Targeting BCR-ABL and Its Downstream Signaling Cascade as Therapy for Chronic Myelogenous Leukemia.

Abstract BCR-ABL is an unregulated tyrosine kinase expressed as a consequence of chromosomal translocation in chronic myelogenous leukemia (CML). The tyrosine kinase activity of BCR-ABL activates signaling cascades that induce cytokine independence and transformation of myeloid progenitor cells. Targeted inhibition of this kinase with specific inhibitors (imatinib or BMS-354825) is a very effective therapy for some CML patients but resistance to these agents (through point mutations and other mechanisms) leads to advanced disease with very few therapeutic options. An alternate therapeutic strategy is to reduce BCR-ABL expression or its critical downstream signaling elements important for transformation. We examined BCR-ABL signaling elements and gene expression changes that occur in CML cells following kinase inhibition by imatinib in newly established imatinib sensitive and resistant cells to identify critical signaling elements involved in CML cell death. Imatinib rapidly and progressively suppressed c-myc expression in imatinib sensitive but not resistant cells prior to the onset of apoptosis. These results suggested that c-myc expression was regulated by BCR-ABL signaling and may play a role in CML tumorigenicity. To confirm a role for c-myc in CML cell growth and/or survival, c-myc expression was specifically down-regulated by siRNA using a novel electroporation instrument (AMAXA) that permits high level gene transfer with limited toxicity in CML cell lines. Jak2 siRNA was used as a control. c-myc, but not Jak2 siRNA, suppressed c-myc expression and cell growth and survival in both imatinib sensitive and resistant CML cells, suggesting that targeted suppression of c-myc may have therapeutic activity against both kinase inhibitor sensitive and resistant CML cells. Since the tyrphostin AG490 was previously shown to inhibit c-myc expression in CML cells through its inhibitory effects on Jak2, we screened a series of > 200 AG490 derivatives for their ability to rapidly reduce c-myc expression in hematological malignancies. After several rounds of testing we synthesized an agent (WP-1066) capable of rapid c-myc downregulation (beginning 1–5 min after treatment with 1–2 microM) but poor Jak2 kinase inhibitory activity (IC50 > 100 microM). These results suggested a more direct effect of WP-1066 on c-myc protein expression than AG490 and mechanistic studies suggest that WP-1066 reduces c-myc protein stability but does not affect c-myc gene expression. In BCR-ABL expressing cells WP-1066 rapidly reduced c-myc protein levels in CML cells and inhibited the growth and survival of cell lines or patient specimens expressing wild-type or mutant forms of BCR-ABL that effect tyrosine kinase inhibitory activity (T315I in BV-173R cells). Equal concentrations of imatinib or WP-1066 reduced BCR-ABL activation and downstream signaling (Stat5 phosphorylation) in CML cells. However, WP-1066 differed from imatinib in its ability to downregulate BCR-ABL protein expression without affects on c-abl or Stat5 expression. Similar results were obtained in clinical specimens taken from patients with BCR-ABL point mutations that mediate imatinib (or BMS-354825) resistance. Nude mouse studies demonstrated that WP-1066 reduced the growth of K562 tumors to an extent similar to that of imatinib. Together these results suggest that WP-1066 downregulates BCR-ABL and c-myc expression, induces apoptosis in CML cells expressing wild-type or mutant BCR-ABL and may have therapeutic activity in imatinib (or BMS-354825) resistant CML tumors.