Gene therapy for BCR‐ABL+ human CML with dual phosphorylation resistant p27Kip1 and stable RNA interference using an EBV vector

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Abstract
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 SCFSkp2 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. Copyright © 2006 John Wiley & Sons, Ltd.