TREATMENT OF PHILADELPHIA LEUKEMIA IN SEVERE COMBINED IMMUNODEFICIENTMICE BY COMBINATION OF CYCLOPHOSPHAMIDE AND BCR ABL ANTISENSE OLIGODEOXYNUCLEOTIDES/

Background: Philadelphia(1) cells are human chronic myelogenous leukem ia (CML) cells that contain the BCR/ABL oncogene (a fusion of the BCR and ABL genes). Selective eradication of these cells in vitro can be a chieved by combined treatment with antisense phosphorothioate oligodeo xynucleotides ([S]ODNs) specifically targeted to this oncogene (bcr/ab l [S]ODNs) and a suboptimal (for use as a single agent) dose of mafosf amide (the in vitro active form of cyclophosphamide). Purpose: We eval uated the ability of bcr/ abl antisense [S]ODNs, alone or subsequent t o treatment with a single injection of cyclophosphamide, to suppress t he leukemic process induced in severe combined immunodeficient (SCID) mice by Philadelphia(1) cells (i.e., primary CML-blast crisis [CML-BC] cells). In addition, we studied potential mechanisms that might expla in the efficacy of the bcr/abl antisense [S]ODN-mafosfamide combinatio n against Philadelphia(1) cells in vitro. Methods: The effects of trea ting leukemic mice with cyclophosphamide (25 mg/kg body weight; 25% of the dose required to eradicate evidence of leukemia in SCID mice) and /or bcr/abl antisense [S]ODNs were assessed by analysis of survival, b y examination of bone marrow for the presence of leukemia cells (using a colony formal ion assay or using coupled reverse transcription and the polymerase chain reaction to screen for bcr/abl messenger RNA), an d by examination of a variety of tissues for the presence of infiltrat ing leukemia cells. The induction of apoptosis (a cell death program) in vitro in primary CML-BC tells following treatment with bcr/abl anti sense [S]ODNs plus or minus prior treatment with mafosfamide was monit ored by use of a commercial assay. Relative cellular uptake of [S]ODNs by CML-BC cells treated in vitro with or without prior treatment with mafosfamide was determined by use of confocal microscopy and flow cyt ometry (for fluorescent [S]ODNs) or by use of blotting techniques that employed radioactively labeled probes (for extracted, unlabeled [S]OD Ns). Levels of specific proteins in treated and untreated cells mere d etermined by use of western blotting methods. Reported P values are tw o-sided Results: The disease process in leukemic mice was retarded sub stantially by combination treatment with cyclophosphamide and specific bcr/abl antisense [S]ODNs (P<.001, relative to treatment with specifi c antisense [S]ODNs alone, cyclophosphamide alone, or cyclophosphamide plus nonspecific [i.e., control] antisense [S]ODNs); 50% of the mice treated with cyclophosphamide and specific antisense [S]ODNs appeal ed to be cured of leukemia. The combination treatment was associated wit h increased induction of apoptosis, in addition, cellular uptake of bc r/abl antisense [S]ODNs appeared to be increased twofold to sixfold by prior treatment with mafosfamide. This increased uptake of [S]ODNs wa s associated with enhanced suppression of p210(bcr/abl) protein levels . Conclusions and Implications: Combination therapy with antisense [S] ODNs targeted to specific oncogenes and less toxic doses of anticancer drugs may represent a rational strategy to pursue for the treatment o f human leukemias.