Multikinase Inhibition by Sorafenib in B and T NHL Cell Lines: Increasing Cytostatic Efficacy after Prolonged Exposure

Introduction: A substantial fraction of patients with relapsed or primarily refractory Non Hodgkin Lymphoma (NHL) can still not be cured with current chemotherapy regimens. Therefore, we evaluated the efficacy of tyrosinkinase inhibition by sorafenib against NHL cells in vitro.

Methods: Phosphorylation of RAF/MEK/ERK pathway members as well as of PDGF and VEGF receptors was analyzed by western blot with phospho-antibodies in a panel of cell lines, i.e. aggressive B-NHL (Balm3, Karpas422, Raji, Ramos, SuDHL4), mantle cell lymphoma (Granta, Jeko, Mino), and T cell lymphoma (CEM, HPB-ALL, Jurkat, Molt4). Cell viability under sorafenib exposure was tested by tetrazolium-based (MTT) assays and the proapoptotic and antiproliferative mechanisms were evaluated by annexin V and cell cycle assays, respectively. Clonogenic potential after long-term (10 days) exposure to sorafenib was analyzed by colony formation in inhibitor-free semisolid media.

Results: Sorafenib reduced cell viability at clinically relevant concentrations with IC₅₀ between 3.7 and 10.9 μM in seven cell lines, whereas four cell lines (Balm3, CEM, HPB-ALL, Granta) were less susceptible requiring IC₅₀ of >15 μM. Most sensitive were the diffuse large B cell lines, SUDHL4 and Karpas422, and the Burkitt cell lines, Ramos and Raji. Induction of apoptotic death through an annexin V positive intermediate was documented in the responding cell lines (range 20–90% at 10.9 μM) after short-term exposure (24 through 72 h). After prolonged exposure (>10 days), apoptosis increased significantly, with proportions of annexin V positive cells at 7.3 μM above 75% in all cell lines except Jeko (39% ± 1.2%). In the remaining cells, we found cell cycle arrest to occur, leading to a complete loss of in vitro clonogenicity after cessation of sorafenib exposure in all cell lines (7.3 μM). Activation of the RAF/MEK/ERK-pathway was inhibited to variable degrees in all cell lines. Intensity of RAF and ERK phosphorylation inhibition alone, however, did not predict susceptibility to sorafenib after short-term exposure, suggesting a heterogeneous and time-dependent pattern of sorafenib targets to be involved.

Conclusions: Sorafenib has significant in vitro efficacy against B and T cell lymphoma cells, especially after prolonged exposure.