1. ¹Department of Oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA
2. ²Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Correspondence: Dr P Brown, Department of Oncology, Johns Hopkins University, 1650 Orleans Street, Room CRB 2M49, Baltimore, MD 21231, USA. E-mail: pbrown2@jhmi.edu

Received 19 January 2006; Revised 28 March 2006; Accepted 4 May 2006; Published online 8 June 2006.

Abstract

Mixed lineage leukemia (MLL) rearrangements occur in 80% of infants and 5% of older children with acute lymphoblastic leukemia (ALL). These cases have a poor prognosis with current therapy. The FLT3 kinase is overexpressed and constitutively activated in MLL-rearranged ALL cells. The FLT3 inhibitor CEP-701 selectively kills these cells, but is unlikely to be curative if used as monotherapy. To identify potentially synergistic combination strategies, we studied CEP-701 and six standard chemotherapeutic agents in three sequences of exposure (S1: chemotherapy followed by CEP-701, S2: simultaneous exposure to both; and S3: CEP-701 followed by chemotherapy) using MLL-rearranged ALL cell lines and patient bone marrow samples. MTT cytotoxicity and annexin V binding apoptosis assays were used to assess antileukemic effects. Combination indices (CI) were calculated for each combination (CI<0.9 – synergistic; CI 0.9–1.1 – additive; CI>1.1 – antagonistic). A striking pattern of sequence-dependent synergy was observed: S1 was markedly synergistic (mean CI=0.590.10), S2 was additive (mean CI=0.990.09) and S3 was antagonistic (mean CI=1.230.10). The sequence dependence is attributable to the effect of CEP-701 on cell cycle kinetics, and is mediated specifically by FLT3 inhibition, as these effects are not seen in control cells without activated FLT3.