Treosulfan Potently Induces Apoptosis in Human Acute Myeloid Leukemia Cells by a Protein Kinase C Dependent Mechanism, Which Can Be Markedly Augmented by the PKC-Activator Bryostatin-1

Schmidmaier, R.; Baumgart, J.; Emmerich, B.; Meinhardt, G.

OBJECTIVES: Despite recent advances in the understanding of the underlying biology of acute myeloid leukemia (AML), the drugs employed have not changed over the past three decades. Primarily, cytosine arabinoside (ara-C) and anthracycline analogs are used in most chemotherapeutic regimens. Although dose-intensification with stem cell rescue and optimized supportive care improved response rates and overall survival, the disease remains incurable in the majority of patients. Thus, alternative or new cytotoxic compounds are warranted. METHODS: We performed chemosensitivity experiments with the alkylating agent treosulfan using AML cell lines (U937, TUR, HL-60, THP-1) and primary leukemia cells from five consecutive patients. Viability, cell cycle distribution, sub-G1-fraction, mitochondrial transmembrane potential and number of annexin V-positive, apoptotic cells were determined. To further evaluate the mechanism of treosulfan-induced apoptosis, we tested for alterations in the levels of pro- and antiapoptotic proteins by immunoblotting. Furthermore, as protein kinase C (PKC) is known to regulate diverse cellular functions, several PKC modulators were tested in conjunction with treosulfan. RESULTS: Treosulfan potently induces cell death in all four cell lines and primary leukemia cells with a LC90 of approximately 100muM, which is severalfold below the clinically achievable plasma levels. Whereas immunoblotting experiments did not reveal significant changes in the intracellular expression levels of common pro- and antiapoptotic proteins (e.g. BCL-2 family, inhibitors of apoptosis IAPs), treosulfan-induced cell death was associated with cellular events indicating apoptosis such as breakdown of the mitochondrial transmembrane potential, the proteolytic activation of caspases-9 and -3 as well as the appearance of a sub-G1 DNA peak. Treosulfan-induced apoptosis was significantly reduced by coincubation with the PKC-inhibitor GF109203X. In contrast, activation of PKC isozymes by TPA (12-O-tetradecanoylphorbol-13-acetate) or bryostatin-1 showed synergistic antileukemic effects with treosulfan in AML cell lines and primary leukemia cells. Since activation of PKCs is followed by proteolytic down-regulation of the kinases, we performed time-course experiments to determine the optimal sequence of PKC activation and treosulfan exposure. Whereas a 72 h preincubation with bryostatin-1 reduced the percentage of apoptotic cells by 61%, short-term preincubation (24 h or less) enhanced the cytotoxic effect of treosulfan exposure by 72%. CONCLUSION: Treosulfan, a drug related to busulfan, potently induces apoptosis in AML cell lines and primary leukemia cells from patients at clinically achievable doses. Thus, our results support the use of treosulfan as cytotoxic conditioning agent for autologous or allogeneic stem cell transplantation in the therapy of AML. Furthermore, bryostatin-1, which has been shown to be applicable in several clinical trials, demonstrates synergistic antileukemic effects with treosulfan when administered in close temporal proximity. Taken together, these data provide a rationale to evaluate treosulfan in combination with bryostatin-1 in the treatment of AML.