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The transcription inhibitor flavopiridol reduces the Bcr-Abl mRNA level and induces cell death in the human Bcr-Abl positive chronic myelogenous leukemia (CML) cell line, K562, and in murine Ba/F3 cells made dependent on its expression. This action of flavopiridol is synergistic with the Bcr-Abl kinase inhibitor imatinib. These results support the sequential blockade hypothesis, which proposes that decreasing the cellular levels of oncogene proteins, that are required for tumor cell survival and proliferation, by inhibitors of transcription and/or translation would augment the action of specific kinase inhibitors (Cancer Res., 66:10959, 2006). This strategy would be most effective in tumor cells that are addicted to the expression of an oncogene, and for oncogenes with short intrinsic half-lives in their mRNAs and proteins. The Bcr-Abl addicted CML is an ideal model system to evaluate this strategy. Here, as an extension of this sequential blockade strategy, we studied another transcription inhibitor, actinomycin D (ActD), a polypeptide antibiotic that is useful for the treatment of sarcomas, Wilms’ tumor, testicular cancer and choriocarcinoma. The action of ActD as a transcription inhibitor was confirmed in the K562 cells. ActD inhibited RNA synthesis as measured by [3H]uridine incorporation with an IC50 of 60 ng/ml after 3 hr of incubation. This was associated with a decrease in the transcript level of Bcr-Abl, measured by real-time quantitative PCR. Consequently, the Bcr-Abl protein level also decreased. As K562 cells are critically dependent on the continuous expression of Bcr-Abl for survival, decreasing the expression of Bcr-Abl induced cell death in the cells. This was demonstrated as induction of PARP cleavage visualized by immunoblots, inhibition of cell growth and clonogenicity. ActD also reduced the transcript and protein levels of c-Myc and Mcl-1, other oncoproteins with short turn over rates. However, siRNA knockdown procedures suggested that c-Myc levels did not affect K562 cell viability. Further, the Ba/F3 cells expressing either the wild-type p210 Bcr-Abl, or the imatinib resistant T315I or E255K mutations of Bcr-Abl were equally sensitive to ActD in clonogenic assays, with IC50 values of 1-2 ng/ml, indicting lack of cross resistance. Thus, these data support the use of ActD for treatment of oncogene-addicted tumors such as CML, both to complement the activities of Bcr-Abl kinase-targeted inhibitors such as imatinib, desatinib, and nilotinib, and to overcome resistance to these kinase inhibitors due to mutations in the Bcr-Abl kinase domain. Other short-lived oncogenic kinases that sustain the tumor phenotype could also be targeted by ActD, including the activated c-Kit in gastrointestinal stromal tumors and platelet-derived growth factor receptor in myeloproliferative disorders.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA