BACKGROUND: BCR-ABL-mediated chronic myelogenous leukemia (CML) CD34(+) cell proliferation mostly depends on the nucleo-cytoplasmic ratio of the cyclin-dependent kinase inhibitor p27. The ubiquitin-ligase SCF(Skp2) promotes degradation of phosphorylated p27 at T187 in the nucleus, resulting in G1/S progression of the cells. On the other hand, phosphatidylinositol-3-kinase (PI3K)-directed T157 nuclear localization signal (NLS) phosphorylation results in cytoplasmic sequestration of p27, leading to abnormal integrin-mediated proliferation of CD34(+) CML cells. METHODS: We demonstrate the generation of an engineered Epstein-Barr virus (EBV) vector with a BAC backbone that has the unique capacity to carry doubly modified (DM) p27 (i.e. T187A, T157A p27) along with the BCR-ABL siRNA expression construct. The HSV-tk suicide gene has also been incorporated in the same vector, which promotes apoptosis in a BCR-ABL-independent pathway. RESULTS: Expression of DM p27 markedly inhibits proliferation of BCR-ABL(+) primary human CML cells. Moreover, DM p27 strongly inhibits the growth of imatinib-resistant CML cells, compared to the T157A p27 (SM p27). The CML growth inhibition is found to be the result of significant G1/S arrest with concomitant increase in hypophosphorylated retinoblastoma (Rb). Moreover, the EBV vector mediated stable RNA interference induces apoptosis in K562 cells and reduces myeloid colony forming units. CONCLUSIONS: We therefore propose a multi-gene delivery strategy for BCR-ABL(+) CML cells by targeting not only the fusion transcript, but also the downstream signaling, to overcome drug resistance in the acute phase of CML.