Combination bortezomib and rituximab treatment affects multiple survival and death pathways to promote apoptosis in mantle cell lymphoma

Abstract

Mantle cell lymphoma (MCL) is a distinct histologic subtype of B cell non-Hodgkins lymphoma (NHL) associated with an aggressive clinical course. Inhibition of the ubiquitin-proteasome pathway modulates survival and proliferation signals in MCL and has shown clinical benefit in this disease. This has provided rationale for exploring combination regimens with B-cell selective immunotherapies such as rituximab. In this study, we examined the effects of combined treatment with bortezomib and rituximab on patient-derived MCL cell lines (Jeko, Mino, SP53) and tumor samples from patients with MCL where we validate reversible proteasome inhibition concurrent with cell cycle arrest and additive induction of apoptosis. When MCL cells were exposed to single agent bortezomib or combination bortezomib/rituximab, caspase dependent and independent apoptosis was observed. Single agent bortezomib or rituximab treatment of Mino and Jeko cell lines and patient samples resulted in decreased levels of nuclear NFkappaB complex(es) capable of binding p65 consensus oligonucleotides, and this decrease was enhanced by the combination. Constitutive activation of the Akt pathway was also diminished with bortezomib alone or in combination with rituximab. On the basis of in vitro data demonstrating additive apoptosis and enhanced NFkappaB and phosphorylated Akt depletion in MCL with combination bortezomib plus rituximab, a phase II trial of bortezomib-rituximab in patients with relapsed/refractory MCL is underway.

Keywords: CD20; apoptosis; mantle cell lymphoma; proteasome inhibition; survival and death pathways.

Figure 1

Proteosome inhibition. A spectrofluorometric assay was used to assess the proteasome activity in both Mino and Jeko cell lines treated with either (A) continuous or (B) 4 hour exposure to bortezomib (10 nM).

Figure 2

Bortezomib and/or rituximab induce apoptosis in MCL cell lines. (A and B) Assessment of cell viability by flow cytometry with Annexin V-FITC and topro 3 staining of Jeko (A) and Mino (B) cell lines, 24 and 48 hours after a continuous exposure to bortezomib (10 nM), alone or in combination with rituximab (1 µg/ml). Percentages of live, apoptotic and dead cells are shown in the lower left, lower right and upper right quadrants, respectively. (C) MCL cell lines Jeko, Mino and SP53 were treated with bortezomib (10 nM) for four hours and/or rituximab (1 µg/ml) plus cross-linker. Percent survival at 24 hours relative to the untreated controls is shown. Data are the mean of three individual experiments, and bars represent the 95% confidence interval. The combination of bortezomib plus rituximab was more effective compared to each drug alone (SP53 p = 0.069, Mino p = 0.003 and Jeko p = 0.02 vs. bortezomib alone; SP53 p = 0.006, Mino p = 0.002 and Jeko p = 0.005 vs. rituximab alone). (D) Percent survival of cells from three MCL patients at 24 hr, following treatment with bortezomib (10 nM, four hour exposure) and/or rituximab (1 µg/ml) plus cross-linker.

Figure 3A

Bortezomib induces caspase cleavage. (A) Jeko and Mino cell lines were cultured in the presence of 10 nM bortezomib (continuous exposure) and/or rituximab (1 µg/ml) plus cross-linker, harvested and lysed at 0 and 24 hours. 20 µg protein per lane was separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with antibodies to caspase 8, 9, 3 and actin. Arrows show molecular weights of caspase fragments and actin.

Figure 3B and C

Jeko (B) and Mino (C) cells were incubated with either DMSO or with 100 µM z-VAD-fmk for one hour at 37°C, then cultured in the presence of 10 nM bortezomib (continuous exposure) and/or rituximab (1 µg/ml) plus cross-linker. Cells were harvested at 24 hours and analyzed by flow cytometry with Annexin V/topro 3 and active caspase 3. Data shown are from three independent experiments.

Figure 4

PARP cleavage induced by bortezomib. Jeko (A) and Mino (B) cell lines were cultured in the presence of 10 nM bortezomib (continuous exposure) and/or rituximab (1 µg/ml) plus cross-linker. Cells were harvested at 0, 24 and 48 hours, and lysates were immunoblotted for PARP (cl-PARP, cleaved PARP; H, herceptin; B, bortezomib; R, rituximab; B + R, bortezomib + rituximab).

Figure 5

Mcl-1 cleavage induced by bortezomib. Jeko (A) and Mino (B) cell lines were cultured in the presence of 10 nM bortezomib (continuous exposure) and/or rituximab (1 µg/ml) plus cross-linker, harvested at 0, 12, 24 and 48 hours and immunoblotted for Mcl-1 (Mcl-1l, pro-form; Mcl-1s, cleaved form; H, herceptin; B, bortezomib; R, rituximab; B + R, bortezomib + rituximab).

Figure 6

NFκB inhibition after treatment with bortezomib and/or rituximab. p65 activity in nuclear extracts collected at 12 hours from treated Jeko (A) and Mino (B) cells, as well as patient samples (C and D). Relative NFκB activity in bortezomib plus rituximab treated Jeko and Mino cell lines was significantly reduced (p = 0.00009 and p = 0.00015 vs. control; p = 0.00035 and p = 0.000006 vs. bortezomib alone; p = 0.000015 and p = 0.000025 vs. rituximab alone, respectively). In MCL patients (C and D), combination treatment also significantly reduced NFκB activity compared to herceptin control (p = 0.015 and p = 0.033, respectively) but not compared to each treatment alone. Nuclear and cytoplasmic protein fractions collected at 6, 12 and 24 hours were prepared from Jeko (E) and Mino (F) cell lines and subjected to immunoblot analysis for p65, α-tubulin (cytoplasmic control) and Brg-1 (nuclear control) (H, herceptin; B, bortezomib; R, rituximab; B + R, bortezomib + rituximab).

Figure 7

Bortezomib alone or in combination with rituximab induces Akt downregulation. Jeko (A) and Mino (B) cell lines were cultured in the presence of 10 nM bortezomib (continuous exposure) and/or rituximab (1 µg/ml), harvested at 0, 12 and 24 hours, and immunoblotted for total (tAkt) and phosphorylated Akt (pAkt) (H, herceptin; B, bortezomib; R, rituximab; B + R, bortezomib + rituximab).