In-Vitro Treatment of Lymphoma Cell Lines with BAY 43-9006 (Sorafenib) Is Followed by Typical Features of Apoptosis and Down-Regulation of MCL-1 Pointing to an Efficacy of Sorafenib in the Teatment of Lymphoma.

BAY 43-9006 (sorafenib) is a multikinase inhibitor that has shown efficacy against a variety of malignancies in preclinical models. In the treatment of lymphoma, however, its place is still to be determined. We treated 7 lymphoma cell lines with sorafenib (10μM) over 24h and 48h respectively: EHEB (B-CLL), JVM-2 (MCL), GRANTA-519 (MCL), JURKAT (T-ALL with p53 mutation), BL-60, NAMALWA and BJAB (all Burkitt’s lymphoma with del 17p and p53 mutation). To determine the rates and type of sorafenib induced apoptosis, 4-colour FACS analyses (CD19, 7AAD, active caspase-3, cytochrome c) were performed. The expression of the following proteins involved in apoptosis, cell cycle regulation and the MAP-kinase pathway was studied by Western blotting: BAX, BCL-2, MCL-1, p53, p21, p27, procaspase-3, procaspase-8, procaspase-9, PARP, ERK, JNK and p38. The posphorylation status of ERK, JNK, and p38 was investigated after 5–120 minutes of treatment. Significant rates of sorafenib induced apoptosis as detected by 7AAD-FACS were seen in EHEB (46% apoptotic rate), JVM-2 (36%), GRANTA (75%), JURKAT (85%) and BL-60 (84%) after 48 hours. Cytochrome c releases, BAX cleavage as well as a down-regulation of MCL-1 and p27 were seen in all cell lines after 48 hours independent of their p53 status (no expression of p27 was detected in JVM-2). No activation of caspase-3 was seen by FACS and no cleavage product of caspase-3 could be detected by Western blotting. However, reduced levels of procaspase-3 were detected for EHEB, BL-60, NAMALWA and JURKAT. A decrease of expression of procaspase 3/8/9 was seen in NAMALWA, BL60 and JURKAT. No regulation of caspases was seen in the MCL cell lines, despite significant rates of sorafenib induced apoptosis. In BL-60 and NAMALWA, both showing a deletion of TP53 and a p53 mutation in the remaining allele, sorafenib treatment resulted in down-regulation of all proteins studied except for BCL-2 that did not show expression change; BJAB also showed reduced levels of p21, JNK and ERK after treatment, but responded with up-regulation of p53 and BCL-2 and unchanged levels for caspases. Down-regulation of BCL-2 was only seen in GRANTA, whereas EHEB responded with an up-regulation. All cell lines except for JURKAT, the only T-cell line, exhibited declining expression of proteins of the MAP-kinase pathway. Sorafenib inhibited the posphorylation of ERK in BL60, NAMALWA and JURKAT at a concentration of 1 and 10μM after 120 minutes of treatment. No change in posphorylation status was seen in the other cell lines. Although the effects of sorafenib on caspases, cell cycle regulating proteins, downstream proteins of the MAP-kinase pathway and their posphorylation status differed among the lymphoma cell lines studied, sorafenib treatment was consistently followed by typical features of apoptosis such as BAX cleavage and cytochrome c release. Induction of cell death did not depend on functional p53 gene. Furthermore, all cell lines investigated responded with down-regulation of the anti-apoptotic protein MCL-1 and the cell cycle regulator p27. Interestingly, not all cell lines responded with activation of caspases. A central role of MCL-1 operating upstream of cytochrome c release and caspase activation as well as induction of cell death by sorafenib through both caspase-dependent and -independent pathways are in line with earlier reports on other malignancies, and point to an efficacy of sorafenib in the treatment of lymphoma.