Sensitivity, Schedule-Dependence and Molecular Effects of the Proteasome Inhibitor Bortezomib in Non-Hodgkin’s Lymphoma Cells.

Bortezomib, the first of a new class of agents called proteasome inhibitors, has shown promising activity in several tumor types, especially in hematological malignancies. To further characterize its activity in Non Hodgkin Lymphoma (NHL), we determined the efficacy of bortezomib, in vitro in 19 cell lines representing five subtypes of lymphoma and in ex vivo culture of patients samples. Using an MTS cell cytotoxicity assay, bortezomib was found to induce apoptosis at lower doses (IC50 ranging from 13.2 to 19.4 nM) in Mantle Cell Lymphoma (MCL) and Diffuse Large B-Cell Lymphoma (DLBCL) cells than in Burkitt lymphoma, Primary Effusion Lymphoma (PEL) and Anaplastic Large Cell Lymphoma (ALCL). In ex vivo culture of patients samples treated with 5 or 10 nM of bortezomib, the percentage of cells undergoing apoptosis as determined by annexin V-FITC staining was greater in MCL (+28.2% ± 12 with 10nM; +12.5% ± 13.8 with 5 nM) than in DLCL samples (+13.4% ± 6.2 with 10nM; +4.9% ± 1.5 with 5nM) when compared to spontaneous apoptosis. Bortezomib also showed a much lower IC50 than conventional cytotoxic agents (doxorubicin, vincristine and 4-hydroperoxycyclophosphamide) in 4 MCL cell lines. The potential synergy between cytotoxic agents and bortezomib was studied using annexin FITC staining as well as PARP cleavage analysis. Co-treatment of MCL cell lines with bortezomib and any of these chemotherapy agents showed a synergistic effect. However, the synergy was greater if the cells were sequentially treated with doxorubicin or vincristine followed by bortezomib. On the opposite the synergistic effect was greatly reduced or abolished when the cells were pretreated with bortezomib before exposure to doxorubicin or vincristine. We then used gene expression profiling to characterize the molecular consequences of bortezomib treatment in both MCL and DLBCL lymphoma cell lines. Using a 2 fold cut-off and a p value of <0.05, a list of 120 genes was generated. The transcriptional profile of bortezomib treated cells identified genes involved in apoptosis and cell cycle in the bortezomib sensitive cell lines we tested; these changes were not observed in a bortezomib resistant PEL cell line. The transcription of several heat-shock proteins (HSP70, 90 and 105) as well as genes involved in the ubiquitin proteasome pathway was significantly increased as previously described. These expression changes were validated on all 37 genes tested using QRT-PCR. We then tested the genes expression changes in the sequential combinations on MCL cell line exposed to subtoxic doses of doxorubicin with pre, concomittant or post treatment with subtoxic dose of bortezomib as well. Results showed that the level of expression of genes involved in key cellular functions (CDK2AP1, TYMS, CHEK1, CD81, RASSF2, CCNB2, AURKB, CNAP1, PIK3CD, TRIB3, CDK2, SQSTM1) was found modified only when MCL cells were sequentially treated with doxorubicin followed by bortezomib. Altogether these results might explain why in a sequential drug administration, pre-treatment of MCL cell lines with bortezomib did not sensitize the cells to doxorubicin or vincristine. These results also provide insight into the mechanism of bortezomib activity in NHL cells as well as a rationale for the use of bortezomib in combination with conventional therapy in future clinical trials.