Abstract

8543 We found that repeated rituximab exposure leads to deregulation of Bcl-2 proteins and concomitant chemotherapy resistance. We demonstrated that obatoclax (O), a potent Bcl-2 inhibitor, enhanced the anti-tumor activity of rituximab or chemotherapy agents. The proteasome is known to regulate Bcl-2 family members expression. In the current work we study the interactions between bortezomib (B) and O against B-cell NHL. Studies were conducted in rituximab sensitive (RSCL) and resistant cell lines (RRCL), as well as in malignant B-cells derived from patients with NHL (n = 20). Cells were exposed in vitro to escalating doses of O with/without B. Cell death was determined by the Cell glow luminescent assay and DNA synthesis by [3H]-Thymidine incorporation. To further study the mechanisms of O and/or B action, RSCL or RRCL were exposed to O or B with or without caspase inhibitors; viability was evaluated as above and apoptosis by flow cytometry and PARP cleavage. Autophagy was detected by LC3 conversion and electron microscopy (EM). Changes in p53 or BH3 domain proteins were evaluated by Western blot. To determine the role of p53 and/or autophagy in O or B activity, SiRNA assays were performed. O or B monotherapy induced time- and dose-dependent cell death of all cells tested. In vitro exposure of RRCL, RSCL and lymphoma specimens to O and B resulted in significant synergistic activity. In vitro exposure of NHL cells to O lead to p53 degradation and Noxa or PUMA induction; while exposure to B resulted in Bax upregulation and Mcl-1 downregulation. The ability of O or B to induce PARP cleavage varied between patient samples and was not observed in RRCL. Inhibition of caspase activity did not affect the ability of O or B to kill NHL cells. Induction of autophagy was detected by LC3 conversion and EM not only in RSCL or RRCL but also in patient-derived tumor cells. Additionally, cell death induced by O could be inhibited by knock-down of Beclin-1 or p53. Our data suggest that O and B have a dual mechanism of action. We also provide data suggesting that both p53 and Beclin-1 have a pivotal role in response to O. Our findings strongly suggest that O added to B may result in a novel and potent therapeutic strategy against aggressive NHL. No significant financial relationships to disclose.

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