S phase by day 2. Mean survival in F₂₅₀A₁ cultures after 72 h exposure to ara-C was 35.6% compared with flavopiridol alone (F₂₅₀A₀, 56.1%; P = 0.0003) and ara-C alone (F₀A₁, 65.2%; P < 0.00001). Conclusions: Flavopiridol induces apoptosis in marrow blasts from patients with refractory acute leukemias. Furthermore, flavopiridol pretreatment increases the proapoptotic and cytotoxic effects of ara-C. The advantage of sequential FP₂₅₀A₁ over either agent alone is seen for both acute myelogenous leukemia and acute lymphoblastic leukemia. These findings support a clinical trial of timed sequential therapy where flavopiridol is given for cytoreduction and subsequent priming of remaining leukemic cells for enhanced cycle-dependent drug cytotoxicity.